ELUCIDATION OF THE MECHANISMS OF INTERACTION BETWEEN BILE ACIDS
AND THE INTERLEUKIN-23/INTERLEUKIN-17 AXIS DURING CHOLESTASIS
By
Kate Maureen O’Brien

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Pharmacology and Toxicology – Environmental Toxicology - Doctor of Philosophy
2014

ABSTRACT
ELUCIDATION OF THE MECHANISMS OF INTERACTION BETWEEN BILE ACIDS
AND THE INTERLEUKIN-23/INTERLEUKIN-17 AXIS DURING CHOLESTASIS
By
Kate Maureen O’Brien
Cholestatic liver disease occurs when bile flow is disrupted, intrahepatically and/or
extrahepatically, leading to neutrophil accumulation and elevated bile acid concentrations
especially taurine-conjugated bile acids. The robust neutrophilic inflammatory response
propagates hepatocellular injury, which is dependent upon macrophage inflammatory protein-2
(MIP-2) expression. Previously, we demonstrated that taurocholic acid (TCA) induces the
expression of MIP-2 via ERK activation of early growth response factor-1 (Egr-1) in
hepatocytes. However, the mechanisms of bile acid induced neutrophilic inflammation are still
not well understood.
Interleukin-17A (IL-17A) is a Th17 cytokine that regulates neutrophil recruitment,
defense against bacterial infection, and inflammation induced injury in autoimmune diseases. IL17A expression is increased in patients with primary biliary cirrhosis and murine models of liver
disease. Therefore, we hypothesized that IL-17A promotes inflammation in cholestatic liver
disease. To test this hypothesis, we performed bile duct ligation (BDL), a murine model of
obstructive cholestasis, on mice treated with an anti-IL-17A antibody. The neutralization of IL17A reduced alanine aminotransferase (ALT) activity and areas of necrosis in mice subjected to
BDL. The expression of pro-inflammatory mediators and neutrophil cell count were decreased in
anti-IL-17A BDL mice. Furthermore, IL-17A synergistically enhanced MIP-2 induction by TCA
in hepatocytes. IL-17F, another member of the IL-17 family, did not affect hepatocellular injury
or inflammation in BDL mice. However, IL-17F had a similar synergistic interaction with TCA

in vitro. These data demonstrate that IL-17A is the predominant Th17 cytokine that contributes
to the pathogenesis of cholestasis.
To determine the molecular mechanisms that underlined this synergistic interaction, we
investigated known signaling pathways (i.e. C/EBPβ, p38, and JNK) that regulate IL-17A
synergistic enhancement of pro-inflammatory mediators by other cytokines. The knockdown of
C/EBPβ did not reduce IL-17A induction of MIP-2, but the upregulation of MIP-2 mRNA levels
was attenuated in TCA treated hepatocytes. However, hepatocellular injury and inflammation
was unaffected in C/EBPβ heterozygous knockout mice subjected to BDL for 3 days. Both p38
and JNK signaling pathways are activated by bile acids; whereas, treatment with IL-17A
inhibited the activation of p38 and JNK. Consistently, inhibition of p38 and JNK signaling
further promoted MIP-2 induction by bile acids. These results demonstrate the transcription
factor, C/EBPβ, as an alternative pathway by which bile acids regulate the inflammatory
response in hepatocytes. Furthermore, these data suggest that IL-17A inhibition of p38 and JNK
signaling promotes the synergistic enhancement of MIP-2 induction by bile acids.
Interleukin-23 (IL-23) is critical for Th17 cellular expansion and secretion of IL-17A.
Mice subjected to BDL had a biphasic induction of IL-23 mRNA levels similar to TCA serum
concentrations. Therefore, we tested the hypothesis that bile acids regulate hepatic IL-23
expression. IL-23 protein expression was increased in mice fed a 0.3% cholic acid diet.
Furthermore, IL-23 protein and mRNA levels were elevated in hepatocytes treated with TCA via
a JNK- and AKT-dependent manner. These data suggest that pathophysiological concentrations
of bile acids promote a positive feedback loop in the IL-23/IL-17A axis during cholestasis.
Overall, these studies demonstrate the molecular mechanisms underlying bile acid
induction of the hepatic Th17 inflammatory response in cholestatic liver disease.

Copyright by
KATE MAUREEN O’BRIEN
2014

ACKNOWLEDGEMENTS

As I look back upon my educational background, I realize that there are many people whom I owe
my success and gratitude. First, I need to thank my PhD mentor, Bryan Copple, for his guidance,
patience, and allowing me to tag along to Michigan State University. Bryan has been an excellent
teacher and friend. I would like to thank my past and present lab mates especially Katryn Allen
and Ryan Albee. Katy, thank you for the wonderful advice and friendship throughout my time at
KUMC and MSU. Ryan, I could not have finished up as soon as I did without your willingness to
help with my final experiments. I need to express my deep gratitude to my fellow graduate
students, Akie Mochizuki, Joseph Zagorski, and Alexandra Turley, for their listening ears and
constant encouragement. Joseph, thank you for reading over my thesis and being my editor. My
lab and family lab (Dr. Cheryl Rockwell) have been invaluable throughout my graduate career.
I could not have made it through my project without my committee members; Jim
Luyendyk, Bob Roth, and Anne Dorrance. Jim, I am in your debt for your encouragement to join
Bryan’s lab and to transfer to Michigan State. Bob, I am so grateful to you for allowing me to use
your lab space as I waited for my lab to be set up during the move. Anne, you are one of the major
reasons why I was brave enough to move to Michigan State with Bryan. Thank you all for
sacrificing your time and giving me your invaluable advice.
I would not have been able to maintain my sanity throughout graduate school without my
family. My parents, Dennis and Susan O’Brien, have been my counselors, cheerleaders, and I
thank God every day for giving me such wonderful parents. My twin brother, John Joseph
O’Brien, is one of the greatest gifts that God has given me. Thank you, John, for spending
1

multiple Christmas vacations in Lansing with me and for always putting me on a pedestal that I do
not deserve. To my sister and brother-in-law, Maggie and John Krisbaugh, thank you for your
support and an amazing trip to Florida.
I would like to thank my two best friends, Elena Sanchez and Nicole Thom, for their
constant support and enduring friendships. Elena, we have been friends our entire lives and I love
you like a sister. Thank you for always being there for me and the endless conversations about
PBS shows. Nicole, I cannot truly express how much your friendship has meant to me throughout
my time in college and graduate school. Finally, thank you to all my friends that I made at KUMC
and MSU for putting up with my crazy schedule and my random scientific talks.
Lastly, I need to thank Michigan State’s Center for Integrative Toxicology, as well as,
both the department of Pharmacology, Toxicology, and Therapeutics at University of Kansas
Medical Center and the department of Pharmacology and Toxicology at Michigan State
University for financial and academic support. I would like to thank all the members of the
Pharm/Tox department at Michigan State for adopting me into the department. Of course, I would
be remiss if I did not thank the financial support: NIH grant DK073566 (B.L.C) and NIH training
grant T32ES007255.

2

TABLES OF CONTENTS

LIST OF TABLES .......................................................................................................................... x
LIST OF FIGURES ....................................................................................................................... xi
KEY TO ABBREVIATIONS ..................................................................................................... xiii
Chapter 1 ......................................................................................................................................... 1
Introduction ..................................................................................................................................... 1
1.1 Anatomy and Physiology of the Liver ......................................................................... 2
1.2 Causes of Cholestasis .................................................................................................... 2
1.3 Pathogenesis of Cholestasis .......................................................................................... 4
1.4 Treatment of Cholestasis .............................................................................................. 6
1.5 Mechanism of Hepatocellular Injury during Cholestasis ......................................... 6
1.5.1 Direct Toxicity of Bile Acids ......................................................................... 6
1.5.2 Role of Inflammation in Hepatocellular Injury
during Cholestasis .......................................................................................... 9
1.5.2.1 Role of Cytokines and Chemokines in Hepatocellular Injury
during Cholestasis ........................................................................... 9
1.5.2.2 Interleukin-17A ............................................................................... 9
1.5.2.3 Interleukin-23 ................................................................................ 11
1.6 Bile Acid Signaling in Hepatocytes ............................................................................ 12
1.7 Purpose......................................................................................................................... 14
Chapter 2 ....................................................................................................................................... 26
Interleukin-17A Synergistically Enhances Bile Acid-induced Inflammation
during Obstructive Cholestasis .................................................................................................... 26
2.1 Abstract ........................................................................................................................ 27
2.2 Introduction ................................................................................................................. 27
2.3 Materials and Methods ............................................................................................... 30
2.3.1 Animal Care ................................................................................................. 30
2.3.2 Bile Duct Ligation ........................................................................................ 30
2.3.3 Hepatocyte Isolation .................................................................................... 31
2.3.4 Serum Chemistry ......................................................................................... 31
2.3.5 Protein Analysis ........................................................................................... 31
2.3.6 Immunofluorescence .................................................................................... 32
2.3.7 Immunohistochemistry ................................................................................ 32
2.3.8 Real-Time PCR ............................................................................................ 32
2.3.9 Statistics ........................................................................................................ 33
2.4 Results .......................................................................................................................... 33
2.4.1 Neutralization of IL-17A reduced neutrophil accumulation
and liver injury after BDL .......................................................................... 33
3

2.4.2 Cytokine gene expression was decreased in anti-IL-17A-treated
BDL mice ...................................................................................................... 34
2.4.3 α–Smooth Muscle Actin (α-SMA) and Type 1 Collagen levels
were unaffected by neutralization of IL-17A ............................................ 34
2.4.4 IL-17A synergistically enhances TCA-induced upregulation
of MIP-2 mRNA in primary mouse hepatocytes ....................................... 35
2.4.5 Upregulation of IL-23 mRNA and protein in bile acid-treated
primary mouse hepatocytes ........................................................................ 35
2.4.6 Signal Transduction Pathways involved in IL-23 induction by TCA ..... 36
2.4.7 Bile Acid feeding promotes hepatic IL-23 expression .............................. 36
2.5 Discussion..................................................................................................................... 37
Chapter 3 ....................................................................................................................................... 65
Role of the Mitogen Activated Protein Kinase Signaling Pathways and C/EBPβ in the
Synergistic Interaction between Bile Acids and the IL-23/IL-17A Axis .................................. 65
3.1 Abstract ........................................................................................................................ 66
3.2 Introduction ................................................................................................................. 67
3.3 Materials and Methods ............................................................................................... 69
3.3.1 Animal Care ................................................................................................. 69
3.3.2 Hepatocyte Isolation .................................................................................... 69
3.3.3 Bile Duct Ligation ........................................................................................ 69
3.3.4 Serum Chemistry ......................................................................................... 70
3.3.5 Immunohistochemistry ................................................................................ 70
3.3.6 Protein Analysis ........................................................................................... 70
3.3.7 Real-Time PCR ............................................................................................ 70
3.3.8 Statistics ........................................................................................................ 71
3.4 Results .......................................................................................................................... 71
3.4.1 Activation of C/EBPβ by IL-17A and TCA ............................................... 71
3.4.2 C/EBPβ is required for upregulation of MIP-2 and IL-23 in TCA
and IL-17A treated hepatocytes ................................................................. 71
3.4.3 C/EBPβ is not required for hepatocellular injury or inflammation
during Cholestasis ........................................................................................ 72
3.4.4 IL-17A synergistically enhances MIP-2 induction by TCA through
inhibition of p38 and JNK activation ......................................................... 72
3.5 Discussion..................................................................................................................... 73
Chapter 4 ....................................................................................................................................... 86
Identification of a Receptor that Regulates Bile Acid Induction of IL-23 ............................... 86
4.1 Abstract ........................................................................................................................ 87
4.2 Introduction ................................................................................................................. 87
4.3 Materials and Methods ............................................................................................... 89
4.3.1 Animal Care ................................................................................................. 89
4.3.2 Hepatocyte Isolation .................................................................................... 89
4.3.3 Real-Time PCR ............................................................................................ 89
4.3.4 Statistics ........................................................................................................ 89
4.4 Results .......................................................................................................................... 90
4

4.4.1 Inhibition of the Epidermal Growth Factor Receptor does not
affect upregulation of IL-23 by TCA ......................................................... 90
4.4.2 Sphingosine-1-Phosphate Receptor 2 is not required for TCA induction
of IL-23.......................................................................................................... 90
4.4.3 Inhibition of Prostaglandin E2 Receptors did not affect upregulation
of IL-23 by TCA ........................................................................................... 90
4.4.4 Lysophosphatidic Acid Receptor inhibition reduces IL-23 expression .. 91
4.5 Discussion..................................................................................................................... 91
Chapter 5 ..................................................................................................................................... 102
Role of Interleukin-17F in Hepatocellular Injury during Cholestatic Liver Disease ........... 102
5.1 Abstract ...................................................................................................................... 103
5.2 Introduction ............................................................................................................... 104
5.3 Materials and Methods ............................................................................................. 105
5.3.1 Animal Care ............................................................................................... 105
5.3.2 Bile Duct Ligation ...................................................................................... 105
5.3.3 Hepatocyte Isolation .................................................................................. 106
5.3.4 Serum Chemistry ....................................................................................... 106
5.3.5 Immunohistochemistry .............................................................................. 106
5.3.6 Real-Time PCR .......................................................................................... 106
5.3.7 Statistics ...................................................................................................... 107
5.4 Results ........................................................................................................................ 107
5.4.1 IL-17F mRNA levels are induced during cholestasis.............................. 107
5.4.2 IL-17F synergistically enhances MIP-2 induction by TCA ................... 107
5.4.3 IL-17F does not affect hepatocellular injury in bile duct ligated mice . 107
5.4.4 Pro-inflammatory cytokine mRNA levels were unaffected in IL-17F
knockout bile duct ligated mice ................................................................ 108
5.4.5 IL-17F knockout mice had elevated mRNA levels of the transcription
factors, C/EBPβ and early growth response factor (Egr-1) ................... 109
5.4.6 The removal of IL-17F promotes the induction of IL-17RC, a subunit
of the IL-17 receptor complex ................................................................... 110
5.5 Discussion................................................................................................................... 110
Chapter 6 ..................................................................................................................................... 123
Discussion..................................................................................................................................... 123
6.1 Summary and Significance....................................................................................... 124
6.2 Future Studies ........................................................................................................... 129
BIBLIOGRAPHY ....................................................................................................................... 134

5

LIST OF TABLES

Table 2.1 Primer Pair Sequences ................................................................................................. 59
Table 2.2 The Neutralization of IL-17A did not affect Markers of Cholestasis ...................... 59
Table 2.3 Additional Replicates for MIP-2 Induction by IL-17A and TCA Timecourse....... 60
Table 2.4 Additional Replicates for MIP-2 Induction by IL-17A and TCA Dose Response . 61
Table 2.5 Additional Replicates for ICAM-1 Induction by IL-17A and TCA ........................ 62
Table 2.6 Additional Replicates for TCA Induction of IL-23 Protein Expression ................. 62
Table 2.7 Additional Replicates for IL-23 Induction by IL-17A and TCA ............................. 63
Table 2.8 Additional Replicates for AKT, p38, and JNK Regulation of IL-23 Induction ..... 64
Table 3.1 Additional Replicates for MIP-2 Regulation in C/EBPβ Heterozygous
Knockout Hepatocytes ................................................................................................. 82
Table 3.2 Additional Replicates for IL-23 Regulation in C/EBPβ Heterozygous
Knockout Hepatocytes ................................................................................................. 83
Table 3.3 Additional Replicates for MIP-2 Induction by JNK Inhibition ............................... 84
Table 3.4 Additional Replicates for MIP-2 Induction by p38 Inhibition................................. 85
Table 4.1 Additional Replicates for the Inhibition of EGFR .................................................. 100
Table 4.2 Additional Replicates for the Inhibition of S1PR2 ................................................. 100
Table 4.3 Additional Replicates for the Inhibition of Prostaglandin E2 Receptors ............. 101
Table 4.4 Additional Replicates for the Inhibition of Lysophosphatidic Acid Receptors .... 101
Table 5.1 Primer Pair Sequences ............................................................................................... 121
Table 5.2 Additional Replicates of the In Vitro Studies........................................................... 122

6

LIST OF FIGURES

Figure 1.1 Basic Liver Anatomy .................................................................................................. 16
Figure 1.2 Outline of the Pathogenesis of Cholestatic Liver Disease ....................................... 18
Figure 1.3 The Mechanism of Murine Th17 Cell Differentiation and Population
Expansion..................................................................................................................... 20
Figure 1.4 Signaling Pathway of Bile Acid Induced Inflammatory Response in
Hepatocytes .................................................................................................................. 22
Figure 1.5 Previously Characterized Bile Acid Signaling in Primary Hepatocytes ............... 23
Figure 1.6 Proposed Studies Investigated in this Dissertation .................................................. 24
Figure 2.1 Role of IL-17A in the Development of Hepatocellular Injury after BDL ............. 41
Figure 2.2 Effect of IL-17A Neutralization on Hepatic Accumulation of Macrophages
in the Liver after BDL ................................................................................................ 43
Figure 2.3 Neutralization of IL-17A decreased Hepatic Neutrophils after BDL .................... 45
Figure 2.4 Neutralization of IL-17A decreased Expression of Pro-inflammatory Mediators
after BDL ..................................................................................................................... 46
Figure 2.5 Neutralization of IL-17A did not affect Liver Fibrosis 9 days after BDL ............. 48
Figure 2.6 Effect of IL-17A on Upregulation of Inflammatory Mediators by TCA ............... 50
Figure 2.7 Upregulation of IL-23 in the Liver and Primary Mouse Hepatocytes................... 52
Figure 2.8 Role of Signal Transduction Pathways in Upregulation of IL-23 by TCA ........... 54
Figure 2.9 Upregulation of IL-23 in the Liver after Bile Acid Feeding ................................... 56
Figure 2.10 Proposed Mechanism of the Interaction between Bile Acids and the
IL-23/IL-17A Axis ..................................................................................................... 58
Figure 3.1 Effect of IL-17A and TCA on C/EBPβ Activation .................................................. 77
Figure 3.2 Effect of C/EBPβ on the Synergistic Interaction between IL-17A and TCA ........ 78
7

Figure 3.3 Hepatocellular Injury and Inflammation was unaffected in C/EBPβ-/+ BDL ....... 80
Figure 3.4 Effect of IL-17A and TCA Co-treatment on p38 and JNK Activation.................. 81
Figure 4.1 Role of EGFR on IL-23 Induction by Bile Acids ..................................................... 96
Figure 4.2 Effect of S1PR2 on IL-23 mRNA Levels in Hepatocytes ........................................ 97
Figure 4.3 Effect of EP1 or EP2 on the Regulation of IL-23 in Primary Mouse
Hepatocytes .................................................................................................................. 98
Figure 4.4 LPA Receptors mediate TCA Induction of IL-23 in Hepatocytes ......................... 99
Figure 5.1 Upregulation of IL-17F mRNA Levels in Bile Duct Ligated Mice....................... 113
Figure 5.2 Effect of IL-17F on Bile Acid Induction of MIP-2 ................................................. 114
Figure 5.3 Deletion of IL-17F did not affect Hepatocellular Injury after BDL .................... 115
Figure 5.4 IL-17F Regulation of Inflammatory Mediators 2 days after BDL ...................... 117
Figure 5.5 Effect of IL-17F on Neutrophil Accumulation in Bile Duct Ligated Mice .......... 118
Figure 5.6 Regulation of Hepatic Expression of C/EBPβ and Egr-1 by IL-17F ................... 119
Figure 5.7 Hepatic Expression of the IL-17 Receptor Complex Subunit, IL-17RC,
after BDL ................................................................................................................... 120
Figure 6.1 Mechanisms of the Interaction between Bile Acids and the IL-23/IL-17A
Axis Outlined in this Dissertation ............................................................................ 132

8

KEY TO ABBREVIATIONS

MIP-2

Macrophage inflammatory protein-2

KC

Keratinocyte-derived chemokine

IL

Interleukin

C/EBP

CCAAT-enhancer binding protein

JNK

c-Jun N-terminal kinase

p38

p38 mitogen-activated kinase

EGFR

Epidermal Growth Factor Receptor

Egr-1

Early growth response factor-1

ERK1/2

Extracellular signal-regulated kinase1/2

FXR

Farnesoid X receptor

ICAM-1

Intercellular adhesion molecule-1

MAPK

Mitogen-activated protein kinase

PAI-1

Plasminogen activator inhibitor-1

PBC

Primary Biliary Cirrhosis

PSC

Primary Sclerosing Cholangitis

ALT

Alanine aminotransferase

ALP

Alkaline phosphatase

PMNs

Polymorphonuclear leukocytes/neutrophils

TCA

Taurocholic acid

CA

Cholic acid

CDCA

Chenodeoxycholic acid
9

Ccl

CC chemokine ligand

BDL

Bile duct ligation

S1PR2

Sphingosine-1-phosphate receptor 2

LPAR

Lysophosphatidic acid receptor

EP

Prostaglandin E2 receptor

CYP7a1

Cytochrome P450 7a1

BSEP

Bile salt export pump

MRP

Multidrug resistance protein

NTCP

Na+-taurocholate cotransporting polypeptide

OATP

Organic anion transport polypeptide

GGT

γ-glutamyl transpeptidase

SHP

Small heterodimer partner

RXR

Retinoid x receptor

LRH-1

Liver receptor homolog-1

HNF-4α

Hepatocyte nuclear factor-4 alpha

FGF15

Fibroblast growth factor 15

ABC

ATP-binding cassette

cAMP

Cyclic adenosine monophosphate

UDCA

Ursodeoxycholic acid

VCAM

Vascular cell adhesion molecule

PECAM

Platelet endothelial cell adhesion molecule

LFA1

Lymphocyte function-associated antigen-1

MAC1

Macrophage-1 antigen
10

CD

Cluster of differentiation

Th

T helper cell

TGF-β

Transforming growth factor-β

ROR

Retinoid-related orphan receptor

STAT

Signal transducer and activator of transcription

TRAF6

TNF receptor associated factor-6

CCl 4

Carbon tetrachloride

Col1a1

Type 1 collagen

TNF-α

Tumor necrosis factor-α

DCA

Deoxycholic acid

PTPase

Protein tyrosine phosphatase

GPCR

G-protein coupled receptors

S1PR

Sphingosine-1-phosphate receptor

PI3K/AKT

Phosphoinositide 3-kinase/AKT

FDA

Food and Drug Administration

HSC

Hepatic stellate cell

ROS

Reactive oxygen species

ASM

Acid sphingomyelinase

FASR

FAS receptor

CREB

cAMP response element-binding protein

MKP-1

Mitogen kinase phosphatase-1

MKK

Mitogen-activated protein kinase kinase

MRP

Multidrug resistance-associated protein
11

Chapter 1
Introduction

12

1.1 Anatomy and Physiology of the Liver
The liver is a multi-functional organ of the hepatobiliary tree that is located in the upper
abdominal cavity beneath the diaphragm and above the stomach, right kidney, and small
intestines. The liver is organized into hexagonal lobules that are further subdivided into three
functional zones based on the principal function of the hepatocytes in those regions. Zone 1 is the
periportal area which consists of the hepatic artery, portal vein, and bile ducts, known as the
portal triad. The functions of periportal hepatocytes are β-oxidation, gluconeogenesis, cholesterol
synthesis, bile formation, and amino acid metabolism. Hepatocytes around the central vein are
located in the centrilobular region, also known as zone 3. Centrilobular hepatocytes have a high
concentration of drug metabolizing enzymes called cytochrome P450s. Glycolysis and
lipogenesis also occur in the centrilobular region. Finally, zone 2 is the midzonal region which is
the region located between zone 1 and 3 (Sendensky and Dufour, 2011).
The flow of blood and bile are in opposite directions in the liver. Blood is supplied from two
sources that enter the liver through the portal triad. The hepatic artery delivers cardiac arterial
blood; whereas, the portal vein carries blood drained from the spleen, gastrointestinal tract, and its
associated organs. Blood flows through the sinusoids toward the central vein and coalesces into
the hepatic veins. The blood then returns to the heart via the vena cava. However, bile flows from
the centrilobular region towards the portal triad where bile is drained into the bile ducts (AbdelMisih and Bloomston, 2010). The liver anatomy is outlined in figure 1.1.
1.2 Causes of Cholestasis
Bile is a heterogeneous fluid containing electrolytes, bilirubin, proteins, phospholipids,
cholesterol, and bile acids. Bile acids are amphipathic molecules in which their formation and
movement into bile is critical for proper nutrient absorption. Cholesterol is converted to the
13

primary bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA), by cytochrome P450
7A1 (CYP7a1) in the hepatic parenchymal cell, the hepatocyte. These primary bile acids are
further metabolized by conjugation to amino acids such as taurine and glycine (Reshetnyak,
2013). Hepatocytes export bile salts into the bile canaliculi by the bile salt export pump (BSEP) or
multidrug resistance protein, MRP2 (Dawson et al., 2009). Bile acids are then stored in the
gallbladder, whereupon the release of cholecystokinin by endocrine cells of the duodenum and
proximal jejunum, elicits the release of bile acids into the small intestine. Bile acids aid in the
breakdown and absorption of fat and vitamins in the small intestine (Reshetnyak, 2013). Bile
acids can be deconjugated in the small intestine through dehydroxylation at the carbon 7 position
and/or epimerization at the carbon 3 position by bacteria. These secondary bile acids are known as
lithocholic acid (LCA) which is derived from CDCA and deoxycholic acid (DCA) which is
derived from CA (Reshetnyak, 2013). Approximately 95% of bile acids are reabsorbed by
enterocytes and recycled back to the liver via the portal vein in a process called enterohepatic
circulation (Dawson et al., 2009). Bile salts are transported into the hepatocytes via the Na+taurocholate cotransporting polypeptide (NTCP) or organic anion transport polypeptides (OATPs)
from the blood (Dawson et al., 2009). If bile transport out of hepatocytes or bile flow through the
biliary tree is disrupted, this results in a condition called cholestasis (Reshetnyak, 2013).
Cholestasis can be classified as obstructive or nonobstructive, which is further subdivided
into intrahepatic or extraheptic (Li and Crawford, 2004). Nonobstructive cholestasis results from
the destruction of intrahepatic and/or extrahepatic bile ducts due to an autoimmune reaction, such
as occurs in primary biliary cirrhosis or primary sclerosing cholangitis. In addition,
nonobstructive cholestasis can be caused by a functional defect in any of the apical or basolateral
bile acid transporters on hepatocytes which occurs in patients with progressive familial genetic
14

disorders, xenobiotic-induced cholestasis, or intrahepatic cholestasis of pregnancy. Obstructive
cholestasis results from a blockage of bile flow through the bile duct either by a tumor
compressing the bile duct or a gallstone obstructing flow. Both obstructive and nonobstructive
cholestasis cause severe hepatocellular and bile duct injury (Beuers, 2009; Li and Crawford, 2004;
Trauner et al., 1998).
1.3 Pathogenesis of Cholestasis
The diagnosis of any form of cholestatic liver disease is dependent upon serum biomarkers
and histological evaluation of a liver biopsy (Li and Crawford, 2004). Though the particular
clinical biomarkers and the histological features may be different among the different cholestatic
liver diseases, there are similar pathophysiological characteristics among the various cholestatic
diseases. Clinically, patients present with jaundice, discolored urine, pale stools, and pruritus (i.e.,
itching). During cholestasis, there is an increase in serum bilirubin, alanine aminotransferase
(ALT), alkaline phosphatase (ALP), γ-glutamyl transpeptidase (GGT), and bile acid
concentrations (Heathcote, 2007). Due to the inability to excrete excess bilirubin and copper,
hepatic histology shows a brown pigmentation from bilirubin and its glucuronides accumulating
in perivenular regions along with copper accumulation in periportal hepatocytes (Li and
Crawford, 2004).
During cholestasis, bile acid concentrations are increased in the serum and liver. Exposure
of the liver to pathophysiological concentrations of bile acids during cholestasis activates a
protective pathway that decreases bile acid synthesis and bile acid uptake into hepatocytes (Gupta
et al., 2001; Trauner et al., 1998). This is accomplished through activation of the bile acid nuclear
receptor, farnesoid X receptor (FXR) (Wang et al., 1999). Activation of FXR suppresses de novo
bile acid synthesis via two mechanisms. First, FXR heterodimerizes with retinoid X receptor
15

(RXR) and increases expression of the co-repressor, small heterodimer partner (SHP) (Chiang et
al., 2000). SHP represses the transcription factors, liver receptor homolog-1 (LRH-1) and
hepatocyte nuclear factor-4 alpha (HNF-4α), which downregulates CYP7a1 (Goodwin et al.,
2000; Gupta et al., 2001; Lee et al., 2000). Secondly, bile acids, through activation of FXR,
upregulate fibroblast growth factor 15 (FGF15) in enterocytes. FGF15 is released into the portal
circulation and activates the fibroblast growth factor receptor 4/β-Klotho complex on hepatocytes,
which represses CYP7a1 in a JNK-dependent manner (Inagaki et al., 2005; Ito et al., 2005; Song
et al., 2009). In addition to affecting bile acid synthesis, activation of FXR modifies bile acid
uptake into hepatocytes. FXR dowregulates the basolateral transporter, NTCP, in a SHPdependent manner which reduces bile acid uptake into hepatocytes (Denson et al., 2001). In
addition, FXR increases bile canalicular expression of BSEP, MDR3, and ABCG5 which promote
bile acid, phospholipid, and cholesterol secretion into the bile (Ananthanarayanan et al., 2001;
Freeman et al., 2004; Huang et al., 2003). These adaptive changes, which include decreased bile
acid synthesis and modified hepatic transport of bile acids, occur to reduce hepatic exposure to
potentially toxic bile acids.
During cholestasis, bile duct epithelial cells proliferate to repair damaged bile ducts and to
expand the biliary tree which promotes bile efflux from the liver (Munshi et al., 2011). During
this process, gastrointestinal and neuroendocrine hormones, as well as paracrine and autocrine
signaling, promote cAMP and Ca2+ mediated mitotic activity in these cells during severe
cholestatic liver injury (Munshi et al., 2011). Furthermore, the regenerative properties of the liver
are initiated at the Canal of Hering and progress throughout the liver to replace dead hepatocytes
(Li and Crawford, 2004). An imbalance between repair and damage can further contribute to the
pathogenesis of cholestasis.
16

If proper bile flow is not reestablished, the liver will become fibrotic (Hirschfield et al.,
2010). Fibrosis occurs when hepatic stellate cells and peribiliary fibroblasts differentiate into
myofibroblasts and begin to proliferate and deposit extracellular matrix, such as type 1 collagen
(Penz-Osterreicher et al., 2011). Persistent deposition of extracellular matrix ultimately leads to
hepatic dysfunction and the development of cirrhosis. Ultimately, breakdown of hepatic
architecture and function in the cirrhotic liver leads to end-stage liver failure. The pathogenesis of
cholestatic liver disease is outlined in figure 1.2.
1.4 Treatment of Cholestasis
Unfortunately, there is no effective therapy for patients with cholestatic liver disease.
Ursodiol®, ursodeoxycholic acid (UDCA), is the only FDA approved drug for patients with
cholestatic liver disease. The beneficial effects of UDCA are due to its anti-oxidant and anticholestatic effects. UDCA prevents cholestasis by stimulating choleresis (i.e., increased bile flow)
via upregulation of transporters on hepatocytes (Jazrawi et al., 1994). UDCA has only been shown
to be effective for the treatment of primary biliary cirrhosis, although the response rate is low in
these patients (Grattagliano et al., 2011; Tsochatzis et al., 2013). Most other types of cholestatic
liver disease do not show improvement with UDCA administration (Carey and Lindor, 2012). In
these patients, the only treatment available is a liver transplant. Considering the limited number of
transplantable livers, better treatment options are needed for cholestatic liver disease.
1.5 Mechanism of Hepatocellular Injury during Cholestasis
1.5.1 Direct Toxicity of Bile Acids. Due to the detergent like properties of bile acids, it
was originally proposed that hepatocellular injury during cholestasis results from direct
cytotoxicity by bile acids. In support of this, treatment of primary rodent hepatocytes with the
secondary bile acids, LCA and DCA stimulated JNK- and FAS-dependent apoptosis, suggesting
17

that hepatocyte apoptosis may be the main mechanism of liver injury during cholestasis (Gupta et
al., 2004; Qiao et al., 2001). However, studies in bile duct ligated mice, an animal model of
obstructive cholestasis, demonstrated that hepatocytes die by necrosis and not apoptosis during
cholestasis (Li and Crawford, 2004; Woolbright et al., 2013). A possible explanation for the
discrepancy between in vitro and in vivo studies is that in the in vitro studies hepatocytes were
treated with secondary conjugated bile acids which stimulate apoptosis. In vivo, however, taurineconjugated and glycine-conjugated primary bile acids are the predominant bile acids that increase
in concentration in cholestatic patients and in murine models of cholestasis, whereas
concentrations of secondary bile acids either decrease or remain unchanged (Trottier et al., 2012;
Zhang et al., 2012). In vitro, pathophysiological concentrations of taurine-conjugated bile acids do
not induce hepatocyte apoptosis (Allen et al., 2011). These studies indicate that hepatocyte injury
during cholestasis does not result from direct toxicity by bile acids. More recent studies have
suggested that inflammatory cells are key mediators of liver injury during cholestasis.
1.5.2 Role of Inflammation in Hepatocellular Injury during Cholestasis. Neutrophilic
inflammation is observed in the livers of patients with obstructive cholestasis and in animal
models of cholestasis (Gujral et al., 2003; Zimmermann et al., 2011). Neutrophils are leukocytes
that freely circulate in the blood (Galli et al., 2011; Pillay et al., 2010). These cells are critical for
host defense against invading pathogens, such as bacteria (Howard et al., 1977). At sites of
inflammation, a chemotactic gradient of various chemokines is formed which recruits neutrophils
(Kolaczkowska and Kubes, 2013). Neutrophil infiltration begins with the process of rolling within
blood vessels, a process which is facilitated by interactions between selectins on the endothelial
surface and ligands on the neutrophil. Next, neutrophils firmly adhere to the endothelium and
transmigrate into the parenchyma via an interaction between adhesion molecules, such as
18

intercellular adhesion molecule (ICAM-1/2), vascular cell adhesion molecule-1 (VCAM-1), and
platelet endothelial cell adhesion molecule (PECAM) on endothelial cells, and the integrins,
LFA1 (CD11a/CD18) and MAC1 (CD11b/CD18) on neutrophils (Kolaczkowska and Kubes,
2013). The membrane expression of LFA1 or MAC1 and the shedding of selectins lead to
neutrophil priming (Galli et al., 2011). Once neutrophils reach a site of infection, they become
activated and release reactive oxygen species and proteases that kill invading pathogens (Gujral et
al., 2003; Kolaczkowska and Kubes, 2013). In conditions where inflammation becomes
dysregulated, neutrophils can damage host tissue (Kolaczkowska and Kubes, 2013; Smith, 1994).
Studies suggest that this occurs during cholestasis, and may be responsible for much of the
hepatocellular injury that occurs.
The β 2 integrin, CD18, is a common subunit among the neutrophil integrins that
regulates the interaction between neutrophils and the endothelium (Kolaczkowska and Kubes,
2013). Studies using CD18 knockout mice have shown that this integrin is essential for
neutrophil-dependent liver injury in several models of liver disease (Gujral et al., 2003; Kodali et
al., 2006). To investigate a role for neutrophils in liver injury during cholestasis, bile duct ligation
(BDL) was performed in CD18 knockout mice. Deletion of CD18 attenuated the increase alanine
aminotransferase (ALT) activity in serum, a biomarker of hepatocellular injury, and decreased
areas of necrosis indicating a key role for neutrophils in liver injury during obstructive cholestasis
(Gujral et al., 2003). In addition to CD18, ICAM-1 has been shown to be critical for neutrophil
extravasation and hepatocyte injury in models of inflammatory liver injury. BDLs in ICAM-1
knockout mice decreased neutrophil extravasation and reduced hepatic injury (Gujral et al., 2004).
Collectively, these studies demonstrated an essential role for neutrophils in the development of
hepatocellular injury during cholestasis.
19

1.5.2.1 Role of Cytokines and Chemokines in Hepatocellular Injury during
Cholestasis. Levels of several cytokines and chemokines are increased in cholestatic patients and
in murine models of cholestasis (Georgiev et al., 2008; Lisboa et al., 2012; Plebani et al., 1999;
Zimmermann et al., 2011). Furthermore, several studies have identified cytokines and chemokines
that are important for inflammatory liver injury during cholestasis. For instance, neutralizing
antibodies against the chemokines, macrophage inflammatory protein-2 (MIP-2) and
keratinocyte-derived chemokine (KC), reduced neutrophil accumulation and hepatocellular injury
in bile duct ligated mice; thereby, indicating a key role for these chemokines in development of
inflammation during cholestasis (Wintermeyer et al., 2009). These findings are in agreement with
clinical observations which show that interleukin-8 (IL-8), the human homolog of MIP-2 and KC,
are at the highest concentrations in the serum of cholestatic patients with greatest neutrophil
infiltration into the liver (Zimmermann et al., 2011).
1.5.2.2 Interleukin-17A. In addition to cells of the innate immune system, such as
neutrophils, cells of the adaptive immune system are recruited to the liver during cholestasis. One
+

of these cell types, CD4 Th17 cells, is increased in the liver of patients with primary biliary
cirrhosis and in murine models of obstructive cholestasis (Meng et al., 2012; Rong et al., 2009).
+

Murine Th17 differentiation from naïve CD4 T cells requires transforming growth factor-β
(TGF-β) with either IL-6 or IL-21 (Korn et al., 2007; Mangan et al., 2006; Veldhoen et al., 2006).
In humans, Th17 differentiation requires TGF-β in combination with IL-1β, IL-6, IL-23, and/or
IL-21 (Liu et al., 2013; Tuomela et al., 2012; Wilson et al., 2007). These cytokines promote
induction of the orphan nuclear receptor, RORγt in mice or RORc in humans, which promote
Th17 differentiation (Ivanov et al., 2006). Furthermore, differentiation and activation of Th17
cells occurs through activation of the transcription factors, RORα and STAT3 (Figure 1.3) (Yang
20

et al., 2007; Yang et al., 2008c). Inhibition of Th17 cell population expansion reduced hepatic
neutrophil accumulation and serum concentrations of ALP and bilirubin, biomarkers of
cholestatic liver disease in CD279, a regulator of T cell proliferation, knockout mice subjected to
bile duct ligation (Licata et al., 2013). These studies demonstrate that Th17 cells also contribute to
inflammation and injury in cholestatic liver. Upon activation, Th17 cells secrete a variety of
interleukins, including IL-17A, that regulate the inflammatory response during injury.
Interleukin-17A (IL-17A) is the best characterized cytokine belonging to the Th17
lymphocyte family (Yao et al., 1995). In addition to IL-17A, the IL-17 family consists of IL-17F,
IL-17E, IL-17D, IL-17B, and IL-17C with IL-17F having the closest homology to IL-17A, and
IL-17E having the most divergent homology (Aggarwal and Gurney, 2002). IL-17A is primarily
produced by Th17 cells, but can also be secreted by natural killer T cells, natural killer cells, γδ T
cells, and neutrophils (Eustace et al., 2011; Pandya et al., 2011). IL-17A acts as a homodimer or
as a heterodimer with IL-17F (Chang and Dong, 2007). The IL-17A receptor is a heterodimer
complex composed of a subunit of IL-17RA and a subunit of IL-17RC (Toy et al., 2006). Upon
ligand binding, the receptor complex signals through the adaptor proteins, ACT1 and TRAF6
(Chang et al., 2006; Liu et al., 2011a; Onishi et al., 2010; Qian et al., 2007). The receptor complex
has a higher affinity for IL-17A homodimer and a lower affinity for IL-17F homodimers with IL17A/IL-17F heterodimers being in between (Korn et al., 2009). IL-17A is critical for host defense
against invading pathogens, such as bacteria. It primarily mediates this effect through recruitment
of neutrophils to sites of infection (Curtis and Way, 2009). Furthermore, IL-17A has been shown
to contribute to inflammation and injury in various autoimmune diseases, such as rheumatoid
arthritis (Gaffen, 2009a; Moran et al., 2009).

21

IL-17A serum concentrations and hepatic IL-17A positive Th17 cells are increased in
cholestatic liver disease patients (Lan et al., 2009; Qian et al., 2013). Similarly, IL-17A
expression is increased in the livers of bile duct ligated mice, and hepatocellular injury is reduced
in IL-17A and IL-17RA knockout mice after bile duct ligation (Meng et al., 2012; O'Brien et al.,
2013). In addition to liver injury, bile duct ligated IL-17A and IL-17RA knockout mice had
reduced liver fibrosis (Hara et al., 2013; Meng et al., 2012). Collectively, these studies
demonstrated an important role for IL-17A in the development of liver injury and fibrosis during
cholestasis. Whether IL-17A contributes to the development of neutrophilic hepatic inflammation
during cholestasis is not known. Furthermore, the mechanism by which cholestasis stimulates
production of IL-17A is not known.
1.5.2.3 Interleukin-23. As stated above, IL-23 is required for maintenance of
Th17 cells. IL-23 is principally secreted by immune cells and belongs to the interleukin-12
family in which it shares a p40 subunit and has a unique p19 subunit (Oppmann et al., 2000). IL23 is not required for Th17 differentiation, but it is critical for the maintenance and stabilization of
the Th17 population (Stritesky et al., 2008; Veldhoen et al., 2006). Similar to IL-17A, IL-23 has
been extensively investigated during autoimmune diseases such as multiple sclerosis and
rheumatoid arthritis (Chen et al., 2006; Murphy et al., 2003). In those autoimmune diseases, IL-23
promotes inflammation and injury by stimulating persistent expression of pro-inflammatory
mediators, such as IL-17A from Th17 cells (Murphy et al., 2003). Recently, the role of IL-23 in
liver diseases has been investigated. Neutralization of IL-23 reduced hepatic lesions and reduced
ALT activity in mice injected with adenovirus, a murine model of viral hepatitis (Hou et al.,
2013). In addition, neutralization of IL-23 attenuated the hepatic inflammatory response in mice
subjected to hepatic ischemia/reperfusion (Husted et al., 2006). In a model of fibrotic liver,
22

fibrosis was reduced in CCl 4 treated mice depleted of bone marrow IL-23 expression (Meng et al.,
2012). These studies demonstrate the ability of IL-23 to propagate injury in multiple disease
states. However, the effect of hepatic IL-23 expression during cholestasis is still not well
understood.
1.6 Bile Acid Signaling in Hepatocytes
The mechanism by which inflammation occurs in the liver during cholestasis is not fully
understood, however, our recent studies suggest that bile acids may be important for initiation of
this process. In cholestatic patients, the predominant bile acids that increase in serum
concentration are either taurine- or glycine- conjugated primary bile acids (Trottier et al., 2012).
Similar observations have been made in an animal model of obstructive cholestasis. Zhang et al
reported that taurocholic acid concentrations reach as high as 3 mM in the serum of bile duct
ligated mice (Zhang et al., 2012). Because bile acids activate signaling pathways in hepatocytes,
we tested the hypothesis that bile acids stimulate hepatocytes to produce pro-inflammatory
cytokines, thereby initiating inflammation during cholestasis. In these studies, primary mouse
hepatocytes were treated with taurocholic acid (TCA), at concentrations that occur in the serum
during cholestasis (Zhang et al., 2012). Although TCA did not affect hepatocyte viability, it did
upregulate various pro-inflammatory cytokines, chemokines, and adhesion molecules in primary
mouse hepatocytes in an ERK-dependent and early growth response factor-1 (Egr-1)-dependent
manner (Figure 1.4) (Allen et al., 2011; Allen et al., 2010). Furthermore, Egr-1 knockout mice
subjected to bile duct ligation had reduced hepatocellular injury and inflammation (Kim et al.,
2006). These studies demonstrated that bile acids activate ERK, which upregulates Egr-1. Egr-1,
a transcription factor, then upregulated pro-inflammatory mediators that promote hepatic

23

inflammation during cholestasis. What remains unknown, however is the mechanism by which
bile acids activate ERK and upregulate Egr-1 in hepatocytes.
Epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor that has been
shown to be activated by both primary and secondary bile acids (Dent et al., 2005). EGFR is
activated by bile acids through the generation of mitochondrial reactive oxygen species (Qiao et
al., 2001). Reactive oxygen species activate EGFR by inhibiting the protein tyrosine phosphatase
(PTPase) that deactivates EGFR (Fang et al., 2004). Ligand independent activation of EGFR in
this way stimulates downstream signaling through activation of the Raf-1/MEK/ERK pathway
(Rao et al., 2002). Whether activation of this signaling pathway is required for upregulation of
inflammatory mediators by bile acids is not known and a focus of my studies.
G-protein coupled receptors (GPCR) are seven transmembrane receptors that signal
through a specific heterotrimeric g-protein coupled to the cytoplasmic tail of the receptor. One of
those G-proteins, G αi , transactivates tyrosine kinase receptors such as EGFR (Dent et al., 2005;
Melien et al., 2000; Melien et al., 1998). For example, hepatocytes treated with pertussis toxin, an
inhibitor of G αi , prevented the phosphorylation of EGFR by conjugated bile acid. (Figure 1.5)
(Dent et al., 2005; Fang et al., 2004). However, the GPCR that promotes activation of EGFR in
hepatocytes has not been elucidated. TGR5 is a GPCR and a well characterized bile acid receptor,
but is not expressed on hepatocytes (Keitel et al., 2008; Keitel et al., 2007). A closely related
GPCR that is coupled to G αi is the spingosine-1-phosphate receptor 2 (S1PR2). Recently,
taurocholic acid was shown to activate ERK and AKT signaling pathways in a S1PR2 dependentmanner (Studer et al., 2012). The role of S1PR2 in bile acid induced inflammation has not been

24

investigated. Furthermore, whether other GPCRs are activated by bile acids and contribute to
inflammatory signaling in hepatocytes has not been studied and will be a focus of my studies.
1.7 Purpose
If proper bile flow is not restored, cholestatic liver disease can result in cirrhosis and
ultimately liver failure (Li and Crawford, 2004). At present, UDCA, the only FDA approved drug
to treat cholestasis, is only moderately effective in a small subset of cholestatic patients
(Tsochatzis et al., 2013). Therefore, it is imperative that we investigate the mechanisms that
underlie the pathogenesis of cholestasis to identify better treatment options. Because of the
importance of inflammation to the development of liver disease during cholestasis, we have
investigated the role of various inflammatory mediators in the pathogenesis of cholestasis.
Neutrophils have been shown to extravasate into the hepatic parenchyma in cholestatic patients
and in animal models of cholestasis. Gujral et al demonstrated that neutrophil infiltration
contributes to hepatocellular injury through the production of reactive oxygen species (Gujral et
al., 2003). Furthermore, the adaptive immune response, through Th17 cellular expansion, has
been shown to be activated in patients with PBC (Lan et al., 2009; Qian et al., 2013).
Differentiation and activation of Th17 cells promotes induction of the IL-23/IL-17A axis
(Aggarwal et al., 2003). What remains unknown is the role of the IL-23/IL-17A axis in cholestatic
liver disease.
Cholestatic liver disease is characterized by an increase in serum and hepatic bile acid
concentrations (Li and Crawford, 2004). We have shown previously that primary conjugated bile
acids promote an inflammatory response in hepatocytes, a process that is dependent upon ERK
activation and upregulation of the transcription factor, Egr-1 (Allen et al., 2011; Allen et al.,
2010). However, the interaction between bile acids and the Th17 cellular response during
25

cholestasis has not been investigated. Collectively, the goal of the present studies is to investigate
the role of IL-17 family members in the pathogenesis of cholestatic liver disease, and to determine
whether there is an interaction between taurine-conjugated primary bile acids and the IL-23/IL17A axis during cholestasis. The studies, organized by chapter, are diagramed in figure 1.6.

26

Figure 1.1 Basic Liver Anatomy. Liver is located in the upper abdominal cavity above the small
intestine. Blood flows from the portal triad (hepatic artery, portal vein, and bile duct) to the
central vein; whereas, bile flows from the central vein to the portal triad. The hepatic parenchymal
cells are the hepatocytes which carry out the principal functions of the liver.
27

Figure 1.1 (cont’d)
Hepatic stellate cells are located in the space of Disse. Other cell types are the Kupffer cells, the
resident macrophage, portal fibroblast, and cholangiocytes (bile duct epithelial cells). Bile acids
generated by the liver are stored in the gallbladder located underneath the liver. Upon endocrine
stimulus, the gallbladder releases bile acids into the small intestine via the common bile duct. Bile
acids are critical for proper nutrient absorption in the small intestines.

28

Figure 1.2 Outline of the Pathogenesis of Cholestatic Liver Disease. A disruption of bile flow
causes cholestatic liver disease leading to an increase in hepatic bile acid concentrations.
29

Figure 1.2 (cont’d)
Pathophysiological concentrations of bile acids induce the hepatic expression of pro-inflammatory
mediators, which promote neutrophilic inflammation and necrotic hepatocellular death.
Furthermore, the activation of the inflammatory response can lead to the differentiation of hepatic
stellate cells which will secrete extracellular matrix. An imbalance of extracellular matrices will
ultimately lead to cirrhosis. In addition, the dysregulation of bile flow promotes bile duct
proliferation. Proliferating cholangiocytes secrete growth factors that propagate the fibrotic
response during cholestasis. Collectively, these pathways will lead to end-stage liver failure.

30

Figure 1.3 The Mechanism of Murine Th17 Cell Differentiation and Population Expansion.
Exposure to transforming growth factor-β (TGF-β) with either IL-6 or IL-21 leads to the
31

Figure 1.3 (cont’d)
+

activation of the transcription factors, RORα and STAT3, which promote murine naïve CD4 T
cell differentiation to the Th17 phenotype. IL-23 maintains and stabilizes the CD4+ Th17 cell into
effector memory Th17 cells. Th17 cells secrete a variety of cytokines such as IL-17A, IL-17F, IL22, and IL-21. These cytokines further propagate the differentiation and expansion of Th17 cells.

32

Figure 1.4 Signaling Pathway of Bile Acid Induced Inflammatory Response in Hepatocytes.
Bile acids upregulate pro-inflammatory mediators such as cytokines, chemokines, and adhesion
molecules via an ERK-dependent activation of the transcription factor, early growth response
factor-1 (Egr-1) in hepatocytes.

33

Figure 1.5 Previously Characterized Bile Acid Signaling in Primary Hepatocytes. The
epidermal growth factor receptor (EGFR) and the Fas receptor are activated by conjugated bile
acids, in a ligand-independent manner. Mitochondrial reactive oxygen species (ROS) inhibits the
protein tyrosine phosphatase (PTPase) activity which will promote EGFR activation. In addition,
bile acid activation of a G αi coupled GPCR will lead to induction of EGFR signaling.
Hepatocellular apoptosis is inhibited by ERK activation. Whereas, the activation of JNK1 by the
FAS receptor promotes apoptotic cell death.

34

Figure 1.6 Proposed Studies Investigated in this Dissertation. In chapter 2, the role of the IL23/IL-17A axis during obstructive cholestasis will be investigated. Chapter 2 will demonstrate
that bile acids induce IL-23 mRNA levels in a JNK- and AKT-dependent manner. Chapter 2 also
35

Figure 1.6 (cont’d)
shows that IL-17A promotes hepatocellular injury and inflammation in a murine model of
obstructive cholestasis, and that IL-17A synergistically enhances the hepatic inflammatory
response by bile acids. Chapter 3 will investigate the mechanism(s) underlying the synergistic
interaction between IL-17A and bile acids. Chapter 4 will elucidate the receptor activated by bile
acids that regulates IL-23 mRNA levels. Lastly, the role of IL-17F during obstructive cholestasis
will be studied in Chapter 5.

36

Chapter 2
Interleukin-17A Synergistically Enhances
Bile Acid-induced Inflammation during
Obstructive Cholestasis
Kate M. O’Brien, Katryn M. Allen, Cheryl E. Rockwell, Keara Towery, James P. Luyendyk,
and Bryan L. Copple American Journal of Pathology. 2013; 183(5): 1498-1507.

37

2.1 Abstract
During obstructive cholestasis, increased concentrations of bile acids activate Erk1/2 in
hepatocytes which upregulates early growth response factor-1 (Egr-1), a key regulator of proinflammatory cytokines, such as macrophage inflammatory protein-2 (MIP-2). Activation of this
inflammatory pathway exacerbates cholestatic liver injury. Recent studies indicated that
interleukin-17A (IL-17A) contributes to hepatic inflammation during obstructive cholestasis,
suggesting that bile acids and IL-17A may interact to regulate hepatic inflammatory responses. To
investigate this, mice were treated with an IL-17A neutralizing antibody or control IgG, and
subjected to bile duct ligation. Neutralization of IL-17A prevented upregulation of proinflammatory cytokines, hepatic neutrophil accumulation, and liver injury, indicating an important
role for IL-17A in neutrophilic inflammation during cholestasis. Treatment of primary, mouse
hepatocytes with taurocholic acid (TCA) increased expression of MIP-2. Interestingly, cotreatment with IL-17A synergistically enhanced upregulation of MIP-2 by TCA. In contrast to
MIP-2, IL-17A did not affect upregulation of Egr-1 by TCA indicating that IL-17A does not affect
bile acid-induced activation of signaling pathways upstream of Egr-1. In addition, bile acids
increased expression of IL-23, a key regulator of IL-17A production, in hepatocytes in vitro and in
vivo. Collectively, these studies identify bile acids as novel triggers of the IL-23/IL-17A axis. In
addition, these studies suggest that IL-17A promotes hepatic inflammation during cholestasis by
synergistically enhancing bile acid-induced production of pro-inflammatory cytokines by
hepatocytes.
2.2 Introduction
Cholestatic liver diseases have many causes such as familial genetic disorders, xenobiotic
exposure, autoimmune disease, and tumors that disrupt bile flow (Li and Crawford, 2004).
38

Common features of most types of cholestatic liver disease is an elevation in pro-inflammatory
cytokines and hepatic accumulation of immune cells (Gehring et al., 2006; Georgiev et al., 2008;
Gujral et al., 2003). Studies in animal models have identified several immune cell types and
immunomodulatory cytokines that are essential for the pathogenesis of cholestatic liver diseases
(Gehring et al., 2006; Gujral et al., 2003; Gujral et al., 2004; Wintermeyer et al., 2009). The
mechanisms that regulate hepatic inflammation during cholestasis, however, are not fully
understood. Elucidation of these pathways could provide important insight pertinent to the
development of therapeutics for cholestatic liver disease. Our recent studies indicated an
important role for bile acids in the development of inflammation during cholestasis.
In obstructive cholestasis, elevated biliary pressure disrupts the integrity of intrahepatic
bile ducts resulting in the leakage of bile into the liver (Fickert et al., 2002). As a result,
concentrations of bile acids, in particular taurine and glycine conjugates, increase in the liver and
serum in patients and in animal models (Trottier et al., 2012; Zhang et al., 2012). During bile duct
ligation (BDL) in mice, serum concentrations of bile acids exceed 3 mM with the highest fraction
of bile acids being conjugated with taurine (Zhang et al., 2012). Modest increases in bile acid
concentrations modulate metabolic pathways in hepatocytes that promote detoxification of bile
acids (Wang et al., 1999). However, pathological concentrations of bile acids trigger a strong
inflammatory response in hepatocytes characterized by induction of diverse classes of cytokines
that promote infiltration of inflammatory cells, including neutrophils, macrophages, and
lymphocytes (Allen et al., 2011; Allen et al., 2010). Accumulating neutrophils extravasate from
sinusoids and adhere to hepatocytes, which stimulates their activation. The activated neutrophils
release toxic mediators that exacerbate hepatocyte injury during cholestasis (Gujral et al., 2003).

39

We recently identified a bile acid-activated signaling network that elicits production of
pro-inflammatory cytokines by hepatocytes. In these studies, pathological concentrations of
taurocholic acid (TCA) activated ERK1/2 and upregulated the transcription factor early growth
response factor-1 (Egr-1) in hepatocytes (Allen et al., 2011; Allen et al., 2010; Kim et al., 2006).
Egr-1 induction by TCA promotes the production of several cytokines, chemokines, and adhesion
molecules that are crucial for neutrophil accumulation and activation during cholestasis (Allen et
al., 2011; Allen et al., 2010). In addition, bile acids increased expression of many proinflammatory mediators in an Egr-1-independent manner, suggesting that bile acids promote
inflammation by multiple mechanisms (Allen et al., 2011). Collectively, these studies indicated
that bile acids are central mediators of hepatic inflammation during cholestasis.
It was recently reported that the cytokine, interleukin-17A (IL-17A), contributes to the
hepatic production of pro-inflammatory mediators and the progression of fibrosis in BDL mice
(Meng et al., 2012). IL-17A is the best characterized member of the IL-17 cytokine family and is
primarily produced and secreted by CD4+ Th17 cells (Korn et al., 2009). Differentiation of
murine naïve T-cells into Th17 effector cells is mediated by the cytokines, transforming growth
factor-β (TGF-β) with either interleukin-6 (IL-6) or interleukin-21 (IL-21) (Gaffen, 2009b; Korn et
al., 2009). In addition, interleukin-23 (IL-23) is required for maintenance and stabilization of
Th17 cells (Stritesky et al., 2008). IL-17A signals via its receptor complex, IL-17RA and IL17RC, as a homodimer or as a heterodimer with IL-17F (Gaffen, 2009b). Through adaptor
proteins, IL-17A elicits production of chemokines and cytokines that promote neutrophil
recruitment and thus an immune reaction to extracellular pathogens (Chang et al., 2006;
Schwandner et al., 2000). It was reported that IL-17A positive cells are increased in the livers of
patients with primary biliary cirrhosis (Lan et al., 2009). Similarly, it was demonstrated that IL40

17A levels are increased in mice subjected to BDL (Meng et al., 2012). In this study, liver injury
and fibrosis were reduced in IL-17RA knockout mice 3 weeks after surgery. Furthermore, there
were reduced levels of the cytokines, TNF-α, IL-1β, and IL-6, indicating an important role for IL17A in the development of liver disease during cholestasis (Meng et al., 2012).
Considering the importance of both bile acids and IL-17A to hepatic inflammation during
cholestasis, there might be a potential link between these inflammatory mediators in the
pathogenesis of hepatocellular injury during cholestasis. Accordingly in the following studies, the
hypothesis was tested that bile acids and the IL-17 signaling axis interact to promote hepatic
inflammation during cholestasis.
2.3 Materials and Methods
2.3.1 Animal Care. Studies were performed on 8-10 week old male C57BL/6 mice
(Jackson Laboratories; Bar Harbor, ME). All of the mice were maintained on a 12-h light/dark
cycle under controlled temperature (18–21°C) and humidity. Food (Rodent Chow; Harlan-Teklad,
Madison, WI) and tap water were allowed ad libitum. For bile acid feeding, mice were given AIN93M diet supplemented with 0.3% cholic acid or AIN-93M alone (Dynets Inc; Bethlehem, PA) for
one week. All of the procedures on animals were carried out in accordance with the Guide for the
Care and Use of Laboratory Animals promulgated by the National Institutes of Health.
2.3.2 Bile Duct Ligation. Mice were treated with 100 µg of anti-IL-17A antibody (clone
50104, R&D systems, Minneapolis, MN; BioCell, West Lebanon, NH) or control IgG2A (clone
54447, R&D systems; BioCell) by intraperitoneal injection 1 hour prior to surgery (Kobayashi et
al., 2010). BDL was then performed on mice as described previously (Kim et al., 2006). The mice
were given an additional 50 µg of anti-IL-17A antibody or control IgG on days 3 and 6 after
surgery. Nine days after surgery, livers and blood were collected from the mice for analysis.
41

2.3.3 Hepatocyte Isolation. Hepatocytes were isolated from mice by collagenase
perfusion as described previously (Kim et al., 2006). Hepatocytes were plated in Williams’
medium E (Invitrogen, Carlsbad, CA) supplemented with10% FBS (Sigma Aldrich; St. Louis,
MO) and penicillin-streptomycin (Sigma Aldrich). After 2 hours, hepatocytes were washed with
1X phosphate buffered saline (PBS) and cultured in serum-free Williams’ medium E. Hepatocytes
were treated with 10 ng/mL of recombinant mouse IL-17A (R&D systems) or 4mM HCl as
vehicle followed by 200 μM TCA dissolved in endotoxin free water for the time indicated. For
signal transduction studies, hepatocytes were treated with 100 nM wortmannin (Cayman Chemical;
Ann Arbor, MI), 30 μM SP600125 (Cayman Chemical), 10 μM SB203580 (Cayman Chemical),
or DMSO as vehicle in the presence or absence of 200 µM TCA (Sigma Aldrich) in endotoxin
free water for 3 hours.
2.3.4 Serum Chemistry. Serum alanine aminotransferase (ALT) activity (Thermo
Scientific; Middletown, VA), total serum bilirubin (Pointe Scientific, Inc; Canton, MI), serum
alkaline phosphatase (ALP) (Pointe Scientific, Inc; Canton, MI), and total serum bile acids
(Colorimetric Total Bile Acids Assay Kit; Bio-Quant, San Diego, CA) were measured using
commercially available kits as per manufactures’ instructions.
2.3.5 Protein Analysis. IL-23 and macrophage inflammatory protein-2 (MIP-2) proteins
were quantified in culture medium by ELISA (Biolegend; San Diego, CA). Total protein was
collected from hepatocytes lysed with RIPA buffer. Protein was separated on a 4-20%
polyacrylamide gel (Bio-Rad, Hercules, and CA). Membranes were incubated with either antiphospho-AKT (Thr308), anti-AKT, anti-phospho-JNK (Thr183/Tyr185), anti-JNK, anti-phosphop38 (Thr180/Tyr182), or anti-p38 antibodies (Cell Signal Technology; Danvers, MA).
Membranes were then incubated with the appropriate secondary conjugated with horseradish
42

peroxidase. Protein bands were detected using ECL detection kit (GE healthcare;
Buckinghamshire, UK) on a LiCor Fc (Odyssey; Lincoln, NE).
2.3.6 Immunofluorescence. Liver pieces were frozen in isopentane cooled in liquid
nitrogen for 8 minutes. Sections of frozen liver were cut and fixed in 4% formalin for 10 minutes.
The sections were incubated with either a rabbit polyclonal anti-IL-23A antibody (Abnova;
Walnut, CA), a rat anti-mouse CD68 antibody (AbD Serotec; Raleigh, NC), or a rat anti-mouse
F4/80 antibody (AbD Serotec; Raleigh, NC) in 10% goat serum diluted with PBS for 3 hours. The
sections were then incubated with the appropriate secondary antibody conjugated with Alexa
Fluor 594 (Invitrogen). CD68 and F4/80 immunostaining were quantified and described by us
previously (Copple et al., 2012).
2.3.7 Immunohistochemistry. Paraffin-embedded liver sections were incubated with
rabbit anti-rat polymorphonuclear 7/4 antigen (PMN) antibody (1:100) (AbD Serotec; Raleigh,
NC) for one hour or rabbit anti-α-smooth muscle actin antibody (1:100; Abcam; Cambridge, MA)
overnight at 4°C. Neutrophils were quantified by counting the number of positively-stained cells
in 20, 200X fields per tissue section. The analysis was performed in a blinded fashion.
2.3.8 Real-Time PCR. Trizol reagent (Sigma Aldrich) was used to isolate RNA.
Contaminating DNA was removed by using the TURBO DNA-free kit (Ambion; Austin, TX). 20
ng of RNA was reverse transcribed into cDNA in a reaction containing 1X MMLV buffer, 0.1mM
dNTPs, 10 mM DTT, 6.4 μg/mL random primers, 5.12 U/μL rRNasin, and 2.56 U/μL MMLV
reverse transcriptase Relative mRNA levels were measured using fast SYBR green reagent
(Applied Biosystems; Foster City, CA) on a 7500 Fast Real-time PCR system (Applied
Biosystems). Primer sequences are shown in Table 2.1. Primers used for these studies were intron
spanning.
43

2.3.9 Statistics. Data are expressed as mean ± SEM. When two or more groups were
analyzed, a two way analysis of variance (ANOVA) was performed. Comparison between groups
was done by using the Holm-Sidak method. A 95% confidence, p<0.05, was the criterion for
significance.
2.4 RESULTS
2.4.1 Neutralization of IL-17A reduced neutrophil accumulation and liver injury
after BDL. Previous studies demonstrated that neutrophils contribute to hepatic injury following
BDL (Gujral et al., 2003). Given that IL-17A is instrumental in driving neutrophilic inflammation,
we characterized the expression of IL-17A in the liver over time following BDL. IL-17A mRNA
levels did not increase until 5 days post-BDL (Figure 2.1A). This increase in expression peaked at
day 7 and remained elevated at 14 days. Next, we determined the role of IL-17A in the
development of hepatocellular injury and neutrophilic inflammation after BDL. Mice were treated
with either an anti-IL-17A antibody or control IgG. The mice were then subjected to BDL or sham
operation for 3 or 9 days. BDL caused an increase in serum ALT activity, which was reduced in
mice treated with anti-IL-17A antibody (Figure 2.1B). Consistent with this, neutralization of IL17A decreased the area of liver necrosis in BDL mice (Figure 2.1C-E). Notably, neutralization of
IL-17A did not affect hepatocellular injury 3 days post-surgery (data not shown), a time-point
before upregulation of IL-17A (Figure 2.1A).
To ascertain whether blocking IL-17A specifically decreased neutrophil-mediated
inflammation or instead caused a reduction in cholestasis in general, the effect of anti-IL-17A on
markers of cholestasis was investigated. Alkaline phosphatase (ALP), total bilirubin, and total bile
acids, were increased in the serum of mice 9 days after BDL (Table 2.2). Neutralization of IL-17A
did not affect the increase in these markers of cholestasis (Table 2.2).
44

Next, we tested the hypothesis that neutralization of IL-17A prevents accumulation of
inflammatory cells in the liver after BDL. Macrophages were detected in the liver by
immunohistochemical staining for F4/80 and CD68 (Figure 2.2A-D). F4/80 and CD68 positive
macrophages increased in the livers of BDL mice treated with control IgG (Figure 2.2E-F).
Neutralization of IL-17A, however, did not affect accumulation of macrophages in the liver
(Figure 2.2E-F). In addition, neutrophil numbers increased in the livers of BDL mice treated with
control IgG (Figure 2.3). In contrast to macrophages, neutralization of IL-17A decreased
neutrophil numbers after BDL (Figure 2.3).
2.4.2 Cytokine gene expression was decreased in anti-IL-17A-treated BDL mice.
Because IL-17A neutralization decreased hepatic accumulation of neutrophils, we investigated the
expression of cytokines and adhesion molecules upon neutralization of IL-17A in BDL mice.
Hepatic mRNA levels of macrophage inflammatory protein-2 (MIP-2), Cxcl5, Ccl7, plasminogen
activator inhibitor-1 (PAI-1), and intercellular adhesion molecule-1 (ICAM-1) were increased in
mice treated with control IgG and subjected to BDL for 9 days (Figure 2.4A-E). Neutralization of
IL-17A in BDL mice attenuated the increase in MIP-2, Cxcl5, Ccl7, and PAI-1 (Figure 2.4A-D).
In contrast, ICAM-1 mRNA levels were unaffected by IL-17A neutralization (Figure 2.4E).
2.4.3 α-Smooth Muscle Actin (α-SMA) and Type I Collagen levels were unaffected by
neutralization of IL-17A. Next, we determined the effect of IL-17A on the expression of
profibrogenic markers 9 days post-surgery. α-SMA and type I collagen mRNA levels were
increased to a similar extent in BDL mice treated with either control IgG or anti-IL-17A (Figure
2.5A-B). Similar to the mRNA levels, α-SMA protein, detected by immunohistochemistry, and
collagen protein, detected by trichrome staining, were not different between control IgG treated
mice and anti-IL17A treated mice subjected to BDL (Figure 2.5C-F).
45

2.4.4 IL-17A synergistically enhances TCA-induced upregulation of MIP-2 mRNA in
primary mouse hepatocytes. We previously demonstrated that TCA promotes pro-inflammatory
mediator production by hepatocytes, which is important for inflammation in the liver during
cholestasis (Allen et al., 2011; Allen et al., 2010; Kim et al., 2006). Considering that IL-17A is
also important for inflammation during cholestasis (Figures 2.3 and 2.4), we investigated whether
IL-17A affects bile acid-induced upregulation of inflammatory mediators by hepatocytes. To
determine this, hepatocytes were treated with IL-17A in the presence or absence of 200 µM TCA.
Both IL-17A and TCA alone increased MIP-2 gene expression, in primary mouse hepatocytes
(Figure 2.6A). Co-treatment with IL-17A and TCA synergistically enhanced induction of MIP-2
mRNA when compared to treatment with IL-17A or TCA alone (Figure 2.6A-B). MIP-2 protein
concentration was increased in the medium from hepatocytes treated with either IL-17A or TCA
(Figure 2.6C). Similar to MIP-2 mRNA levels, co-treatment with IL-17A and TCA substantially
increased MIP-2 protein levels when compared to hepatocytes treated with either IL-17A or TCA
alone (Figure 2.6C). We previously demonstrated that upregulation of MIP-2 in bile acid-treated
hepatocytes is Egr-1-dependent (Allen et al., 2011). Therefore, we determined the effect of IL17A on upregulation of Egr-1 by bile acids. As expected, TCA increased Egr-1 mRNA levels in
hepatocytes (Figure 2.6D). IL-17A did not affect upregulation of Egr-1 by TCA (Figure 2.6D).
Unlike MIP-2, neutralization of IL-17A in mice did not affect upregulation of ICAM-1 in
the liver after BDL (Figure 2.4E). Accordingly, we determined whether IL-17A would affect
upregulation of ICAM-1 in bile acid-treated hepatocytes. Consistent with in vivo (Figure 2.4E),
IL-17A did not enhance TCA-induced upregulation of ICAM-1 (Figure 2.6E).
2.4.5 Upregulation of IL-23 mRNA and protein in bile acid-treated primary mouse
hepatocytes. Because IL-23 promotes IL-17A production, IL-23 gene expression was measured
46

in wild-type mice subjected to BDL over a 14 day time course. IL-23 gene expression was
biphasic with an initial peak at 6 hours followed by a second peak at 5 days (Figure 2.7A). Our
previous studies demonstrated that serum and liver bile acid concentrations (Zhang et al., 2012)
followed a similar time course as IL-23 mRNA levels; thereby, suggesting that bile acids may
contribute to upregulation of IL-23 after BDL. Therefore, we tested the hypothesis that TCA
increases IL-23 production by primary mouse hepatocytes. Treatment of hepatocytes with 200 µM
TCA increased IL-23 mRNA and protein levels (Figure 2.7B-C). Similar to its effects on MIP-2,
IL-17A enhanced upregulation of IL-23 in hepatocytes by TCA (Figure 2.7D).
2.4.6 Signal Transduction Pathways involved in IL-23 induction by TCA. In primary
mouse hepatocytes, 200 µM TCA activated AKT (Figure 2.8A), c-Jun N-terminal
kinase (JNK) (Figure 2.8B), and p38 mitogen-activated kinase (p38) (Figure 2.8C). Inhibition of
PI3K/AKT or JNK signaling by wortmannin or SP600125 respectively attenuated upregulation of
IL-23 by TCA (Figure 2.8D-E). Inhibition of p38 by SB203580, however, enhanced IL-23
induction by TCA (Figure 2.8F).
2.4.7 Bile Acid feeding promotes hepatic IL-23 expression. To determine whether bile
acids increase IL-23 expression in vivo, mice were fed a diet containing 0.3% cholic acid or a
control diet for one week. By 1 week of feeding, this diet increased serum bile acid
concentrations, including TCA (Song et al., 2011). IL-23 mRNA levels were increased in the
livers of mice fed the 0.3% cholic acid diet (Figure 2.9A). Immunohistochemical staining for IL23 in the liver demonstrated increased IL-23 protein in hepatocytes and sinusoidal cells (Figure
2.9B-C).

47

2.5 Discussion
During obstructive cholestasis, neutrophils accumulate in the liver, become activated, and
exacerbate hepatocellular injury (Gujral et al., 2003). A recent study demonstrated that IL-17RA
null mice had reduced liver injury 3 weeks after BDL, suggesting that IL-17A signaling is a
prerequisite for hepatic neutrophil accumulation during obstructive cholestasis (Meng et al.,
2012). Consistent with this, our current study demonstrated that neutralization of IL-17A reduced
neutrophil numbers in the liver after BDL (Figure 2.3), which was associated with a reduction in
ALT and area of hepatic necrosis (Figure 2.1B-E). Hepatic neutrophil accumulation depends upon
upregulation of various inflammatory cytokines and adhesion molecules (Gehring et al., 2006;
Gujral et al., 2004; Wintermeyer et al., 2009). In our studies, neutralization of IL-17A attenuated
upregulation of several pro-inflammatory cytokines, including MIP-2, PAI-1, Ccl7, and Cxcl5
(Figure 2.4A-D), suggesting that IL-17A promotes hepatic neutrophil accumulation through
upregulation of pro-inflammatory mediators which stimulate neutrophil migration to the liver
during obstructive cholestasis. Unlike a previous report, however, we did not see a reduction in
markers of liver fibrosis (Figure 2.5). The reason for this discrepancy may be because we
investigated a much earlier time-point after BDL (i.e., 9 days in our study vs. 3 weeks in the
previous study) (Meng et al., 2012).
We recently demonstrated that bile acids upregulate several cytokines, chemokines, and
adhesion molecules, in hepatocytes, in an Egr-1-dependent manner (Allen et al., 2011). In
addition, we demonstrated that this process is important for neutrophil-dependent liver injury
during cholestasis (Allen et al., 2010; Kim et al., 2006). Our current studies demonstrate that IL17A is also required for upregulation of inflammatory cytokines and neutrophil-dependent liver
injury similar to bile acids. In many cell types, IL-17A alone is not a potent inflammatory
48

cytokine (Maione et al., 2009). However, IL-17A modifies the inflammatory response produced
by other cytokines, such as tumor necrosis factor-α (TNF-α) and IL-6. For example, IL-17A
synergistically enhances upregulation of pro-inflammatory mediators by TNF-α or IL-6 in
alveolar type II cells, endothelial cells, mesangial cells, astrocytes, and fibroblasts (Griffin et al.,
2012; Iyoda et al., 2010; Liu et al., 2011b; Ma et al., 2010; Ogura et al., 2008). This suggests that
IL-17A may interact with other inflammatory mediators, such as bile acids, to produce
inflammation during cholestasis. Our results demonstrate that IL-17A and TCA do interact to
promote robust production of inflammatory cytokines by hepatocytes. IL-17A alone modestly
increased MIP-2; however, MIP-2 production by hepatocytes was markedly increased in the
presence of IL-17A and TCA (Figure 2.6A-C). Bile acids activate Erk1/2 in hepatocytes, which
stimulate upregulation of Egr-1. Egr-1 then regulates production of MIP-2. Interestingly, IL-17A
did not affect upregulation of Egr-1 (Figure 2.6D), suggesting that IL-17A does not enhance
upregulation of MIP-2, by modulating bile acid signaling upstream of Egr-1.
The synergistic enhancement of MIP-2 expression by IL-17A and TCA may not only be
important for obstructive cholestasis, but could be critical in other liver diseases, including
primary biliary cirrhosis (PBC) and alcoholic hepatitis where IL-17A and bile acid concentrations
are increased (Harada et al., 2009; Lemmers et al., 2009; Takiguchi and Koga, 1988). In these
diseases, IL-17A may promote modest hepatic inflammation during early stages. When these
diseases become severe and hepatic bile acid concentrations increase, however, the interaction
between IL-17A and bile acids could severely exacerbate hepatic inflammation and disease
progression.
Bile acids may not only modify the response of hepatocytes to IL-17A but may alter the
response of other cell types, in particular Kupffer cells. Studies have shown that IL-17A
49

stimulates production of inflammatory mediators by Kupffer cells (Meng et al., 2012). In contrast
to hepatocytes, however, bile acids inhibit production of inflammatory cytokines by Kupffer cells
(Keitel et al., 2008; Sheen-Chen et al., 1998). It has been proposed that this occurs through
activation of the bile acid receptor, TGR5 (Keitel et al., 2008). Accordingly, it is possible that bile
acids may inhibit IL-17A-induced production of inflammatory cytokines by Kupffer cells during
obstructive cholestasis, which may explain the lack of TNF-α production by hepatic macrophages
during obstructive cholestasis, even though concentrations of the macrophage activators, IL-17A
and lipopolysaccharide, are increased (Gehring et al., 2006). A similar effect could occur with
other cell types that express TGR5, including bile duct epithelial cells and sinusoidal endothelial
cells (Keitel et al., 2008; Keitel et al., 2007). Our in vitro data demonstrate that hepatocytes
express higher levels of inflammatory mediators in the presences of bile acids and IL-17A
suggesting that in liver diseases, such as cholestasis, where bile acid concentrations are increased,
the production of inflammatory mediators may shift from prototypical inflammatory cells
towards hepatocytes. This type of inflammation may be more resistant to commonly used antiinflammatory drugs, such as glucocorticoids, which may explain the resistance of many types of
cholestatic liver disease to anti-inflammatory therapies (Silveira and Lindor, 2008).
IL-23 is critical for the maintenance and stabilization of CD4+ Th17 cells into effector T
helper cells (Korn et al., 2009). After BDL, IL-23 concentrations are increased, and this increase
is required for production of IL-17A (Figure 2.7A) (Meng et al., 2012). Interestingly, our studies
demonstrated that the time-course of IL-23 upregulation in the liver after BDL was biphasic and
identical to the time-course of bile acid concentration changes in the serum and liver (Figure
2.7A) (Zhang et al., 2012). This suggested that bile acids may be important for upregulation of IL23 in the liver. Consistent with this, TCA increased expression of IL-23 in primary mouse
50

hepatocytes (Figure 2.7B-C), and IL-23 protein levels were increased in hepatocytes from mice
fed a diet containing 0.3% cholic acid (Figure 2.9). This indicates that bile acids could sustain
and exacerbate a Th17 response by stimulating production of IL-23, a process that was AKT- and
JNK-dependent (Figure 2.8D-E). Similar to its effects on MIP-2, IL-17A enhanced TCA-induced
production of IL-23 by hepatocytes (Figure 2.7D). This could initiate a positive feedback loop
whereby bile acids stimulate production of IL-23 by hepatocytes which enhances IL-17A
production by Th17 cells. IL-17A then enhances bile acid-induced production of IL-23 and other
inflammatory mediators by hepatocytes. This process further enhances IL-17A production and
ultimately IL-23 and other inflammatory cytokines that exacerbate hepatic inflammation and
injury. This process is illustrated in Figure 2.10.
Our results demonstrate that IL-17A synergistically enhances bile acid-induced
production of inflammatory mediators by hepatocytes. Furthermore, we demonstrated for the first
time that bile acids stimulate hepatocytes to produce IL-23. Although our studies suggest that
inhibition of IL-17A in patients with cholestatic liver disease may be a logical therapeutic option,
systemic inhibition of IL-17A could negatively impact the ability of patients to clear bacterial
infections, which may be particularly dangerous in patients with cholestasis who are at risk for
developing infections. Accordingly, elucidation of the signaling mechanisms involved in the
interaction between IL-17A and bile acids may provide important insight into specifically
targeting the pathways involved in hepatic inflammation during cholestasis without affecting host
defense against invading pathogens.

51

Figure 2.1 Role of IL-17A in the Development of Hepatocellular Injury after BDL. (A) IL17A mRNA levels were quantified by real-time PCR in the livers of mice subjected to BDL for
a

the indicated time. Significantly different from day 0 (p<0.05). Mice were treated with either an
52

Figure 2.1 (cont’d)
anti- IL-17A antibody or isotype control. The mice were then subjected to BDL (n=10, n=11) or
sham operation (n=3,n=3). (B) ALT activity and (C-E) percent area of liver necrosis were
a

quantified at 9 days after surgery. The bar represents 100 μm. Significantly different from shamb

operated mice (p<0.05). Significantly different from isotype control BDL mice (p<0.05).

53

Figure 2.2 Effect of IL-17A Neutralization on Hepatic Accumulation of Macrophages in the
Liver after BDL. Mice were treated with either an anti-IL-17A antibody or isotype control, and
then subjected to BDL (n=4,n=5) or sham operation (n=3,n=3). At 9 days after surgery, F4/80
54

Figure 2.2 (cont’d)
positive macrophages were identified in the liver by immunostaining in (A) control IgG BDL
mice and (B) anti-IL-17A antibody BDL mice. In addition, (C) isotype control BDL and (D) antiIL-17A antibody BDL livers were stained for CD68 positive macrophages. The bar represents 100
μM. The area of positive staining was then quantified in sections of liver and expressed as a
a

fraction of the total area (E- F). Significantly different from sham-operated control mice
(p<0.05).

55

Figure 2.3 Neutralization of IL-17A decreased Hepatic Neutrophils after BDL. Neutrophils
were stained in sections of liver by immunohistochemistry. (A) Section of liver from a mouse
treated with isotype control and subjected to BDL. (B) Section of liver from a mouse treated with
anti-IL-17A antibody and subjected to BDL. Positive cells appear red in the photomicrographs.
For interpretation of the references to color in this and all other figures, the reader is referred to
the electronic version of this dissertation. The bar represents 50 μm. (C) Neutrophils were counted
a

in sections of liver (sham: n=3,n=3) (IgG BDL n=9, anti-IL-17A BDL n=10). Significantly
b

different from sham-operated mice (p<0.05). Significantly different from isotype control treated
mice subjected to BDL (p<0.05).
56

Figure 2.4 Neutralization of IL-17A decreased Expression of Pro-inflammatory Mediators
after BDL. Mice were treated with anti-IL-17A antibody or control IgG and subjected to BDL

57

Figure 2.4 (cont’d)
(n=10, n=11) or sham surgery (n=3,n=3) as detailed in the Materials and Methods. At 9 days after
surgery, mRNA levels of the indicated pro-inflammatory mediator were measured by real-time
a

b

PCR. Significantly different from sham operated mice (p<0.05). Significantly different from
BDL mice treated with control IgG (p<0.05).

58

Figure 2.5 Neutralization of IL-17A did not affect Liver Fibrosis 9 days after BDL. Mice
were treated with anti-IL-17A antibody or control IgG and subjected to BDL (n=10, n=11) or

59

Figure 2.5 (cont’d)
sham surgery (n=3, n=3) as detailed in the Materials and Methods. At 9 days after surgery, mRNA
a

levels of (A) type I collagen and (B) α-SMA were quantified. Significantly different from sham
operated mice (p<0.05). α-SMA protein was detected in the livers of BDL mice treated with (C)
control IgG or (D) anti-IL-17A by immunohistochemistry. Positive staining appears dark brown.
Sections of liver from BDL mice treated with (E) control IgG or (F) anti-IL-17A were stained
with Masson’s trichrome. The bars represent 50 μM.

60

Figure 2.6 Effect of IL-17A on Upregulation of Inflammatory Mediators by TCA. (A)
Primary mouse hepatocytes were treated with 10 ng/mL of IL-17A. The cells were then treated
with 200 µM TCA for the indicated time. MIP-2 mRNA levels were measured by real-time PCR.
61

Figure 2.6 (cont’d)
a

b

Significantly different from vehicle treated hepatocytes (p<0.05). Significantly different from
c

TCA-treated hepatocytes (p<0.05). Significantly different from IL-17A-treated hepatocytes
(p<0.05). (B) Primary mouse hepatocytes were treated with 10 ng/mL of IL-17A for 16 hours.
The cells were then treated with various concentrations of TCA. MIP-2 mRNA levels were
a

measured by real-time PCR. Significantly different from hepatocytes treated with vehicle and
TCA (p<0.05). (C) For quantification of MIP-2 protein, hepatocytes were treated with 10 ng/mL
IL-17A. The cells were then treated with 200 µM TCA for 12 hours. MIP-2 protein was quantified
a

in the medium by ELISA. Significantly different from vehicle-treated hepatocytes
b

(p<0.05). Significantly different from hepatocytes treated with either TCA or IL-17A alone
(p<0.05). Primary mouse hepatocytes were treated with 10 ng/mL of IL-17A followed by
treatment with 200 μM TCA. (D) Egr-1 and (E) ICAM-1 mRNA levels were measured by reala

time PCR. Significantly different from vehicle treated hepatocytes (p<0.05).

62

Figure 2.7 Upregulation of IL-23 in the Liver and Primary Mouse Hepatocytes. (A) Mice
were subjected to BDL or sham operation. IL-23 mRNA levels were measured in in the
a

liver. Significantly different from day 0 (p<0.05). Primary mouse hepatocytes were treated with
200 µM TCA. IL-23 (B) mRNA and (C) protein were measured after 3 and 24 hours of TCA
a

treatment. Significantly different from vehicle- treated hepatocytes (p<0.05). (D) Hepatocytes
were treated with 10 ng/mL of IL-17A followed by treatment with 200 µM TCA. IL-23 mRNA
was measured
63

Figure 2.7 (cont’d)
a

by real-time PCR (D). Significantly different from vehicle-treated hepatocytes
b

(p<0.05). Significantly different from hepatocytes treated with either IL-17A or TCA (p<0.05).

64

Figure 2.8 Role of Signal Transduction Pathways in Upregulation of IL-23 by TCA.
Hepatocytes were treated with 200 μM TCA for 30 minutes. Western blot was used to detect (A)
phospho-and total-AKT (B) phospho-and total-JNK and (C) phospho- and total-p38. Hepatocytes
65

Figure 2.8 (cont’d)
were treated with (D) 100 nM wortmannin, (E) 30 μM SP600125, or (F) 10 μM SB203580
a

without 200 μM TCA. IL-23 mRNA levels were measured by real-time PCR. Significantly
b

different from vehicle treated hepatocytes (p<0.05). Significantly different from hepatocytes
treated with TCA in the absence of inhibitor (p<0.05).

66

Figure 2.9 Upregulation of IL-23 in the Liver after Bile Acid Feeding. Mice were given either
AIN-93M diet supplemented with 0.3% cholic (n=8) or AIN-93M diet alone (n=9). (A) IL-23
67

Figure 2.9 (cont’d)
mRNA was measured in the liver by real-time PCR. Immunohistochemistry was used to detect IL23 in sections of liver from mice fed (B) AIN-93M control diet or (C) AIN-93M diet
supplemented with 0.3% cholic acid. Positive staining for IL-23 appears red in the
photomicrographs. The block arrows indicate IL-23 staining in hepatocytes. The thin arrow
indicates area of IL-23 staining in the sinusoid. The bar represents 50 μm. DAPI, which appears
blue in the photomicrographs, was used to stain nuclei.

68

Figure 2.10 Proposed Mechanism of the Interaction between Bile Acids and the IL-23/IL17A Axis. Bile acids upregulate MIP-2 and other cytokines in hepatocytes by an Egr-1-dependent
mechanism. Bile acids also upregulate IL-23 in hepatocytes through AKT and JNK activation. IL23 maintains Th17 cellular expansion and promotes production of IL-17A. Through an unknown
signaling cascade, IL-17A synergistically enhances TCA-induced production of MIP-2 and IL-23
by hepatocytes. Enhanced production of IL-23 leads to the formation of a positive feed-back loop
which further amplifies inflammation during cholestasis.

69

Table 2.1 Primer Pair Sequences
Gene
MIP-2
IL-17A
IL-23
Cxcl5
Ccl7
PAI-1
RPL13a
ICAM-1
Egr-1

Forward
5’-CCT CAA CGG AAG AAC CAA
AGA G-3’
5’-CCG CAA TGA AGA CCC TGA
TAG A-3’
5’-GTG ACC CAC AAG GAC TCA
AGG A-3’
5'-GCT GGC ATT TCT GTT GCT
GTT-3'
5’-AAG ATC CCC AAG AGG AA
TCTC A-3’
5’AGT CTT TCC GAC CAA GAG
CA-3’
5’-ACA AGA AAA AGC GGA TGG
TG-3’
5’-AAC AGT TCA CCT GCA CGG
AC-3’
5’-TGG GAT AAC TCG TCT CCA
CC-3’

Reverse
5’-CTC AGA CAG CGA GGC ACA
TC-3’
5’-TCA TGT GGT GGT CCA GCT
TTC-3’
5’-AGC AGG CTC CCC TTT GAA
GAT-3’
5'-CGG TTA AGC AAA CAC AAC
GCA-3'
5’-CAG ACT TCC ATG CCC TTC
TTT-3’
5’-ATC ACT TGC CCC ATG AAG
AG-3’
5’-TTC TCC TCC AGA GTG GCT
GT-3’
5’- GTC ACC GTT GTG ATC CCT
G-3’
5’-GAG CGA ACA ACC CTA TGA
GC-3’

Table 2.2 The Neutralization of IL-17A did not affect Markers of Cholestasis

Measurement
ALP (U/L)
Bilirubin
(mg/dL)
Serum Bile
Acid (uM)

Control IgG
Sham

Anti-IL-17A
Sham

Control IgG
Bile Duct
Ligation

146.7 ± 2.2

155.4 ± 14.3

973.6 ± 142.5

0.1 ± 0.1

0.07 ± 0.04

16.8 ± 1.2

13.6 ± 1.5

11.5 ± 0.5

274.3 ± 7.6

a

a
a

Anti-IL-17A
Bile Duct
Ligation
a

916.6 ± 51.9
a

17.4 ± 1.0

a

277.0 ± 10.9

The mice were treated with 100ug of anti-IL-17A antibody or control IgG. After 24 hours, the
mice were subjected to bile duct ligation or sham surgery. The mice were further treated with
50ug of anti-IL-17A antibody or control IgG 3 and 6 days post-surgery. The mice were sacrificed
9 days post-surgery. ALP, bilirubin, and total bile acid were measured using commercially
a

available kits. Significantly different from sham operated mice (p<0.05).
70

Table 2.3 Additional Replicates for MIP-2 Induction by IL-17A and TCA Timecourse

10 ng/mL IL17

200 μM TCA

Time (hr)

Vehicle

1

1.01±0.12,
1.00± 0.04

4.54±0.60 ,

2

1.01±0.13,
1.00±0.01

3.29±0.84 ,

5.62±1.09 ,

a

a

1.00±0.07,
1.01±0.07

3

1.18±0.49,
1.02±0.16

4

1.12±0.38,
1.04±0.21

6

a

1.29±0.08,
1.48±0.54

a

7.73± 1.13

a

a

5.53±0.80

3.10±0.24

a

a

5.33±0.72 ,

24.41±4.78 ,

a

a

5.74±1.00

6.55±2.25

a

a

6.88±1.74 ,

20.04±5.33 ,

a

a

8.76±0.90

6.83±0.70

a

a

4.53±1.91 ,

14.53±5.76 ,

a

11.65±1.47

2.92±0.11

a

10 ng/mL IL17A + 200 μM
TCA
b,c
12.26±1.70 ,
b,c

10.15±0.74

b,c

31.19±3.05

,

b,c

9.94±1.72

b,c

76.53±10.71

,

b,c

12.43±1.60

b,c

86.36±29.56

,

b,c

25.60±2.80

b,c

21.92±3.27

,

b,c

22.11±4.09

Hepatocytes were treated with 10 ng/mL of IL-17A in the presence or absence of 200 μM TCA
for the time indicated. Total RNA was isolated and reverse transcribed into cDNA. MIP-2
a

mRNA levels were measured by real-time PCR. Significantly different from vehicle treated
b

hepatocytes (p<0.05). Significantly different from IL-17A alone treated hepatocytes
c

(p<0.05) Significantly different TCA alone treated hepatocytes (p<0.05).

71

Table 2.4 Additional Replicates for MIP-2 Induction by IL-17A and TCA Dose Response
Treatment

Mean ± SEM

Vehicle

1.01±0.08, 1.03±0.17

10ng/mL IL-17A

7.21±0.36, 6.15±0.22

10 µM TCA

1.17±0.05, 1.21±0.03

50 µM TCA

2.55±0.14, 1.24±0.15

100 µM TCA

12.18±0.77, 1.13±0.08

200µM TCA

27.95±3.86, 1.65±0.14

300 µM TCA

18.88±0.09, 1.55±0.08

10ng/mL IL-17A + 10 µM TCA

8.34±1.12, 7.30±0.65

10ng/mL IL-17A + 50 µM TCA

17.66±1.52 , 7.62±0.56

10ng/mL IL-17A + 100 µM TCA

41.52±5.65 , 9.17±0.87

10ng/mL IL-17A + 200 µM TCA

36.18±15.37 , 13.83±1.44

10ng/mL IL-17A + 300 µM TCA

132.86±18.20 , 20.33±1.15

a

a

a

a

a

a

a

Hepatocytes were treated with 10ng/mL IL-17A for 16 hours. Next, hepatocytes were treated
with increasing concentrations of TCA (10-300 μM) for 3 hours. MIP-2 mRNA levels were
a

measured by real-time PCR. Significantly different from vehicle and TCA alone treated
hepatocytes (p<0.05).

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Table 2.5 Additional Replicates for ICAM-1 Induction by IL-17A and TCA
Treatment

Mean ± SEM

Vehicle

1.00±0.01, 1.00±0.06

10ng/mL IL-17A

1.34±0.11, 1.13±0.27

200 µM TCA

2.04±0.24 , 1.92±0.11

10ng/mL IL-17A + 200 µM TCA

2.33±0.03 , 2.19±0.19

a

a

a

a

Hepatocytes were pretreated with 10 ng/mL IL-17A for 16 hours and then treated with 200 μM
a

TCA for 3 hours. ICAM-1 mRNA levels were measured by real-time PCR. Significantly
different from vehicle treated hepatocytes (p<0.05).
Table 2.6 Additional Replicates for TCA Induction of IL-23 Protein Expression
Treatment

Mean ± SEM

Vehicle

18.64±2.34, 10.18±0.70

200 µM TCA

126.09±5.82 , 23.95±3.81

a

a

Hepatocytes were treated with 200 μM TCA for 24 hour. IL-23 protein concentrations were
a

measured in the media by ELISA. Significantly different from vehicle treated hepatocytes
(p<0.05).

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Table 2.7 Additional Replicates for IL-23 Induction by IL-17A and TCA
Treatment

Mean ± SEM

Vehicle

1.11±0.31

10ng/mL IL-17A

1.54±0.28

200 µM TCA

6.46±0.48

a

a,b

12.40±2.0

10ng/mL IL-17A + 200 µM TCA

Hepatocytes were pretreated with 10 ng/mL IL-17A for 16 hours, and then treated with 200 μM
a

TCA for 3 hours. IL-23 mRNA levels were measured by real-time PCR. Significantly different
b

vehicle treated hepatocytes (p<0.05). Significantly different from TCA treated hepatocytes
(p<0.05).

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Table 2.8 Additional Replicates for AKT, p38, and JNK Regulation of IL-23 Induction
Treatment

Mean ± SEM

Vehicle

1.03±0.18, 1.00±0.04

200 µM TCA

21.40±3.99 , 6.38, 0.85

100nM Wortmannin

1.05±0.08, 1.14±0.22

a

a,b

9.29±0.75

100nM Wortmannin + 200 µM TCA

a,b

, 3.06±0.93

a

a

7.49±0.55 , 4.54±0.48

10 µM SB203850
10 µM SB203850 + 200 µM TCA

a

a,b

111.79±12

.78, 23.57±4.07a,b
a

4.63±0.62 , 1.18±0.25

30 µM SP600125

a,b

16.23±1.82

30 µM SP600125 + 200 µM TCA

a,b

, 2.20±0.16

Primary murine hepatocytes were pretreated with either 100 nM Wortmannin, 30 μM SP600125,
or 10 μM SB203850 for 30 minutes. Next, they were treated with 200 μM TCA for 3 hours. ILa

23 mRNA levels were measured by real-time PCR. Significantly different for vehicle treated
b

hepatocytes (p<0.05). Significantly different from TCA treated hepatocytes (p<0.05).

75

Chapter 3
Role of the Mitogen Activated Protein Kinase
Signaling Pathways and C/EBPβ in the
Synergistic Interaction between Bile Acids
and the IL-23/IL-17A Axis

76

3.1 Abstract
During cholestasis, there is an increase in hepatic bile acid concentrations and
interleukin-17A (IL-17A) expression. Recently, we demonstrated that IL-17A synergistically
enhances bile acid induction of the chemokine, macrophage inflammatory protein-2 (MIP-2).
However, the mechanisms that underlie this synergistic interaction have not been elucidated.
The transcription factor, CCAAT-enhancer binding protein-β (C/EBPβ), is highly
expressed in hepatocytes and has been shown to mediate IL-17A enhancement of proinflammatory mediators in murine hepatocytes. Therefore, we tested the hypothesis that C/EBPβ
expression is required for the synergistic interaction between IL-17A and taurocholic acid
(TCA). Knockdown of C/EBPβ reduced MIP-2 mRNA levels in TCA treated hepatocytes;
whereas, MIP-2 expression was unaffected in IL-17A treated C/EBPβ heterozygous knockout
(C/EBPβ

-/+

) hepatocytes compared to wild-type. The synergistic induction of MIP-2 was

partially attenuated in the IL-17A and TCA treated C/EBPβ

-/+

hepatocytes. Interestingly,

hepatocellular injury and inflammation were unchanged in C/EBPβ heterozygous knockout mice
subjected to bile duct ligation, a murine model of obstructive cholestasis. These results
demonstrate that C/EBPβ is a transcriptional regulator of the bile acid induced inflammatory
response in hepatocytes.
To further evaluate the mechanism underlying the synergistic interaction between bile
acids and IL-17A, we investigated the p38 mitogen-activated kinase (p38) and c-Jun N-terminal
kinase (JNK) signaling pathways which have been shown to mediate IL-17A synergistic
interactions with various cytokines. TCA-induced activation of p38 and JNK was inhibited by
IL-17A. Pharmacological inhibition of p38 and JNK activation synergistically enhanced MIP-2

77

induction by TCA. These data suggest that the IL-17A synergistically enhances MIP-2 induction
by TCA via the inhibition of p38 and JNK activation.
3.2 Introduction
Neutrophils are required for hepatocellular injury during obstructive cholestasis (Gujral et
al., 2003). The inflammatory mediators, macrophage inflammatory protein-2 (MIP-2),
keratinocyte-derived chemokine (KC), and intercellular adhesion molecule-1 (ICAM-1) are
critical for neutrophil recruitment to the liver during cholestasis (Gujral et al., 2004;
Wintermeyer et al., 2009). We showed recently that conjugated primary bile acids, at
pathophysiological concentrations, do not affect cellular viability of hepatocytes but promote
ERK1/2-dependent upregulation of the transcription factor, early growth response factor-1 (Egr1), which upregulates MIP-2 and ICAM-1 (Allen et al., 2011; Allen et al., 2010). Interestingly,
we also demonstrated that IL-17A synergistically enhances bile acid-induced inflammatory
signaling in hepatocytes (O'Brien et al., 2013).
Interleukin-17A (IL-17A) is a Th17 cytokine that promotes neutrophil recruitment and
host defense against bacterial infections (Curtis and Way, 2009; Kono et al., 2011; Maione et al.,
2009). IL-17A positive cells are increased in patients with primary biliary cirrhosis and in the
murine models of cholestasis, bile duct ligation (BDL) and α-naphthylisothiocyanate (ANIT)
(Kobayashi et al., 2010; Lan et al., 2009). Inhibition of IL-17A with a neutralizing antibody in
bile duct ligated and ANIT treated mice reduced liver injury, hepatic expression of inflammatory
mediators, and hepatic neutrophil accumulation. These data indicate a key role for IL-17A in the
development of liver inflammation, during cholestasis (Kobayashi et al., 2010; O'Brien et al.,
2013). We further showed that IL-17A synergistically enhanced upregulation of inflammatory
mediators in bile acid-treated hepatocytes, suggesting that IL-17A promotes inflammation during

78

cholestasis by interacting with bile acid inflammatory signaling in hepatocytes (O'Brien et al.,
2013). What remains unknown, however, is the mechanism by which bile acids and IL-17A
synergistically interact.
Others have shown that IL-17A synergizes with multiple inflammatory cytokines, such as
tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), to stimulate production of
inflammatory mediators by alveolar type II cells and astrocytes, which is similar to the
interaction we observed between IL-17A and bile acids (Liu et al., 2011b; Ma et al., 2010). The
mechanisms that underlie these synergistic interactions are varied and include modulation of
mitogen activate protein kinase (MAPK) signaling by IL-17A, alterations in cytokine mRNA
stability by IL-17A, and upregulation of transcription factors (Nf-kB and C/EBPβ) by IL-17A
(Iyoda et al., 2010; Liu et al., 2011b; Patel et al., 2007; Sun et al., 2011). For example, inhibition
of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated kinase (p38) signaling prevented
the synergistic interaction between IL-6 and IL-17A in astrocytes (Ma et al., 2010). Since bile
acids upregulate inflammatory mediators in hepatocytes through MAPK signaling, it is possible
that IL-17A may enhance bile acid-induced inflammatory signaling by modulating MAPK
signaling (Allen et al., 2010; Rao et al., 2002). Another mechanism by which this may occur is
through upregulation of various transcription factors by IL-17A.
IL-17A signaling is mediated through the receptor complex, IL-17RA/IL-17RC, which
activates the transcription factors, NF-kB and C/EBPβ (Maitra et al., 2007; Sonder et al., 2011).
In most cell types, however, IL-17A is a weak activator of NF-kB (Granet et al., 2004; Shen and
Gaffen, 2008). Nuclear translocation of C/EBPβ requires phosphorylation of regulatory domain 2
(RD2) (Williams et al., 1995). IL-17A promotes phosphorylation of C/EBPβ on two different
threonine residues, Thr188 and Thr179, via ERK and glycogen synthase kinase-3β (GSK3β)

79

(Shen et al., 2009). It has been demonstrated that C/EBPβ expression is critical for the
synergistic induction of IL-6 by TNF-α and IL-17A, in murine embryonic fibroblasts (Ruddy et
al., 2004). Considering the potential of C/EBPβ to mediate synergistic interactions between IL17A and other mediators, we also investigated its role in the interaction between bile acids and
IL-17A.
3.3 Materials and Methods
3.3.1 Animal Care. All experiments were performed on either male C57BL/6 wild-type,
C/EBPβ heterozygous knockouts or C/EBPβ wild-type littermates (Jackson Laboratories; Bar
Harbor, ME). All of the mice were maintained on a 12-h light/dark cycle under controlled
temperature (18–21°C) and humidity. Food (Rodent Chow; Harlan-Teklad, Madison, WI) and
tap water were allowed ad libitum. All of the procedures on animals were carried out in
accordance with the Guide for the Care and Use of Laboratory Animals promulgated by the
National Institutes of Health.
3.3.2 Hepatocyte Isolation. Hepatocytes were isolated by collagenase H retrograde
perfusion as described previously (Kim et al., 2006). Hepatocytes were treated with 10 ng/mL of
mouse recombinant IL-17A reconstituted in 4 mM HCl (R&D systems; Minneapolis, MN) for
either 16 hours or 30 minutes followed by treatment with 200 μM taurocholic acid (TCA) in
endotoxin free water for the time indicated. For signal transduction studies, hepatocytes were
treated with 10 µM SB203850, 30 µM SP600125, or DMSO as vehicle for 30 minutes followed
by treatment with 200 µM TCA for 3 hours.
3.3.3 Bile Duct Ligation. C/EBPβ heterozygous knockouts or C/EBPβ wild-type
littermates (Jackson Laboratories; Bar Harbor, ME) were anesthetized with isoflurane. A midline
laparotomy was performed and the bile duct ligated with 4-0 surgical silk. The abdominal

80

incision was closed with sutures, and the mice received 0.2 mg/kg buprenex by subcutaneous
injection twice daily. Mice were sacrificed 72 hours after surgery.
3.3.4 Serum Chemistry. Serum alanine aminotransferase (ALT) activity was measured
by a commercially available kit (Thermo Scientific; Middletown, VA) per manufacturers’
instructions.
3.3.5 Immunohistochemistry. Paraffin-embedded liver sections were incubated with a
rat anti-mouse polymorphonuclear leukocytes (PMN) 7/4 antibody (1:2500) (AbD Serotec;
Raleigh, NC) for one hour. Neutrophils were quantified by counting the number of positivelystained cells in 10, 200X fields per tissue section. The analysis was performed in a blinded
fashion.
3.3.6 Protein Analysis. Hepatocytes were lysed in RIPA buffer containing protease and
phosphatase inhibitors (Thermo Scientific; Rockford, IL). Protein was separated on a 4-20%
polyacrylamide gel and transferred to PVDF membrane. The membranes were incubated with
either an anti-p38, anti-phosphorylated p38 (Thr180/Tyr182), anti-JNK, anti-phosphorylated
JNK (Thr183/Tyr185), anti-C/EBPβ, or anti-phosphorylated C/EBPβ (Thr188) antibodies (all
from Cell Signal Technology; Danvers, MA). The membranes were then incubated with the
appropriate secondary antibody conjugated to horseradish peroxidase. The bands were detected
using the ECL chemiluminescence detection kit (GE Healthcare; Buckinghamshire, UK) on a
LiCor Fc (Odyssey; Lincoln, NE).
3.3.7 Real-Time PCR. Total RNA was isolated by Trizol extraction, and contaminating
DNA was removed using the Turbo DNA-free kit (Ambion; Austin, TX) as per manufacturer’s
instructions. RNA was reverse transcribed into cDNA as described in 2.3.8. mRNAs were
analyzed on a 7500 Fast Real-time PCR system (Applied Biosystems; Foster City, CA) by using

81

the fast SYBR green reagent (Bio-rad; Hercules, CA). Primer sequences are outlined in Table
2.1. All primers used were intron spanning.
3.3.8 Statistics. Data are expressed as mean ± SEM. When two or more groups were
analyzed, a two way analysis of variance (ANOVA) was performed. When the data set did not
pass normality, a log transformation was utilized. Comparison between groups was done by
using the Holm-Sidak method. A 95% confidence, p<0.05, was the criterion for significance.
3.4 Results
3.4.1 Activation of C/EBPβ by IL-17A and TCA. There are multiple downstream
pathways activated by IL-17A in various cells, including C/EBPβ (Shen et al., 2009). Therefore,
we evaluated whether C/EBPβ is activated in primary murine hepatocytes treated with IL-17A
and TCA. Treatment of hepatocytes with either IL-17A or TCA stimulated phosphorylation of
C/EBPβ, indicating activation. Surprisingly, co-treatment with IL-17A and TCA did not
stimulate phosphorylation of C/EBPβ. Instead, there was a reduction of phosphorylated C/EBPβ
after 10 minutes in the co-treated hepatocytes (Figure 3.1).
3.4.2 C/EBPβ is required for upregulation of MIP-2 and IL-23 in TCA and IL-17A
treated hepatocytes. We next tested the hypothesis that activation of C/EBPβ is required for the
synergistic interaction between IL-17A and TCA. To test this hypothesis, hepatocytes were
isolated from C/EBPβ heterozygous knockout mice or wild-type littermates. For confirmation,
we measured the protein expression of C/EBPβ in these livers. Hepatic C/EBPβ expression was
decreased in the C/EBPβ heterozygous knockout mice, compared to their wild-type littermates
(Figure 3.2A). Knockdown of C/EBPβ did not affect upregulation of MIP-2 or IL-23 by IL-17A
(Figure 3.2B-C). However, MIP-2 and IL-23 mRNA levels were reduced in TCA treated
C/EBPβ heterozygous knockout hepatocytes, when compared to wild-type hepatocytes.

82

Furthermore, the synergistic enhancement of MIP-2 and IL-23 was partially attenuated in
C/EBPβ heterozygous knockout hepatocytes treated with IL-17A and TCA (Figure 3.2B-C).
These results demonstrate a critical role for C/EBPβ in upregulation of MIP-2 and IL-23 in
hepatocytes by TCA.
3.4.3 C/EBPβ is not required for hepatocellular injury or inflammation during
cholestasis. Considering the importance of C/EBPβ for upregulation of inflammatory mediators
by bile acids in vitro, we performed bile duct ligation in C/EBPβ heterozygous knockout mice
and their wild-type littermates to determine its regulation of inflamed-induced hepatocellular
injury during cholestasis. Alanine aminotransferase (ALT) activity, a clinical biomarker of
hepatocellular injury, was increased in wild-type mice subjected to BDL. Partial deletion of
C/EBPβ did not affect the increase in ALT activity in BDL mice (Figure 3.3A).
Next, we determined the effect of C/EBPβ on the inflammatory response during
cholestasis. MIP-2 mRNA levels were increased in wild-type mice subjected to BDL.
Upregulation of MIP-2 trended lower in C/EBPβ heterozygous knockout mice subjected to bile
duct ligation; however, it was not significant (Figure 3.3B). IL-23 was not increased in either
wild-type or C/EBPβ heterozygous knockout mice subjected to BDL (Figure 3.3C). Consistent
with the cytokine mRNA levels, there was no difference in neutrophil cell count in the C/EBPβ
heterozygous knockout BDL mice compared to their wild-type counterparts (Figure 3.3D).
3.4.4 IL-17A synergistically enhances MIP-2 induction by TCA through inhibition
of p38 and JNK activation. Studies have shown that bile acids activate the p38 and JNK
signaling pathways (Gupta et al., 2004; O'Brien et al., 2013; Qiao et al., 2003; Schoemaker et al.,
2004). Therefore, we investigated whether these pathways are required for upregulation of MIP2 by bile acids and whether their activation was affected by IL-17A. Treatment of hepatocytes

83

with IL-17A partially decreased phosphorylation of JNK and p38. Treatment with TCA
increased phosphorylation of JNK and p38. Interestingly, IL-17A prevented phosphorylation of
JNK and p38 by TCA (Figure 3.4A-B). Since IL-17A inhibited activation of JNK and p38 by
TCA, we next determined the impact of blocking the signaling of these pathways with
pharmacological inhibitors on upregulation of MIP-2 by TCA. Inhibition of JNK and p38
enhanced MIP-2 mRNA production by TCA; thereby, suggesting that IL-17A may induce
upregulation of MIP-2 by TCA through the inhibition of JNK and p38 (Figure 3.4C-D).
3.5 Discussion
Pathophysiological concentrations of bile acids produce hepatic inflammation via the
induction of MIP-2 expression in hepatocytes (Allen et al., 2011). IL-17A is a pro-inflammatory
cytokine, produced by Th17 cells, that has been shown to maintain and prolong neutrophilic
inflammatory responses through a synergistic interaction between cytokines in various disease
states, including cholestasis (Griffin et al., 2012; Liu et al., 2011b; Maione et al., 2009). We
showed previously that there is a synergistic induction of MIP-2 in hepatocytes exposed to IL17A and TCA (O'Brien et al., 2013). However, the signaling pathway(s) that mediate the synergy
between hepatocytes and Th17 cells is not understood.
Previous studies have shown that IL-17A synergistically enhances production of
inflammatory mediators by TNF-α. The synergistic interaction between IL-17A and TNF-α was
shown to require the activation of the transcription factor, C/EBPβ, in mouse embryonic
fibroblasts (Ruddy et al., 2004). A previous study demonstrated that IL-17A promotes the
phosphorylation of the full length liver activating protein (LAP) isoform of C/EBPβ (Maitra et
al., 2007) (Figure 3.1). Shen et al demonstrated that IL-17A induces the phosphorylation of
C/EBPβ on two threonine residues in a ERK- and GSK3β-dependent manner, which represses

84

gene transcription of IL-6, Ccl7, and Cxcl5, in stromal cells (Shen et al., 2009). ERK is a known
regulator of the transcription factor, Egr-1 (Allen et al., 2010). Previously, we have shown that
Egr-1 is critical for TCA induction of MIP-2 mRNA levels and inflammation induced injury, in a
murine model of cholestasis (Allen et al., 2010; Kim et al., 2006). C/EBPβ and Egr-1 are known
to have a protein-protein interaction that prolongs the transcription of target genes (Jakobsen et
al., 2013; Zhang et al., 2003). Furthermore, C/EBPβ has been studied in hepatocyte proliferation,
liver repair, and steatosis (fatty liver disease) due to its high expression in the liver (Birkenmeier
et al., 1989; Greenbaum et al., 1998; Rahman et al., 2007). Therefore, we investigated the role of
C/EBPβ in the synergistic enhancement of MIP-2 by IL-17A and TCA in murine hepatocytes.
The knockdown of C/EBPβ decreased MIP-2 mRNA levels after TCA treatment and attenuated
the synergistic induction of MIP-2 by TCA and IL-17A (Figure 3.2B). Interestingly, there was a
complete prevention of the synergistic induction of IL-23 in C/EBPβ heterozygous knockout
hepatocytes (Figure 3.2C). Our results suggest a critical role for C/EBPβ in bile acid induction of
the inflammatory response by hepatocytes. However, C/EBPβ was not required for IL-17A
signaling in hepatocytes (Figure 3.2B-C). Therefore, bile acid and IL-17A synergistic induction
of the inflammatory response could require the interaction of both C/EBPβ and Egr-1 on the
response elements of the inflammatory genes. Our in vitro results suggest that there are either
distinct pathways that mediate the synergistic interaction, or the enhancement of MIP-2 requires
the sequential activation of another transcription factor, such as Egr-1.
To further investigate C/EBPβ expression in bile acid regulation of the inflammatory
response, we subjected C/EBPβ heterozygous knockout mice to bile duct ligation for 72 hours.
Surprisingly, the knockdown of C/EBPβ did not affect hepatocellular injury (Figure 3.3A). In
bile duct ligated mice, hepatocyte proliferation and repair begins around day 2 post-surgery

85

(Georgiev et al., 2008). It has been demonstrated that the deletion of C/EBPβ delays entrance
into S phase of the cell cycle in mice that have undergone partial hepatectomy (Greenbaum et al.,
1998; Jakobsen et al., 2013). Due to the fact that the C/EBPβ heterozygous knockout mice
underwent BDL for 3 days, the impairment of liver regeneration could explain the similar serum
ALT activity between the two groups. In addition, inflammation was unaffected in C/EBPβ
heterozygous knockout mice compared to wild-type (Figure 3.3B-D). Although, MIP-2 mRNA
levels had a lower trend in the C/EBPβ heterozygous BDL mice (Figure 3.3B). The difference
between our in vivo and in vitro results could be explained by a compensatory mechanism
promoted by C/EBPδ, another isoform that is also induced by IL-17A in mouse embryonic
fibroblast and mediates IL-17A synergistic enhancement of IL-6 (Maitra et al., 2007; Ruddy et
al., 2004; Shen et al., 2005). However, TCA may not induce C/EBPδ in primary hepatocytes,
which may explain why we observed a C/EBPβ-dependent regulation of MIP-2 induction.
Hepatocyte specific C/EBPβ knockout mice subjected to BDL would better indicate the role of
C/EBPβ in the inflammatory response. Overall, these results further suggest that IL-17A
synergistic interaction with TCA requires the activation of additional signaling cascades besides
C/EBPβ.
Next, we examined known MAPK signaling pathways that are activated by either IL-17A
or bile acids (Chen et al., 2011; Patel et al., 2007; Qiao et al., 2003). The synergistic interaction
between IL-17A and multiple cytokines (TNF-α and IL-6) requires activation of either p38 or
JNK in mesangial cells and astrocytes (Iyoda et al., 2010; Ma et al., 2010). Furthermore, bile
acid activation of the JNK pathway regulates activation of the pro-apoptotic cascade in rodent
hepatocytes (Qiao et al., 2003). Therefore, we determined the protein expression of phosphoJNK and phospho-p38, in hepatocytes treated with IL-17A in the presence or absence of TCA

86

(Figure 3.4A-B). Contrary to other cell types, IL-17A inhibited activation of JNK and p38
(Figure 3.4A-B). In addition, inhibition of JNK and p38 with pharmacological inhibitors
synergistically enhanced MIP-2 mRNA levels in TCA treated hepatocytes (Figure 3.4C-D).
These studies suggest that the synergistic enhancement of MIP-2 occurs through IL-17A
inhibition p38 and JNK signaling. An underlying cause of this inhibition might be that IL-17A
promotes activation of a protein phosphatase that would deactivate either p38 or JNK signaling.
IL-17A inhibits the activity of MAPK phosphatase-1 (MKP-1) in cardiac fibroblasts (Valente et
al., 2012). Due to the divergent cellular response from our results and those in other cells, IL17A may activate MKP-1 in hepatocytes, which promotes the deactivation of the p38 or JNK
signaling pathways. The role of p38 and JNK in MIP-2 synergistic induction still needs further
investigation.
In conclusion, we have demonstrated that C/EBPβ mediates upregulation of proinflammatory chemokines in primary mouse hepatocytes by TCA in vitro. Furthermore, our
results suggest an involvement of the MAPK signaling pathways in the synergistic interaction
between IL-17A and bile acids in hepatocytes.

87

Figure 3.1 Effect of IL-17A and TCA on C/EBPβ Activation. Hepatocytes were pretreated
with 10 ng/mL IL-17A for 30 minutes. Then, hepatocytes were treated with 200 µM TCA for the
time indicated. Western blots were incubated with either an anti-phospho-C/EBPβ or an antiC/EBPβ. Bands were detected using ECL chemiluminescence detection kit.

88

Figure 3.2 Effect of C/EBPβ on the Synergistic Interaction between IL-17A and TCA.
Whole liver from either C/EBPβ heterozygous knockout or wild-type mice were homogenized in
RIPA buffer consisting of protease and phosphatase inhibitors to collect total protein. Total
protein was separated on a 4-20% polyacrylamide gel and transferred to a PVDF membrane. (A)
The membrane was incubated with either an anti-C/EBPβ (LAP) antibody or an anti-β-actin
antibody. Bands were detected using an ECL chemiluminescence detection kit. Hepatocytes
were isolated from C/EBPβ heterozygous knockout mice or their wild-type littermates.

89

Figure 3.2 (cont’d)
Hepatocytes were pretreated with 10 ng/mL IL-17A for 16 hours and then treated with 200 µM
TCA for 3 hours. (B) MIP-2 and (C) IL-23 mRNA levels were measured by real-time
a

b

PCR. Significantly different from wild-type vehicle treated hepatocytes (p<0.05). Significantly
c

different from wild-type TCA treated hepatocytes (p<0.05). Significantly different from wildtype IL-17A treated hepatocytes (p<0.05).

90

Figure 3.3 Hepatocellular Injury and Inflammation was unaffected in C/EBPβ

-/+

BDL.

C/EBPβ heterozygous (n=3, n=7) and wild-type littermates (n=3, n=6) were subjected to sham or
bile duct ligation. (A) Serum ALT activity was quantified 72 hours post-surgery. (B) MIP-2 and
a

(C) IL-23 mRNA was measured by real time PCR. Significantly different from wild-type sham
operated mice (p<0.05). Neutrophils were stained in paraffin embedded liver sections by
immunohistochemistry. (D) Neutrophils were counted in sections of liver.

91

Figure 3.4 Effect of IL-17A and TCA Co-treatment on p38 and JNK Activation.
Hepatocytes were pretreated with 10 ng/mL IL-17A for 30 minutes and then treated with 200 μM
TCA for 15 minutes. Western blots were incubated with either (A) anti-phospho-JNK or antiJNK and (B) anti-phospho-p38 or anti-p38. Hepatocytes were pretreated with either (C) 30 µM
SP600125 or (D) 10 µM SB203580 for 30 minutes and then treated with 200 µM TCA for 3
a

b

hours. Significantly different from vehicle treated hepatocytes (p<0.05). Significantly different
c

from inhibitor treated hepatocytes (p<0.05). Significantly different from TCA treated
hepatocytes.
92

Table 3.1 Additional Replicates for MIP-2 Regulation in C/EBPβ Heterozygous Knockout
Hepatocytes
Treatment

Genotype

Mean ± SEM

Vehicle

Wild-type

1.01±0.11, 1.00±0.07

10ng/ml IL-17A

Wild-type

4.66±0.52 , 4.04±0.22

200 μM TCA

Wild-type

8.91±0.54 , 8.24±0.09

a

a

a

a

a,b,c

36.55±2.89

200 μM TCA + 10ng/mL IL-

,

Wild-type
a,b,c

17A

35.27±3.04

Vehicle

C/EBPβ Heterozygous

1.03±0.19, 1.02±0.13

10ng/mL IL-17A

C/EBPβ Heterozygous

1.94±0.15, 4.35±0.35

200 μM TCA

C/EBPβ Heterozygous

200 μM TCA + 10ng/mL IL-

a

a,b

4.82±0.63

, 4.33±0.32

a,c

C/EBPβ Heterozygous

a,b

a,b,c

10.62±0.99 , 18.55±1.10

17A

Hepatocytes were isolated from C/EBPβ heterozygous knockout mice or their wild-type
littermates. Hepatocytes were pretreated with 10 ng/mL IL-17A for 16 hours and then treated
with 200 µM TCA for 3 hours. MIP-2 mRNA levels were measured by real-time
a

b

PCR. Significantly different from wild-type vehicle treated hepatocytes (p<0.05). Significantly
c

different from wild-type TCA treated hepatocytes (p<0.05). Significantly different from wildtype IL-17A treated hepatocytes (p<0.05).

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Table 3.2 Additional Replicates for IL-23 Regulation in C/EBPβ Heterozygous Knockout
Hepatocytes
Treatment

Genotype

Mean ± SEM

Vehicle

Wild-type

1.11±0.39, 1.04±0.20

10ng/ml IL-17A

Wild-type

3.10±1.11, 1.19±0.15

200 μM TCA

Wild-type

8.42±0.66 , 10.99±1.02

a

200 μM TCA + 10ng/mL ILWild-type

a

a,b,c

15.60±1.35

a

, 10.20±2.64

17A
Vehicle

C/EBPβ Heterozygous

1.00±0.06, 1.02±0.17

10ng/mL IL-17A

C/EBPβ Heterozygous

0.93±0.28, 1.14±0.11

200 μM TCA

C/EBPβ Heterozygous

1.54±0.59 , 1.69±0.03

C/EBPβ Heterozygous

1.38±0.43 , 1.18±0.16

200 μM TCA + 10ng/mL IL-

b

b

b

b

17A

Hepatocytes were pretreated with 10 ng/mL IL-17A for 16 hours and then treated with 200 µM
a

TCA for 3 hours. IL-23 mRNA levels were measured by real-time PCR. Significantly different
b

from wild-type vehicle treated hepatocytes (p<0.05). Significantly different from wild-type
c

TCA treated hepatocytes (p<0.05). Significantly different from wild-type IL-17A treated
hepatocytes (p<0.05).

94

Table 3.3 Additional Replicates for MIP-2 Induction by JNK Inhibition
Treatment

Mean ± SEM

DMSO

1.00±0.02, 1.00±0.06
a,b

200 μM TCA

9.31±0.24

a,b

, 15.12±1.44

a

30 μM SP600125

a

5.01±0.11 , 2.68±0.24
a,b,c

200 μM TCA + 30 μM SP600125

14.55±1.11

a,b,c

, 26.16±1.93

Hepatocytes were pretreated with 10 ng/mL IL-17A for 30 minutes and then treated with 200 μM
TCA for 15 minutes. Hepatocytes were pretreated with either 30 µM SP600125 for 30 minutes
a

and then treated with 200 µM TCA for 3 hours. Significantly different from vehicle treated
b

hepatocytes (p<0.05). Significantly different from inhibitor treated hepatocytes
c

(p<0.05). Significantly different from TCA alone treated hepatocytes.

95

Table 3.4 Additional Replicates for MIP-2 Induction by p38 Inhibition
Treatment

Mean ± SEM

DMSO

1.00±0.06, 1.00±0.07
a,b

200 μM TCA

15.12±1.44

a

10 μM SB203580

a,b

, 5.74±0.13

a

3.55±0.12 , 3.31±0.14
a,b,c

200 μM TCA + 30 μM SB203580

37.96±4.08

a,b,c

, 13.44±0.99

Hepatocytes were pretreated with 10 ng/mL IL-17A for 30 minutes and then treated with 200 μM
TCA for 15 minutes. Hepatocytes were pretreated with 10 µM SB203580 for 30 minutes and
a

then treated with 200 µM TCA for 3 hours. Significantly different from vehicle treated
b

hepatocytes (p<0.05). Significantly different from inhibitor treated hepatocytes
c

(p<0.05). Significantly different from TCA alone treated hepatocytes.

96

Chapter 4
Identification of a Receptor that Regulates
Bile Acid Induction of IL-23

97

4.1 Abstract
Interleukin-23 (IL-23) is critical for the maintenance and stabilization of the Th17
inflammatory response. Recently, we showed that pathophysiological concentrations of bile
acids upregulate IL-23 expression in an AKT- and JNK- dependent manner. However, the
mechanisms that mediate bile acid induction of IL-23 are not well known. Epidermal growth
factor receptor (EGFR) and sphinogsine-1-phosphate receptor 2 (S1PR2) have been shown to be
activated by taurocholic acid (TCA) in primary rodent hepatocytes. Therefore, we tested the
hypothesis that EGFR or S1PR2 regulate IL-23 expression. Treatment of primary mouse
hepatocytes with TCA increased expression of IL-23. Inhibition of EGFR or S1PR2, with
pharmacological inhibitors, did not prevent induction of IL-23 by TCA. Next, we investigated
prostaglandin E2 receptors (EP) and lysophosphatidic acid receptors (LPAR) which are related to
S1PR2 and share structural homology to the bile acid receptor, TGR5. Hepatocytes treated with
EP inhibitors had no effect on IL-23 expression, in the presence of TCA. However, IL-23 mRNA
levels were reduced in hepatocytes pretreated with Ki16425, a LPAR1/3 competitive antagonist.
Collectively, our results suggest that bile acid activation of LPARs may be important for
induction of IL-23 expression in hepatocytes.
4.2 Introduction
Interleukin-23 (IL-23) is a member of the IL-12 family that is predominantly secreted by
antigen presenting cells such as dendritic cells (Oppmann et al., 2000). IL-23 is required for
maintenance of CD4+ Th17 population expansion which stimulates them to produce Th17
cytokines, such as IL-17A and IL-17F (Stritesky et al., 2008). Patients with primary biliary
cirrhosis have increased serum concentrations of IL-23 (Rong et al., 2009). Similarly, hepatic
mRNA levels of IL-23 are elevated in mice subjected to bile duct ligation, a murine model of

98

cholestasis (O'Brien et al., 2013). Profibrogenic markers are reduced in mice that have been
depleted of IL-23 in the bone marrow and subjected to bile duct ligation (Meng et al., 2012). We
have demonstrated that pathophysiological concentrations of bile acids increase hepatic
expression of IL-23 in a JNK- and AKT-dependent manner (O'Brien et al., 2013). However, the
receptor that mediates the activation of these pathways, leading to bile acid induction of IL-23, is
not known.
Epidermal Growth Factor Receptor (EGFR) is a tyrosine kinase receptor that activates
ERK1/2 in hepatocytes treated with secondary conjugated bile acids in a ligand-independent
manner (Rao et al., 2002). EGFR activation by secondary bile acids requires generation of
mitochondrial reactive oxygen species (Fang et al., 2004; Qiao et al., 2001; Rao et al., 2002).
The role of EGFR activation in the bile acid induction of IL-23 has not been investigated.
TGR5, a G protein-coupled receptor, is a well characterized bile acid receptor expressed
on various cells, including macrophages and bile duct epithelial cells (Keitel et al., 2008; Keitel
et al., 2010). However, studies have shown that TGR5 is not expressed by hepatocytes (Keitel et
al., 2007). Recently, treatment of hepatocytes with taurine-conjugated bile acids has been shown
to activate sphingosine-1-phosphate receptor 2 (S1PR2), a G protein coupled receptor with close
homology to TGR5 (Hama and Aoki, 2010; Studer et al., 2012). In these studies, inhibition of
S1PR2 with the selective antagonist, JTE-013, reduced bile acid activation of the ERK and AKT
signaling pathways, and bile acid induction of small heterodimer partner (SHP), in primary
rodent hepatocytes (Studer et al., 2012). Since AKT activation is critical for bile acid mediated
upregulation of IL-23, it is possible that S1PR2 may be required for upregulation of IL-23 by
TCA. Accordingly, in the following studies, the hypothesis was tested that EGFR or S1PR2
mediate the induction of hepatic IL-23 by bile acids.

99

4.3 Materials and Methods
4.3.1 Animal Care. All experiments were performed on 8-12 week old male C57BL/6
mice (Jackson Laboratories; Bar Harbor, ME). All of the mice were maintained on a 12-h
light/dark cycle under controlled temperature (18–21°C) and humidity. Food (Rodent Chow;
Harlan-Teklad, Madison, WI) and tap water were allowed ad libitum. All of the procedures on
animals were carried out in accordance with the Guide for the Care and Use of Laboratory
Animals promulgated by the National Institutes of Health.
4.3.2 Hepatocyte Isolation. Hepatocytes were isolated by collagenase H retrograde
perfusion as described previously (Kim et al., 2006). Hepatocytes were pretreated with 5 µM
AG1478, 10 µM JTE-013, 10 µM Ki16425, 10 µM tetradecyl phosphonate, 10 μM AH6809, 10
μM SC19220, or DMSO as vehicle (all from Cayman Chemicals; Ann Arbor, MI) for 30
minutes. Hepatocytes were then treated with 200 μM taurocholic acid (TCA) (Sigma Aldrich; St.
Louis, MO) for 3 hours.
4.3.3 Real-Time PCR. Total RNA was isolated from isolated hepatocytes by Trizol
extraction, and contaminating DNA was removed by Turbo DNA-free kit (Ambion; Austin, TX)
per manufacturers’ instructions. RNA was reverse transcribed into cDNA as described in 2.3.8.
mRNA was analyzed on a 7500 Fast Real-time PCR system (Applied Biosystems; Foster City,
CA) through the utilization of fast SYBR green reagent (Bio-rad; Hercules, CA). Primer
sequences are outlined in Table 2.1. All primers used were intron spanning.
4.3.4 Statistics. Data are expressed as mean ± SEM. When two or more groups were
analyzed, a two way analysis of variance (ANOVA) was performed. When the data set did not
pass normality, a log transformation was utilized. Comparison between groups was done by
using the Holm-Sidak method. A 95% confidence, p<0.05, was the criterion for significance.

100

4.4 Results
4.4.1 Inhibition of the Epidermal Growth Factor Receptor does not affect
upregulation of IL-23 by TCA. Both conjugated and unconjugated bile acids activate the EGFR
in a ligand independent manner (Fang et al., 2004; Qiao et al., 2001). However, EGFR activation
has only been investigated in the regulation of apoptotic hepatocyte death and not in the
upregulation of inflammatory cytokines (Qiao et al., 2003; Rao et al., 2002). Treatment of
hepatocytes with TCA increased expression of IL-23. Pretreatment with AG1478, at a
concentration that prevents bile acid activation of ERK1/2, did not affect upregulation of IL-23
by TCA (Figure 4.1) (Rao et al., 2002). These data suggest that bile acid induction of IL-23 does
not require EGFR activation.
4.4.2 Sphingosine-1-Phosphate Receptor 2 is not required for TCA induction of IL23. Activation of ERK1/2 and AKT by TCA requires S1PR2 in rodent hepatocytes (Studer et al.,
2012). We recently demonstrated that bile acid induction of IL-23 is AKT-dependent. Therefore,
hepatocytes were pretreated with JTE-013, a specific antagonist for S1PR2, at a concentration
that prevents activation of AKT, in bile acid treated hepatocytes (Studer et al., 2012). TCA
increased IL-23 mRNA levels. Inhibition of S1PR2 with JTE-013 did not prevent upregulation of
IL-23 by bile acids (Figure 4.2).
4.4.3 Inhibition of Prostaglandin E2 Receptors did not affect upregulation of IL-23
by TCA. Prostaglandin E2 receptors (EP) are GPCRs that are structurally related to TGR5, a bile
acid activated GPCR (Hama and Aoki, 2010). There are four different subtypes of the
prostaglandin E2 receptor (EP1-4). The antagonists, AH6809 or SC19220, which inhibit EP1 and
EP2 were used to test the role of these receptors in upregulation of IL-23 by TCA (Kiriyama et
al., 1997). TCA induced IL-23 gene expression in hepatocytes. This induction was unaffected in

101

hepatocytes pretreated with either AH6809 or SC19220 indicating no role for these receptors in
upegulation of IL-23 by TCA (Figure 4.3).
4.4.4 Lysophosphatidic Acid Receptor inhibition reduces IL-23 expression.
Lysophosphatidic acid receptors (LPAR) are GPCRs that are closely related to TGR5 (Hama and
Aoki, 2010). Hepatic LPAR1, LPAR2, and LPAR3 protein expression is induced in a model of
chronic alcoholic liver disease (Sautin et al., 2002). Furthermore, LPAR1 and LPAR2 are
expressed at low levels in freshly isolated hepatocytes and might regulate hepatocyte
differentiation from oval cells (Sautin et al., 2002; Svetlov et al., 2002). To evaluate a role for
LPA receptors in upregulation of IL-23 by TCA, hepatocytes were pretreated with a selective
antagonist of LPAR1 and LPAR3, Ki16425 (Ohta et al., 2003). Inhibition of these receptors
partially prevented upregulation of IL-23 by TCA (Figure 4.4). To determine if TCA stimulated
hepatocytes to generate lysophosphatidic acid (LPA) which contribute to IL-23 induction, we
utilized an autotaxin inhibitor, tetradecyl phosphonate (Durgam et al., 2005). Autotaxin, also
known as lysophospholipase D, converts lysophosphatidylcholine to lysophosphatidic acid
(Stracke et al., 1992; Umezu-Goto et al., 2002). Pretreatment with tetradecyl phosphonate, had
no effect on upregulation of IL-23 by TCA (Figure 4.4).
4.5 Discussion
We recently showed that bile acid induction of hepatocyte interleukin-23 (IL-23) requires
AKT and JNK signaling pathways (O'Brien et al., 2013). However, the receptor that regulates
bile acid induction of the inflammatory response has not been elucidated. In these preliminary
studies, we investigated the role of EGFR and the GPCRs: S1PR2, EP, and LPAR in the
regulation of IL-23 expression in hepatocytes.

102

Epidermal growth factor receptor (EGFR) and sphingosine-1-phosphate receptor 2
(S1PR2) are activated by TCA treatment in primary rodent hepatocytes (Rao et al., 2002; Studer
et al., 2012). Inhibition of either EGFR or S1PR2 did not prevent induction of IL-23 in
hepatocytes (Figure 4.1-4.2). Similarly, prevention of EGFR and S1PR2 activation did not
prevent upregulation of MIP-2 by bile acids (data not shown). Previously, we showed that TCA
induction of MIP-2 is ERK-dependent (Allen et al., 2010). Therefore, the result that S1PR2
inhibition did not prevent MIP-2 induction was unexpected. However, previous studies
evaluating activation of S1PR2 by TCA were performed in hepatocytes cultured in media
containing dexamethasone. S1PR2 expression is not detected in hepatocytes in vivo under basal
conditions, but mRNA levels are elevated in vitro in those studies (Kageyama et al., 2012). We
observed that treatment with dexamethasone enhances the induction of MIP-2 by TCA.
Therefore, dexamethasone may upregulate S1PR2 expression in primary hepatocytes, which will
mediate TCA downstream signaling. Though TCA has been shown to activate EGFR, those
studies investigated the apoptosis signaling cascade (Qiao et al., 2001; Rao et al., 2002). Our
studies demonstrate that EGFR and S1PR2 do not mediate the bile acid induction of the
inflammatory response (Figure 4.1-4.2). Therefore, the hepatic inflammatory response is
regulated by an uncharacterized bile acid receptor.
Prostaglandin E2 receptors (EP1-4) are GPCRs that share similar homology to the known
bile acid receptor, TGR5 (Hama and Aoki, 2010). There are four subtypes of prostaglandin E2
receptors, EP1-4, which are all expressed on primary murine hepatocytes (Kataoka et al., 2005).
EP1 knockout mice treated with endotoxin had a reduction of hepatic injury and apoptotic
hepatocyte death (Han et al., 2008). Furthermore, hepatocytes treated with an EP4 agonist
promoted the upregulation of the anti-apoptotic protein, Bcl-xL, through the transactivation of

103

EGFR (Kataoka et al., 2005). These studies demonstrate protective and detrimental effects of
prostaglandin E2 receptors in hepatic disease. Due to these studies and prostaglandin E2
receptors’ relationship to TGFR, we investigated the potential role of these receptors in the
regulation of the hepatic inflammatory response by bile acids. Inhibition of EP1 and EP2 had no
effect on the upregulation of IL-23 in TCA treated hepatocytes (Figure 4.4). These data
demonstrate that these prostaglandin E2 receptors are not involved in the upregulation of IL-23
by bile acids.
Lysophosphatidic acid receptors (LPARs) are another family of GPCRs that are closely
related to the bile acid receptor, TGR5 (Hama and Aoki, 2010). There are six LPA receptors with
three LPA receptors (LPAR1-3) that belong to the endothelial differentiation gene family with
approximately 50% homology between them (Choi et al., 2010). LPAR1 and LPAR2 have a
moderate to low expression in freshly isolated and cultured primary hepatocytes and whole liver;
whereas, LPAR3 has not been shown to be expressed on hepatocytes (Choi et al., 2010; Svetlov
et al., 2002). LPAR1 is induced in hepatocellular carcinoma and mediates the upregulation of
matrix metalloproteinase-9 (MMP-9) via a PI3K/AKT- and p38-depedent manner in a
hepatocarinoma cell line (Park et al., 2011). In this study, we used a selective inhibitor for
LPAR1, LPAR3, and autotaxin to determine their involvement in bile acid induction of IL-23.
Hepatocytes treated with a selective antagonist (Ki16425) for LPAR1 and LPAR3 partially
prevented the induction of IL-23 by bile acids (Figure 4.4). To further investigate LPARs, we
pretreated hepatocytes with tetradecyl phosphonate, an inhibitor of autotaxin enzymatic activity.
Tetradecyl phosphonate treatment did not reduce IL-23 expression in hepatocytes exposed to
TCA (Figure 4.4). These results suggest that bile acids do not promote LPA generation in
hepatocytes, which in turn activates LPARs in autocrine and paracrine manner.

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Both Ki16425 and tetradecyl phosphonate have been shown to modestly activate PPARγ
with tetradecyl phosphonate having a higher affinity than Ki16425 (Durgam et al., 2005). PPARγ
is a nuclear receptor that mediates an anti-inflammatory response. In addition, PPARγ
downregulates IL-8, the human homolog of MIP-2 and KC, in human colon cancer cells
(Nakajima et al., 2001). Considering that only Ki16425 prevented IL-23 induction, our results
suggest that this off target effect is not the mechanism underlying the upregulation of IL-23 gene
expression. At present, we are employing a spectrum of LPAR antagonists to further determine
the effect of LPAR activation on the hepatic inflammatory response.
Autotaxin activity and LPA concentration are increased in cholestatic liver disease
patients (Kremer et al., 2012). Mice injected intradermally with LPA had an increase scratch
response in a dose dependent manner (Kremer et al., 2010). Furthermore, there is strong
correlation between pruritus intensity and autotaxin enzymatic activity in PBC patients (Kremer
et al., 2010). The potential role of LPA and autotaxin in pruritus is further demonstrated in
cholestatic patients who have undergone nasobiliary drainage, a procedure that removes excess
bile through the placement of catheter via the nasal cavity. After nasobiliary drainage, these
patients have a drastic decrease in serum bile acid concentrations, LPA concentrations, and
autotaxin activity within 24 hours. However, nasobiliary drainage is a temporary treatment for
pruritus as bile acid, LPA, and autotaxin concentrations will increase in the patients over time
(Kremer et al., 2010). Our results suggest that pathophysiological concentrations of bile acids do
not promote pruritus via the generation of LPA by autotaxin. Therefore, the mechanisms and
mediators of pruritus still need further investigation.
Overall, our results demonstrate that LPAR1 is activated by bile acids in hepatocytes and
regulates the inflammatory response. As these studies are preliminary, each receptor class needs

105

further investigation to determine their role in bile acid promotion of the hepatic inflammatory
response.

106

Figure 4.1 Role of EGFR on IL-23 Induction by Bile Acids. Hepatocytes were treated with 5
μM AG1478 in the presence or absence of 200 μM TCA. IL-23 mRNA levels were measured by
a

b

real-time PCR. Significantly different from vehicle treated hepatocytes (p<0.05). Significantly
different from TCA treated hepatocytes (p<0.05).

107

Figure 4.2 Effect of S1PR2 on IL-23 mRNA Levels in Hepatocytes. Hepatocytes were
pretreated with a specific antagonist for SIPR2, JTE-013, for 30 minutes. Hepatocytes were then
treated with 200 µM TCA for 3 hours. IL-23 mRNA was measured by real-time
a

b

PCR. Significantly different from vehicle treated hepatocytes (p<0.05). Significantly different
from TCA treated hepatocytes (p<0.05).

108

Figure 4.3 Effect of EP1 or EP2 on the Regulation of IL-23 in Primary Mouse Hepatocytes.
Primary hepatocytes were treated with 10 μM AH6809 or 10 μM SC19220, selective antagonist
to EP2 or EP1, in the presence or absence of 200 µM TCA. IL-23 mRNA levels were measured
a

by real-time PCR. Significantly different from vehicle treated hepatocytes (p<0.05).

109

Figure 4.4 LPA Receptors mediate TCA Induction of IL-23 in Hepatocytes. Hepatocytes
were treated with two different inhibitors of LPA signaling, Ki16425 and tetradecyl phosphonate
a

for 30 minutes. Hepatocytes were then treated with 200 μM TCA for 3 hours. Significantly
b

different from vehicle treated hepatocytes (p<0.05). Significantly different from TCA treated
hepatocytes (p<0.05).

110

Table 4.1 Additional Replicates for the Inhibition of EGFR
Treatment

Mean ± SEM
1.04±0.18, 1.00±0.06
a
a
6.56±1.34 , 10.05±0.21
1.09±0.40, 1.03±0.20
a
a,b
7.27±0.21 , 14.03±0.87

DMSO
200 μM TCA
5 μM AG1478
200 μM TCA + 5 μM AG1478

Hepatocytes were pretreated with 5 μM AG1478 for 30 minutes and then treated with 200 μM
a

TCA for 3 hours. IL-23 mRNA levels were measured by real-time PCR. Significantly different
b

from vehicle treated hepatocytes (p<0.05). Significantly different from TCA treated hepatocytes
(p<0.05).
Table 4.2 Additional Replicates for the Inhibition of S1PR2
Treatment
DMSO
200 μM TCA
10 μM JTE-013
200 μM TCA + 10 μM JTE-013

Mean ± SEM
1.04±0.18, 1.00±0.06
a
a
6.56±1.34 , 10.05±0.21
1.52±0.50, 1.60±0.40
a,b
a,b
10.54±0.80 , 22.04±0.83

Hepatocytes were treated with 10 μM JTE-013 for 30 minutes. Hepatocytes were then treated
with 200 μM TCA for 3 hours. IL-23 mRNA levels were measured by real-time
a

b

PCR. Significantly different from vehicle treated hepatocytes (p<0.05). Significantly different
from TCA treated hepatocytes (p<0.05).

111

Table 4.3 Additional Replicates for the Inhibition of Prostaglandin E2 Receptors
Treatment
DMSO
200 μM TCA
10 μM AH6809
200 μM + 10 μM AH6809
10 μM SC19220
200 μM TCA + 10 μM SC19220

Mean ± SEM
1.04±0.22, 1.00±0.09
a
a
11.40±2.87 , 8.84±0.22
0.80±0.12, 0.83±0.17
a
a
6.13±2.13 , 7.50±1.06
0.96±0.34, 1.11±0.18
a
a
6.56±1.30 , 5.88±0.70

Hepatocytes were pretreated with either 10 μM AH6809 or 10 μM SC19220 for 30 minutes;
a

then, hepatocytes were treated with 200 μM TCA for 3 hours. Significantly different from
vehicle treated hepatocytes (p<0.05).
Table 4.4 Additional Replicates for the Inhibition of Lysophosphatidic Acid Receptors
Treatment
DMSO
200 μM TCA
10 μM Ki16425
200 μM TCA + 10 μM Ki16425
10 μM Tetradecyl Phosphonate
200 μM TCA + 10 μM Tetradecyl
Phosphonate

Mean ± SEM
1.02±0.16, 1.02±0.14
a
a
5.30±0.52 , 6.08±0.70
0.66±0.09, 0.89±0.21
a,b
a,b
3.40±0.18 , 2.19±0.65
0.82±0.10, 0.94±0.05
a
a
4.64±0.75 , 3.32±0.12

Hepatocytes were treated with either 10 μM Ki16425 or 10 μM tetradecyl phosphonate in the
presence or absence of 200 μM TCA. IL-23 mRNA levels were quantified after 3 hours of TCA
a

treated by real-time PCR. Significantly different from vehicle treated hepatocytes
b

(p<0.05). Significantly different from TCA treated hepatocytes (p<0.05).

112

Chapter 5
Role of Interleukin-17F in Hepatocellular
Injury during Cholestatic Liver Disease

113

5.1 Abstract
Activation of the inflammatory response, predominantly mediated by neutrophils, has
been shown to be critical for the propagation of hepatocellular injury in cholestatic liver disease
caused by a disruption of bile flow. Pathophysiological concentrations of bile acids promote the
hepatic expression of macrophage inflammatory protein (MIP-2) which regulates neutrophilic
inflammation. However, the critical cytokines that promote neutrophilic inflammation are not
well understood. A Th17 cytokine, IL-17A, expression has been shown to be upregulated in
cholestatic patients and to contribute to hepatocellular injury, inflammation, and fibrosis in a
murine model of cholestasis. Interleukin-17F (IL-17F) shares 50% homology to IL-17A, but its
role during cholestasis has not been elucidated. We tested the hypothesis that IL-17F promotes
inflammation and hepatocellular injury during cholestatic liver disease. First, we treated
hepatocytes with IL-17F in the presence or absence of taurocholic acid (TCA). There was a
synergistic enhancement of MIP-2 in hepatocytes treated with IL-17F and TCA. To further
evaluate the role of IL-17F during cholestasis, wild-type and IL-17F knockout mice were
subjected to bile duct ligation (BDL) and markers of hepatocellular injury and inflammation
were measured. 48 hours after BDL, IL-17F mRNA levels were increased in wild-type mice.
There was no reduction in alanine aminotransferase (ALT) activity, a clinic biomarker of
hepatocellular injury, or percent area of necrosis in IL-17F knockout mice, compared to wildtype. Furthermore, the deletion of IL-17F did not affect the expression of pro-inflammatory
mediators, but there was a reduction in neutrophils in the IL-17F knockout BDL mice.
Collectively, these studies demonstrate that IL-17F does not regulate the inflammatory response
in the early stages of cholestasis, but IL-17F does enhance the bile acid induction of the
hepatocyte inflammatory response in vitro.

114

5.2 Introduction
Cholestasis is characterized by a robust inflammatory response, hepatocellular injury, bile
duct proliferation, and dysregulation of bile acid homeostasis. Persistent cholestasis stimulates
differentiation of hepatic stellate cells and portal fibroblast into myofibroblasts, which produce
extracellular matrix causing fibrosis (Beuers, 2009; Hirschfield et al., 2010; Li and Crawford,
2004; Trauner et al., 1998). In obstructive cholestasis, the inflammatory response is primarily
neutrophilic (Gujral et al., 2003; Li and Crawford, 2004). Neutrophil accumulation during
cholestasis is dependent upon the chemokines, macrophage inflammatory protein-2 (MIP-2) and
keratinocyte-derived chemokine (KC), and the adhesion molecule, intercellular adhesion
molecule-1 (ICAM-1) (Gujral et al., 2004; Wintermeyer et al., 2009). The mechanisms that
regulate the inflammatory response during cholestasis are not completely understood.
Previous studies demonstrated that the cytokine, IL-17A, is increased in patients with
primary biliary cirrhosis (Harada et al., 2009; Lan et al., 2009). IL-17A is a Th17 cytokine that
regulates neutrophil recruitment to sites of inflammation (Maione et al., 2009). Recently, we
demonstrated that the neutralization of IL-17A reduces levels of pro-inflammatory cytokines and
prevents neutrophil accumulation into the livers of mice subjected to bile duct ligation.
Furthermore, mice treated with IL-17A neutralizing antibody had less hepatocellular injury after
bile duct ligation (O'Brien et al., 2013). We have also demonstrated that IL-17A synergistically
enhances bile acid dependent upregulation of MIP-2 in hepatocytes in a p38- and JNKdependent manner. Although these studies indicate a clear role for IL-17A in the pathogenesis of
cholestatic liver disease, whether other IL-17 family members also play a part has not been
investigated.

115

Interleukin-17F (IL-17F) is the closest family member of interleukin-17A with 50%
homology (Hymowitz et al., 2001). IL-17F can be secreted by differentiated Th17 cells, lamina
propria T cells, memory CD4+ T cells, γδ T cells, natural killer T cells, and bronchial epithelial
cells (Fujita et al., 2012; Yang et al., 2008b). Interleukin-17F and interleukin-17A activate the
same heterodimer receptor complex which consists of IL-17RA and IL-17RC (Toy et al., 2006).
Murine IL-17F behaves as a homodimer or as a heterodimer with IL-17A (Chang and Dong,
2007). Similar to IL-17A, IL-17F promotes inflammatory responses in various autoimmune
diseases, such as asthma (Oda et al., 2005; Yang et al., 2008a). Whether it is increased in the
liver during cholestasis and promotes inflammation and injury is not known. Therefore, we
tested the hypothesis that IL-17F promotes inflammation and hepatocellular injury in cholestatic
liver disease.
5.3 Materials and Methods
5.3.1 Animal Care. These studies were performed on 8-12 weeks old male C57BL/6
(Jackson Laboratories; Bar Harbor, ME) or IL-17F knockout mice provided by Dr. Chen Dong at
M.D. Anderson Cancer Center. Generation of IL-17F knockout mice described in (Yang et al.,
2008a). All of the mice were maintained on a 12-hr light/dark cycle under controlled temperature
(18-21 °C) and humidity. Food (Rodent Chow; Harlan-Teklad, Madison, WI) and tap water were
allowed ad libitum. All of the procedures on animals were carried out in accordance with the
Guide for the Care and Use of Laboratory Animals promulgated by the National Institutes of
Health.
5.3.2 Bile Duct Ligation. Mice were anesthetized with isoflurane. A midline laparotomy
was performed and the bile duct ligated with 4-0 surgical silk. The abdominal incision was

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closed with sutures, and the mice received 0.2 mg/kg buprenex by subcutaneous injection twice
daily. Mice were sacrificed 48 hours after surgery.
5.3.3 Hepatocyte Isolation. Livers were perfused by retrograde collagenase H (Sigma
Aldrich; St. Louis, MO) perfusion as described previously (Kim et al., 2006). Hepatocytes were
cultured in Williams’ Media E supplemented with 10% fetal bovine serum and penicillinstreptomycin. Hepatocytes were treated with 10 ng/mL recombinant mouse IL-17F (R&D
systems; Minneapolis, MN) or 4 mM HCl as vehicle for 16 hours. Hepatocytes were then treated
with 200 µM taurocholic acid (Sigma Aldrich) or endotoxin free water as vehicle for 3 hours.
5.3.4 Serum Chemistry. Serum alanine aminotransferase (ALT) activity was measured
by a commercially available kit (Thermo Scientific; Middletown, VA) per manufacturers’
instructions.
5.3.5 Immunohistochemistry. Paraffin-embedded liver sections were incubated with a
rat anti-mouse polymorphonuclear leukocytes (PMN) 7/4 antibody (1:2500) (AbD Serotec;
Raleigh, NC) for one hour. Neutrophils were quantified by counting the number of positivelystained cells in 10, 200X fields per tissue section. The analysis was performed in a blinded
fashion.
5.3.6 Real-Time PCR. Total RNA was isolated from whole liver and isolated
hepatocytes by Trizol extraction. Contaminating DNA was removed by the Turbo DNA-free kit
(Ambion; Austin, TX). RNA was reverse transcribed into cDNA as described in 2.3.8. mRNA
levels were evaluated using fast SYBR green reagent (Bio-rad; Hercules, CA) on a 7500 Fast
Real-time PCR system (Applied Biosystems; Foster City, CA). Primer sequences are outlined in
Table 5.1. All primers used were intron spanning.

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5.3.7 Statistics. Data are expressed as mean ± SEM. When two or more groups were
analyzed, a two way analysis of variance (ANOVA) was performed. A log transformation was
performed when the data set did not pass normality. Comparison between groups was done by
using the Holm-Sidak method. A 95% confidence, p<0.05, was the criterion for significance.
5.4 Results
5.4.1 IL-17F mRNA levels are induced during cholestasis. First, we determined the
gene expression of IL-17F during cholestatic liver disease. Mice were subjected to bile duct
ligation (BDL), a murine model of obstructive cholestasis, and IL-17F mRNA levels were
measured by real-time PCR. IL-17F mRNA levels had an initial peak at 2 days post-surgery.
Though IL-17F mRNA levels were back to basal levels at 3 days, the gene expression was
increased and remained elevated after 4 days of BDL (Figure 5.1).
5.4.2 IL-17F synergistically enhances MIP-2 induction by TCA. IL-17A
synergistically enhances bile acid induction of the hepatic inflammatory response via negative
regulation of JNK and p38 activation. Considering the relationship between IL-17A and IL-17F,
we treated hepatocytes with IL-17F in the presence or absence of TCA. Similar to IL-17A, IL17F treatment promoted the upregulation of MIP-2 expression in hepatocytes. Co-treatment of
IL-17F and TCA synergistically enhanced MIP-2 fold induction (Figure 5.2).
5.4.3 IL-17F does not affect hepatocellular injury in bile duct ligated mice.
Neutralization of IL-17A reduced hepatocellular injury in a murine model of cholestasis
(O’Brien et al., 2013). IL-17F is the closest family member to IL-17A, in terms of homology,
and has the same physiological functions as IL-17A (Hymowitz et al., 2001). Therefore, we
tested the hypothesis that injury would be reduced in IL-17F knockout mice subjected to bile
duct ligation. Alanine aminotransferase (ALT) activity, a clinical biomarker for hepatocellular

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injury, was increased in wild-type mice subjected to BDL. Serum ALT activity was unaffected in
IL-17F knockout BDL mice (Figure 5.3A). Furthermore, there was no difference in the area of
necrotic regions in the IL-17F knockout mice subjected to BDL, compared to their wild-type
counterparts (Figure 5.3B-F).
5.4.4 Pro-inflammatory cytokine mRNA levels were unaffected in IL-17F knockout
bile duct ligated mice. IL-17A contributes to the induction of pro-inflammatory cytokines and
chemokines expression in liver diseases (O'Brien et al., 2013). Furthermore, it has been shown
that both IL-17A and IL-17F are critical for neutrophil recruitment to sites of injury in
autoimmune diseases (Kono et al., 2011; Liang et al., 2007). Therefore, we measured the mRNA
levels of cytokines and chemokines involved in the inflammatory response during cholestasis
(Bergheim et al., 2006; Gehring et al., 2006; Wintermeyer et al., 2009). MIP-2 was induced in
wild-type BDL mice, but the induction was unaffected in IL-17F null BDL mice (Figure 5.4A).
Plasminogen activator inhibitor-1 (PAI-1) not only regulates fibrinolysis, but PAI-1 has been
shown to inhibit integrin- and vitronectin mediated cell migration (Kjoller et al., 1997). In
addition, the deletion of PAI-1 reduces neutrophil infiltration and hepatocellular injury in bile
duct ligated mice (Bergheim et al., 2006; Wang et al., 2005). Interestingly, mice lacking IL-17F
had a higher basal level of PAI-1 gene expression than their wild-type counterparts. PAI-1
mRNA was increased in mice subjected to bile duct ligation. However, PAI-1 mRNA levels
were not different in the IL-17F knockout BDL mice compared to the wild-type BDL mice
(Figure 5.4B). Interleukin-6 (IL-6) is a pleiotropic cytokine that has both pro-inflammatory and
anti-inflammatory functions (Scheller et al., 2011). IL-6 knockout mice subjected to BDL had
exacerbating hepatocellular injury and reduced hepatocyte proliferation (Gehring et al., 2006).

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IL-6 mRNA levels were increased in IL-17F null BDL mice, but it was unaffected in wild-type
mice subjected to BDL (Figure 5.4C).
During cholestasis, the initiation of the inflammatory response is the recruitment of
neutrophils to the hepatic parenchyma (Georgiev et al., 2008). In addition, the inhibition of
neutrophil extravasation partially attenuates liver injury (Gujral et al., 2003; Gujral et al.,
2004).Therefore, we counted the numbers of neutrophils in the livers of mice subjected to BDL.
As expected, bile duct ligated mice had an elevation of neutrophil cell count. Bile duct ligated,
IL-17F knockout mice had a reduction of neutrophil cell count compared to wild-type mice
(Figure 5.5A-C).
5.4.5 IL-17F knockout mice had elevated mRNA levels of the transcription factors,
C/EBPβ and early growth response factor (Egr-1). The transcription factor, CCAAT/enhancer
binding protein-β (C/EBPβ), is a downstream target of the IL-17RA/IL-17RC receptor complex
(Maitra et al., 2007). IL-17F knockout sham-operated mice had elevated mRNA levels of
C/EBPβ, compared to wild-type mice. Subjecting mice to bile duct ligation induced C/EBPβ
mRNA levels, but the deletion of IL-17F did not affect the upregulation of C/EBPβ in bile duct
ligated mice (Figure 5.6A). Next, we investigated IL-17F regulation of another transcription
factor, early growth response factor-1 (Egr-1). Previously, we have shown that Egr-1 is required
for the induction of cytokines, chemokines, and adhesion molecule by taurocholic acid in
hepatocytes (Allen et al., 2011). It has been shown that C/EBPβ activation enhances Egr-1
activity in a hepatocarinoma cell line (Zhang et al., 2003). Similar to C/EBPβ, IL-17F knockout
mice had elevated levels of Egr-1 compared to wild-type mice subjected to sham-operation. Bile
duct ligation increased the mRNA levels of Egr-1, but Egr-1 had the same fold induction in IL17F knockout BDL mice compared their sham counterparts (Figure 5.6B).

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5.4.6 The removal of IL-17F promotes the induction of IL-17RC, a subunit of the
IL-17 receptor complex. IL-17F activates the receptor complex, IL-17RA/IL-17RC (Toy et al.,
2006). It has been shown that IL-17RC has a higher affinity for IL-17F than IL-17A and is
induced by IL-17F (Kuestner et al., 2007). Bile duct ligation induced the expression of IL-17RC
in wild-type mice. The deletion of IL-17F promoted a higher basal level of IL-17RC mRNA
levels compared to wild-type, but IL-17RC was not further elevated in IL-17F null mice
subjected to BDL (Figure 5.7).
5.5 Discussion
During cholestasis, neutrophilic inflammation propagates hepatocellular injury through
the release of reactive oxygen species and proteases (Gujral et al., 2003). Patients with
cholestatic liver disease have elevated concentrations of conjugated primary bile acids, which
promote the expression of pro-inflammatory cytokines, chemokines, and adhesion molecules via
the activation of the transcription factor, Egr-1, in hepatocytes (Allen et al., 2011; Trottier et al.,
2012). We recently demonstrated that IL-17A, a Th17 neutrophil recruitment cytokine,
synergistically enhances bile acid induction of the hepatic inflammatory response. Furthermore,
we have shown that IL-17A contributes to inflammation and hepatocellular injury in obstructive
cholestasis (O'Brien et al., 2013). IL-17F shares 50% homology to IL-17A and binds to the same
heteromeric receptor complex (IL-17RA/IL-17RC) as IL-17A (Akimzhanov et al., 2007; Toy et
al., 2006). Therefore, we investigated the role of IL-17F during cholestatic liver disease.
First, we determined the gene expression of IL-17F during obstructive cholestasis.
Hepatic IL-17F mRNA levels are induced in bile duct ligated mice, a model of obstructive
cholestasis with an initial peak at 48 hours post-surgery (Figure 5.1). Consistent with these
findings, IL-17F mRNA levels are increased in mice treated with carbon tetrachloride (CCl 4 ), a

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model of fibrotic liver (Meng et al., 2012). Through immunohistochemistry, we have observed
the co-localization of portal fibroblast and IL-17F expression in the portal triad (data not shown).
Therefore, portal fibroblast could be the cellular source of the initial peak of IL-17F expression
at 48 hours post-surgery. To evaluate the physiological function of IL-17F in the hepatic
inflammatory response, we treated hepatocytes with IL-17F with or without TCA. Similar to the
IL-17A and bile acid interaction, IL-17F synergistically enhanced MIP-2 induction by TCA
(Figure 5.2) but not to the same extent as IL-17A. These data suggest that IL-17F would have a
similar pathophysiological function as IL-17A during cholestasis. Therefore, we subjected IL17F knockout mice to bile duct ligation or sham-operation for 48 hours.
Hepatocellular injury and the expression of pro-inflammatory mediators were unaffected
in IL-17F null mice subjected to BDL for 48 hours (Figure 5.3-5.4). However, neutrophil
accumulation in the necrotic regions was drastically reduced in bile duct ligated IL-17F knockout
mice (Figure 5.5). IL-17A has been shown to promote granulopoiesis via the induction of
granulocyte-colony stimulating factor (G-CSF) (Schwarzenberger et al., 2000; Schwarzenberger
et al., 1998). Therefore, deletion of IL-17F may suppress G-CSF dependent neutrophil
granulopoiesis and the dysregulation of bone marrow neutrophil trafficking. Similarly, the
neutralization of IL-17A also did not affect hepatocellular injury or inflammation in mice
subjected to BDL for 3 days. Collectively, these results demonstrate the neither Th17 cytokine
contributes to hepatocellular injury or inflammation at the early stages of cholestasis.
Considering that T lymphocytes are the major cellular source of IL-17F, a later timepoint needs
to be investigated to ensure for T cell infiltration into the parenchyma of the liver.
Histological evaluation of IL-17F knockout mice indicated steatosis (i.e., fatty liver)
(Figure 5.3B-C). Though the role of IL-17F in adipose homeostasis has not been elucidated,

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studies have demonstrated that the IL-23/IL-17A axis is upregulated in obese patients and
contributes to the regulation of adipose tissue accumulation and adipogenesis (SumaracDumanovic et al., 2009; Zuniga et al., 2010). Furthermore, IL-17RA knockout mice fed a high
fat diet had increased hepatic triglycerides and white adipose tissue but reduced hepatocellular
injury (Harley et al., 2013). These observations of increased fatty acid content in IL-17F
knockout mice would suggest that IL-17F also contributes to adipose regulation and obesity. An
induction of steatosis would account for the high basal level of the transcription factors, Egr-1
and C/EBPβ, and the pro-inflammatory mediator, PAI-1 (Figure 5.6, Figure 5.4B). Others have
shown that C/EBPβ is induced in mice fed a high fat diet, and that deletion of C/EBPβ reduces
injury, inflammation, and fatty acid formation during steatosis (Millward et al., 2007; Rahman et
al., 2007). The interaction between C/EBPβ and Egr-1 regulates the low density lipoprotein
receptor (LDLR) expression in a hepatocarinoma cell line which will regulate cholesterol
homeostasis (Zhang et al., 2003). Furthermore, it has been shown that serum PAI-1 activity is
closely correlated to the degree of steatosis in obese patients (Alessi et al., 2003). These results
imply that antibodies against IL-17A or IL-17F should not be considered for the treatment of
cholestatic liver disease due to the risk of exacerbating inflammation caused by the dysregulation
of fatty acid metabolism.
Collectively, these studies demonstrate that neither IL-17F nor IL-17A contributes
to the initiation of inflammation and hepatocellular injury in the early stages of cholestasis.
However, IL-17F and IL-17A synergistically enhanced bile acid induction of MIP-2 in
hepatocytes. The role IL-17A/IL-17F heterodimer needs to be elucidated to properly evaluate the
effect of Th17 cells during cholestasis and other liver diseases.

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Figure 5.1 Upregulation of IL-17F mRNA Levels in Bile Duct Ligated Mice. Wild-type mice
were subjected to bile duct ligation. IL-17F mRNA levels were measured in the liver at various
a

times after bile duct ligation by real-time PCR in wild-type mice. Significantly different from
day 0 (p<0.05).

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MIP-2 mRNA Levels
(Relative to Control)

60
50

Vehicle
10 ng/mL IL-17F

a,b

40
a

30
20
10

a

0

TCA

Vehicle

Figure 5.2 Effect of IL-17F on Bile Acid Induction of MIP-2. Primary hepatocytes were
treated with 10 ng/mL of IL-17F for 16 hours. Hepatocytes were then treated with 200 µM TCA
for 3 hours. MIP-2 mRNA levels were measured by real-time PCR. Graph is a representative of
a

an n of 3. Table 2 shows MIP-2 mRNA levels of the other two sample groups. Significantly
b

different from TCA alone treated hepatocytes (p<0.05). Significantly different from IL-17F
alone treated hepatocytes (p<0.05).

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Figure 5.3 Deletion of IL-17F did not affect Hepatocellular Injury after BDL. Wild-type or
IL-17F knockout mice were subjected to either sham (n=3, n=3) or bile duct ligation (n=5, n=3).
(A) Alanine aminotransferase activity and (D-F) percent area of necrosis were quantified after 48
126

Figure 5.3 (cont’d)
hours of surgery. (B) H&E liver section from wild-type mice subjected to sham operation. (C)
H&E liver section from IL-17F knockout mice subjected to sham operation. (D) H&E liver
section from wild-type mice subjected to bile duct ligation. (E) H&E liver section from IL-17F
a

knockout mice subjected to bile duct ligation. Bar represents 50 µm. Significantly different
from sham-operated wild-type mice (p<0.05).

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Figure 5.4 IL-17F Regulation of Inflammatory Mediators 2 days after BDL. Sham (n=3,
n=3) or bile duct ligation (n=5, n=3) was performed on either wild-type or IL-17F knockout mice
for 48 hours. (A) MIP-2, (B) PAI-1, and (C) IL-6 mRNA levels were measured by real-time
a

b

PCR. Significantly different from wild-type sham-operated mice (p<0.05). Significantly
different from IL-17F knockout sham-operated mice (p<0.05).

128

A

B

Average Neutrophil Count
Per High Power Field

C

35
30
25
20
15
a

10
5
0
Wild-type

IL-17F Knockout

Figure 5.5 Effect of IL-17F on Neutrophil Accumulation in Bile Duct Ligated Mice. Wildtype (n=5) or IL-17F knockout mice (n=3) were subjected to bile duct ligation for 48 hours.
Neutrophils were stained in paraffin embedded liver sections by immunohistochemistry. (A)
Section of liver from wild-type mice subjected to BDL. (B) Section of liver from IL-17F
knockout mice subjected to BDL. The bar represents 50 µm. Positive cells appear red in the
a

photomicrograph. (C) Neutrophils were counted in sections of liver. Significantly different from
wild-type subjected to BDL (p<0.05).

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Figure 5.6 Regulation of Hepatic Expression of C/EBPβ and Egr-1 by IL-17F. Wild-type or
IL-17F knockout mice were subjected to either sham-operation (n=3, n=3) or BDL (n=5, n=3)
for 48 hours. (A) C/EBPβ or (B) Egr-1 mRNA levels were measured by real-time
a

PCR. Significantly different from wild-type sham-operated mice (p<0.05).

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Figure 5.7 Hepatic Expression of the IL-17 Receptor Complex Subunit, IL-17RC, after
BDL. Sham (n=3, n=3) or bile duct ligation (n=5, n=3) was performed on either wild-type or IL17F knockout mice for 48 hours. IL-17RC mRNA levels were measured by real-time
a

b

PCR. Significantly different from wild-type sham-operated mice (p<0.05). Significantly
different from IL-17F knockout sham-operated mice (p<0.05).

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Table 5.1: Primer Pair Sequences
Gene

Forward

Reverse

5’-CCT CAA CGG AAG AAC CAA

5’-CTC AGA CAG CGA GGC ACA

AGA G-3’

TC-3’

5’ACC AGA GGA AAT TTT CAA TAG

5’-TGA TGC ACT TGC AGA AAA

GC-3’

CA-3’

5’ACA AGA AAA AGC GGA TGG TG-

5’-TTC TCC TCC AGA GTG GCT

3’

GT-3’

5’-AGT CTT TCC GAC CAA GAG CA-

5’-ATC ACT TGC CCC ATG AAG

3’

AG-3’

5’-CCT GCT CCT CAG AGA CAT CC-

5’-ATC TGG TCC TAC ACG AAG

3’

CC-3’

MIP-2

IL-6

RPL13a

PAI-1

IL-17RC

C/EBPβ

5’-GTT TCG GGA CTT GAT GCA
5’-CCC CGC AGG AAC ATC TTT A-3’
AT-3’
5’-GAG CGA ACA ACC CTA TGA

Egr-1

5’-TGG GAT AA TCG TCT CCA CC-3’
GC-3’
5’-TTG ATG CAG CCT GAG TGT CT-

5’-AAT TCC AGA ACC GCT CCA

5’

GT-3’

IL-17F

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Table 5.2: Additional Replicates of the In Vitro Studies
Treatment

Mean ± SEM

Vehicle

1.01±0.11, 1.00±0.09

10 ng/mL IL-17F

2.98±0.44, 2.67±0.34

200 µM TCA

6.15±0.21 , 21.80±3.9

a

a,b

9.91±1.6

10 ng/mL IL-17F + 200 µM TCA

a

a,b

, 69.04±6.7

Hepatocytes were pretreated with 10 ng/mL of IL-17F for 16 hours. Hepatocytes were then
a

treated with 200 μM TCA for 3 hours. Significantly different from vehicle treated hepatocytes
b

(p<0.05). Significantly different from TCA treated hepatocytes (p<0.05).

133

Chapter 6
Discussion

134

6.1 Summary and Significance
There is only one FDA approved drug, ursodeoxycholic acid (UDCA), for the treatment
of cholestatic liver disease (Beuers, 2009; Calmus and Poupon, 1991). However, only a few
cholestatic patients show any improvement under an UDCA treatment regimen (Heathcote,
2007). The study of the mechanisms underlying the inflammatory response during cholestasis
could provide a therapeutic target for cholestatic liver disease. Therefore, we have begun to focus
on the mechanisms regulating the interaction between the innate and adaptive immune response
during cholestasis. In these studies, we investigated the contribution of the Th17 cellular
response by elucidating the role of the IL-23/IL-17 axis during cholestasis.
There is some clinical evidence that pathophysiological concentrations of bile acids
promote hepatic injury. Cholestatic patients that have undergone biliary drainage have a
reduction in portal inflammation and hepatocellular injury markers (Hammel et al., 2001).
Furthermore, decompressed (e.g. excess bile acid removed) cholestatic patients showed an
overall improvement and an increase chance of remission (Stapelbroek et al., 2006).
Consistently, we have recently shown that bile acids promote the expression of pro-inflammatory
cytokines, chemokines, and adhesion molecules that are critical for the recruitment of neutrophils
into the liver parenchyma (Allen et al., 2011; Wintermeyer et al., 2009). Furthermore, we have
demonstrated that taurine-conjugated bile acids induce the hepatic expression of IL-23, which is
+

required to maintain the expansion of the Th17 cells. CD4 Th17 cells are the principal cellular
source of IL-17A during cholestatic liver disease (Stritesky et al., Meng et al 2012) (Figure 2.7BD). IL-17A expression is increased in cholestatic patients and murine models of cholestasis
(Ichikawa et al., 2010; O'Brien et al., 2013). These results suggest a cross-talk between bile acids
and Th17 cells that propagate the neutrophilic inflammatory response in cholestatic liver disease.
135

In addition, our results suggest bile acids as novel regulators of both the innate and adaptive
immune response.
To investigate the relationship between bile acids and Th17 cellular response, we injected
a neutralizing antibody against IL-17A and subjected mice to bile duct ligation, a murine model
of obstructive cholestasis. The neutralization of IL-17A decreased ALT activity and areas of
necrosis in BDL mice, after 9 days of surgery (Figure 2.1B-E). In addition, pro-inflammatory
cytokine mRNA levels and total neutrophil cell count is reduced in anti-IL-17A BDL mice
compared to wild-type BDL (Figure 2.3-2.4). To further elucidate the role of IL-17A, we treated
primary hepatocytes in the presence or absence of TCA. IL-17A enhanced MIP-2 and IL-23
induction by TCA (Figure 2.6A, 2.7D). First, these data suggest the importance of Th17 cellular
expression of IL-17A to sustain bile acid promotion of neutrophil recruitment and infiltration
into the parenchyma. Second, the results demonstrate that bile acids promote a positive feedback
loop between hepatocytes and the Th17 cells during cholestasis.
At present, secukinumab, a monoclonal antibody against IL-17A, has completed a phase
III clinical trial for the treatment of psoriasis and rheumatoid arthritis (Genovese et al., 2013;
Rich et al., 2013). Patients treated with secukinumab have shown a better improvement in their
condition than those treated with entanercept, an antibody against TNF-α (Hueber et al., 2012).
However, Crohn’s disease patients treated with secukinumab had severe adverse effects. Most
prominent of these adverse effects were neutropenia, leucopenia, infections, and candidiasis
(Hueber et al., 2012). These clinical studies suggest that secukinumab would not be a valuable
treatment for cholestatic patients due to their high risk of infections. Therefore, the elucidation of
the mechanism underlying the synergistic interaction between bile acids and IL-17A could
provide a treatment for cholestatic patients without the severe adverse effects.

136

To determine the mechanism of bile acids and IL-17A synergistic interaction, we studied
the transcription factor (C/EBPβ) that has been shown to regulate IL-17A signaling via the IL17RA/IL-17RC adaptor proteins, ACT1 and TRAF6 (Chang et al., 2006; Liu et al., 2009). First,
hepatocytes isolated from C/EBPβ heterozygous knockout mice had a reduction in MIP-2 and
IL-23 fold change after TCA treatment (Figure 3.2B-C). MIP-2 synergistic induction is partially
attenuated in IL-17A and TCA co-treated C/EBPβ heterozygous knockout hepatocytes (Figure
3.2B). In addition, the knockdown of C/EBPβ returns IL-23 mRNA levels to basal levels, in cotreated hepatocytes (Figure 3.2C). Our in vitro results shown that C/EBPβ expression is critical
for the promotion of the positive feedback loop of the IL-23/IL-17A axis during cholestasis.
Furthermore, our results demonstrate that hepatic C/EBPβ activity is not required for the
upregulation of MIP-2 by IL-17A.
Next, we subjected either C/EBPβ heterozygous knockout or wild-type mice to bile duct
ligation for 3 days. Hepatocellular injury was unaffected in C/EBPβ heterozygous knockout BDL
mice (Figure 3.3A). Pro-inflammatory mediator mRNA levels and total neutrophils were not
significantly different in the C/EBPβ heterozygous BDL mice compared to wild-type (Figure
3.3B-D). However, MIP-2 mRNA levels had lower trend in C/EBPβ heterozygous BDL mice
(Figure 3.3B). Though these results demonstrate that C/EBPβ does not contribute to bile acid
induced neutrophilic inflammation, C/EBPβ activation is required for hepatocytes proliferation
and repair as well as adipose homeostasis (Greenbaum et al., 1998; Tanaka et al., 1997). The
knockdown of whole body C/EBPβ could inhibit the initiation of hepatocyte proliferation by
mitogens secreted from other cellular sources. A hepatocyte specific knockout of C/EBPβ would
better demonstrate C/EBPβ regulation of the synergistic interaction between bile acids and IL17A during obstructive cholestasis.

137

To further evaluate the mechanism underlying the synergy between IL-17A and TCA,
we studied the known MAPK pathways that are activated by bile acid signaling (Gupta et al.,
2004; Schoemaker et al., 2004). IL-17A treatment prevented the phosphorylation of p38 or JNK
in hepatocytes (Figure 3.4A-B). This inhibition of p38 and JNK was also observed in
hepatocytes co-treated with IL-17A and TCA (Figure 3.4A-B). Similarly, hepatocytes treated
with either p38 or JNK inhibitors had a further enhancement of MIP-2 induction (Figure 3.4CD). These data suggest that IL-17A prevents the activation of p38 and/or JNK which promotes
the synergistic enhancement of MIP-2 by bile acids. However, hepatocytes were pretreated with
IL-17A for 30 minutes before TCA treatment. Our results might also suggest an autoregulation
of p38 and JNK activation by which a phosphatase deactivates these pathways rapidly after IL17A activation. The role of IL-17A in the activation of p38 and JNK needs further investigation.
As stated above, we have shown that bile acids promote the hepatic expression of IL-23
which requires the activation of AKT and JNK (Figure 2.7B-C, 2.8A-E, 2.9). Whereas, p38
downregulates IL-23 fold induction in TCA-treated hepatocytes (Figure 2.8F). Our data suggest
that MIP-2 and IL-23 induction by bile acids are regulated by two independent pathways.
However, it is possible that MIP-2 and IL-23 upregulation are receptor-specific as are the MAPK
pathways that regulate those cytokines. The receptor that regulates bile acid promotion of the
inflammatory response is not known. Therefore, we investigated a known bile acid activated
receptor, EGFR (Qiao et al., 2001). The inhibition of EGFR did not prevent the upregulation of
IL-23 by TCA (Figure 4.1). Next, we determined the role of GPCRs (S1PR2, LPAR, and EP)
that are closely related to the bile acid receptor, TGR5, which is not expressed on hepatocytes
(Hama and Aoki, 2010; Keitel et al., 2007). Neither S1PR2 nor EP inhibition affected IL-23
expression in hepatocytes (Figure 4.2-4.3). LPAR1 and LPAR2 have 50% homology between

138

them and are expressed on primary mouse hepatocytes. Consistently, human hepatocytes express
low levels of LPAR1 but do not express LPAR2 (Svetlov et al., 2002). The inhibition of LPAR1
and LPAR3 by the selective inhibitor, Ki16425, attenuated IL-23 mRNA levels (Figure 4.4).
Interestingly, it has been demonstrated that the inhibition of LPAR1 reduces fibrotic markers,
alanine aminotransferase activity, and hepatocyte proliferation in murine models of hepatic
injury (Choi et al., 2010; Murch et al., 2007; Sautin et al., 2002). Our results suggest that LPAR1
also mediates the Th17 inflammatory response by upregulating IL-23 expression in hepatocytes.
Interestingly, Th17 cellular differentiation was suppressed in LPAR1 deficient naïve CD4+ T
cells (Miyabe et al., 2013). Collectively, these data suggest that LPAR1 could be an excellent
therapeutic target for the treatment of cholestatic liver disease.
Lastly, we studied the inflammatory role of another Th17 cytokine family member, IL17F. IL-17F is the closest family member to IL-17A (Hymowitz et al., 2001). IL-17F mRNA
levels have an initial peak at 2 days post-BDL, but it returns to basal level within 3 days.
However, its induction is reestablished and remains elevated after 4 days of obstruction (Figure
5.1). To determine if IL-17F would have the same pathophysiological function as IL-17A, we
treated hepatocytes with IL-17F in the presence or absence of TCA. Similar to IL-17A, IL-17F
synergistically enhanced MIP-2 induction by TCA (Figure 5.2). Next, we performed either
sham-operation or bile duct ligation on IL-17 knockout mice for 48 hours. The deletion of IL17F did not affect hepatocellular injury or pro-inflammatory mediator mRNA levels in mice
subjected to BDL, but there was attenuation of neutrophil cell count in the areas of necrosis
(Figure 5.3-5.5). These results demonstrate a critical role of IL-17F to mediate neutrophil
accumulation in cholestatic liver disease. Interestingly, IL-17F knockout mice had a higher basal
level of PAI-1, Egr-1, and C/EBPβ than their wild-type counterparts (Figure 5.4B, 5.6). This is

139

likely due to the increase in fatty acid content in the liver of IL-17F knockout mice. Overall,
these results demonstrate the potential of IL-17F to regulate adipose homeostasis and
granulopoiesis. These results also further suggest the impracticality of the administration of an
antibody against the Th17 cytokines, IL-17A and IL-17F, due to the risk of uncontrollable
weight gain and the inability to actively regulate neutrophil production in the bone marrow.
Overall, we have identified bile acids as novel inducers of the IL-23/IL-17A axis during
cholestasis. The molecular mechanisms that regulate this interaction between bile acids and the
IL-23/IL-17A axis are outlined in Figure 6.1. Furthermore, these studies could be extrapolated to
other liver diseases that have abnormally high concentrations of bile acids such as alcoholic liver
disease (Trinchet et al., 1994). Though our studies demonstrate that antibody against IL-17A or
IL-17F could cause more detrimental effects than beneficial, the discovery of receptor regulating
the upstream mediator of the Th17 cellular response could be a plausible option.
6.2 Future Studies
We have shown that the MAPK pathways, p38 and JNK, activation negatively regulates
MIP-2 induction by TCA. In addition, IL-17A treatment reduced the protein expression of
phospho-p38 and phospho-JNK indicating activation. However, it still needs to be demonstrated
that IL-17A inhibition of p38 and/or JNK is the underlying cause of the synergistic enhancement
of MIP-2. First, we need to investigate mitogen kinase phosphatase-1 (MKP-1) activity in IL17A treated hepatocytes. IL-17A has been shown to inhibit the activation of MKP-1, which will
promote the activation of p38 and JNK in cardiac fibroblast (Valente et al., 2012). Therefore, it is
possible that there is a cellular divergent, and IL-17A is activating MKP-1 in hepatocytes.
Furthermore, IL-17A has been shown to activate the protein kinase, MAPK kinase (MKK3/6),
that regulates the phosphorylation of p38 in synovial fibroblast (Faour et al., 2003).

140

Measurement of protein expression of MKP-1 or MKK3/6 would suggest a pathway by which
IL-17A prevents the activation of p38 and JNK. MKP-1 or MKK3/6 deficient hepatocytes
treated with IL-17A in the presence or absence of TCA would illustrate a direct effect by IL17A. These studies would provide further evidence to support the hypothesis that the synergistic
interaction between IL-17A and bile acid requires the inhibition of p38 and JNK activity.
We have shown that either LPAR1 and/or LPAR3 mediate bile acid induction of IL-23
expression in hepatocytes (Figure 4.4). However, LPAR3 has not been shown to be expressed on
hepatocytes. Another family member that shares 50% homology to LPAR1 is LPAR2 that has a
low expression on primary hepatocytes (Svetlov et al., 2002). At present, we are employing a
spectrum of inhibitors for the LPARs. As our results are preliminary, we need to further evaluate
these receptors in the cell culture before we elucidate their role in vivo.
The LPA receptors are GPCR that have the ability to couple to G αi (Fukushima et al.,
1998). Bile acids activation of the ERK pathway has been shown to be pertussis toxin sensitive;
thereby, suggesting that a G(αi)PCR is the underlying receptor (Dent et al., 2005). Therefore,
treatment of hepatocytes with pertussis toxin would demonstrate LPAR coupled with G αi
promotes the regulation of the hepatic inflammatory response. Our results suggest that LPA
generation does not activate these receptors in an autocrine or paracrine loop. However,
tetradecyl phosphonate has been shown to have off target effects (Durgam et al., 2005).
Therefore, we need to further evaluate the role of these receptors in the regulation of hepatic IL23 expression. We could utilize mice deficient in LPAR1 or autotaxin. LPAR1 deficient
hepatocytes should have a reduction in IL-23 mRNA levels when treated with TCA. LPAR1
deficient mice subjected to BDL would demonstrate the beneficial effects of treating cholestatic
patients with an LPAR antagonist.
141

An interesting observation in this dissertation is the appearance of steatosis in IL-17F
knockout mice (Figure 5.3B-C). Recent publications have shown that IL-17A is increased in
obese patients and regulates adipose differentiation, but the mechanisms underlying this effect
are not well understood (Sumarac-Dumanovic et al., 2009; Zuniga et al., 2010). Furthermore, IL17F has not been shown to mediate adipose formation and accumulation. Therefore, the role of
IL-17F during obesity needs to be determined.

142

Figure 6.1 Mechanisms of the Interaction between Bile Acids and the IL-23/IL-17A Axis
Outlined in this Dissertation. Bile acids promote the induction of IL-23 mRNA levels via
lysophosphatidic acid receptors (LPARs). AKT and JNK signaling pathways are required for the
induction of IL-23; whereas, p38 inhibits the upregulation of IL-23 mRNA levels in hepatocytes.
IL-23 expression maintains the population expansion of Th17 cells, which promotes the
secretion of the cytokines, IL-17A and IL-17F. IL-17A inhibits the activation of p38 and JNK

143

Figure 6.1 (cont’d)
signaling; thereby, leading to the synergistic enhancement of MIP-2 induction by bile acids.
Furthermore, C/EBPβ is a novel pathway that mediates bile acid induction of MIP-2 in
hepatocytes.

144

BIBLIOGRAPHY

145

BIBLIOGRAPHY

Abdel-Misih, S.R., and Bloomston, M. (2010). Liver anatomy. Surg Clin N Am 90, 643-653.
Aggarwal, S., Ghilardi, N., Xie, M.H., de Sauvage, F.J., and Gurney, A.L. (2003). Interleukin-23
promotes a distinct CD4 T cell activation state characterized by the production of interleukin-17.
J Biol Chem 278, 1910-1914.
Aggarwal, S., and Gurney, A.L. (2002). IL-17: prototype member of an emerging cytokine
family. J Leukocyte Biol 71, 1-8.
Akimzhanov, A.M., Yang, X.O., and Dong, C. (2007). Chromatin remodeling of interleukin-17
(IL-17)-IL-17F cytokine gene locus during inflammatory helper T cell differentiation. J Biol
Chem 282, 5969-5972.
Alessi, M.C., Bastelica, D., Mavri, A., Morange, P., Berthet, B., Grino, M., and Juhan-Vague, I.
(2003). Plasma PAI-1 levels are more strongly related to liver steatosis than to adipose tissue
accumulation. Arterioscl Throm Vas 23, 1262-1268.
Allen, K., Jaeschke, H., and Copple, B.L. (2011). Bile acids induce inflammatory genes in
hepatocytes: a novel mechanism of inflammation during obstructive cholestasis. Am J Pathol
178, 175-186.
Allen, K., Kim, N.D., Moon, J.O., and Copple, B.L. (2010). Upregulation of early growth
response factor-1 by bile acids requires mitogen-activated protein kinase signaling. Toxicol Appl
Pharm 243, 63-67.
Ananthanarayanan, M., Balasubramanian, N., Makishima, M., Mangelsdorf, D.J., and Suchy,
F.J. (2001). Human bile salt export pump promoter is transactivated by the farnesoid X
receptor/bile acid receptor. J Biol Chem 276, 28857-28865.
Bergheim, I., Guo, L., Davis, M.A., Duveau, I., and Arteel, G.E. (2006). Critical role of
plasminogen activator inhibitor-1 in cholestatic liver injury and fibrosis. J Pharm Exp Ther 316,
592-600.
Beuers (2009). EASL Clinical Practice Guidelines: management of cholestatic liver diseases. J
Hepatol 51, 237-267.
Birkenmeier, E.H., Gwynn, B., Howard, S., Jerry, J., Gordon, J.I., Landschulz, W.H., and
McKnight, S.L. (1989). Tissue-specific expression, developmental regulation, and genetic
mapping of the gene encoding CCAAT/enhancer binding protein. Gene Dev 3, 1146-1156.
Calmus, Y., and Poupon, R. (1991). Ursodeoxycholic acid (UDCA) in the treatment of chronic
cholestatic diseases. Biochimie 73, 1335-1338.

146

Carey, E.J., and Lindor, K.D. (2012). Current pharmacotherapy for cholestatic liver disease.
Expert Opin Pharmaco 13, 2473-2484.
Chang, S.H., and Dong, C. (2007). A novel heterodimeric cytokine consisting of IL-17 and IL17F regulates inflammatory responses. Cell Res 17, 435-440.
Chang, S.H., Park, H., and Dong, C. (2006). Act1 adaptor protein is an immediate and essential
signaling component of interleukin-17 receptor. J Biol Chem 281, 35603-35607.
Chen, Y., Kijlstra, A., Chen, Y., and Yang, P. (2011). IL-17A stimulates the production of
inflammatory mediators via Erk1/2, p38 MAPK, PI3K/Akt, and NF-kappaB pathways in ARPE19 cells. Mol Vis 17, 3072-3077.
Chen, Y., Langrish, C.L., McKenzie, B., Joyce-Shaikh, B., Stumhofer, J.S., McClanahan, T.,
Blumenschein, W., Churakovsa, T., Low, J., Presta, L., et al. (2006). Anti-IL-23 therapy inhibits
multiple inflammatory pathways and ameliorates autoimmune encephalomyelitis. J Clin Invest
116, 1317-1326.
Chiang, J.Y., Kimmel, R., Weinberger, C., and Stroup, D. (2000). Farnesoid X receptor responds
to bile acids and represses cholesterol 7alpha-hydroxylase gene (CYP7A1) transcription. J Biol
Chem 275, 10918-10924.
Choi, J.W., Herr, D.R., Noguchi, K., Yung, Y.C., Lee, C.W., Mutoh, T., Lin, M.E., Teo, S.T.,
Park, K.E., Mosley, A.N., et al. (2010). LPA receptors: subtypes and biological actions. Annu
Rev Pharmacol 50, 157-186.
Curtis, M.M., and Way, S.S. (2009). Interleukin-17 in host defence against bacterial,
mycobacterial and fungal pathogens. Immunology 126, 177-185.
Dawson, P.A., Lan, T., and Rao, A. (2009). Bile acid transporters. J Lipid Res 50, 2340-2357.
Denson, L.A., Sturm, E., Echevarria, W., Zimmerman, T.L., Makishima, M., Mangelsdorf, D.J.,
and Karpen, S.J. (2001). The orphan nuclear receptor, shp, mediates bile acid-induced inhibition
of the rat bile acid transporter, ntcp. Gastroenterology 121, 140-147.
Dent, P., Fang, Y., Gupta, S., Studer, E., Mitchell, C., Spiegel, S., and Hylemon, P.B. (2005).
Conjugated bile acids promote ERK1/2 and AKT activation via a pertussis toxin-sensitive
mechanism in murine and human hepatocytes. Hepatology 42, 1291-1299.
Durgam, G.G., Virag, T., Walker, M.D., Tsukahara, R., Yasuda, S., Liliom, K., van Meeteren,
L.A., Moolenaar, W.H., Wilke, N., Siess, W., et al. (2005). Synthesis, structure-activity
relationships, and biological evaluation of fatty alcohol phosphates as lysophosphatidic acid
receptor ligands, activators of PPARgamma, and inhibitors of autotaxin. J Med Chem 48, 49194930.

147

Eustace, A., Smyth, L.J.C., Mitchell, L., Williamson, K., Plumb, J., and Singh, D. (2011).
IDentification of cells expressing il-17a and il-17f in the lungs of patients with copd. CHEST
139, 1089-1100.
Fang, Y., Han, S.I., Mitchell, C., Gupta, S., Studer, E., Grant, S., Hylemon, P.B., and Dent, P.
(2004). Bile acids induce mitochondrial ROS, which promote activation of receptor tyrosine
kinases and signaling pathways in rat hepatocytes. Hepatology 40, 961-971.
Faour, W.H., Mancini, A., He, Q.W., and Di Battista, J.A. (2003). T-cell-derived interleukin-17
regulates the level and stability of cyclooxygenase-2 (COX-2) mRNA through restricted
activation of the p38 mitogen-activated protein kinase cascade: role of distal sequences in the 3'untranslated region of COX-2 mRNA. J Biol Chem 278, 26897-26907.
Fickert, P., Zollner, G., Fuchsbichler, A., Stumptner, C., Weiglein, A.H., Lammert, F.,
Marschall, H.U., Tsybrovskyy, O., Zatloukal, K., Denk, H., et al. (2002). Ursodeoxycholic acid
aggravates bile infarcts in bile duct-ligated and Mdr2 knockout mice via disruption of
cholangioles. Gastroenterology 123, 1238-1251.
Freeman, L.A., Kennedy, A., Wu, J., Bark, S., Remaley, A.T., Santamarina-Fojo, S., and Brewer,
H.B., Jr. (2004). The orphan nuclear receptor LRH-1 activates the ABCG5/ABCG8 intergenic
promoter. J Lipid Res 45, 1197-1206.
Fujita, J., Kawaguchi, M., Kokubu, F., Ohara, G., Ota, K., Huang, S.K., Morishima, Y., Ishii, Y.,
Satoh, H., Sakamoto, T., et al. (2012). Interleukin-33 induces interleukin-17F in bronchial
epithelial cells. Allergy 67, 744-750.
Fukushima, N., Kimura, Y., and Chun, J. (1998). A single receptor encoded by vzg-1/lpA1/edg-2
couples to G proteins and mediates multiple cellular responses to lysophosphatidic acid. P Natl
Acad Sci USA 95, 6151-6156.
Gaffen, S.L. (2009a). The role of interleukin-17 in the pathogenesis of rheumatoid arthritis.
Current rheumatology reports 11, 365-370.
Gaffen, S.L. (2009b). Structure and signalling in the IL-17 receptor family. Nat Rev Immunol 9,
556-567.
Galli, S.J., Borregaard, N., and Wynn, T.A. (2011). Phenotypic and functional plasticity of cells
of innate immunity: macrophages, mast cells and neutrophils. Nat Immunol 12, 1035-1044.
Gehring, S., Dickson, E.M., San Martin, M.E., van Rooijen, N., Papa, E.F., Harty, M.W., Tracy,
T.F., Jr., and Gregory, S.H. (2006). Kupffer cells abrogate cholestatic liver injury in mice.
Gastroenterology 130, 810-822.
Genovese, M.C., Durez, P., Richards, H.B., Supronik, J., Dokoupilova, E., Mazurov, V., Aelion,
J.A., Lee, S.H., Codding, C.E., Kellner, H., et al. (2013). Efficacy and safety of secukinumab in

148

patients with rheumatoid arthritis: a phase II, dose-finding, double-blind, randomised, placebo
controlled study. Ann Rheum Dis 72, 863-869.
Georgiev, P., Jochum, W., Heinrich, S., Jang, J.H., Nocito, A., Dahm, F., and Clavien, P.A.
(2008). Characterization of time-related changes after experimental bile duct ligation. Brit J Surg
95, 646-656.
Goodwin, B., Jones, S.A., Price, R.R., Watson, M.A., McKee, D.D., Moore, L.B., Galardi, C.,
Wilson, J.G., Lewis, M.C., Roth, M.E., et al. (2000). A regulatory cascade of the nuclear
receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis. Mol Cell 6, 517-526.
Granet, C., Maslinski, W., and Miossec, P. (2004). Increased AP-1 and NF-κB activation and
recruitment with the combination of the proinflammatory cytokines IL-1β, tumor necrosis factor
alpha and IL-17 in rheumatoid synoviocytes. Arthritis Res Ther 6, 1-9.
Grattagliano, I., Palmieri, V.O., Portincasa, P., Minerva, F., and Palasciano, G. (2011). Longterm ursodeoxycholate improves circulating redox changes in primary biliary cirrhotic patients.
Clin Biochem 44, 1400-1404.
Greenbaum, L.E., Li, W., Cressman, D.E., Peng, Y., Ciliberto, G., Poli, V., and Taub, R. (1998).
CCAAT enhancer- binding protein beta is required for normal hepatocyte proliferation in mice
after partial hepatectomy. J Clin Invest 102, 996-1007.
Griffin, G.K., Newton, G., Tarrio, M.L., Bu, D.X., Maganto-Garcia, E., Azcutia, V., Alcaide, P.,
Grabie, N., Luscinskas, F.W., Croce, K.J., et al. (2012). IL-17 and TNF-alpha sustain neutrophil
recruitment during inflammation through synergistic effects on endothelial activation. J Immunol
188, 6287-6299.
Gujral, J.S., Farhood, A., Bajt, M.L., and Jaeschke, H. (2003). Neutrophils aggravate acute liver
injury during obstructive cholestasis in bile duct-ligated mice. Hepatology 38, 355-363.
Gujral, J.S., Liu, J., Farhood, A., Hinson, J.A., and Jaeschke, H. (2004). Functional importance
of ICAM-1 in the mechanism of neutrophil-induced liver injury in bile duct-ligated mice. Am J
Physiol-Gastr L 286, G499-507.
Gupta, S., Natarajan, R., Payne, S.G., Studer, E.J., Spiegel, S., Dent, P., and Hylemon, P.B.
(2004). Deoxycholic acid activates the c-Jun N-terminal kinase pathway via FAS receptor
activation in primary hepatocytes. Role of acidic sphingomyelinase-mediated ceramide
generation in FAS receptor activation. J Biol Chem 279, 5821-5828.
Gupta, S., Stravitz, R.T., Dent, P., and Hylemon, P.B. (2001). Down-regulation of cholesterol
7alpha-hydroxylase (CYP7A1) gene expression by bile acids in primary rat hepatocytes is
mediated by the c-Jun N-terminal kinase pathway. J Biol Chem 276, 15816-15822.
Hama, K., and Aoki, J. (2010). LPA(3), a unique G protein-coupled receptor for
lysophosphatidic acid. Prog Lipid Res 49, 335-342.
149

Hammel, P., Couvelard, A., O'Toole, D., Ratouis, A., Sauvanet, A., Flejou, J.F., Degott, C.,
Belghiti, J., Bernades, P., Valla, D., et al. (2001). Regression of liver fibrosis after biliary
drainage in patients with chronic pancreatitis and stenosis of the common bile duct. New Engl J
Med 344, 418-423.
Han, C., Li, G., Lim, K., DeFrances, M.C., Gandhi, C.R., and Wu, T. (2008). Transgenic
expression of cyclooxygenase-2 in hepatocytes accelerates endotoxin-induced acute liver failure.
J Immunol 181, 8027-8035.
Hara, M., Kono, H., Furuya, S., Hirayama, K., Tsuchiya, M., and Fujii, H. (2013). Interleukin17A plays a pivotal role in cholestatic liver fibrosis in mice. J Surg Res 183, 574-582.
Harada, K., Shimoda, S., Sato, Y., Isse, K., Ikeda, H., and Nakanuma, Y. (2009). Periductal
interleukin-17 production in association with biliary innate immunity contributes to the
pathogenesis of cholangiopathy in primary biliary cirrhosis. Clin Exp Immunol 157, 261-270.
Harley, I.T., Stankiewicz, T.E., Giles, D.A., Softic, S., Flick, L.M., Cappelletti, M., Sheridan, R.,
Xanthakos, S.A., Steinbrecher, K.A., Sartor, R.B., et al. (2013). IL-17 signaling accelerates the
progression of nonalcoholic fatty liver disease in mice. Hepatology in press.
Heathcote, E.J. (2007). Diagnosis and management of cholestatic liver disease. Clin
Gastroenterol H 5, 776-782.
Hirschfield, G.M., Heathcote, E.J., and Gershwin, M.E. (2010). Pathogenesis of cholestatic liver
disease and therapeutic approaches. Gastroenterology 139, 1481-1496.
Hou, L., Jie, Z., Desai, M., Liang, Y., Soong, L., Wang, T., and Sun, J. (2013). Early IL-17
production by intrahepatic T cells is important for adaptive immune responses in viral hepatitis. J
Immunol 190, 621-629.
Howard, M.W., Strauss, R.G., and Johnston, R.B., Jr. (1977). Infections in patients with
neutropenia. Ama Am J Dis Child 131, 788-790.
Huang, L., Zhao, A., Lew, J.L., Zhang, T., Hrywna, Y., Thompson, J.R., de Pedro, N., Royo, I.,
Blevins, R.A., Pelaez, F., et al. (2003). Farnesoid X receptor activates transcription of the
phospholipid pump MDR3. J Biol Chem 278, 51085-51090.
Hueber, W., Sands, B.E., Lewitzky, S., Vandemeulebroecke, M., Reinisch, W., Higgins, P.D.,
Wehkamp, J., Feagan, B.G., Yao, M.D., Karczewski, M., et al. (2012). Secukinumab, a human
anti-IL-17A monoclonal antibody, for moderate to severe Crohn's disease: unexpected results of
a randomised, double-blind placebo-controlled trial. Gut 61, 1693-1700.
Husted, T.L., Blanchard, J., Schuster, R., Shen, H., and Lentsch, A.B. (2006). Potential role for
IL-23 in hepatic ischemia/reperfusion injury. Inflamm Res 55, 177-178.

150

Hymowitz, S.G., Filvaroff, E.H., Yin, J.P., Lee, J., Cai, L., Risser, P., Maruoka, M., Mao, W.,
Foster, J., Kelley, R.F., et al. (2001). IL-17s adopt a cystine knot fold: structure and activity of a
novel cytokine, IL-17F, and implications for receptor binding. EMBO J 20, 5332-5341.
Ichikawa, M., Takahashi, N., Yada, Y., Koike, Y., Kawamata, R., Kono, Y., Honma, Y.,
Yotsumoto, S., and Momoi, M.Y. (2010). Selectively high levels of serum interleukin 17 in a
newborn infant with progressive severe cholestasis. Pediatrics 126, e247-250.
Inagaki, T., Choi, M., Moschetta, A., Peng, L., Cummins, C.L., McDonald, J.G., Luo, G., Jones,
S.A., Goodwin, B., Richardson, J.A., et al. (2005). Fibroblast growth factor 15 functions as an
enterohepatic signal to regulate bile acid homeostasis. Cell Metab 2, 217-225.
Ito, S., Fujimori, T., Furuya, A., Satoh, J., Nabeshima, Y., and Nabeshima, Y. (2005). Impaired
negative feedback suppression of bile acid synthesis in mice lacking betaKlotho. J Clin Invest
115, 2202-2208.
Ivanov, II, McKenzie, B.S., Zhou, L., Tadokoro, C.E., Lepelley, A., Lafaille, J.J., Cua, D.J., and
Littman, D.R. (2006). The orphan nuclear receptor RORgammat directs the differentiation
program of proinflammatory IL-17+ T helper cells. Cell 126, 1121-1133.
Iyoda, M., Shibata, T., Kawaguchi, M., Hizawa, N., Yamaoka, T., Kokubu, F., and Akizawa, T.
(2010). IL-17A and IL-17F stimulate chemokines via MAPK pathways (ERK1/2 and p38 but not
JNK) in mouse cultured mesangial cells: synergy with TNF-alpha and IL-1beta. Am J PhysiolRenal 298, F779-787.
Jakobsen, J.S., Waage, J., Rapin, N., Bisgaard, H.C., Larsen, F.S., and Porse, B.T. (2013).
Temporal mapping of CEBPA and CEBPB binding during liver regeneration reveals dynamic
occupancy and specific regulatory codes for homeostatic and cell cycle gene batteries. Genome
Res 23, 592-603.
Jazrawi, R.P., de Caestecker, J.S., Goggin, P.M., Britten, A.J., Joseph, A.E., Maxwell, J.D., and
Northfield, T.C. (1994). Kinetics of hepatic bile acid handling in cholestatic liver disease: effect
of ursodeoxycholic acid. Gastroenterology 106, 134-142.
Kageyama, Y., Ikeda, H., Watanabe, N., Nagamine, M., Kusumoto, Y., Yashiro, M., Satoh, Y.,
Shimosawa, T., Shinozaki, K., Tomiya, T., et al. (2012). Antagonism of sphingosine 1-phosphate
receptor 2 causes a selective reduction of portal vein pressure in bile duct-ligated rodents.
Hepatology 56, 1427-1438.
Kataoka, K., Takikawa, Y., Lin, S.D., and Suzuki, K. (2005). Prostaglandin E2 receptor EP4
agonist induces Bcl-xL and independently activates proliferation signals in mouse primary
hepatocytes. J Gastroenterol 40, 610-616.
Keitel, V., Donner, M., Winandy, S., Kubitz, R., and Häussinger, D. (2008). Expression and
function of the bile acid receptor TGR5 in Kupffer cells. Biochem Bioph Res Co 372, 78-84.

151

Keitel, V., Reinehr, R., Gatsios, P., Rupprecht, C., Gorg, B., Selbach, O., Haussinger, D., and
Kubitz, R. (2007). The G-protein coupled bile salt receptor TGR5 is expressed in liver sinusoidal
endothelial cells. Hepatology 45, 695-704.
Keitel, V., Ullmer, C., and Haussinger, D. (2010). The membrane-bound bile acid receptor
TGR5 (Gpbar-1) is localized in the primary cilium of cholangiocytes. Biol Chem 391, 785-789.
Kim, N.D., Moon, J.O., Slitt, A.L., and Copple, B.L. (2006). Early growth response factor-1 is
critical for cholestatic liver injury. Toxicol Sci 90, 586-595.
Kiriyama, M., Ushikubi, F., Kobayashi, T., Hirata, M., Sugimoto, Y., and Narumiya, S. (1997).
Ligand binding specificities of the eight types and subtypes of the mouse prostanoid receptors
expressed in Chinese hamster ovary cells. Brit J Pharmacol 122, 217-224.
Kjoller, L., Kanse, S.M., Kirkegaard, T., Rodenburg, K.W., Ronne, E., Goodman, S.L.,
Preissner, K.T., Ossowski, L., and Andreasen, P.A. (1997). Plasminogen activator inhibitor-1
represses integrin- and vitronectin-mediated cell migration independently of its function as an
inhibitor of plasminogen activation. Exp Cell Res 232, 420-429.
Kobayashi, M., Higuchi, S., Mizuno, K., Tsuneyama, K., Fukami, T., Nakajima, M., and Yokoi,
T. (2010). Interleukin-17 is involved in alpha-naphthylisothiocyanate-induced liver injury in
mice. Toxicology 275, 50-57.
Kodali, P., Wu, P., Lahiji, P.A., Brown, E.J., and Maher, J.J. (2006). ANIT toxicity toward
mouse hepatocytes in vivo is mediated primarily by neutrophils via CD18. Am J Physiol-Gastr L
291, G355-363.
Kolaczkowska, E., and Kubes, P. (2013). Neutrophil recruitment and function in health and
inflammation. Nature reviews. Immunology 13, 159-175.
Kono, H., Fujii, H., Ogiku, M., Hosomura, N., Amemiya, H., Tsuchiya, M., and Hara, M. (2011).
Role of IL-17A in neutrophil recruitment and hepatic injury after warm ischemia-reperfusion
mice. J Immunol 187, 4818-4825.
Korn, T., Bettelli, E., Gao, W., Awasthi, A., Jager, A., Strom, T.B., Oukka, M., and Kuchroo,
V.K. (2007). IL-21 initiates an alternative pathway to induce proinflammatory T(H)17 cells.
Nature 448, 484-487.
Korn, T., Bettelli, E., Oukka, M., and Kuchroo, V.K. (2009). IL-17 and Th17 Cells. Annu Rev
Immunol 27, 485-517.
Kremer, A.E., Martens, J.J., Kulik, W., Rueff, F., Kuiper, E.M., van Buuren, H.R., van Erpecum,
K.J., Kondrackiene, J., Prieto, J., Rust, C., et al. (2010). Lysophosphatidic acid is a potential
mediator of cholestatic pruritus. Gastroenterology 139, 1008-1018, 1018 e1001.

152

Kremer, A.E., van Dijk, R., Leckie, P., Schaap, F.G., Kuiper, E.M., Mettang, T., Reiners, K.S.,
Raap, U., van Buuren, H.R., van Erpecum, K.J., et al. (2012). Serum autotaxin is increased in
pruritus of cholestasis, but not of other origin, and responds to therapeutic interventions.
Hepatology 56, 1391-1400.
Kuestner, R.E., Taft, D.W., Haran, A., Brandt, C.S., Brender, T., Lum, K., Harder, B., Okada, S.,
Ostrander, C.D., Kreindler, J.L., et al. (2007). Identification of the IL-17 receptor related
molecule IL-17RC as the receptor for IL-17F. J Immunol 179, 5462-5473.
Lan, R.Y., Salunga, T.L., Tsuneyama, K., Lian, Z.X., Yang, G.X., Hsu, W., Moritoki, Y., Ansari,
A.A., Kemper, C., Price, J., et al. (2009). Hepatic IL-17 responses in human and murine primary
biliary cirrhosis. J Autoimmun 32, 43-51.
Lee, Y.K., Dell, H., Dowhan, D.H., Hadzopoulou-Cladaras, M., and Moore, D.D. (2000). The
orphan nuclear receptor SHP inhibits hepatocyte nuclear factor 4 and retinoid X receptor
transactivation: two mechanisms for repression. Mol Cell Biol 20, 187-195.
Lemmers, A., Moreno, C., Gustot, T., Marechal, R., Degre, D., Demetter, P., de Nadai, P.,
Geerts, A., Quertinmont, E., Vercruysse, V., et al. (2009). The interleukin-17 pathway is
involved in human alcoholic liver disease. Hepatology 49, 646-657.
Li, M.K., and Crawford, J.M. (2004). The pathology of cholestasis. Semin Liver Dis 24, 21-42.
Liang, S.C., Long, A.J., Bennett, F., Whitters, M.J., Karim, R., Collins, M., Goldman, S.J.,
Dunussi-Joannopoulos, K., Williams, C.M., Wright, J.F., et al. (2007). An IL-17F/A heterodimer
protein is produced by mouse Th17 cells and induces airway neutrophil recruitment. J Immunol
179, 7791-7799.
Licata, L.A., Nguyen, C.T., Burga, R.A., Falanga, V., Espat, N.J., Ayala, A., Thorn, M.,
Junghans, R.P., and Katz, S.C. (2013). Biliary obstruction results in PD-1-dependent liver T cell
dysfunction and acute inflammation mediated by Th17 cells and neutrophils. J Leukocyte Biol
94, 813-823.
Lisboa, L.F., Asthana, S., Kremer, A., Swain, M., Bagshaw, S.M., Gibney, N., and Karvellas,
C.J. (2012). Blood cytokine, chemokine and gene expression in cholestasis patients with
intractable pruritis treated with a molecular adsorbent recirculating system: a case series. Can J
Gastroenterol 26, 799-805.
Liu, C., Qian, W., Qian, Y., Giltiay, N.V., Lu, Y., Swaidani, S., Misra, S., Deng, L., Chen, Z.J.,
and Li, X. (2009). Act1, a U-box E3 ubiquitin ligase for IL-17 signaling. Science Signaling 2,
ra63.
Liu, C., Swaidani, S., Qian, W., Kang, Z., Sun, P., Han, Y., Wang, C., Gulen, M.F., Yin, W.,
Zhang, C., et al. (2011a). A CC' loop decoy peptide blocks the interaction between Act1 and IL17RA to attenuate IL-17- and IL-25-induced inflammation. Science Signaling 4, ra72.

153

Liu, Y., Mei, J., Gonzales, L., Yang, G., Dai, N., Wang, P., Zhang, P., Favara, M., Malcolm,
K.C., Guttentag, S., et al. (2011b). IL-17A and TNF-alpha exert synergistic effects on expression
of CXCL5 by alveolar type II cells in vivo and in vitro. J Immunol 186, 3197-3205.
Liu, Y., Yu, S., Li, Z., Ma, J., Zhang, Y., Wang, H., Yang, B., and Wu, C. (2013). TGF-beta
enhanced IL-21-induced differentiation of human IL-21-producing CD4+ T cells via Smad3.
PloS one 8, e64612.
Ma, X., Reynolds, S.L., Baker, B.J., Li, X., Benveniste, E.N., and Qin, H. (2010). IL-17
enhancement of the IL-6 signaling cascade in astrocytes. J Immunol 184, 4898-4906.
Maione, F., Paschalidis, N., Mascolo, N., Dufton, N., Perretti, M., and D'Acquisto, F. (2009).
Interleukin 17 sustains rather than induces inflammation. Biochem Pharmacol 77, 878-887.
Maitra, A., Shen, F., Hanel, W., Mossman, K., Tocker, J., Swart, D., and Gaffen, S.L. (2007).
Distinct functional motifs within the IL-17 receptor regulate signal transduction and target gene
expression. P Natl Acad Sci USA 104, 7506-7511.
Mangan, P.R., Harrington, L.E., O'Quinn, D.B., Helms, W.S., Bullard, D.C., Elson, C.O.,
Hatton, R.D., Wahl, S.M., Schoeb, T.R., and Weaver, C.T. (2006). Transforming growth factorbeta induces development of the T(H)17 lineage. Nature 441, 231-234.
Melien, O., Sandnes, D., Johansen, E.J., and Christoffersen, T. (2000). Effects of pertussis toxin
on extracellular signal-regulated kinase activation in hepatocytes by hormones and receptorindependent agents: evidence suggesting a stimulatory role of G(i) proteins at a level distal to
receptor coupling. J Cell Physiol 184, 27-36.
Melien, O., Thoresen, G.H., Sandnes, D., Ostby, E., and Christoffersen, T. (1998). Activation of
p42/p44 mitogen-activated protein kinase by angiotensin II, vasopressin, norepinephrine, and
prostaglandin F2alpha in hepatocytes is sustained, and like the effect of epidermal growth factor,
mediated through pertussis toxin-sensitive mechanisms. J Cell Physiol 175, 348-358.
Meng, F., Wang, K., Aoyama, T., Grivennikov, S.I., Paik, Y., Scholten, D., Cong, M., Iwaisako,
K., Liu, X., Zhang, M., et al. (2012). Interleukin-17 signaling in inflammatory, Kupffer cells, and
hepatic stellate cells exacerbates liver fibrosis in mice. Gastroenterology 143, 765-776 e761-763.
Millward, C.A., Heaney, J.D., Sinasac, D.S., Chu, E.C., Bederman, I.R., Gilge, D.A., Previs,
S.F., and Croniger, C.M. (2007). Mice with a deletion in the gene for CCAAT/enhancer-binding
protein beta are protected against diet-induced obesity. Diabetes 56, 161-167.
Miyabe, Y., Miyabe, C., Iwai, Y., Takayasu, A., Fukuda, S., Yokoyama, W., Nagai, J., Jona, M.,
Tokuhara, Y., Ohkawa, R., et al. (2013). Necessity of lysophosphatidic acid receptor 1 for
development of arthritis. Arthritis Rheum 65, 2037-2047.
Moran, E.M., Mullan, R., McCormick, J., Connolly, M., Sullivan, O., Fitzgerald, O., Bresnihan,
B., Veale, D.J., and Fearon, U. (2009). Human rheumatoid arthritis tissue production of IL-17A
154

drives matrix and cartilage degradation: synergy with tumour necrosis factor-alpha, Oncostatin
M and response to biologic therapies. Arthritis Res Ther 11, R113.
Munshi, M.K., Priester, S., Gaudio, E., Yang, F., Alpini, G., Mancinelli, R., Wise, C., Meng, F.,
Franchitto, A., Onori, P., et al. (2011). Regulation of biliary proliferation by neuroendocrine
factors: implications for the pathogenesis of cholestatic liver diseases. Am J Pathol 178, 472-484.
Murch, O., Collin, M., and Thiemermann, C. (2007). Lysophosphatidic acid reduces the organ
injury caused by endotoxemia-a role for G-protein-coupled receptors and peroxisome
proliferator-activated receptor-gamma. Shock 27, 48-54.
Murphy, C.A., Langrish, C.L., Chen, Y., Blumenschein, W., McClanahan, T., Kastelein, R.A.,
Sedgwick, J.D., and Cua, D.J. (2003). Divergent pro- and antiinflammatory roles for IL-23 and
IL-12 in joint autoimmune inflammation. J Exp Med 198, 1951-1957.
Nakajima, A., Wada, K., Miki, H., Kubota, N., Nakajima, N., Terauchi, Y., Ohnishi, S.,
Saubermann, L.J., Kadowaki, T., Blumberg, R.S., et al. (2001). Endogenous PPAR gamma
mediates anti-inflammatory activity in murine ischemia-reperfusion injury. Gastroenterology
120, 460-469.
O'Brien, K.M., Allen, K.M., Rockwell, C.E., Towery, K., Luyendyk, J.P., and Copple, B.L.
(2013). IL-17A Synergistically Enhances Bile Acid-Induced Inflammation during Obstructive
Cholestasis. Am J Pathol 183, 1498-1507.
Oda, N., Canelos, P.B., Essayan, D.M., Plunkett, B.A., Myers, A.C., and Huang, S.K. (2005).
Interleukin-17F induces pulmonary neutrophilia and amplifies antigen-induced allergic response.
Am J Resp Crit Care 171, 12-18.
Ogura, H., Murakami, M., Okuyama, Y., Tsuruoka, M., Kitabayashi, C., Kanamoto, M.,
Nishihara, M., Iwakura, Y., and Hirano, T. (2008). Interleukin-17 promotes autoimmunity by
triggering a positive-feedback loop via interleukin-6 induction. Immunity 29, 628-636.
Ohta, H., Sato, K., Murata, N., Damirin, A., Malchinkhuu, E., Kon, J., Kimura, T., Tobo, M.,
Yamazaki, Y., Watanabe, T., et al. (2003). Ki16425, a subtype-selective antagonist for EDGfamily lysophosphatidic acid receptors. Mol Pharmacol 64, 994-1005.
Onishi, R.M., Park, S.J., Hanel, W., Ho, A.W., Maitra, A., and Gaffen, S.L. (2010). SEF/IL-17R
(SEFIR) is not enough: an extended SEFIR domain is required for il-17RA-mediated signal
transduction. J Biol Chem 285, 32751-32759.
Oppmann, B., Lesley, R., Blom, B., Timans, J.C., Xu, Y., Hunte, B., Vega, F., Yu, N., Wang, J.,
Singh, K., et al. (2000). Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with
biological activities similar as well as distinct from IL-12. Immunity 13, 715-725.
Pandya, A.D., Al-Jaderi, Z., Hoglund, R.A., Holmoy, T., Harbo, H.F., Norgauer, J., and
Maghazachi, A.A. (2011). Identification of human NK17/NK1 cells. PloS one 6, e26780.
155

Park, S.Y., Jeong, K.J., Panupinthu, N., Yu, S., Lee, J., Han, J.W., Kim, J.M., Lee, J.S., Kang, J.,
Park, C.G., et al. (2011). Lysophosphatidic acid augments human hepatocellular carcinoma cell
invasion through LPA1 receptor and MMP-9 expression. Oncogene 30, 1351-1359.
Patel, D.N., King, C.A., Bailey, S.R., Holt, J.W., Venkatachalam, K., Agrawal, A., Valente, A.J.,
and Chandrasekar, B. (2007). Interleukin-17 stimulates C-reactive protein expression in
hepatocytes and smooth muscle cells via p38 MAPK and ERK1/2-dependent NF-kappaB and
C/EBPbeta activation. J Biol Chem 282, 27229-27238.
Penz-Osterreicher, M., Osterreicher, C.H., and Trauner, M. (2011). Fibrosis in autoimmune and
cholestatic liver disease. Best practice & research. Clin Gastroenterol 25, 245-258.
Pillay, J., den Braber, I., Vrisekoop, N., Kwast, L.M., de Boer, R.J., Borghans, J.A., Tesselaar,
K., and Koenderman, L. (2010). In vivo labeling with 2H2O reveals a human neutrophil lifespan
of 5.4 days. Blood 116, 625-627.
Plebani, M., Panozzo, M.P., Basso, D., De Paoli, M., Biasin, R., and Infantolino, D. (1999).
Cytokines and the progression of liver damage in experimental bile duct ligation. Clin Exp
Pharmacol P 26, 358-363.
Qian, C., Jiang, T., Zhang, W., Ren, C., Wang, Q., Qin, Q., Chen, J., Deng, A., and Zhong, R.
(2013). Increased IL-23 and IL-17 expression by peripheral blood cells of patients with primary
biliary cirrhosis. Cytokine 64, 172-180.
Qian, Y., Liu, C., Hartupee, J., Altuntas, C.Z., Gulen, M.F., Jane-Wit, D., Xiao, J., Lu, Y.,
Giltiay, N., Liu, J., et al. (2007). The adaptor Act1 is required for interleukin 17-dependent
signaling associated with autoimmune and inflammatory disease. Nat Immunol 8, 247-256.
Qiao, L., Han, S.I., Fang, Y., Park, J.S., Gupta, S., Gilfor, D., Amorino, G., Valerie, K., Sealy,
L., Engelhardt, J.F., et al. (2003). Bile acid regulation of C/EBPbeta, CREB, and c-Jun function,
via the extracellular signal-regulated kinase and c-Jun NH2-terminal kinase pathways, modulates
the apoptotic response of hepatocytes. Mol Cell Biol 23, 3052-3066.
Qiao, L., Studer, E., Leach, K., McKinstry, R., Gupta, S., Decker, R., Kukreja, R., Valerie, K.,
Nagarkatti, P., El Deiry, W., et al. (2001). Deoxycholic acid (DCA) causes ligand-independent
activation of epidermal growth factor receptor (EGFR) and FAS receptor in primary hepatocytes:
inhibition of EGFR/mitogen-activated protein kinase-signaling module enhances DCA-induced
apoptosis. Mol Biol Cell 12, 2629-2645.
Rahman, S.M., Schroeder-Gloeckler, J.M., Janssen, R.C., Jiang, H., Qadri, I., Maclean, K.N.,
and Friedman, J.E. (2007). CCAAT/enhancing binding protein beta deletion in mice attenuates
inflammation, endoplasmic reticulum stress, and lipid accumulation in diet-induced nonalcoholic
steatohepatitis. Hepatology 45, 1108-1117.

156

Rao, Y.P., Studer, E.J., Stravitz, R.T., Gupta, S., Qiao, L., Dent, P., and Hylemon, P.B. (2002).
Activation of the Raf-1/MEK/ERK cascade by bile acids occurs via the epidermal growth factor
receptor in primary rat hepatocytes. Hepatology 35, 307-314.
Reshetnyak, V.I. (2013). Physiological and molecular biochemical mechanisms of bile
formation. World J Gastroentero 19, 7341-7360.
Rich, P., Sigurgeirsson, B., Thaci, D., Ortonne, J.P., Paul, C., Schopf, R.E., Morita, A., Roseau,
K., Harfst, E., Guettner, A., et al. (2013). Secukinumab induction and maintenance therapy in
moderate-to-severe plaque psoriasis: a randomized, double-blind, placebo-controlled, phase II
regimen-finding study. British J Dermatol 168, 402-411.
Rong, G., Zhou, Y., Xiong, Y., Zhou, L., Geng, H., Jiang, T., Zhu, Y., Lu, H., Zhang, S., Wang,
P., et al. (2009). Imbalance between T helper type 17 and T regulatory cells in patients with
primary biliary cirrhosis: the serum cytokine profile and peripheral cell population. Clin Exp
Immunol 156, 217-225.
Ruddy, M.J., Wong, G.C., Liu, X.K., Yamamoto, H., Kasayama, S., Kirkwood, K.L., and
Gaffen, S.L. (2004). Functional cooperation between interleukin-17 and tumor necrosis factoralpha is mediated by CCAAT/enhancer-binding protein family members. J Biol Chem 279,
2559-2567.
Sautin, Y.Y., Jorgensen, M., Petersen, B.E., Saulnier-Blache, J.S., Crawford, J.M., and Svetlov,
S.I. (2002). Hepatic oval (stem) cell expression of endothelial differentiation gene receptors for
lysophosphatidic acid in mouse chronic liver injury. J Hematoth Stem Cell 11, 643-649.
Scheller, J., Chalaris, A., Schmidt-Arras, D., and Rose-John, S. (2011). The pro- and antiinflammatory properties of the cytokine interleukin-6. Biochim Biophys acta 1813, 878-888.
Schoemaker, M.H., Conde de la Rosa, L., Buist-Homan, M., Vrenken, T.E., Havinga, R.,
Poelstra, K., Haisma, H.J., Jansen, P.L., and Moshage, H. (2004). Tauroursodeoxycholic acid
protects rat hepatocytes from bile acid-induced apoptosis via activation of survival pathways.
Hepatology 39, 1563-1573.
Schwandner, R., Yamaguchi, K., and Cao, Z. (2000). Requirement of tumor necrosis factor
receptor-associated factor (TRAF)6 in interleukin 17 signal transduction. J Exp Med 191, 12331240.
Schwarzenberger, P., Huang, W., Ye, P., Oliver, P., Manuel, M., Zhang, Z., Bagby, G., Nelson,
S., and Kolls, J.K. (2000). Requirement of endogenous stem cell factor and granulocyte-colonystimulating factor for IL-17-mediated granulopoiesis. J Immunol 164, 4783-4789.
Schwarzenberger, P., La Russa, V., Miller, A., Ye, P., Huang, W., Zieske, A., Nelson, S., Bagby,
G.J., Stoltz, D., Mynatt, R.L., et al. (1998). IL-17 stimulates granulopoiesis in mice: use of an
alternate, novel gene therapy-derived method for in vivo evaluation of cytokines. J Immunol 161,
6383-6389.
157

Sendensky, A., and Dufour, J.-F. (2011). Liver Physiology. In Chronic Liver Failure, P. Ginès,
P.S. Kamath, and V. Arroyo, eds. Humana Press, pp. 33-45.
Sheen-Chen, S.M., Chau, P., and Harris, H.W. (1998). Obstructive jaundice alters Kupffer cell
function independent of bacterial translocation. J Surg Res 80, 205-209.
Shen, F., and Gaffen, S.L. (2008). Structure-function relationships in the IL-17 receptor:
implications for signal transduction and therapy. Cytokine 41, 92-104.
Shen, F., Li, N., Gade, P., Kalvakolanu, D.V., Weibley, T., Doble, B., Woodgett, J.R., Wood,
T.D., and Gaffen, S.L. (2009). IL-17 receptor signaling inhibits C/EBPbeta by sequential
phosphorylation of the regulatory 2 domain. Science Signaling 2, ra8.
Shen, F., Ruddy, M.J., Plamondon, P., and Gaffen, S.L. (2005). Cytokines link osteoblasts and
inflammation: microarray analysis of interleukin-17- and TNF-alpha-induced genes in bone
cells. J Leukocyte Biol 77, 388-399.
Silveira, M.G., and Lindor, K.D. (2008). Treatment of primary biliary cirrhosis: therapy with
choleretic and immunosuppressive agents. Clin Liver Dis12, 425-443; x-xi.
Smith, J.A. (1994). Neutrophils, host defense, and inflammation: a double-edged sword. J
Leukocyte Biol 56, 672-686.
Sonder, S.U., Saret, S., Tang, W., Sturdevant, D.E., Porcella, S.F., and Siebenlist, U. (2011). IL17-induced NF-kappaB activation via CIKS/Act1: physiologic significance and signaling
mechanisms. J Biol Chem 286, 12881-12890.
Song, K.H., Li, T., Owsley, E., Strom, S., and Chiang, J.Y. (2009). Bile acids activate fibroblast
growth factor 19 signaling in human hepatocytes to inhibit cholesterol 7alpha-hydroxylase gene
expression. Hepatology 49, 297-305.
Song, P., Zhang, Y., and Klaassen, C.D. (2011). Dose-response of five bile acids on serum and
liver bile Acid concentrations and hepatotoxicty in mice. Toxicol Sci 123, 359-367.
Stapelbroek, J.M., van Erpecum, K.J., Klomp, L.W., Venneman, N.G., Schwartz, T.P., van
Berge Henegouwen, G.P., Devlin, J., van Nieuwkerk, C.M., Knisely, A.S., and Houwen, R.H.
(2006). Nasobiliary drainage induces long-lasting remission in benign recurrent intrahepatic
cholestasis. Hepatology 43, 51-53.
Stracke, M.L., Krutzsch, H.C., Unsworth, E.J., Arestad, A., Cioce, V., Schiffmann, E., and
Liotta, L.A. (1992). Identification, purification, and partial sequence analysis of autotaxin, a
novel motility-stimulating protein. J Biol Chem 267, 2524-2529.
Stritesky, G.L., Yeh, N., and Kaplan, M.H. (2008). IL-23 promotes maintenance but not
commitment to the Th17 lineage. J Immunol 181, 5948-5955.
158

Studer, E., Zhou, X., Zhao, R., Wang, Y., Takabe, K., Nagahashi, M., Pandak, W.M., Dent, P.,
Spiegel, S., Shi, R., et al. (2012). Conjugated bile acids activate the sphingosine-1-phosphate
receptor 2 in primary rodent hepatocytes. Hepatology 55, 267-276.
Sumarac-Dumanovic, M., Stevanovic, D., Ljubic, A., Jorga, J., Simic, M., StamenkovicPejkovic, D., Starcevic, V., Trajkovic, V., and Micic, D. (2009). Increased activity of
interleukin-23/interleukin-17 proinflammatory axis in obese women. Int J Obesity 33, 151-156.
Sun, D., Novotny, M., Bulek, K., Liu, C., Li, X., and Hamilton, T. (2011). Treatment with IL-17
prolongs the half-life of chemokine CXCL1 mRNA via the adaptor TRAF5 and the splicingregulatory factor SF2 (ASF). Nat Immunol 12, 853-860.
Svetlov, S.I., Sautin, Y.Y., and Crawford, J.M. (2002). EDG receptors and hepatic
pathophysiology of LPA and S1P: EDG-ology of liver injury. Biochim Biophys acta 1582, 251256.
Takiguchi, S., and Koga, A. (1988). Effects of bile acids and endotoxin on the function and
morphology of cultured hamster Kupffer cells. Virchows Arch B 54, 303-311.
Tanaka, T., Yoshida, N., Kishimoto, T., and Akira, S. (1997). Defective adipocyte differentiation
in mice lacking the C/EBP[beta] and/or C/EBP[delta] gene. EMBO J 16, 7432-7443.
Toy, D., Kugler, D., Wolfson, M., Vanden Bos, T., Gurgel, J., Derry, J., Tocker, J., and Peschon,
J. (2006). Cutting edge: interleukin 17 signals through a heteromeric receptor complex. J
Immunol 177, 36-39.
Trauner, M., Meier, P.J., and Boyer, J.L. (1998). Molecular pathogenesis of cholestasis. New
Engl J Med 339, 1217-1227.
Trinchet, J.C., Gerhardt, M.F., Balkau, B., Munz, C., and Poupon, R.E. (1994). Serum bile acids
and cholestasis in alcoholic hepatitis. Relationship with usual liver tests and histological features.
J Hepatol 21, 235-240.
Trottier, J., Bialek, A., Caron, P., Straka, R.J., Heathcote, J., Milkiewicz, P., and Barbier, O.
(2012). Metabolomic profiling of 17 bile acids in serum from patients with primary biliary
cirrhosis and primary sclerosing cholangitis: a pilot study. Digest Liver Dis 44, 303-310.
Tsochatzis, E.A., Feudjo, M., Rigamonti, C., Vlachogiannakos, J., Carpenter, J.R., and
Burroughs, A.K. (2013). Ursodeoxycholic acid improves bilirubin but not albumin in primary
biliary cirrhosis: further evidence for nonefficacy. BioMed Res Int 2013, 139763.
Tuomela, S., Salo, V., Tripathi, S.K., Chen, Z., Laurila, K., Gupta, B., Aijo, T., Oikari, L.,
Stockinger, B., Lahdesmaki, H., et al. (2012). Identification of early gene expression changes
during human Th17 cell differentiation. Blood 119, e151-160.

159

Umezu-Goto, M., Kishi, Y., Taira, A., Hama, K., Dohmae, N., Takio, K., Yamori, T., Mills,
G.B., Inoue, K., Aoki, J., et al. (2002). Autotaxin has lysophospholipase D activity leading to
tumor cell growth and motility by lysophosphatidic acid production. J Cell Biol 158, 227-233.
Valente, A.J., Yoshida, T., Gardner, J.D., Somanna, N., Delafontaine, P., and Chandrasekar, B.
(2012). Interleukin-17A stimulates cardiac fibroblast proliferation and migration via negative
regulation of the dual-specificity phosphatase MKP-1/DUSP-1. Cell Signal 24, 560-568.
Veldhoen, M., Hocking, R.J., Atkins, C.J., Locksley, R.M., and Stockinger, B. (2006). TGFbeta
in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17producing T cells. Immunity 24, 179-189.
Wang, H., Chen, J., Hollister, K., Sowers, L.C., and Forman, B.M. (1999). Endogenous bile
acids are ligands for the nuclear receptor FXR/BAR. Mol Cell 3, 543-553.
Wang, H., Vohra, B.P., Zhang, Y., and Heuckeroth, R.O. (2005). Transcriptional profiling after
bile duct ligation identifies PAI-1 as a contributor to cholestatic injury in mice. Hepatology 42,
1099-1108.
Williams, S.C., Baer, M., Dillner, A.J., and Johnson, P.F. (1995). CRP2 (C/EBP beta) contains a
bipartite regulatory domain that controls transcriptional activation, DNA binding and cell
specificity. EMBO J 14, 3170-3183.
Wilson, N.J., Boniface, K., Chan, J.R., McKenzie, B.S., Blumenschein, W.M., Mattson, J.D.,
Basham, B., Smith, K., Chen, T., Morel, F., et al. (2007). Development, cytokine profile and
function of human interleukin 17-producing helper T cells. Nat Immunol 8, 950-957.
Wintermeyer, P., Cheng, C.W., Gehring, S., Hoffman, B.L., Holub, M., Brossay, L., and
Gregory, S.H. (2009). Invariant natural killer T cells suppress the neutrophil inflammatory
response in a mouse model of cholestatic liver damage. Gastroenterology 136, 1048-1059.
Woolbright, B.L., Antoine, D.J., Jenkins, R.E., Bajt, M.L., Park, B.K., and Jaeschke, H. (2013).
Plasma biomarkers of liver injury and inflammation demonstrate a lack of apoptosis during
obstructive cholestasis in mice. Toxicol Appl Pharm 273(3), 524-531.
Yang, X.O., Chang, S.H., Park, H., Nurieva, R., Shah, B., Acero, L., Wang, Y.H., Schluns, K.S.,
Broaddus, R.R., Zhu, Z., et al. (2008a). Regulation of inflammatory responses by IL-17F. J Exp
Med 205, 1063-1075.
Yang, X.O., Nurieva, R., Martinez, G.J., Kang, H.S., Chung, Y., Pappu, B.P., Shah, B., Chang,
S.H., Schluns, K.S., Watowich, S.S., et al. (2008b). Molecular antagonism and plasticity of
regulatory and inflammatory T cell programs. Immunity 29, 44-56.
Yang, X.O., Panopoulos, A.D., Nurieva, R., Chang, S.H., Wang, D., Watowich, S.S., and Dong,
C. (2007). STAT3 regulates cytokine-mediated generation of inflammatory helper T cells. J Biol
Chem 282, 9358-9363.
160

Yang, X.O., Pappu, B.P., Nurieva, R., Akimzhanov, A., Kang, H.S., Chung, Y., Ma, L., Shah,
B., Panopoulos, A.D., Schluns, K.S., et al. (2008c). T helper 17 lineage differentiation is
programmed by orphan nuclear receptors ROR alpha and ROR gamma. Immunity 28, 29-39.
Yao, Z., Fanslow, W.C., Seldin, M.F., Rousseau, A.M., Painter, S.L., Comeau, M.R., Cohen, J.I.,
and Spriggs, M.K. (1995). Herpesvirus Saimiri encodes a new cytokine, IL-17, which binds to a
novel cytokine receptor. Immunity 3, 811-821.
Zhang, F., Lin, M., Abidi, P., Thiel, G., and Liu, J. (2003). Specific interaction of Egr1 and
c/EBPbeta leads to the transcriptional activation of the human low density lipoprotein receptor
gene. J Biol Chem 278, 44246-44254.
Zhang, Y., Hong, J.Y., Rockwell, C.E., Copple, B.L., Jaeschke, H., and Klaassen, C.D. (2012).
Effect of bile duct ligation on bile acid composition in mouse serum and liver. Liver Int 32, 5869.
Zimmermann, H.W., Seidler, S., Gassler, N., Nattermann, J., Luedde, T., Trautwein, C., and
Tacke, F. (2011). Interleukin-8 is activated in patients with chronic liver diseases and associated
with hepatic macrophage accumulation in human liver fibrosis. PloS one 6, e21381.
Zuniga, L.A., Shen, W.J., Joyce-Shaikh, B., Pyatnova, E.A., Richards, A.G., Thom, C., Andrade,
S.M., Cua, D.J., Kraemer, F.B., and Butcher, E.C. (2010). IL-17 regulates adipogenesis, glucose
homeostasis, and obesity. J Immunol 185, 6947-6959.

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