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EFFECTS OF N-3 POLYUNSATURATED FATTY AICDS ON
ADIPOSE STEM CELL PRODUCTION OF lNTERLEUKlN-6
(IL-6)

presented by

HUI WEN HSUEH

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5/08 K'IProlecc8-PresICIRC/DaleDue.indd

EFFECTS OF N-3 POLYUNSATURATED FATTY ACIDS ON ADIPOSE STEM CELL
PRODUCTION OF INTERLEUKIN-6 (IL-6)

By

HUI WEN HSUEH

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Human Nutrition

2010

ABSTRACT
EFFECTS OF N-3 POLYUNSATURATED FATTY ACIDS ON ADIPOSE STEM CELL
PRODUCTION OF INTERLEUKIN-6 (IL-6)
By
HUI WEN HSUEH
Accumulating evidence has suggested that adipose tissue plays a key role in

obesity-associated increases in systemic inflammatory responses. It has been shown that
adipose tissue mass positively correlates with increased circulating inflammatory
cytokine IL-6 and n-3 polyunsaturated fatty acids (n-3 PUFA) reduce obesity-associated
inflammation. Recently results from our laboratory showed that subset of adipose tissue
cells including adipose stem cells secrete significantly higher levels of inflammatory
factors that can be accounted for an increase in systemic inflammation. However, in
obese mice no studies have addressed whether n-3 PUFA has an inhibitory role in IL-6
secretion and IL-6 gene expression in adipose tissue derived stem cells. Thus, we treated
adipose stem cells with stearidonic acid (SDA, l8:4 n-3, EPA precursor) and
eicosapentaenoic acid (EPA, 20:5 n-3) to determine if IL-6 secretion and gene expression
were altered. Results demonstrated that EPA and SDA significantly reduced
lipopolysaccharide (LPS)-induced IL-6 secretion and IL-6 mRNA expression levels in the
adipose tissue stem cells. Toll-like-receptor 2 (TLR2) mediates this inhibitory effect in
reducing IL-6 mRNA by inhibiting NF-KB activation and not via MAPK activation

pathways.

This work is dedicated to my dearest parents, brother, and uncle.

iii

ACKNOWLEDGEMENTS

It is an honor for me to express my sincere gratitude to my advisor, Dr. Kate J.
Claycombe, for her continuous support of my M.S. study and research. Her patience,
encouragement, and immense knowledge helped me to overcome many difficult
situations. Her guidance helped me throughout my thesis research and writing process.

In addition to my advisor, I would like to thank my thesis committee members: Dr.
Won Song, Dr. P.S. MohanKumar and Dr. Eun-Kyoung Kim, for their assistance and
suggestions. My sincere thanks also goes to Dr. Zhou Zhou for her kind directions in
experiment skills.

Last but not least, I would like to thank the graduate school for their support during
my summer research and the FSHN department for giving me an opportunity to acquire

an advanced degree in Human Nutrition.

TABLE OF CONTENTS

List of Figures .................................................................................................................... vi
Key to Abbreviations ........................................................................................................ vii

Chapter 1. Introduction

1.1 Overview ................................................................................................. 1
1.2 Specific Aims and Hypotheses (Ho) ........................................................ 3
1.3 Rationale for Study Aims ........................................................................ 4
Chapter 2. Background
2.1 Dysregulation of Immune Function and Chronic diseases ...................... S
2.2 Interrelationship between Adipose Tissue, Adipose Stem Cell,
Inflammation and Chronic Disease Development ................................... 6
2.3 Polyunsaturated Fatty Acid Structures and Physiological Functions ...... 7
2.4 Toll-Like Receptor (TLR) Family: Mediators of Anti-Inflammatory
Function of n-3 PUFA ............................................................................ 11
Chapter 3. Methodology
3.1 Study Design .......................................................................................... 13
3 .2 Materials ................................................................................................ 1 5
3.3 Methods ................................................................................................. 15
3 .4 Statistical Analysis ................................................................................. l 8
Chapter 4. Results
4.1 Results for Hol ...................................................................................... 19
4.2 Results for H02 ...................................................................................... 22
4.3 Results for H03 ...................................................................................... 24
Chapter 5: Discussion and Conclusion ............................................................................. 30
Appendix.......... .............................................................................................................. 36
References. . . . . . ................................................................................................................. 39

Figure 2.1

Figure 4.1

Figure 4.2

Figure 4.3

Figure 4.4
Figure 4.5

Figure 4.6

Figure 4.7

Figure 5.1

Figure Al.

Figure A2.

LIST OF FIGURES

Images in this thesis are presented in color

Biosynthesis and metabolism of fatty acids. ................................................ 9

Effects of LA, AA, SDA and EPA on LPS-induced IL~6 secretion in CD34+

cells. ............................................................................................................ 2O
Dose response effects of EPA in CD34+ cells ............................................. 21
Effects of EPA, SDA and LA on LPS-Induced IL-6 mRNA level in CD34+

cells. ............................................................................................................ 23
Effects of SDA and EPA on TLR2 protein expression in CD34+ cells ....... 26

Effects of SDA and EPA on IRAKl protein expression in CD34+ cells. 27

Effects of SDA and EPA on phospho-ERKl/2 protein expression in CD34+
cells. ............................................................................................................ 28

LPS-induced NF-KB translocation and inhibitory effects of SDA and EPA.
”u"unuuuuu”nuuuuununnnuuununuunnuu". ............................................. 29

Proposed mechanism of n-3 PUFA inhibition IL-6 mRN A in the adipose
stem cells. ................................................................................................... 35

MTT assays for testing viability of CD34+ adipose stem cells by EPA
treatment. .................................................................................................... 37

Time course of LPS-induced IL-6 mRNA level in CD34+ cells. ............... 38

vi

KEY TO ABBREVIATIONS

AP-l
cox
CRP
CVD
EPA
IKKB
IL-6
IRAKI
1x13

LA

LOX

LPS

LT
MACS
MyD88
NF-KB
P-ERK1/2
PG

SDA

SVF

TAKl

Activating protein-1
Cyclooxygenase

C-reactive protein
Cardiovascular disease;
Eicosapentaenoic acid;
Inhibitor of NF -KB kinase-B
Interleukin—6

IL-l receptor-associated kinase
Inhibitor of NF-KB

Linoleic acid

Lipoxygenase
Lipopolysaccharide
Leukotriene

Magnetic cell sorting

Myeloid differentiation primary response gene (88)

Nuclear factor-kappa B

Phosphorylation of extracellular signal regulated protein kinase 1 and 2

Prostaglandin
Stearidonic acid

Stromal vascular fraction

Transforming growth factor-B-activated kinase 1

vii

TLR Toll-like receptor;

TRAF Tumor necrosis factor receptor-associated factor

viii

Chapter 1: Introduction

1.1 Overview:

Currently, over 60% of US. adults are overweight [1]. It has been shown that
obesity increases the risk for developing a number of chronic diseases such as type 2
diabetes mellitus, cardiovascular disease (CVD), and cancer [2, 3]. Obesity positively
correlates with adipose tissue mass and increased circulating inflammatory biomarker
such as interleukin (IL)-1, IL-6, IL-8, IL-1 8, tumor necrosis factor-a (TNF-a), and
monocyte chemoattractant protein-1 (MCP-l) [4-6]. Recent studies have suggested that
increased inflammatory response or inflammatory biomarker levels mediate, at least in
part, chronic disease development processes [7, 8]. Therefore, it is important to determine
what nutrients can be used to reduce inflammatory biomarker levels in obesity that results
in decreased risk for chronic diseases.

Clinical studies have shown that obese humans have increased adipose tissue and
serum IL-6 concentration [9]. Reduction in body weight and adipose tissue mass resulted
in decrease in serum IL-6 concentration [10-12]. IL-6 is a pleiotropic proinflarnmatory
cytokine that has been shown to be secreted from activated leukocytes, endothelial cells,
adipocytes adipose stem cells [13], and preadipocytes [14, 15]. Once secreted, IL-6 acts
synergistically with other regulatory factors to decrease insulin signaling pathway [16],
increase acute phase protein production [17], and serve as an activator of immune cell
differentiation [17].

Moreover, several recent prospective studies have demonstrated the beneficial
effects of n-3 polyunsaturated fatty acids (PUFA) in lowering CVD risk [18-20], and risk

of sudden cardiac death in normal weight men [21]. Since adipose tissue has been shown

to produce significant amount of IL-6 [22], and IL-6 has been an major activator of
C-reactive protein (CRP) which is a major CVD biomarker and one of the major causes
of CVD, it is plausible that one of the possible mechanisms of CVD risk-lowering effects
of n-3 PUFA may be due to decreased serum IL-6 [23]. Currently, the preventative effects
of n-3 PUFA on CVD risk have been generally attributed to the fish oils, eicosa—
pentaenoic acid [EPA; 20:5(n-3)] and docosahexaenoic acid [DHA; 22:6 (n-3)] [24, 25].
Elongation of shorter chain n-3 PUFA such as a—linolenic acid [ALA; 18:3 (n-3)] to EPA
is regulated by the key regulatory enzyme A-6 desaturase. To increase cellular EPA levels,
several recent studies have addressed using stearidonic acid [SDA; 18:4 (n-3)] which
bypasses regulated A-6 desaturase step in conversion of ALA to EPA and showed that
SDA significantly enrich cellular EPA levels [26-28].

Since adipose tissue contributes significantly toward overall increase in serum
IL-6 [22], reducing adipose tissue cell production of IL-6 may significantly reduce
systemic IL-6 levels in obesity. Previous studies from our laboratory also showed that
IL-6 is produced at highest level from adipose stem cells when compared to other cell
types in the adipose tissue including adipocyte, preadipocyte, and adipose tissue resident
macrophages [29].

No studies to date have addressed whether n-3 PUFAs have an anti-inflammatory
effect on stem cells, or particularly stem cells from adipose tissue. Taken together these
findings, we propose to study whether n-3 PUFAs inhibit IL-6 production from the
adipose stem cells that are currently characterized to have very high degree of

inflammatory function.

1.2 Specific Aims and Hypotheses:

Aim 1: To test the inhibitory role of n—3 FAs (SDA and EPA) and n—6 FAs (LA and AA)
on LPS-induced IL-6 secretion from adipose stem cell. We used n-3 PUFAs which are
shown to have its anti-inflammatory effects to address whether n-3 PUFA have
anti-inflammatory effect on specific type of cell that produce very high levels of IL-6
such as adipose tissue stem cells. LPS was used to induce IL—6 secretion in this study.

Hypothesis 1: LA, AA, SDA and EPA would decrease IL-6 secretion.

Aim 2: To evaluate the inhibitory role of LA, SDA, and EPA on LPS-induced IL—6
mRNA expression on adipose stem cell. We used n-3 PUFAs, which have been shown to
have anti-inflammatory effects to address whether n-3 PUFA have anti-inflammatory
effect on specific type of cell that induce very high expression of IL-6 mRNA such as
adipose tissue stem cells. LPS is used to induce IL-6 mRNA expression in this study.

Hypothesis 2: LA, SDA and EPA would decrease IL-6 mRNA expression.

Aim 3: To investigate the underlying mechanism of inhibiting IL-6 mRNA expression by
SDA and EPA, the involvement of TLR5 and downstream mediators of TLR5 signaling
pathways. TLR 2 and TLR 4 are used as target protein in our experiment. In addition, It
was examined if our target proteins mediates the inhibitory effect of SDA and EPA in
reducing IL-6 mRNA by inhibiting NF -i<B activation.

Hypothesis 3: SDA and EPA would decrease IL-6 mRN A expression via TLR mediated

pathway.

1.3 Rationale for Study Aims:

The specific aims of this study are to evaluate the inhibitory effect of n—3 PUFA
on adipose stem cell production of IL-6 and IL-6 gene expression and to investigate the
mechanism underlying the inhibitory effect of n-3 PUFA. To our best of knowledge, no
studies to date have addressed whether 11-3 PUFA have anti-inflammatory effect on stem
cells or particularly stem cells from adipose tissue.

Therefore, we studied whether n-3 PUFA inhibits IL-6 production in the adipose
stem cells. In the analyses for Aim 1 and Aim 2 we will compare the inhibitory effect of
n-3 and n-6 PUFA by ELISA and real time RT-PCR methods. Results obtained from Aim
1 and Aim 2 will then lead to our next study in which we will study how intracellular
signaling molecules mediate inhibitory effect of IL-6 mRNA expression by SDA and EPA.
As TLR has been shown to be involved in mediating inflammatory responses of adipose
tissue cell types [30], we also investigate the potential link between the TLR and n-3
PUFAs’ action on lowering IL-6.

Studies have suggested that the elevated CRP concentrations might be due to
elevated cytokine IL-6 in adipose tissue [22, 31]. This is because increased levels of IL-6
from adipose tissue may induce elevated CRP in people with excess body fat. Elevated
levels of CRP observed in obesity, type 2 diabetes mellitus, and CVD [32] suggested that
systemic inflammation plays a significant role in chronic disease. Successful completion
of our research would ultimately increase the understanding of how n-3 PUFA reduces

serum IL-6 levels from one of the potent IL-6 secretion.

Chapter 2: Background

2.1 Dysregulation of Immune Function and Chronic diseases

Inflammation is the body’s response to infection or injury [33]. The symptoms are
accompanied by pain, redness, swelling, and heat [34]. This occurs as a result of
increased blood flow as well as increased permeability of blood capillaries, which
permits large molecule to pass through the blood stream and cross the endothelial wall.
This allows increasing movement of leukocytes from blood stream to surrounding tissue.
Inflammation is a part of innate immune response that is essential to health. For example,
immune response is necessary to protect the host from pathogenic organism. However,
chronic over activation may lead to adverse effects, which is characterized by
overproduction of proinflammatory cytokines by monocytes, macrophages, and
lymphocytes [3 5]. Over activation can also lead to the development of autoimmune
diseases such as type 1 diabetes, rheumatoid arthritis, Crohn’s disease, psoriasis, and
multiple sclerosis [3 5].

Alternatively, the immune system of some individuals can become sensitized to a
benign antigen from food or the environment called as allergens that can lead to allergies
[36]. Allergenic responses are often due to chronic inflammation which is accompanied
by inappropriate recognition and response to the antigen [3 6] Allergenic diseases are
characterized by a dysregulated T cell activation and over production of IL-4, IL-5, and
IL-10 [35]. Chronic inflammation and overproduction not only leads to chronic disease

development but also result in organ damages and loss of organ functions.

2.2 Interrelationship between Adipose Tissue, Adipose Stem Cell, Inflammation
and Chronic Disease Development

Adipose tissue is comprised of several different cell types such as fibroblasts,
nondifferentiated mesenchymal cells, preadipocytes, and adipocytes [3 7]. The stromal
vascular fraction (SVF) cells of adipose tissue are comprised of heterogeneous cell types,
including macrophages, preadipocytes, fibroblasts, and nondifferentiated mesenchymal
stem cells. Adipose tissue SVF cells secrete higher levels of IL-6 and MCP-l compared
to adipocytes [13, 38]. Our lab showed that adipose stem cells secrete higher levels of
IL-6 compared to adipocytes [13]. Obesity-associated increases in circulating
proinflarnmatory cytokine levels, decrease with a reduction in body weight, especially in
decreasing body adipose tissue mass [4, 5]. This suggests adipose tissue is an important
source of obesity-associated increases inflammatory responses. Several cytokine and
chemokines, including IL-l , IL-6, IL-8, IL-18, tumor necrosis factor (TNF-a) and
monocyte chemoattractant protein-1 (MCP-l), have been shown to be regulated
according to the changes in body adiposity [6].

An increasing number of studies have shown that circulating levels of IL-6 are
significantly higher in obese humans and animals [10, 39]. IL-6 is a proinflammatory
cytokine and is secreted from a variety of tissue such as endothelial cells, activated
leukocytes, and adipocytes. [14, 15]. Once secreted, IL-6 acts synergistically with other
regulatory factors to affect the development of several chronic diseases such as '
cardiovascular disease (CVD) and Type 2 diabetes [14, 40]. IL-6 decreases lipoprotein
lipase (LPL) activity, which in turn results in increased circulating lipid levels [40]. The

study also showed that IL-6 induces hepatic C-reactive protein which is the marker of

CVD risk. Furthermore, obesity-associated increases in IL-6 levels were shown to be

linked to decreased insulin-induced glucose uptake [41, 42].

2.3 Polyunsaturated Fatty Acid Structures and Physiological Functions

The two polyunsaturated fatty acids (PUFAs) in n-3 family, which were used in
this study are stearidonic acid (SDA; 18:4n-3) and eicosapentaenoic acid (EPA; 20:5 n-3).
A fatty acid is a hydrocarbon chain with a carboxyl group at one end and a methyl group
at the other end. Natural fatty acids contain 2 to 30 (even number) carbon atoms. Fatty
acids can be saturated (n=0), monounsaturated (n=1) or polyunsaturated (n22), based on
the number (n) of double bonds in the carbon chain. PUFA can be classified as n-3 and
n-6 fatty acid with the double bond in the third and sixth carbon position from the methyl
terminal, respectively.

In humans, there are two essential fatty acids, linoleic acid (LA; 18:2n-6) and a-
linolenic acid (ALA; 18:3n-3), which are not synthesized endogenously. Humans do not
have A-l 5-desaturase, which interconvert n-6 to n-3 fatty acid. Plant tissues and plant
oils tend to be rich sources of LA and ALA. LA contributes over 50% and often up to
80% of the fatty acid found in corn, safflower, sunflower and soybean oils [43]. In
rapeseed and soybean oils, ALA contributes between 5 and 15% of total fatty acids
present [43]. In flaxseed oil ALA contributes as much as 60% of total fatty acids.
Currently, the preventive effects of n—3 PUFA on CVD risk have been generally attributed
to the fish oils, EPA and docosahexaenoic acid (DHA; 22:6n-3) [14]. Elongation of
shorter chain n-3 PUFA such as ALA to EPA is regulated by the key regulatory enzyme

A-6 desaturase. To increase cellular EPA levels, several recent studies have used SDA,

which bypass A-6 desaturase step in conversion of ALA to EPA indicating that SDA
significantly enriched cellular EPA levels [26-28]. Thus, in our study, we also
investigated the effects of SDA on adipose stem cell production of IL-6. Long chain n—6
PUFA such as arachidonic acid [3] (AA; 20:4n-6) are synthesized form LA. An outline of

the pathways of biosynthesis and metabolism of PUFA is shown in Figure 2.1 [43].

 

 

12:0 +1420 +16z0 +18:0

Diet or de novo synthesis / A9-desaturase

18:1n—9

 

 

 

A12-desaturase
in plants only y

 

 

 

A1 5-desaturase
in plants only

 

Diet

 

 

(LA)18:2n—6 b 18:3n-3 (ALA)
A6-desaturase I
18:3n-6 l8:4n—3 (SDA)
20:3n-6 20:4n—3
l
‘ AS-desaturase COX
/' 3-series PG
20:4n—6 (AA) 20:5n—3 (EPA)
I 5-series LT

cox / \LOX

2-series PG

1

4-series LT

l

V

 

 

Inflammation and immunity

 

 

LOX

l
l

l

22:6n-3

Figure 2.1 Biosynthesis and metabolism of fatty acids.

Fatty acids are crucial components of cell membranes. The exact proportion of
fatty acids in human immune cells varies according to cell type and the lipid fraction
examined. Although ALA can be converted to EPA and DHA, humans still need an
exogenous supply of long chain PUFAs [43]. The biosynthetic pathways of both n-3 and
n-6 families share the same enzyme called A-6-desaturase [43]. A-6—desaturase is
important for the conversion of LA to ALA and ALA to SDA, EPA, and DHA. High
levels of plasma LA due to high n-6 PUFA intake shifts its actions toward the n-6
pathway [43]. Moreover, the conversion of ALA to EPA and DHA is inefficient [44].
Thus, it is plausible that adequate intakes of long chain n-3 PUFAs may reduce the risk
for cardiovascular disease [45], inflammation [46], cancer [47] and neurological disorders
[48].

One of the ways in which n-3 PUFAs can lower chronic disease risk is via
production of eicosanoids that are counter-regulatory to n-6 PUFAs derived eicosanoids.
For example, eicosanoids generated from 20-carbon by cyclooxygenase-2 (COX-2)
action gives rise to the 2-series prostaglandins (PG) [35]. Metabolism of AA by the
5-lipoxygenase (5-LOX) pathway gives rise to 4-series LT [35]. EPA gives rise to the
3-series PGs and TXs, and 5-series of LTs [43] that have opposing effects of AA-derived
eicosanoids. For example, PGIZ is an activator and PGI3 is an inhibitor of platelet
aggregation will lead to increase and decrease CVD risk, respectively [49].

Moreover, n-3 PUFA have been shown to inhibit proinflammatory gene
expression. Fish oil feeding decreased ex-vivo production of TNF-a, IL-10 and IL-6, in
cultured mice macrophages [50] and monocytes [51, 52]. In addition, in-vivo feeding

studies showed that fish oil feeding resulted in lower peak plasma TNF-u, IL-lB and IL-6

concentrations [53].

Additionally, n-3 PUFAs may directly affect transcription factor activity and
modulate gene expression [54]. Non-esterified fatty acids and their metabolites are able
to bind the transcription factor, for example, peroxisome proliferator—activated receptors
(PPARs) act as ligands to activate them [55]. Oleic acid and PUFAs have been shown to
decrease the transcription factor sterol regulatory element-binding protein (SREBP) in the
liver and consequently change the expression of enzyme for lipogenesis [56]. Studies
have also shown that potent anti—inflammatory functions of E- and D-series resolvins

derived from EPA and DHA respectively as well as protectins derived from DHA [57].

2.4 Toll-Like Receptor (TLR) Family: Mediators of Anti-Inflammatory Function
of n-3 PUFA

Toll-like receptors (TLRs) are membrane proteins characterized by an ectodomain
composed of leucine rich repeats (LRR) that are responsible for recognition of pathogens.
There are 13 mouse TLRs that have been indentified, and each of them can recognize
distinct pathogens or pathogen-associated molecules such as bacteria, viruses, fungi and
protozoa [58].

Among 13 isoforrns of TLR5, lipid is recognized by TLR], 2, 4, and 6. For
instance, TLR4 and TLR2 together with its extracellular components such as CD14 and
MD-2, recognize lipopolysaccharide (LPS) from gram-negative bacteria [59]. TLR2 has
been shown to form heterodimers with TLRl , TLR6 or non-TLR5 such as CD36 [59].
TLR5 and TLRll recognize protein ligands. TLR5 is expressed abundantly in intestinal

CD1 1c-positive lamina propria cells where it recognizes bacterial flagellin [59]. TLR3

ll

has been shown to recognize double stranded RNA (dsRNA), which is produced by many
viruses during replication. Binding of ligands to TLR activates intracellular signaling
pathways that leads to induction of inflammatory cytokine genes. For example, IL-6,

IL-1 [3, IL-12 and Tumor Necrosis Factor (1 (TNFa) [5 8] via recruitment of intracellular
Toll/Interleukin-l receptor (TIR) domain-containing adaptors, such as myeloid
differentiation factor 88 (MyD88), TIR-containing adaptor inducing IFN-B (Trif), and
(TriO-related adaptor molecule (TRAM) [60]. Recruitment of MyD88 leads to activation
of Mitogen-activated protein kinases (MAPKs), which include ERK (extracellular
signal-regulated kinases), c-Jun N-terminal kinases (IN K), and p38. MyD88 also
activates NF -KB to control the inflammatory cytokine genes [61]. TIR domain containing
adaptor protein (TIRAP) mediates MyD88-dependent pathway downstream of TLR2 and
TLR4 [58].

MyD88 stimulates recruitment of Interleukin-1 receptor-associated kinase (IRAK)
family, such as IRAKl , IRAK2, IRAK4, and IRAK-M. Once phosphorylated, IRAK
dissociates from MyD88 and interact with TRAF6, which is a TNF receptor-associated
factor (TRAF) family [58]. Next, TRAF forms a complex with ch13 and Uele to
synthesize the polyubiquitin chains, which activates Transforming growth
factor-B—activated kinase 1 (TAKl) [62]. TAKl activates two downstream pathways
involving IKK (IKB kinase) complex composed of IKKu , IKKB and a regulatory protein
IKKy/NEMO which catalyzes the phosphorylation of IKB proteins. IKB is degraded after
phosphorylation and the subsequent NF-KB translocation from cytoplasm to nucleus [58].
NF-KB controls the expression of various inflammatory cytokine genes [63]. MAPK

phosphorylates and activates the transcription factor activator protein-1 (AP- 1) [63].

12

LPS is a cell wall component of gram negative bacteria [64]. LPS is a well known
ligand for TLR4 and to lesser extent TLR2 [65]. The binding of LPS to TLR on the cell
surface stimulates the proinflammatory cytokine production [66] via activation and
recruitment of MyD88 and other downstream intermediates of TLR signaling pathway as

described above.

13

Chapter 3: Methodology

3.1 Study Design

For the preparation of adipose tissue derived stem cell (CD34+), white adipose
tissue was excised from leptin deficient obese C57BL/6J-ob/ob (ob/ob) mice purchased
from Jackson Laboratory (Bar Harbor, ME), minced and digested using 0.02%
colagenase containing 2mg/ml of collagenase type I (Worthington Biochemical
Corporation, Lakewood, NJ) in Hank’s Balanced Salt Solution (HBSS) supplemented
with sodium bicarbonate (0.3 5 g/L) at 37 °C in shaking water for 1 h. Adipose tissue was
collected from the subcutaneous inguinal (SI) region. The digested adipose tissue cells
were filtered through 100 um nylon cell strainers (BD Biosciences, Bedford, MA). The
stromal vascular fraction (SVF) cells were isolated by centrifugation (450 x g for 1 min),
treated with red blood cell lysis buffer for 5 min at room temperature, washed twice with
Dulbecco’s Modification of Eagle’s Medium (DMEM) (Collgro Mediatech Inc., Hemdon.
VA), and resuspended with fresh DMEM supplemented with 10% heat inhibited Fetal
Bovine Serum (HIFBS) (GIBCO, Grand Island, New York), 100 IU penicillin and 100
[rig/ml streptomycin. CD34+ cells were isolated from SVF cells using Magnetic Cell
Sorting (MACS) according to the manufacturer’s instructions (Miltenyi Biotec, Auburn,
CA) as we previously reported [13].

For hypothesis 1, which is to investigate lL-6 secretion mediated by PUFAs,
CD34+ cells were cultured and treated in 48 well plate with 0.5 X 106 cells/ml per well.
CD34+ cells were pretreated with LA, AA, SDA or EPA for 4 h prior to co-stimulation of
these cells with the same dose of these reagents in the presence of LPS 200 ng/ml for an

additional 24 h. The supernatant was collected and frozen immediately at —80°C for

14

measuring IL-6 concentration by Enzyme-Linked lmmunosorbent Assay (ELISA)
method.

For IL-6 mRNA expression, CD34+ cells were plated and treated in 6-well cell
culture plates with 5 x 106 cells/ml per well for IL-6 mRNA detected by real time PCR.
Total RNA was extracted from CD34+ cells using RNeasy Mini Kit (Qiagen, Valencia,

CA). 100 ng total RNA was used to measure IL-6 mRNA by real time RT-PCR.

3.2 Materials

EPA and SDA were generously provided by Dr. Kenneth Allen (Colorado State
University, Fort Collins, Colorado) and LA was purchased from Sigma Chemical Co. (St.
Louis, MO). These fatty acids were dissolved in ethanol prior to treatment. For the
control treatments, the same concentration of ethanol as in EPA and SDA treatment

groups were added as vehicle control. LPS was purchased from Sigma (St. Louis, M0).

3.3 Methods

Isolation of CD34+ cells. CD34+ cells were stained with a R-phycoerythrin
(PE)-conjugated anti-CD34+ antibody (eBioscience, San Diego, CA). Subsequently, the
cells were magnetically labeled with anti-PE microbeads. The cells suspension were
loaded in a column which was placed in the magnetic field of a MACS separator. The
magnetically labeled cells were retained in the column, while the unlabeled cells run
through. After removal of the column from the magnetic field, the cells were eluted with
MACS buffer, and resuspended in 10% HIFBS DMEM P/S medium.

IL-6 measurement. IL-6 secretion was measured by ELISA method. Briefly,

15

Purified rat anti-mouse IL—6 antibody (BD pharmingen, San Diego, CA) was
immobilized in microtiter plates. IL-6 standard solutions and the supematants from
different treatments were applied to the pre-coated wells and incubated for l h at 37°C.
Unbound protein was washed away and the Biotinylated IL-6 antibody (BD pharmingen,
San Diego, CA) was added to each well and incubated for l h at room temperature. Next,
after washing, Streptavidin-hourseradish Peroxidase (Strep-HRP) (BD pharmingen, San
Diego, CA) was added to each well and incubated for l h at room temperature. Then
Tetramethylbenzidine (TMB) substrate (Neogen, Lexington, KY) was added and further
incubated at room temperature for 15 min followed by washing steps. The reaction was
stopped with the addition of sulfitric acid (1 mol/l) and absorbance was measured in the
microtiter plate reader (Molecular Devices Corporation, Menlo Park, CA) at 450 nm.
IL-6 concentrations in the samples were re-calculated using the IL-6 standard curve.

IL-6 mRNA analysis. CD34+ cells were cultured in 6-well cell culture plates
with 5 x 106 cells/ml per well for IL-6 mRNA measurement by real time RT-PCR. The
primers, probe and endogenous control (1 SS-rRN A) were purchased as Taqman assay
reagents (Applied Biosystems, Foster City, CA). TaqMan One-Step RT-PCR Master Mix
Regent (Applied Biosystems) was used to quantify IL-6 and 18S-rRNA following
manufacturer’s instructions on an ABI Prism 7900 Sequences Detection System (PE
Applied Biosystems). 188-rRNA was used to normalize target gene expression. Target
gene expression levels were calculated relative to the control group.

Western Blot Analysis. CD34+ cells were washed in the 100mm dish with 3 ml
ice-cold PBS and added with 200ul lyses buffer (Sigma-Aldrich, St. Louis, MO) and

protease inhibitor cocktail (Roche, Mannheim, Germany). The cells were scraped and

16

transfered to 1.5ml tube. The cells were then sheared with a sonic dismembrator (Model
60; Fisher Scientific, Pittsburgh, PA) and centrifuged at 13,000 X g for 15 min before the

supernatant was recovered. The relative protein concentration was determined by BCA
method (Pierce, Rockford, IL). Protein (60 ug) was loaded onto 7.5 % sodium dodecyl
sulfate polyacrylamide gel for electrophoresis. The gel was transferred to a
polyvinylidene difluoride membrane and incubated in Tris buffered saline-Tween (TBST),
containing Tris base, NaCl and 0.05% Tween 20 (pH 7.6) and blocked by immersion in
5% nonfat dried milk for 1 h followed by washing. The membrane was then incubated
with anti-mouse TLR2 antibody (R&D Systems), anti-mouse TLR4 antibody (Santa Cruz
Biotechnology, Santa Cruz, CA), anti-mouse IRAK] (Cell Signaling, Danvers, MA) and
anti-mouse phoshpho-p44/42 MAPK (Cell Signaling, Danvers, MA) in TBST buffer
overnight, followed by washing and incubation with secondary antibody coupled to
horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h. The
membrane was exposed on an x-ray film (Kodak Co., Rochester, NY) using ECL
Western blot detection reagents (Bio-Rad Laboratories, Hercules, CA).

NF-kB assay. NF-KB assay kit (Thenno scientific, Rockford, IL) was used to
image translocation of NF-icB from the cell cytosol to the nucleus according to
manufacturer’s instruction. Briefly, CD 34+ cells were pretreated for 4h with 100 uM EPA
and 100 uM SDA followed by treatment with 200ng/ml LPS for 1 h. The cells were fixed
with 16% parafonnaldehyde solution for 10 min and permeabilized for 20 min. The cells
were then incubated with rabbit primary antibody specific for NF -KB antibody for 1 h at
room temperature and then incubated with DyLight 488-conjugated secondary antibody

for 1 h. The NF-KB activation was visualized with use of the fluorescent microscope

(Leica DM IL, Wetzlar, Germany). Digital images of random microscopic fields were
taken to count cells that showed translocation of NF -KB from the cytosol to nucleus.

These numbers were normalized with total cell counts for each of the treatment groups.

3.4 Statistical Analysis

Data were analyzed using Sigma Stat for Windows (J andel Scientific, San Rafael,
CA). IL-6 secretion, IL-6 mRNA expression as well as NF-KB translocation data were
subjected to general linear model (GLM) ANOVA and pairwise comparisons were made
by Bonferroni method. Data reported as means i SEM. Differences were considered

significant at P < 0.05.

18

Chapter 4: Results

4.1 Results for Hypothesis 1: LA, AA, SDA and EPA decreased IL-6 secretion. To
compare effects of n-3 and n-6 PUFA, the cells were preincubated for 4 h with 100 uM
LA, AA, SDA, and EPA and treated with 200ng/ml LPS for 24 h. Data showed that LPS
induced significantly higher IL-6 secretion compared to control group (Figure 4.1). This
LPS-induced IL-6 secretion levels were significantly reduced with treatment of adipose
stem cells with LA, AA, SDA and EPA. Moreover, the inhibitory effects of n-3 PUFA
(SDA and EPA) were significantly higher than these of n-6 PUFA (LA and AA) was
added at the same concentration (100 uM) (Figure 4.1). This data supports that n-3 PUFA
has greater inhibitory effects on adipose stem cell secretion of IL-6 compared to n-6
PUFA. To show dose-dependent decreases with increased n-3 PUFA, the cells were
pre-treated with 30, 60, 90, and 120uM EPA for 4 h followed by 200ng/ml LPS for
additional 24 h. Results showed that EPA reduced the LPS-induced IL-6 secretion in a
dose dependent manner up to 90 uM concentration (Figure. 4.2) Importantly, we also
conducted Methylthiazolyldiphenyl-tetrazolium bromide (MTT, Sigma, St. Louis, MO)
assays to test viability of adipose stem cells at 30, 60, 90 and 120 uM EPA. Results of our
MTT assay, there was no significant difference in control, 30, 60, 90 and 120 uM EPA
group cells indicating that EPA used up to 120 uM concentration results in no toxicity to

the adipose stem cells.

19

 

2.0

1.8 - b

IL-6 (ng/ml)

 

 

 

Ctrl LPS LA SDA AA EPA
+LPS

Figure 4.1 Effects of LA, AA, SDA and EPA on LPS-induced IL-6 secretion in
CD34+ cells. CD34+ adipose stem cells were plated at 5><105 cells/ml, pretreated with 100
uM of LA, SDA, AA or EPA for 4 h, prior to co-stimulation of these cells with LPS (200
ng/ml) in the same doses of LA, AA, SDA or EPA for an additional 24 h. Cell culture
media were then collected to measure IL-6 secretion using ELISA method. Ctrl: control;
LPS: cells treated with 200 ng/ml of LPS; LA, AA, SDA or EPA 100 uM: cells treated
with 100 uM of LA, AA, SDA or EPA. Bars that share the same letter are not
significantly different from each other at p<0.05, n=3 independent experiments with
triplicate wells per treatment for each experiment.

20

 

2.5

2.0 a

lL-6 (ng/ml)

 

 

 

Ctrl LPS EPA EPA EPA EPA
30 uM 6O uM 90nM 120 uM
+LPS

Figure. 4.2 Dose response effects of EPA in CD34+ cells. CD34+ adipose stem cells
were plated at 5><10S cells/ml, pretreated with 30, 60, 90 and 120 uM of EPA for 4 h,
prior to co-stimulation of these cells with LPS (200 ng/ml) in the same doses of EPA and
for an additional 24 h. Cell culture media were then collected to measure IL-6 secretion
using ELISA method. Ctrl: control; LPS: cells treated with 200 ng/ml of LPS. Bars that
share the same letter are not significantly different from each other at p<0.05, n=3
independent experiments with triplicate wells per treatment for each experiment.

21

4.2 Results for Hypothesis 2: LA, SDA and EPA decreased IL-6 mRNA

expression. To determine if n-3 and n-6 PUFA inhibit IL-6 mRNA expression in the
adipose stem cells, we pretreated the cells with LA, SDA and EPA for 4 h and then
stimulated cells with LPS for O, 2, 4 and 6 h and showed that LPS stimulated IL-6
maximally at 4 h stimulation time point. Accordingly, using 4 h LPS stimulation time, we
tested inhibitory effects of LA, SDA and EPA on IL-6 mRNA expression using real time
RT-PCR methods. LPS significantly increased IL-6 mRNA levels compared to control
and LA, SDA and EPA significantly reduced LPS-induced IL-6 mRNA levels (Figure
4.3). The data suggest that inhibitory effects of LA, SDA and EPA on IL-6 secretion are,

in part mediated via decreased IL-6 mRN A levels.

22

 

16

lL-6 mRNA relative expression

 

 

 

Ctrl LPS LA SDA EPA
+LPS

Figure. 4.3 Effects of EPA, SDA and LA on LPS-Induced IL-6 mRNA level in CD34+
cells. CD34+ adipose stem cells were plated as 5 x 106/5 ml, pretreated with 60 M of
EPA, SDA and LA for 4 h, prior to co-stimulation of these cells with the same doses of
EPA, SDA, LA and LPS (200 ng/ml) for an additional 4 h. Total RNA were extracted and
relative IL-6 gene expression were determined by real time RT—PCR and normalized
against 18S rRNA. Bars that share the same letter are not significantly different from each
other at p<0.05, n=3 independent experiments with triplicate wells per treatment for each
experiment.

23

4.3 Results for Hypothesis 3: SDA and EPA decreased IL-6 mRNA expression via
TLR mediated pathway. To determine intracellular signaling pathway underlying n-3

PUF A inhibition of IL-6 gene expression, we hypothesized that TLR family of proteins
and their downstream signaling molecules are affected by n-3 PUF A treatments in
adipose stem cells. Our preliminary data showed that protein expression of TLR2 and not
TLR4 was induced with LPS treatment. Based on this observation, we tested if SDA or
EPA reduced TLR2 expression. As shown in Figure 4.4, LPS significantly increased
TLR2 expression and both SDA (Figure 4.4A) and EPA (Figure 4.4B) treatment reduced
LPS-induced TLR2 expression. A recent study has showed the, post activation of TLR5,
LPS-induced increases in interleukin-1 receptor associated kinase-1 (IRAKl) degradation
results in activation of transcriptopn factor NF -KB [67] that is important in increasing
IL-6 gene expression. Thus, we tested if IRAK-l protein expression of the downstream
signaling pathway intermediate of TLR2 such as is decreased. As shown in Figure 4.5,
our results showed no significant increases in IRAK] protein expression with 30 min
SDA (Figure 4.5A) and EPA (Figure 4.5B) treatments. However, modest increases in
IRAK] protein expression were shown with 1 h treatment of cells with EPA and SDA
(Figure 4.5A and 4.5B).

In addition, studies have shown that increases in lL-6 mRNA are mediated by
increasing via activation of the Mitogen-Activated Protein Kinase (MAPK) intermediates
such as p42/44 MAPK (Erk1/2) [68]. Thus, we tested involvement of MAPK
intermediate ERK activation by testing phospho-ERK1/2 protein expression levels with
SDA and EPA treatments. As shown in Figure 4.6, our data showed that LPS increases

phospho-ERKl/Z while SDA (Figure 4.6A) and EPA (Figure 4.6B) had no effects on

24

reducing LPS-induced ERK1/2. These data suggested that SDA- and EPA- induced
decreases in IL-6 gene expression are not mediated via decreased activation of ERK1/2 in
adipose stem cells.

Since transcription factor NF-KB has been shown to play an important role in
increasing IL-6 gene transcription [5 8], we investigated whether SDA and EPA decrease
LPS-induced NF-icB activation. Studies have shown that cytosolic NF-icB translocate
from the cytosol to the nucleus upon stimulation by LPS. To test if SDA and EPA reduce
LPS—induced NF-KB activation in the adipose stem cells, we pretreated CD34+ cells with
100 uM of SDA and EPA for 4 h and then stimulated cells with LPS for 1 h. As shown in
Figure 4.7A, results from our fluorescence microscopic images showed that NF-KB
translocate with LPS stimulation and SDA and EPA reduce LPS-induced NF-KB
translocation from cytosol to nucleus. The number of cells that showed NF-KB
translocation from cytosol to nucleus was quantitated by calculating the ratio of cells
positive for NF -icB translocation to total (both positive and negative for NF-KB
translocation) (Figure 4.7B). As shown in figure 4.7B, our data showed that LPS
significantly increased NF -KB translocation and SDA and EPA inhibit LPS-induced

NF-KB translocation.

25

(A).

(B).

Ctrl LPS SDA SDA+LPS

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B-actin

Figure 4.4 Effects of SDA and EPA on TLR2 protein expression in CD34+ cells. (A).
CD34+ adipose stem cells were plated at 10><106 cells/10ml, pretreated with 100 [AM of
SDA for 4 h, prior to co-stimulation of these cells with the same doses of SDA and LPS
(200 ng/ml) for an additional 12 h. To evaluate TLR2 protein expression, total protein
was collected. TLR2 was measured by Western blot method and normalized against
[i-actin expression levels. (B). CD34+ adipose stem cells were plated at 10><106 cells/10ml,
pretreated with 100 uM of EPA for 4 h, prior to co-stimulation of these cells with the
same doses of EPA and LPS (200 ng/ml) for an additional 12 h. To evaluate TLR2 protein
expression, total protein was collected. TLR2 was measured by Western blot method and
normalized against B-actin expression levels. For both 4A and 4B, results are
representative of three independent experiments.

26

(A)-
30 min 1 h

 

Ctrl LPS SDA SDA+LPS LPS SDA SDA+LPS
i 'j_ _j * ’ .... ”W WW“

    

 

 

 

   

(B).
30 min 1 h
EPA EPA+LPS LPS EPA ”EPA+LPS

B—actin up 4“ “van—- “ in... all!

Figure 4. 5 Effects of SDA and EPA on IRAK] protein expression in CD34 cells. (A).
CD34 adipose stem cells were plated at 10><106 cells/10ml, pretreated with 100 11M of
SDA for 4 h, prior to co- -stimulation of these cells with the same doses of SDA and LPS
(200 ng/ml) for an additional 30 min or 1 h. IRAKl was measured by Western blot
method and normalized against B-actin expression levels. (B). CD34+ adipose stem cells
were plated at 10><106 cells/10ml, pretreated with 100 pM of EPA for 4 h, prior to
co-stimulation of these cells with the same doses of EPA and LPS (200 ng/ml) for an
additional 30 min or 1 h. IRAK] was measured by Western blot method and normalized
against B-actin expression levels. For both 5A and 5B, results are representative of three
independent experiments.

27

(A)-

. --.__.

Ctrl LPS SDA SDA+LPS

phospho-ERK 1/2

B-actin

   

(B).

Ctrl LPS EPA EPA+LPS

phospho-ERK 1/2

 

B-actin

Figure 4.6 Effects of SDA and EPA on phospho-ERK1/2 protein expression in CD34+
cells. (A). CD34+ adipose stem cells were plated at 10><106 cells/10ml, pretreated with
100 11M of SDA for 4 h, prior to co-stimulation of these cells with the same doses of SDA,
and LPS (200 ng/ml) for an additional 1 h. To evaluate p-ERK1/2 protein expression,
total protein was collected. p-ERK1/2 was measured by Western blot method and
normalized against B-actin expression levels. (B). CD34+ adipose stem cells were plated
at 10><106 cells/10ml, pretreated with 100 uM of EPA for 4 h, prior to co-stimulation of
these cells with the same doses of EPA, and LPS (200 ng/ml) for an additional 1 h. To
evaluate p-ERK1/2 protein expression, total protein was collected. p-ERKl/2 was
measured by Western blot method and normalized against B-actin expression levels. For
both 4.6A and 4.7B, results are representative of two independent experiments.

28

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29

Chapter 5: Discussion and Conclusion

IL-6 is secreted from a variety of tissues, such as endothelial cells, activated
leukocytes and adipocytes [14]. Once secreted, IL-6 acts synergistically with other
regulatory factors to affect the development of several chronic diseases such as Type 2
diabetes and CVD [14, 40]. IL-6 reduces the activity of lipoprotein lipase (LPL), thereby
increasing the level of circulating lipid and induces hepatic C-reactive protein (CRP) that
is one of the biomarkers of CVD risk [40, 69]. IL-6 also has been shown to decrease
insulin signaling pathway thereby increasing insulin resistance and risk for type 2
diabetes [14].

Increasing numbers of studies have shown that adipose tissue secretes a number
of proinflammatory cytokine including IL-6 [22, 70]. Among those, adipose tissue
contributes significantly to the total circulation of IL-6 [22]. Studies have demonstrated
that obesity is associated with increase in proinflammatory cytokines including IL-6 [22,
70]. Obesity-associated increase in circulating proinflammatory cytokines, particularly
IL-6, can be decreased after a substantial reduction in adipose tissue mass [4, 5, 9-12, 39].
Data from our current study demonstrate that n-3 PUFA have potent anti-inflammatory
effects on adipose tissue stem cells. We further showed that this inhibitory effect of n-3
PUFA is mediated via TLR2 pathway. Since one of the first studies showing n-3 PUFA’s
beneficial health effects in Greenland Eskimos [71], increasing number of studies have
demonstrated the protective effect of fish oil compounds such as EPA and DHA[14] for
reducing risk for CVD [24, 25] particularly by inflammatory cytokine production [72-74]

EPA exists in normal human body at 33.33 ug/ml (=105 uM). When we intake

EPA (2700 mg per day, 3 times per day) everyday, the effective serum concentration of

30

EPA reaches 159.79 ug/ml (502.95 uM) [75]. Thus, the range of concentrations used in
this study is within biologically achievable ranges to have anti-inflammatory effects.
Although ALA has been shown to have cardiovascular protective role [76], ALA is poorly
converted to EPA [77] due to rate-limiting A6-desaturase steps in the conversion of ALA
to EPA [26, 27]. We have used SDA that can be converted into EPA [26-28] in—vivo
bypass rate-limiting A‘s-desaturase step. Importantly, concerns have been raised about the
contamination of fish by mercury and pesticides [78]. Therefore, our current study of
SDA of which can be produced in land based agricultural plants potentially contribute
significantly toward much increasing our knowledge in the agricultural-based sources of
n-3 PUFA that can be used in reducing chronic disease risks.

Another noteworthy finding of our study has shown that n-3 PUFA can lower
inflammatory cytokine production from adipose stem cells. As we have show that these
cells secret very high levels of proinflammatory factors previously [13], stem cells can
differentiate into inflammatory factor secreting adipocytes, and numbers of stems cells
and adipocytes are increased in obese humans and animals, determining whether any
dietary nutrient can be used to reduce adipose tissue specific inflammation would be
beneficial in reducing choric disease risks in obesity.

It has been shown that adipose tissue play an important source of
obesity-associated increases in plasma IL-6 [29]. Particularly, the stromal vascular
fractions (SVF) cells including stem cells also have significantly higher levels of IL-6
compared with adipocytes [29]. Thus, in our current study, we used adipose stem cells to
show that EPA and SDA significantly decreased LPS-induced IL-6 secretion.

LPS is a cell wall component of gram-negative bacteria [64]. It is well known that

31

LPS is better extent ligand for TLR4 and to lesser extent TLR2 [65]. In addition, it has
been shown that TLR2 is also activated by LPS by depending on LBP and CD14 [79, 80].
LPS binding protein (LBP) first recognizes LPS and brings LPS to the TLR forming a
ternary complex with CD14 [81]. The binding of LPS to TLR on the cell surface
stimulates the proinflammatory cytokine production [66] via activation and recruitment
of myeloid differentiation factor 88 (MyD88) and other downstream intermediates of
TLR signaling pathway such as MyD88 adaptor-like protein (Mal, aka TIRAP),
TIR-containing adaptor inducing [EN —13 (TRIP), etc [60, 82, 83]. Activation of these
proteins leads to recruitment of IRAK4, which phosphorylate IRAKl resulting in
eventual activation of NF -icB translocation into nucleus and upregulation of IL-6 gene
expression [84, 85].

As shown in figure 5.1, we suggest that the inhibitory effects of n-3 PUFA on
LPS-induced IL-6 mRNA expression in adipose stem cells are mediated via activation of
TLR2 and not TLR4. We further suggest that this TLR2 mediated inhibition of IL-6
mRNA in the adipose stem cells is mediated via regulation of IRAK-1 and NF-icB
pathway and not through ERK1/2 pathway (Figure 5.1). Although we have not shown in
our current study, other studies have shown that TLR2 heterodimerize with TLR6 in the
differentiated adipocytes [86]. Thus, it is possible that TLR2 heterodimerize with TLR6
in the adipose stem cells to mediate LPS effects on IL-6 and that n-3 PUFA inhibit this
activation. To our best knowledge, this is first study to show that TLR2 is involved in
adipose stem cell signaling, leading to increased IL-6 gene expression. However, Lin, et
al., showed that TLR4 mediate LPS effects in differentiated murine adipocyte cell line

cell 3T3L1 [87]. Since, other study showed that TLR2 is upregulated by TLR4

32

signaling upon LPS stimulation [88], it is also possible that LPS induced TLR2 mediated
upregulation of IL-6 mRNA in the adipose stem cells is mediated in part by indirect
activation of TLR4. However, it is unlikely that TLR4 is involved in n-3 PUFA inhibition
of adipose stem cell IL-6 expression because our results did not show activation of TLR4
while TLR2 was significantly upregulated. Furthermore, our results also showed that EPA
and SDA did not have any effect in TLR4 protein expression. TLR5 including TLR4 and
TLR2 are over-expressed in atherosclerotic lesions [89]. Further, TLR4-deficient mice
showed a decreased aortic plaque size in apoE-null mice [90]. These results suggest that
TLR4 signaling mechanism may be significantly associated with cardiovascular disease
risk by linking lipid metabolism, inflammation, and atherosclerosis. Identification of
these genes may lead to the development of a new molecular level approach to design
CVD treatment.

In our current study, we showed that n-3 PUFA significantly decreased
LPS-induced IL-6 mRN A expression and IL-6 secretion from adipose tissue derived
CD34+ stem cells. As we have shown that adipose stromal vascular cells [29] and adipose
stem cells in particular play important roles in increasing serum IL-6 levels in obese
animals [13], reducing these highly inflammatory cells’ production of IL-6 may
contribute toward reducing systemic IL-6 levels in obese animals. Accordingly, in our
future studies, we will include in-vivo feeding experiments to assess the effects of n-3
PUFAs on adipose tissue and isolated adipose stem cell IL-6 levels. Several possible
mechanisms to explain n-3 PUFAs’ benefits are (i) via substrate competition, preventing -
conversion of AA into proinflammatory eicosanoids such as PG and LT [65]; (ii) serving

as an alternative substrate to produce less potent 5-series LTs and 3-series PGs and TX

33

[91]. EPA derived endogenous resolvins have been shown to decrease IL-6 during
inflammatory response mostly in respiratory tracks [92, 93]. Currently, no studies
addressed if resolvin levels are increased with EPA or SDA treatment and if resolvin
decrease adipose stem cell or adipose tissue inflammation/IL-6 levels. Thus, future

studies are also needed for determination effects of resolvin in adipose IL-6 levels.

34

n-3 PUFA

IL-6 protein

   

Plasma membrane

Cytoplasm

IL-6 protein
it

 

IL-6 mRNA

 

 

Nucleus ® NF-KB .' r—o
IL-6 gene

Figure 5.1 Proposed mechanism of n-3 PUFA inhibition IL-6 mRN A in the adipose
stem cells. LPS binds to TLR2/TLR6 complex via CD14. The binding of LPS to TLR2
on the adipose stem cell surface stimulates IL-6 mRN A gene expression via activation
and recruitment of myeloid differentiation factor 88 (MyD88) and IRAK4 which
phosphorylates IRAK] resulting in eventual activation of NF-KB which translocates into
nucleus to upregulate IL-6 mRNA expression leading to increased IL-6 protein and IL-6
secretion. N-3 PUFA such as SDA and EPA inhibit LPS-induced IL-6 mRNA expression
in adipose stem cells via regulation of IRAKl and NF-K‘B pathways and not through
ERK1/2 pathway.

 

35

Appendix

As mentioned in the result, to show dose-dependent decreases with increased n-3
PUFA, the cells were pre-treated with 30, 60, 90, and 120uM EPA for 4 h followed by
200ng/ml LPS for additional 24 h. Results showed that EPA reduced the LPS-induced
IL-6 secretion in a dose dependent manner up to 90 uM concentration (Figure. 4.2)
Importantly, we also conducted Methylthiazolyldiphenyl-tetrazolium bromide (MTT,
Sigma, St. Louis, MO) assays to test viability of adipose stem cells at 30, 60, 90 and 120
uM EPA. Results of our MTT assay showed no significant difference in control, 30, 60,
90 and 120 11M EPA group cells indicating that EPA used up to 120 uM concentration
results in no toxicity to the adipose stem cells (Figure A1).

To determine if n-3 and n-6 PUFA inhibit IL-6 mRNA expression in the adipose
stem cells, we pretreated the cells with LA, SDA and EPA for 4 h and then stimulated
cells with LPS for 0, 2, 4 and 6 h and showed that LPS stimulated IL-6 maximally at 4 h

stimulation time point (Figure A2).

36

200

 

180 — a
160 -
14o -
120 4

100 ~

(yo Of ctrl

80')

60a

 

40—

201

 

 

 

 

 

Ctrl EPA EPA EPA EPA
3011M 6011M 9011M 12011M

Figure Al. MTT assays for testing viability of CD34+ adipose stem cells by EPA
treatment. CD34+ adipose stem cells were plated at 5><10S cells/ml, treated with 30. 60, 90
and 120 uM of EPA for 24 h. Cell culture media were then collected to measure OD value.
Ctrl: control. Bars that share the same letter are not significantly different from each other

at p<0.05, n=2 independent experiments with triplicate wells per treatment for each
experiment.

37

 

14

lL-6 mRNA relative expression

 

 

 

 

O 2 4 6
Time (h)

Figure A2. Time course of LPS-induced H.-6 mRNA level in CD34+ cells. CD34+
adipose stem cells were plated as 5 x 106/5 ml, treated with LPS (200 ng/ml) for different
time point. Total RNA were extracted and relative IL-6 gene expression were determined
by real time RT-PCR and normalized against 183 rRNA. n=1 independent experiment
with triplicate wells per treatment for the experiment.

38

10.

11.

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