CRITICAL ROLE OF G-PROTEIN COUPLED RECEPTOR KINASE 5
IN SEPSIS
By
Nandakumar Packiriswamy

A DISSERTATION
Submitted to
Michigan State University
In partial fulfillment of the requirements
for the degree of
Comparative Medicine and Integrative Biology- Doctor of Philosophy
2014

ABSTRACT
CRITICAL ROLE OF G-PROTEIN COUPLED RECEPTOR KINASE 5
IN SEPSIS
By
Nandakumar Packiriswamy
G-protein coupled receptor kinase-5 (GRK5) is a member of the GRK family of kinases,
originally discovered in the context of G-protein coupled receptor (GPCR) desensitization.
Recent studies from our lab and others have demonstrated critical role of GRK5 in inflammatory
pathways particularly involving Toll-like receptors. Because Toll-like receptor signaling
pathways are critical in the regulation of sepsis pathogenesis, it was hypothesized that GRK5 is a
critical regulator of inflammatory pathways in sepsis pathogenesis. Thus the overall goal of the
thesis project was to decipher the role of GRK5 in sepsis pathogenesis using mouse models of
sepsis. In initial studies, we demonstrated GRK5 regulates cytokine production in vivo by both
MyD88 and TRIF dependent pathways, suggesting that GRK5 can regulate a broad range of
TLRs. In the next set of studies, we used poly-microbial and mono-microbial sepsis models to
further understand the role of GRK5. Indeed, our studies using polymicrobial sepsis delineates
key roles for GRK5 in modulating sepsis outcome. We demonstrated that GRK5 is a critical
positive regulator of inflammatory mediator production; sepsis induced thymic apoptosis and
development of immune suppression. More importantly, we established that GRK5 deficiency
protected mice from polymicrobial sepsis-induced mortality suggesting that GRK5 could be
amenable for therapeutic targeting in sepsis. Having demonstrated the role of GRK5 in
polymicrobial sepsis, we further examined the role of GRK5 in a mono-microbial acute lung
infection model. Here we demonstrated that GRK5 has distinct roles in regulating progression of
inflammation depending on the dose of bacteria. Our study revealed GRK5 as a crucial regulator

of neutrophil chemotaxis early during infection likely via modulation of CXCL1/KC levels. In
addition, we further show that GRK5 modulates bacterial burden in lungs depending on the
initial dose of E. coli infection. And the regulation of bacterial burden was further dependent on
the in vivo neutrophil activation status. Overall, our studies have uncovered novel roles of GRK5
during acute inflammatory disease processes and further work in discerning the involved
molecular mechanisms will likely lead to therapeutic strategies to target GRK5 during sepsisinduced inflammatory conditions.

ACKNOWLEDGEMENTS

Foremost, I would like to express my sincere gratitude to my advisor Dr. Narayanan
Parameswaran for his continuous support, generous time and sincere guidance throughout my
program and for critical reading of this dissertation. His enthusiasm for science helped me
overcome some of the frustrations invariably associated with research. He was and remains my
best role model for a scientist and a great mentor. I’m extremely fortunate to have Dr.Nara
Parameswaran as my major advisor. I specially thank Drs. Jack Harkema, Linda Mansfield and
Jean Nemzek, for serving on my guidance committee and for their constructive and insightful
comments about my research.
In my daily work, I have been blessed with friendly and cheerful lab members. I wish to
recognize the past and present members of Dr. Nara Parameswaran laboratory who have made it
an exciting and fun place to work. My special thanks to Haritha Durairaj, Michael steury and Ian
McCabe for helping me out with major experiments, and Deepika sharma, Taehyung Lee and
Eunhee lee for their constructive ideas and comments.
I, thank the Comparative Medicine and Integrative Biology (CMIB) program for
accepting me into the program and providing me a great opportunity to enhance my scientific
career. I sincerely thank Dr. Vilma Yuzbasiyan-Gurkan’s for her constant encouragement and
unwavering support.
Finally, I thank my family and friends for their unflagging love and support to
successfully complete my Ph.D. degree.

iv

TABLE OF CONTENTS

LIST OF FIGURES ..................................................................................................................... viii
KEY TO ABBREVIATIONS ..........................................................................................................x
CHAPTER ONE: A LITERATURE REVIEW ON G-PROTEIN COUPLED RECEPTOR
KINASE 5 (GRK5) AND SEPSIS ..................................................................................................1
1.1: Introduction: G-protein-coupled receptor kinases (GRKs) ......................................................3
1.2: The G-protein coupled receptor kinase family: Identification, organization and structure ......5
1.3: Regulation and activation of GRKs ..........................................................................................9
1.4: Phenotypes of GRK deficient mouse ......................................................................................10
1.5: GRKs in immune system and inflammation ..........................................................................11
1.5.1: Inflammatory signaling and GRKs .........................................................................11
1.5.1.1: Toll like receptor (TLR) signaling, NFκB and GRK5 ..............................13
1.5.1.2: Mitogen Activated Protein Kinase (MAPK) signaling ............................17
1.5.1.2.1: Extracellular Signal Regulated Kinases (ERK) and GRK5 .......18
1.5.1.2.2: p38 MAPK and GRK5 ...............................................................18
1.5.1.2.3: c-Jun N-terminal kinase (JNK) and GRK5 ................................19
1.5.2: Chemotaxis and GRKs ............................................................................................20
1.5.3: Apoptosis and GRKs ..............................................................................................23
1.6: Pathological role of GRKs .....................................................................................................26
1.7: Acute Inflammatory diseases .................................................................................................27
1.7.1: Sepsis - Incidences and causes ................................................................................27
1.7.2: Pneumonia- Incidences and causes .........................................................................28
1.7.3: Inflammatory response and NFκB signaling during sepsis/pneumonia .................29
1.7.4: Cytokines and their cellular sources .......................................................................31
1.7.5: Immunosuppression & Apoptosis ...........................................................................32
1.7.6: Coagulation and Endothelial response ....................................................................34
1.7.7: Cardiac dysfunction ................................................................................................35
1.7.8: CLP as a model for polymicrobial sepsis ...............................................................35
1.7.9: Per-oral inoculation of E. coli- as a model for pneumonia .....................................36
1.8: Significance ...........................................................................................................................37
HYPOTHESIS AND SPECIFIC AIMS .......................................................................................39
CHAPTER TWO: OVERLAPPING AND DISTINCT ROLES OF GRK5 IN TLR2-, AND
TLR3-INDUCED INFLAMMATORY RESPONSE IN VIVO ....................................................40
2.1: Abstract ..................................................................................................................................41
2.2: Introduction ............................................................................................................................42
2.3: Materials and Methods ...........................................................................................................43
2.3.1: Animals and protocol ..............................................................................................43
2.3.2: Cytokine analysis ....................................................................................................44
2.3.3: Statistical method ....................................................................................................44
v

2.4: Results and discussion ...............................................................................................44
CHAPTER THREE: GRK5 MEDIATES INFLAMMATION BUT DOES NOT REGULATE
CELLULAR INFILTRATION OR BACTERIAL LOAD IN POLYMICROBIAL SEPSIS
MODEL IN MICE ........................................................................................................................51
3.1: Abstract ..................................................................................................................................52
3.2: Introduction ............................................................................................................................53
3.3: Materials and Methods ...........................................................................................................54
3.3.1: Materials .................................................................................................................54
3.3.2: Mice ........................................................................................................................55
3.3.3: Sepsis model ...........................................................................................................55
3.3.4: Peritoneal Lavage fluid and blood collection .........................................................55
3.3.5: Cytokine/chemokine measurements .......................................................................56
3.3.6: Bacterial counts .......................................................................................................56
3.3.7: Determination of thymic cell numbers and apoptosis ............................................56
3.3.8: Caspase activity assays ...........................................................................................57
3.3.9: In vitro stimulation of septic peritoneal cells ..........................................................57
3.3.10: Restraint stress ......................................................................................................57
3.3.11: Corticosterone measurements ...............................................................................58
3.3.12: RNA extraction and Real-time Q-PCR .................................................................58
3.3.13: Western blot analysis ............................................................................................58
3.3.14: Statistical analysis .................................................................................................59
3.4: Results ....................................................................................................................................59
3.4.1: GRK5 mediates sepsis-induced cytokine production .............................................60
3.4.2: GRK5 mediates NFкB signaling in polymicrobial sepsis ......................................60
3.4.3: GRK5 does not regulate chemotaxis to the local site of injury/infection ...............61
3.4.4: Deficiency of GRK5 does not affect bacterial load following sepsis .....................63
3.4.5: Regulation of thymocyte numbers by GRK5 in sepsis ...........................................63
3.4.6: Regulation of thymocyte apoptosis by GRK5 in vivo ............................................66
3.4.7: Mechanism of GRK5-mediated thymic apoptosis ..................................................66
3.4.8: GRK5 inhibits immunoresponsiveness of peritoneal cells in sepsis ......................71
3.4.9: Role of GRK5 in sepsis-induced mortality .............................................................73
3.5: Discussion ..............................................................................................................................74
CHAPTER FOUR: CRITICAL INVOLVEMENT OF GRK5 IN E. COLI INDUCED
PNEUMONIA ...............................................................................................................................80
4.1: Abstract ..................................................................................................................................81
4.2: Introduction ............................................................................................................................82
4.3: Materials and Methods ...........................................................................................................83
4.3.1: Materials .................................................................................................................83
4.3.2: Experimental animals .............................................................................................83
4.3.3: E. coli infection .......................................................................................................83
4.3.4: Bacterial load determination ...................................................................................84
4.3.5: In vitro Bacterial killing assay ................................................................................84
4.3.6: Immune cell infiltration ..........................................................................................85
4.3.7: Chemokine receptor expression ..............................................................................85
vi

4.3.8: Cytokine/Chemokine measurements ......................................................................86
4.3.9: Immuno Blot Analysis ............................................................................................86
4.3.10: Statistical Analysis ................................................................................................86
4.4: Results ....................................................................................................................................87
4.4.1: GRK5 modulates bacterial clearance but doesn’t mediate intracellular bacterial
killing.......................................................................................................................87
4.4.2: GRK5 mediates early neutrophil recruitment into lungs ........................................90
4.4.3: GRK5 regulates CXCL1 but does not regulate CXCR2, CXCR4 and CCR1 cell
surface expression ..................................................................................................93
4.4.4: GRK5 modulates inflammatory status ....................................................................96
4.4.5: GRK5 confers survival advantage during E. coli induced pneumonia at higher
infection dose ..........................................................................................................96
4.5: Discussion ............................................................................................................................103
CHAPTER FIVE: SUMMARY AND CONCLUSION ...........................................................107
5.1: Role of GRK5 in MyD88-dependent and -independent cytokine production in vivo .........108
5.1.1: Results ...................................................................................................................109
5.1.2: Conclusion ............................................................................................................109
5.1.3: Limitations and future directions ..........................................................................110
5.2: Role of GRK5 in Polymicrobial sepsis ................................................................................110
5.2.1: Results ...................................................................................................................110
5.2.2: Conclusion ............................................................................................................111
5.2.3: Limitations and future directions ..........................................................................111
5.3: Role of GRK5 in E. coli induced pneumonia ......................................................................112
5.3.1: Results ...................................................................................................................112
5.3.2: Conclusion ............................................................................................................113
5.3.3: Limitations and future directions ..........................................................................113
BIBLIOGRAPHY .....................................................................................................................115

vii

LIST OF FIGURES

Figure 1.1: Schematic summary of the role of GRKs/arrestins in carrying out G-protein
dependent and independent functions .........................................................................4
Figure 1.2: Domain structure GRK family of proteins ...................................................................6
Figure 2.1: Plasma IL-12p40 and G-CSF are regulated by GRK5 in both Pam3CSK4 and Poly
(I:C) treatment groups ...............................................................................................45
Figure 2.2: Plasma IL-5, MCP-1, MIP-1β and IL-6 production are regulated by GRK5 only in
the Pam3CSK4 treatment group ................................................................................46
Figure 2.3: Plasma TNF and RANTES are regulated by GRK5 only in the Poly (I:C) treatment
group ..........................................................................................................................48
Figure 2.4: Cytokines/chemokines differentially regulated by GRK5 in response to
activation of TLR2 and TLR3 signaling pathway .....................................................49
Figure 3.1: GRK5 mediates inflammatory response in cecal ligation and puncture model of
polymicrobial sepsis ..................................................................................................62
Figure 3.2: GRK5 mediates IB phosphorylation and expression of NF-κB dependent
genes in liver and lung ...............................................................................................64
Figure 3.3: Role of GRK5 in peritoneal cell infiltration following cecal ligation and puncture ..67
Figure 3.4: Role of GRK5 on bacterial load following cecal ligation and puncture ....................68
Figure 3.5: GRK5 mediates thymocyte apoptosis in polymicrobial sepsis ..................................69
Figure 3.6: Sepsis-induced plasma corticosterone levels are diminished in
GRK5 knockout mice .................................................................................................70
Figure 3.7: GRK5 suppresses immuno-responsiveness of peritoneal cells in sepsis ...................72
Figure 3.8: Role of GRK5 on survival following cecal ligation and puncture .............................75
Figure 4.1: Decreased bacterial burden in GRK5 deficient mice at low dose E. coli ..................88
Figure 4.2: GRK5 does not play a role in intra cellular killing of bacteria .................................89

viii

Figure 4.3: GRK5 regulates early recruitment and activation of neutrophil upon E.coli
infection .......................................................................................................................91
Figure 4.4: Increased proportions of efferocytosing macrophages in GRK5 KO lungs at
low dose E. coli ...........................................................................................................92
Figure 4.5: GRK5 deficiency induced CXCL1 production early in low dose E. coli
induced pneumonia .....................................................................................................94
Figure 4.6: GRK5 does not regulate expression of neutrophil CXCR2, CXCR4 and CCR1 .......95
Figure 4.7: GRK5 deficiency induced decreased inflammatory response in low dose
E. coli induced pneumonia .................................................................................... 97-98
Figure 4.8: Increased mortality observed in GRK5 deficient mouse infected with
E.coli at 5x106 CFU/mouse .......................................................................................100
Figure 4.9: Increased Bacterial burden in GRK5 deficient mice at high dose E. coli ...............101
Figure 4.10: GRK5 deficiency mediates early neutrophil recruitment into lungs in high
dose E. coli infected mice ........................................................................................102

ix

KEY TO ABBREVIATIONS

AdGRK5-NT: Adenoviral GRK5-N-terminal construct
Akt: Protein Kinase B (PKB)
ANF: Atrial natriuretic factor
ANOVA: Analysis of Variance
AP-1: Activator protein-1
Apaf1: Apoptosis protease-activating factor 1
ATCC: American type culture collection
β2AR: β2 -adrenergic receptor
BAD: Bcl-2 associated death promoter
Bcl-2: B cell lymphoma protein 2
Bcl-3: B cell lymphoma protein
Bcl-xl: B cell lymphoma-extra large protein
CaM: Calmodulin
cAMP: cyclic AMP
dsRNA: double stranded RNA
E. coli: Escherichia coli
ERK: Extracellular signal-regulated kinase
GPCR: G protein-coupled receptor
GRK: G protein-coupled receptor kinase
GRK5-NT: G protein-coupled receptor kinase 5 – amino terminal
GTP: Guanosine triphosphate
HDAC: Histone deacetylase
HEK293: Human embryonic kidney 293
HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid
x

HPRT: Hypoxanthine-guanine phosphoribosyl transferase
IB: Immunoblot
ICAM: Intracellular adhesion molecule
IκB: Inhibitor of κB
IKK: Inhibitor of κB kinase
iNOS: inducible nitric oxide synthase
IRAK1: Interleukin-1 receptor-associated kinase-1
IRAK4: Interleukin-1 receptor-associated kinase-4
IRF3: Interferon regulatory factor 3
Jaks: Janus kinases
LPS: Lipopolysaccharide
MAPK: Mitogen-activated protein kinase
Mdm2: Murine double minute oncogene 2
MEK1: MAPK/ERK kinase
MyD88: Myeloid differentiation primary response gene 88
n: Amino
NADPH-oxidase: Nicotinamide adenine dinucleotide phosphate-oxidase
NES: Nuclear export sequence
NFκB: Nuclear factor kappa enhancer of activated B cells
NLS: Nuclear localization sequence
NT: Amino-terminal region
p: phosphorylated
p38-MAPK: p38 mitogen activated protein kinase 5
p65: Transcription factor p65 (RelA)
p50: NF-KappaB1
PAMPs: pathogen-associated molecular patterns
PBS: Phosphate-buffered saline
xi

PCR: Polymerase chain reaction
PH: Pleckstrin homology
PIP2: Phosphatidylinositol 4,5-bisphosphate
PI3K: phosphoinositide 3-kinase
PKA: cAMP-dependent protein kinase
PKC: Protein kinase C
PKD: Protein kinase D
PLC: Phospholipase C
PLK1: Polo-like kinase 1
PMSF: Phenylmethanesulfonyl fluoride
qRT-PCR: Quantitative reverse transcription – polymerase chain reaction
RGS: Regulator of G protein signaling
RH: RGS homology
ROS: reactive oxygen species
SDS: Sodium dodecyl sulphate
SDS-PAGE: Sodium dodecyl sulphate polyacrylamide gel electrophoresis
TAK1: Transforming growth factor- β-activated kinase 1
TBK1: TANK binding kinase 1
TEMED: Tetramethylethylenediamine
TIR: Toll-interleukin-1 receptor
TIRAP: TIR domain-containing adaptor protein
TLRs: Toll-like receptors
TNFα: Tumor necrosis factor-α
TNFR: Tumor necrosis factor receptor
TPL2: Tumor progression locus 2 TAB: TAK1-binding protein
TRAF6: TNF receptor-associated factor 6
TRIF: TIR domain-containing adaptor inducing IFN-β
xii

CHAPTER ONE

A LITERATURE REVIEW ON G-PROTEIN COUPLED RECEPTOR KINASE 5
(GRK5) AND SEPSIS

1

G protein coupled receptor kinases (GRKs) are key modulators of G-protein coupled
receptor (GPCR) signaling. GRKs are serine/threonine kinases that phosphorylate agonist bound
GPCRs, desensitize them and regulate their downstream GPCR signaling. In addition to their
classical role in GPCR desensitization, GRKs are known to carry out GPCR and/or G-protein
independent roles including phosphorylation dependent regulation of cellular signaling,
modifying transcription of genes by virtue of nuclear localizing signal (NLS), or by
phosphorylation independent protein-protein interactions. Recent studies emphasized on this
GPCR- and G-protein independent role of GRKs kindled a renewed interest in using GRKs as
drug targets in inflammatory diseases. Thus, it was hypothesized that GRK5 (one of the GRKs)
by virtue of its role in mediating inflammatory signaling can modulate the outcome of
inflammatory disease pathogenesis. This thesis investigates, the role of GRK5, a member of
GRK family in the pathophysiology of sepsis using mouse microbial sepsis models.

This brief introduction provides an overview of the hypothesis tested in this thesis with a
glance on the focus of subsequent chapters. Chapter one provides a comprehensive literature
review pertaining to the current knowledge on GRKs and its involvement in inflammatory
diseases. Chapters two through four are organized based on the specific aims of this thesis, each
consisting of an abstract, introduction, materials and methods, results and discussion. Chapter
two focuses on the overlapping and distinct roles of GRK5 in MyD88 dependent and
independent pathways using TLR2-, and TLR3- ligand induced inflammatory response in vivo.
Chapter three focuses on the critical role of GRK5 in mediating polymicrobial sepsis. Chapter
four focuses on understanding the critical involvement of GRK5 in modulating severity and

2

outcome of E. coli induced acute pulmonary inflammation. Chapter five highlights the results
obtained in this project with a discussion on how these findings contribute to our current
understanding of the role of GRK5. Also, discussed are some of the limitations and future
directions of this project.

1.1: Introduction: G-protein-coupled receptor kinases (GRKs)
The superfamily of hepta-helical transmembrane proteins (also called G-protein-coupled
receptors (GPCRs)) modulate a variety of physiological processes including sense of vision,
olfaction, hormonal signal transduction, cellular proliferation, differentiation, survival etc. Upon
activation, GPCRs activate heterotrimeric G-proteins to dissociate into Gα and Gβγ subunits.
These subunits modulate various effector mechanisms to bring about the physiological
responses. Most GPCRs display a rapid loss of responsiveness even in the continuing presence of
stimuli; this phenomenon is now widely appreciated and known as desensitization. The
mechanism of GPCR desensitization is a specific, controlled two-step mechanism (Figure 1).
First, GPCR kinases (GRKs) phosphorylate the activated receptors, marking those targets for
binding to arrestins (intracellular scaffolding protein). Secondly, the arrestin bound receptor
prevents the GPCR from G-protein-dependent signaling by not only steric interference but also
by mediating endocytosis of these receptors1,2.

3

A)

1.

GPCR activation leads to recruitment of
various second messengers.

2.

Second messengers activate downstream
signaling, referred as G-protein dependent
GPCR functions.

3.

GPCR activation recruits GRKs

B)
4. GRKs phosphorylate intracellular
domains of GPCRs.

C)

5.

Phosphorylated GPCR signals arrestin
recruitment and binding.

6.

Arrestin binding inhibits G-protein
signaling. Arrestin and GPCR form a
signalosome complex.

7.

Arrestin signalosome activates Gprotein-independent signaling.

Figure 1.1: Schematic summary of the role of GRKs/arrestins in carrying out GPCR
dependent and independent functions. a) GPCR activation and G-protein dependent function.
b) GRK recruitment and phosphorylation of GPCR intracellular domains. c) Binding of arrestin,
formation of signalosome complexes leading to G-protein independent functions.

4

Sustained research in the GPCR field yielded remarkable insights on the GPCR
regulation and function leading to development of various pharmacological agents3,4. And of all
the pharmaceutical drugs out on market more than 50% target GPCRs. In addition to the
desensitization functions, recent research indicates that GRKs and arrestins are involved in
signaling functions that are desensitization- and G-protein independent. In many cases, it has
been shown that arrestins and GRKs mediate these G-protein independent effects by recruiting
several signaling mediators or by phosphorylation of signaling molecules by GRKs. In addition
to mediating G-protein-independent effects of GPCRs, there is now convincing evidence that
GRKs regulate non-GPCR families including tyrosine kinase receptors, TNF receptors and toll
like receptors in both phosphorylation dependent and independent manner. In this review, we
discuss these emerging functions of GRKs and their potential implications in both physiological
and pathological conditions.

1.2: The G-protein coupled receptor kinase family: Identification, organization and
structure
During the 1970s and mid-1980s, agonist induced dampening of G protein-mediated
signaling was discovered on two receptors, rhodopsin and β2-adrenergic receptor via
phosphorylation of their cytoplasmic c-terminal domain. The enzyme phosphorylating rhodopsin
receptor, which controls vision, was identified in the late 1970s and aptly termed “Rhodopsin
kinase” (modern name: GRK1). During 1986, a novel kinase was identified that phosphorylated
β2- adrenergic receptor and dampened G protein-mediated signaling5. This kinase was initially
named β-adrenergic receptor kinase or βARK (modern name: GRK2). In the

5

Figure 1.2: Domain structure GRK family of proteins: GRK family of proteins contains a
GRK family specific short N-terminal domain and catalytic domain, with variations in Cterminal domains. Regulator of G-protein signaling homology domain (RH) is present in Nterminal region represented in red. Structural differences in the C-terminal domain differentiate
GRKs. The Phosphatidylinositol 4,5-bisphosphate (PIP2) sites in C-terminal domain of GRK2/3
family (represented in blue), polybasic and palmitoylation sites in C-terminal domain of
GRK4/5/6 family, short farnesylated C-termini of GRK1/7 aids in membrane translocation.
GRK5 contains a nuclear localization signal represented in red. Numbers above domains
represent amino acid residue based on Lodowski et al. (2006)6.

6

same year, cloning of the mammalian beta-adrenergic receptor revealed a sequence similarity
with the photo receptor, rhodopsin and led to the recognition of a GPCR family, with rhodopsin
as its founding member. Another protein (termed S-antigen) was discovered initially for its role
in allergic uveitis7, was found to associate with rhodopsin and dampen G-protein mediated
retinal signaling8,9. In 1986, with the discovery of this “arresting” function in retinal signaling,
the protein was renamed “arrestin”. Further functional characterization of GRKs and arrestin
firmly established the idea of two-step inactivation of GPCRs. Meanwhile, other members of the
GRK family were identified including GRK3

10

, GRK4

11

, GRK5

12

, GRK6

13

and GRK714,15.

GRKs are grouped functionally into two classes: Visual and non-visual GRKs. Expression of the
visual GRKs comprising GRK1 and GRK7 is restricted to the eyes and pineal gland. The nonvisual GRKs can be grouped into two sub-groups: GRK2-like [GRK2 and GRK3, otherwise
known as βARK1 and 2 (β-adrenergic receptor kinases 1 and 2)] and GRK4-like (GRK4, GRK5
and GRK6) based on their structural similarity16. Unlike visual GRKs, the non-visual GRKs are
widely distributed throughout the body with the exception of GRK4, which is found restricted in
the testis and proximal tubule of the kidney. Therefore, most GPCRs in the body are invariably
regulated by four GRKs namely GRK2, GRK3, GRK5 and GRK6. And each of the four GRKs
may regulate more than one GPCR (there are approximately 800 GPCRs in the mammalian
body), increasing the complexity of understanding the role of GRKs.

All GRKs are multi domain proteins, sharing a 25 residue n-terminal region (unique for
GRKs) followed by a regulator of G-protein signaling homology domain (RH) and a catalytic
ser/thr protein kinase domain responsible for phosphorylating substrates (Figure 1). The nterminal basic region in the GRK4- like family (GRK4-6) aid in membrane translocation.

7

However, the c-terminal domain is the most essential domain for targeting the GRKs to the
plasma membrane. GRK1 and 7 are membrane associated by virtue of their short farnesylated ctermini. The c-termini of GRK2 like kinases are comparatively longer than the GRK4 like family
and contain a 125 amino acid pleckstrin homology (PH) domain, which has binding sites for
PIP2 and free Gβγ. Hence, on GPCR activation these primarily cytosolic GRKs (GRK2 and 3)
are targeted to the membranes. GRK4 and GRK6 are exclusively membrane associated by their
palmitoylated residues within the last 15-20 amino acids of the c-terminus and also by their nterminal basic region. GRK5 is also predominantly membrane bound, but not through lipidation
of c-termini, but through the poly basic regions found in both the c- and n- termini17.

Early research on GRKs primarily focused on GRK2, GRK3 and it has been widely
assumed that GRK4, GRK5 and GRK6 might be just redundant kinases. However, studies
focusing on the GRK4- like family have remarkably changed the view of GRKs plainly acting
for GPCRs. Recent studies have appreciated the intrinsic kinase activity possessed by these
GRKs to regulate non GPCR substrates. In particular, GRK5 and GRK6 of the GRK4 like sub
family have been shown to phosphorylate p53, HDAC5, Lrp6, tubulin etc. Interestingly, GRK5
also contains a nuclear localization signal and has been shown to accumulate inside the nucleus.
Indeed, GRK5 can phosphorylate class II histone deacetylase and therefore mediate gene
transcription18. Interestingly, GRK6A, one of the three splice variants of GRK6, was also
detected inside the nucleus; however, its physiological role is yet to be ascertained19. These
unforeseen roles of GRKs and their ability to initiate alternate signaling pathways have invoked a
tremendous interest in the field.

8

1.3: Regulation and Activation of GRKs
Similar to other kinases, GRK activity is regulated by both protein-protein interactions
and phosphorylation events. Gβγ subunits were the earliest identified proteins known to interact
with and activate GRKs. Other studies have demonstrated that lipids can also bind and activate
GRKs. Interestingly, phospholipids (PIP2) binding to the c-terminal PH domain of GRK2 family
activates their kinase activity, whereas GRK4 like family members are activated by binding of
phospholipids (PIP2) to their n-terminal polybasic regions20. Furthermore, calmodulin, caveolin1, and actin were shown to affect GRK activity by direct binding.

Phosphorylation can lead to either activation or deactivation of GRKs. Visual GRKs
(GRK 1 and 7) tend to get deactivated by both auto phosphorylation and phosphorylation
mediated by PKA 21. In contrast, non-visual GRKs tend to get activated or deactivated depending
on the stimuli. GRK5 auto phosphorylated by phospholipid binding enhances its ability to
phosphorylate receptors, but GRK5 phosphorylated by protein kinase C (PKC) tends to inhibit it
22

. However, the same PKC tends to enhance the activity of GRK2 by inducing

phosphorylation23. Apart from phosphorylation, GRK2 can also be s-nitrosylated by nitric oxide
synthase to inhibit its activity24. These findings suggest that multiple pathways are involved in
regulating the activities of GRKs.

Recently25, crystallographic structures of GRK1, GRK2 and GRK6 have been elucidated
to give certain vital information on how these GRKs act to cause receptor desensitization. These
studies revealed GRKs in a closed conformation in which the n-termini had conserved 18-20
residues that formed an alpha (αN) helix and interacted with the kinase domain. The formation of

9

this αN helix stabilized the closed (active) conformation of the kinase domain leading to
allosteric activation and favored catalysis by the kinase domain. Furthermore, the αN helix is
proposed to act as a docking site for the activated receptors and removal of the αN helix
abolished GPCR phosphorylation suggesting αN helix formation is indeed the primary step in the
activation of GRKs.

1.4: Phenotypes of GRK deficient mouse
Phenotypes of GRK knockout mice enabled researchers to identify the unknown
physiological and pathological roles of GRKs. In some cases, GRKs were able to compensate for
each other, and were able to produce less obvious phenotypes. The most obvious phenotype was
from that of homozygous knockout of GRK2 that led to embryonic lethality due to defective
cardiac development26. Using targeted knockouts and heterozygous mice, GRK2 has been shown
to be important in the development of heart, lymphocyte chemotaxis, experimental auto immune
encephalomyelitis, sepsis, atherosclerosis etc. Closer examination of other GRK knockout mice
surprisingly revealed distinctive phenotypes- GRK3 in olfaction27; GRK6 in behavioral
responses, locomotor stimulating effects of cocaine etc.28

GRK5 was found to regulate cholinergic responses. GRK5 deficiency caused
hypothermia, hypo activity, tremors and enhanced salivation with oxotremorine (a muscarinic
receptor agonist)29. In addition β2 adrenergic receptor-induced airway smooth muscle relaxation
was reduced in GRK5 deficient mouse. Transgenic mice overexpressing GRK5 in cardiac tissues
and vascular smooth muscle showed increased β adrenergic receptor desensitization and increase
in blood pressure respectively.

10

Research over the last few years shows, GRKs have multiple functions in different organ
systems. However, their role in immune system and its consequences are only beginning to be
understood. Here, we will focus on the role of GRKs on the regulation of the immune system and
its effects on function.

1.5: GRKs in immune system and inflammation
GPCRs are known to modulate immune functions including inflammatory responses30-32,
chemotaxis30-32, and apoptosis33 . GRKs are the turn off signals for G-protein dependent
signaling, and along with its function on non- GPCR signaling such as TLR signaling, makes
them important proteins to study with respect to the immune system.

1.5.1: Inflammatory signaling and GRKs
Inflammation is a complex biological response of host tissue to any inciting stimuli
ranging from pathogens, irritants and even damaged host cells. Inflammatory processes are
generally considered to be beneficial in most cases, however, when not controlled (acute
inflammation) or persisting for a long duration of time (chronic inflammation) are considered
harmful. It is for that reason the inflammation process is tightly regulated by the host. Broadly
inflammation can be categorized into three stages: a) onset stage, b) propagation stage and c)
resolution stage. Any defect in regulating these stages can have major consequences in the final
outcome of the response. During the onset stage, different classes of receptor molecules,
recognize the inciting stimuli and activate downstream effector molecules leading to propagation
of inflammation via production of inflammatory mediators like cytokines and chemokines. These
inflammatory mediators amplify the inflammatory process by recruiting immune cells to the site,

11

which results in prompt removal of inciting stimuli. Once the stimulus is removed, antiinflammatory mediators are produced which result in resolution of inflammation. If this is not
the case, the inflammation will propagate itself causing destruction of host tissues by
inflammatory mediators and also by the inciting stimuli eventually leading to death of the host.
Sepsis or septic syndromes classically manifest in this manner, wherein the inciting cause is a
microbe. Since, my research project, mostly focuses on bacterial inflammation, from here on I
will focus on bacteria/ bacterial products induced inflammatory signaling and the role of GRKs
in modulating those signaling pathways.

Once the skin or mucosal barrier is breached by bacteria, the phagocytic cells of the
innate immune system interact with the pathogens via recognizing evolutionarily conserved
patterns on the surface of microbes, called PAMP’s – pathogen-associated molecular patterns.
PAMPs include a wide variety of microbial components ranging from Lipopolysaccharide (LPS)
from gram-negative bacteria, flagellin, lipoteichoic acid from Gram positive bacteria,
peptidoglycan from the bacterial cell wall, zymosan from fungi, and nucleic acid from viruses.
PAMPs bind to pattern recognition receptors (PRR) on or within innate immune cells. Broadly
PRR´s are classified into: 1) the toll like receptors (TLR’s); 2) the intracellular nucleotidebinding oligomerization domain (NOD) leucine-rich repeat proteins; and 3) the retinoic-acidinducible gene I (RIG-I)-like helicases. Upon engagement these receptors activate intra cellular
signaling proteins like NFκB, AP1, CREB, and c/EBPβ, which will eventually lead to gene
transcription and inflammatory mediator production.

12

1.5.1.1: Toll like receptor (TLR) signaling, NFκB and GRK5
Toll-like receptors are molecular sensors for microorganisms including viruses, bacteria,
and fungi. TLR2 and TLR4 recognizes components from Gram positive and negative bacteria
respectively, TLR3 senses viral dsRNA, and TLR9 is activated by unmethylated bacterial DNA.
Lipopolysaccharide (LPS) binds to the CD14-TLR4/MD2 complex and results in activation of
TLR4. Intracellular adaptor molecules containing conserved Toll/interleukin-1 receptor (TIR)
domain are necessary to recruit and activate downstream kinases and transcription factors that
regulate the host inflammatory response and type I IFN production. LPS/TLR4 signaling has
been divided into two pathways: a MyD88-dependent and a MyD88-independent pathway (Also
known as TRIF pathway).

Activation of the MyD88-dependent pathway leads to the early phase of NFκB
activation and pro-inflammatory cytokine expression, while MyD88-independent pathway
mediates the late phase of NFκB activation and the induction of interferon-inducible genes in
TLR4 signaling34. Upon binding of ligand to the receptor, MyD88 recruits and activates a death
domain-containing kinase IRAK4 which in turn phosphorylates IRAK1. Phosphorylated IRAK1
dissociates from the receptor complex and binds TRAF6 which in turn interact with TAK1.
TAK1 then activates downstream IKK (IκB kinase) and MAPK pathways. IKK complex (IKKα,
IKKβ, IKKγ/NEMO), induces phosphorylation and subsequent degradation of IκB and
translocation of the transcription factor NFκB to the nucleus, which controls the expression of
pro-inflammatory cytokines such as IL-1, IL-6, IL-8, TNFα and other inflammatory mediators.

13

The MyD88-independent (TRIF) pathway utilizes the adaptor protein TRAM together
with TRIF leading to the activation interferon regulatory factor (IRF) 3 and the subsequent
induction of IRF3-dependent gene expression such as interferon-β (IFN-β). Studies using TRIFdeficient macrophages demonstrate that TRIF plays a crucial role in the activation of IRF3, and
the late-phase activation of NFκB and MAPK35.

Nuclear factor κB (NFκB) was first described in 1986 as a nuclear protein binding to the
kappa immunoglobulin-light chain enhancer in B cells36. NFκB is the central mediator in both
MyD88 and TRIF pathway. Most inflammatory signaling pathways converge onto NFκB and so
its components are often targeted to treat, inflammatory diseases. NFκB is a transcriptional
regulator and their families consist of five members: p65 (RelA), Rel B, c-Rel, p50 and p5237.
All NFκB family members share a highly conserved Rel homology domain (RHD) that is
responsible for DNA binding, dimerization, and interaction with a family of inhibitory proteins
known as inhibitors of NFκB (IκBs). Except RelB which only forms heterodimers, the other
NFκB proteins can combine as homodimers or heterodimers and activate specific target genes.
p50/RelA heterodimer being the common dimer complex is very important in the coordination of
inflammatory and innate immune responses38. Under resting state, IκBs (IκBα, β, ε, Bcl-3 and
the precursor p100 or p105 Rel proteins)39 by masking the nuclear localization signal (NLS) on
the NFκB subunits restricts their entry into nucleus. However, upon phosphorylation of IκBs by
IKK or other kinases leads to their ubiquitination and proteasomal degradation releasing NFκB.
This process translocates NFκB subunits into nucleus to start gene transcription. The NFκB
activation is self-limiting, and is switched off when the inflammatory signal terminates. IκBs, by
virtue of their nuclear export sequence (NES) removes the NFκB subunits from nucleus. In

14

addition, NFκB activation induced IκBα synthesis can in turn prevent further NFκB activation.
Also, IκBß and IκBε, are synthesized constitutively and reestablish NFκB inhibition.

NFκB signaling pathway can be activated by a variety of stimuli including bacterial
products, growth factors, UV irradiation and oxidative stress. In addition to the aforementioned
stimuli, pro-inflammatory cytokines such as IL-1 and TNF induced by NFκB, can amplify NFκB
activation via IL-1 and TNF receptor mediated pathways39. Recent data indicates that NFκB
activation and resultant cytokine production contributes to the pathogenesis of organ injury and
lethality during sepsis. However, using NFκB inhibitors as a drug therapy in sepsis can have
negative effects owing to their involvement in other physiological responses which can be
beneficial to the host.

GRKs and NFB pathway: GRKs, in particular GRK2 and GRK5 have been reported to
regulate molecules of NFκB signaling pathway. GRK5, in macrophages was shown to negatively
regulate TLR4-induced ERK activation. Knockdown of GRK5 in macrophages significantly
enhanced IKK-mediated p105 phosphorylation and degradation40. NFBp105 normally
sequesters the MAP3K kinase, tumor progression locus 2 (TPL2) in an inactive state and
following phosphorylation and subsequent degradation of p105, TPL2 is free to initiate the
MAPK signaling cascade that culminates in ERK1/2 activation. GRK5 was shown to directly
interact with phosphorylate p105 and prevent its phosphorylation and degradation by IKK. In
this way, GRK5 was shown to inhibit TLR4-induced ERK activation.

15

Later Iaccarino et al41., reported that GRK5 binds to IκBα and stabilizes it from
degrading and this led to nuclear accumulation of IκBα into nucleus, by covering the nuclear
export signal (NES) sequence. This effect in turn led to decreased NFκB activation since, IκBs in
the nucleus bind to NFκB preventing it from transcription of genes. The IκBα binding site is
mapped to the RH domain in the N-terminal of GRK5 and over expression of this domain alone
(GRK5-RH) is sufficient to inhibit NFκB. Also, the catalytic function of GRK5 doesn’t seem to
play a role in inhibiting NFκB as overexpression of a kinase dead GRK5 mutant, GRK5∆K215R,
in bovine aortic endothelial cells, behaved like GRK5 wild type with regards to IκBα nuclear
accumulation and the phosphorylation.

However, in contrast to the previous studies GRK5 can also positively regulate NFκB
signaling in human cells (HeLa cell line and HEK293 cells), mouse macrophages, zebra fish
embryos and drosophila42. Also, GRK5 is reported to be required for normal microbial resistance
in vivo in zebra fish and drosophila42. Additionally, in vitro studies

43

in macrophages have

shown that GRK5 acts as an IκB kinase and phosphorylates the same residues (serine 32/36) on
IκBα as that of the canonical IKKβ. An IκBα S32A/S36A mutant exhibited approximately 60%
reduced in vitro GRK5-mediated phosphorylation, compared to wild type IκBα suggesting that
serine32/36 sites are phosphorylated by GRK5. Cytokine and chemokine production was largely
attenuated in GRK5 knockout mice and p65 nuclear translocation and IκBα phosphorylation
were significantly reduced in LPS treated GRK5-/- macrophages, compared to GRK5+/+
macrophages. Overall these data suggest, GRK5 regulates both positively and negatively,
depending on the cell type and disease models involved. GRK5 modulates inflammatory
signaling depending on the cell type involved. This is intriguing because, NFκB signaling can be

16

beneficial and modulating its signal depending on the cell type might prove beneficial.
Additionally, results from chapter 2 and chapter 3 suggest that in a whole animal system, GRK5
primarily plays a positive regulatory role in NFκB signaling and its eventual inflammatory
responses. In summary, GRK5 modulates NFκB signaling either by phosphorylating (IκBs or
NFκB1-p105) or stabilizing (IκBs) components of NFκB signaling and regulates downstream
events which can have a multitude of effects on inflammatory processes.

GRK2 has also been shown to interact with IκBα via the n-terminal domain of IκBα and
phosphorylates IκBα at very low stoichiometric ratios compared to IKKβ and GRK544. However,
GRK2 seems to negatively regulate NFκB signaling via interaction with NF-κB1p105, and by
down regulating the MEK-ERK pathway45 leading to decreased inflammatory response.
Incidentally, in vitro studies carried out on macrophages treated with TLR2, TLR4 ligands46 and
lymphocyte samples from septic patients47 show GRK2 levels are up regulated, whereas GRK5
levels are down regulated. These results suggest that GRK2 and GRK5 might have opposing
roles in inflammation via their action on NFκB signaling pathways.

1.5.1.2. Mitogen Activated Protein Kinase (MAPK) signaling
Mitogen-activated protein kinases (MAPKs) are a family of serine/threonine protein
kinases that mediate fundamental biological processes and cellular responses to external stress
signals. Three major groups of distinctly regulated MAP kinase cascades are known that lead to
altered gene expression: ERK1/2, JNK, and p38 MAP kinase. ERK is activated by MAP kinase
kinase (MKK) and MKK2, JNK by MKK4 and MKK7, and p38 MAP kinase by MKK3, MKK4,
and MKK6. Upon activation of the MAP kinases, transcription factors present in the cytoplasm

17

or nucleus are phosphorylated and activated, leading to expression of target genes resulting in a
biological response.

1.5.1.2.1: Extracellular Signal Regulated Kinases (ERK) and GRK5
ERK1/2 is induced under inflammatory conditions by both LPS and TNFα and is known
to regulate production of cytokines like TNFα, IL-1, IL-8 and PGE2. ERK inhibitors are found to
be useful in abrogating inflammatory responses in various disease models. GRK5 and 2 can
negatively regulate NFκB1 p105-ERK pathway in macrophages stimulated with LPS40,45. Also
overexpression of GRK5 and/or GRK6 has been found to enhance β-arrestin 2-mediated ERK1/2
activation, whereas overexpression of GRK2 and/or GRK3 abolishes β-arrestin 2-mediated
ERK1/2 activation48. This response is observed with activation of β2 adrenergic receptor,
cannabinoid receptor 249, Angiotensin 1A receptor43 and Insulin like growth factor 1 receptor50.

1.5.1.2.2: p38 MAPK and GRK5
The p38 MAP kinase pathway shares many similarities with the other MAP kinase
cascades, being associated with inflammation, cell growth, cell differentiation, and cell death. A
number of pathogenic stimuli, including LPS, staphylococcal peptidoglycan and enterotoxin B,
echovirus 1 and herpes simplex virus 1, activate p38 through different toll like receptors. The
main biological response of p38 activation involves the production and activation of
inflammatory mediators to initiate leucocyte recruitment and activation. p38 MAPK positively
regulates expression of many genes involved in inflammation, such as those coding for TNF-α,
IL-1β, IL-6, IL-8, cyclooxygenase-2, and collagenase-1, -3. Inhibition of p38 MAPK with
SB203580

reduced

pro-inflammatory

cytokine

18

production

in

monocyte/macrophages,

neutrophils, and T lymphocytes. Expression of pro-inflammatory cytokines can be regulated at
both the transcriptional and post-transcriptional levels. Also, p38 directly affects arachidonate
signaling, which can stimulate and suppress inflammation. Several studies have implicated p38
MAPK in polymicrobial sepsis. p38 MAPK activity markedly increases in splenic and peritoneal
macrophages and inhibiting it improved survival rate during sepsis. GRK5 and GRK2 are shown
to regulate a constitutively active virally encoded GPCR (US28)51. GRK2 and GRK5
phosphorylate the active receptor and inhibit GPCR activation which is favorable for viral
propagation, and increases activation of p38 MAPK which is favorable for the host. This result is
predicted to increase cellular detection of viral particle by producing p38 MAPK dependent
factors.

1.5.1.2.3: c-Jun N-terminal kinase (JNK) and GRK5
JNKs, while activated by mitogens, are also activated by a variety of environmental
stresses (heat shock, ionizing radiation, and oxidants), genotoxins (topoisomerase inhibitors and
alkylating agents), ischemic reperfusion injury, mechanical shear stress, vasoactive peptides, proinflammatory cytokines and PAMPs/DAMPs. JNK induces transcription of AP-1, c-Jun, ATF-2,
and ELK-1, all of which are important mediators of inflammatory gene transcription. JNK
activation of AP-1 is important for synthesis of TNFα, as well as proliferation and differentiation
of lymphocytes and hence plays a vital role in immune system. The role of GRK5 in JNK
signaling is not well characterized. However, studies with transgenic mice overexpressing
cardiac specific GRK5 and a constitutively active mutant of the α1B-adenergic receptor showed
attenuation of JNK activation compared to controls. GRK5 had variable effects on α1BAR

19

signaling, and the complexity of GRK5 regulation of in vivo α1BAR signaling remains to be
fully elucidated52.

1.5.2: Chemotaxis and GRKs
Chemotaxis is an important function, which enable immune cells to arrive at the site of
inflammation. Cells producing chemokines, act on the chemokine receptor, and initiate a
chemotactic response. The chemotactic response depends on the amount of chemokines
produced and also on the expression levels of chemokines receptor. Chemotaxis would depend
on the integrated modulation of different steps of the chemotactic process (receptor sensing, cell
polarization, membrane protrusion, adhesion/ de-adhesion cycles) in given cell types and in
response to specific stimuli. Chemokine receptors being GPCRs also undergo desensitization
upon continuous presence of stimuli. It is likely that GRKs, which regulate GPCRs, can also
modulate chemotaxis and thereby modulate immune responses. Intriguingly, GRK2,-3,-5 and -6
are expressed in high levels in immune cells suggesting modulation of these GRKs during
disease might change the outcome or progression of the disease.

1.5.2.1: GRKs and cell migration
Of the GRKs, GRK2 is highly studied and more characterized. Studies have shown that
GRK2 regulation of cell migration is not straightforward; it depends on the stimuli and also on
the cell type. In most cell types53-55, GRK2 negatively regulates chemotactic responses consistent
with its canonical negative role in GPCR signaling. However, in a few cell types 56, GRK2
positively regulates chemotactic responses. These roles of GRK2 might depend on the cell type
and its polarization state. In more polarized cells like epithelial cells, GRK2 positively regulates

20

chemotactic responses, whereas in the less polarized cells such as immune cells, GRK2 does the
opposite. Interestingly, the positive regulation of chemotactic responses by GRK2 doesn’t
require catalytic functions, rather requires recruitment of GRK2 to the plasma membrane
denoting a protein –protein interaction. In keeping with this notion, a membrane-targeted kinase
mutant strongly enhances cell motility, and GRK2 indeed interacts with GIT1 (GRK2 interacting
factor), and is present at the leading edge of polarized/migrating epithelial cells in wound-healing
assays. Also, this transient association of GRK2 with GIT1 is critical for proper ERK1/2
activation and efficient cell migration. Also, GRK2 directly phosphorylates histone deacetylase 6
(HDAC6), a cytoplasmic histone deacetylase responsible for deacetylation of tubulin and other
substrates involved in cell migration. Furthermore, GRK2 phosphorylates ERM proteins ezrin
and radixin, which contribute to the F-actin polymerization dependent motility. These novel roles
of GRK2 in cell migration might throw some light on comprehending the non-canonical positive
regulation of motility by GRK2
.
GRK5 regulates GPCRs that are critical in chemotaxis in both a canonical and noncanonical manner. GRK5 is shown to regulate migration in very few cell types. GRK5 regulates
monocyte migration by regulating the CC chemokine receptor-2 (CCR2), a GPCR for the
monocyte chemo attractant protein-157. GRK5 in also shown to modulate monocyte chemotaxis
in a non-GPCR dependent fashion by regulating the colony-stimulating factor-1 receptor (CSF1R), a receptor tyrosine kinase. GRK5 is reported to attenuate atherosclerosis by desensitizing
CCR2 in monocytes and inhibiting their migration57. An in vitro study suggests1 that GRK5 can
regulate CXCR4 (CXC chemokine receptor-4) desensitization indirectly, by phosphorylating
HIP (HSP70 interacting protein), a co-chaperone protein which phosphorylates CXCR4 and

21

mediates desensitization. However, in vivo evidence of GRK5-mediated desensitization of
CXCR4 is currently lacking. Furthermore, in a model of polymicrobial sepsis2, GRK5 plays a
role in modulating inflammation and mortality but doesn’t seem to play a role in chemotaxis of
immune cells. GRK5 regulates prostate cancer cell migration and invasion58. GRK5 forms a
complex with moesin (ERM (ezrin–radixin–moesin) proteins and plays important roles in the
cytoskeleton remodeling and cell adhesions to neighboring cells and to matrix), phosphorylates
moesin principally on threonine 66 residue, and regulates cellular distribution of moesin58. In
addition, GRK5 is required for the in vivo growth, invasion, and metastasis of prostate cancer.
GRK6 is shown to regulate chemokine receptors like CXCR259, CXCR460 and LTB461. By
desensitizing these receptors, GRK6 regulates neutrophil and lymphocyte recruitment in vivo in
different disease models62-64. In epithelial cells, a functional screening identified GRK6 as a
critical mediator in integrin-mediated cell adhesion and migration of tumor cells65, and GRK6
deficiency reportedly promotes CXCR2 receptor-mediated tumor progression and metastasis in a
lung carcinoma model66. These roles of GRK6 are mostly related to their canonical role of GPCR
desensitization, and considering the non-canonical roles played by GRK2, it is likely for GRK6
to have non canonical functions in chemotaxis. However, currently there are no sufficient studies
to suggest a non-canonical role for GRK6 in chemotaxis.

Although, the role of GRK5 in chemotaxis seems less well-studied compared to GRK2
and GRK6, its role may be relevant to the cell type and the disease models in question. A recent
study demonstrated that GRK5 functions as an actin-bundling scaffold and promotes neuronal
filopodial formation, neurite outgrowth, etc67. These functions in neuronal tissues closely
resemble the initiation events of chemotaxis in immune cells. GRK5 may replicate these

22

functions in other cells. Also, it is also possible that other GRKs might play a redundant role in
chemotaxis of immune cells and further study is needed to assess the role of GRK5 in other cells
including epithelial cells.

1.5.3: Apoptosis and GRKs
Apoptosis or programmed cell death regulates development, selection, maturation of
immune cells at various stages of their life cycle. Hence, normal rates of apoptosis are
considered important in immune system development. Inappropriate apoptosis (either too little or
too much) is a mediating factor in many human conditions including sepsis, neurodegenerative
diseases, ischemic damage, autoimmune disorders and many types of cancer. In addition to the
role in development of immune system, apoptotic bodies/cells can alter the course of the
inflammation. Apoptotic cells are usually taken up by macrophages and Dendritic cells (DC). In
response to the uptake, macrophages induce production of and release immunosuppressive
cytokines such as IL-10, TGF-β, prostaglandin E2 and PAF

68

and suppress production of

proinflammatory cytokines IL-1β, IL-12 and TNFα 69 . Similarly, ingestion of apoptotic cells by
DCs also results in suppression of IL-12 and IFN-γ expression, up regulation of co-inhibitory
molecules and production of anti-inflammatory cytokines70. Thus, apoptotic cells have
significant impact on the function of these phagocytes and in turn modulate inflammatory
disease pathogenesis.

The mechanisms of apoptosis are highly complex and sophisticated, involving an energydependent cascade of molecular events. There are two major apoptotic pathways: the extrinsic or
death receptor pathway and the intrinsic or mitochondrial pathway. The extrinsic, intrinsic, and

23

granzyme B pathways converge on the same terminal, or execution pathway. This pathway is
initiated by the cleavage of caspase-3 and results in DNA fragmentation, degradation of
cytoskeletal and nuclear proteins, cross-linking of proteins, formation of cytoplasmic blebs and
apoptotic bodies and finally phagocytosis of the apoptotic bodies by adjacent parenchymal cells,
neoplastic cells or macrophages.

GRKs are reported to play a role in apoptosis of immune cells. GRK5 was shown to
phosphorylate p53 and regulate irradiation-induced apoptosis71. p53 is crucial in determining the
cellular response to stress, not only by inducing apoptosis but by also having a role in growth
arrest. Induction of p53-dependent apoptosis occurs via extrinsic or intrinsic pathways depending
on apoptosis signals and converges on the activation of caspase proteins. P53 triggers the
expression of Apaf1 and BH3 proteins (proteins involved in the intrinsic pathway) and up
regulates caspase expression. P53 also up regulates the expression of extrinsic pathway
receptors, Fas, DR5 and PERP, by increasing mRNA levels, and promoting trafficking of the Fas
receptor to the cell surface from the Golgi. GRK5 phosphorylates p53 in vitro and in vivo at
Thr55, which results in reduced p53 levels. Treatment with the proteasome inhibitor, MG132,
prevented the reduction in p53, thus confirming its degradation is via the proteasome. GRK5
knockout mice displayed tissue-wide aberrant up regulation of p53 suggesting GRK5 negatively
regulates p53 in vivo. GRK5-mediated p53 degradation directly affects apoptosis, as knockdown
of GRK5 in the osteosarcoma cell line, U2OS, increases apoptosis by 40% following cisplatin
treatment. Apoptosis of Saos-2 cells (sarcoma osteogenic cell line), which are p53 null, were
unaffected following GRK5 knockdown, thus suggesting that GRK5 induces apoptosis via p5371.
In contrast, results from chapter 3 suggest GRK5 positively regulates thymocyte apoptosis in

24

vivo in polymicrobial model of sepsis2. Specifically, lack of GRK5 caused decreased apoptosis
of CD4CD8++ cells in thymus, which is attributed to decreased sepsis induced corticosterone
levels in GRK5 deficient mice. Additionally, in concordance with the results in chapter 3, GRK5
has been shown to negatively regulate Bcl-2 transcription (anti apoptotic protein) in SH-SY5Y
(neuroblastoma cell line) cells72. This result is predicted to increase cell death thereby shows a
positive role for GRK5 in apoptosis, although the impact of this regulation on apoptosis is yet to
be elucidated72. Even though, the above studies provide an opposing role for GRK5 in apoptosis,
the disease context is different and hence the role might differ. However, these results suggest
GRK5 modulates apoptosis of cells through multiple mechanisms and also plays an important
role in inflammatory disease conditions.

Other GRKs have also been reported to modulate apoptosis of cells. GRK2
overexpression increased caspase 3 levels and induced increased cardiomyocyte apoptosis
following, ischemia/reperfusion injury to myocardium. Conversely, GRK2 inhibition reduced
apoptosis by increased NOS activity, NO production and AKT levels in cardiomyocytes73. Also,
GRK2 levels were increased in apoptotic lymphocytes obtained from heart failure patients.
These data suggest a positive regulatory role of apoptosis by GRK2

74

. GRK6 is reported to

regulate clearance of apoptotic cells75. GRK6 cooperates with GIT1 to activate Rac1, which
promotes apoptotic engulfment. GRK6 greatly contributed to removing unnecessary B cells by
splenic white pulp macrophages and removing senescent red blood cells by splenic red pulp
macrophages. GRK6-deficient mice also have increased iron stores in splenic red pulp in
which F4/80+ macrophages are responsible for senescent red blood cell clearance. As a
consequence, GRK6-deficient mice develop autoimmune disease.

25

In summary, the above studies suggest, GRKs by their GPCR-dependent and independent
signaling pathways regulate apoptosis of immune and non-immune cells and the functional
consequences regulates the overall outcome of inflammatory disease conditions.

1.6: Pathological role of GRKs
Altered activity/expression of GRKs might critically contribute to deregulate chemotaxis
of immune cells, inflammatory signaling and the apoptotic pathway. And overall these functions
have greater impact in altering the course of many diseases. GRKs expression levels vary in
many immune mediated diseases and are known to play a significant role in many pathologies
including, sepsis, cardiovascular diseases, neurodegenerative disorders and auto immune
diseases. In particular

GRK2 and GRK5 are known to play an significant role in the

pathogenesis of sepsis47,76-79, cystic fibrosis of lung80, neurodegenerative diseases81-83,
hypertension84,

myocardial

ischemia/infarct85,

heart

failure86,87

and

hypothyroidism88.

Additionally GRK5 also regulates development of atherosclerosis 57, colitis (unpublished data) in
mice, GRK2 in secondary bacterial infections89 and

tuberculosis90, GRK2 and GRK6 in

rheumatoid arthritis64, GRK6 in colitis63 and auto immune diseases75. These roles are attributed
both to their canonical GPCR dependent functions and to their role in non-GPCR dependent
functions. As more and more novel interactions of GRKs with non-GPCR substrates are
uncovered, GRKs are becoming identified as diverse targets for many disease conditions. It is
very important to take into consideration of their interactions with diverse targets, when GRKs
are targeted for therapeutic purposes.

26

Altogether these studies provide a broad overview and rationale for looking at the role of
GRK5 in inflammation. In this dissertation, I present evidence that GRK5 plays critical roles in
TLR induced inflammatory response, polymicrobial sepsis and E. coli induced pneumonia.

1.7: Acute Inflammatory diseases
1.7.1: Sepsis - Incidences and causes
Severe sepsis and septic shock - the serious manifestations of infectious diseases are in
the majority of cases caused by bacteria. Gram positive and gram negative infections each
constitute about half of verified bacterial infections. Fungi are emerging as important pathogens
in subgroups of patients and viruses underlie sporadic cases. The term sepsis, severe sepsis and
septic shock have been used for decades, but it was not until 199291 that a consensus meeting
agreed on universal definitions of these syndromes. Sepsis was defined as systemic inflammatory
response syndrome together with a clinical suspicion of infection; severe sepsis as sepsis in
addition to signs of acute reduction of organ perfusion (not related to primary septic focus or
underlying chronic disease); and septic shock as severe sepsis in addition to hypotension
requiring vasopressor support, or lack of response to adequate fluid resuscitation.

Overall in the United States, 2% of patients admitted to hospital end up with severe sepsis
representing 10% of all ICU admissions. The number of cases in the United States exceeds 750,
000 per year and was recently reported to be rising92. In an analysis of four large sepsis trials, 14day mortality averaged 26% and 28 day mortality 42%, which is equivalent to 215, 000 deaths
per annum in the United States. In this study, mortality in childhood was around 10%, rising to
38.4% in those over age 85.

27

Severe sepsis occurs as a result of both community- acquired and health care–associated
infections. Pneumonia is the most common cause, accounting for about half of all cases,
followed by intra-abdominal and urinary tract infections. Staphylococcus aureus and
Streptococcus pneumoniae are the most common gram-positive isolates, whereas Escherichia
coli, Klebsiella species, and Pseudomonas aeruginosa predominate among gram-negative
isolates93-95. A recent epidemiologic study involving 14,000 ICU patients in 75 countries showed
gram-negative bacteria predominate in patients with sepsis96.

1.7.2: Pneumonia- Incidences and causes
Pneumonia is an inflammation of the lung often caused by infection with bacteria,
viruses, and other organisms, although there are also non-infectious causes. It is the number one
cause of sepsis-induced mortality in humans. Most septic cases originate as a mild lower
respiratory tract infection, which progresses to pneumonia and eventually spread the etiological
agent to other organs and causes sepsis. The World Health Organization estimates that lower
respiratory tract infection is the most common infectious cause of death in the world (the third
most common cause overall), and has estimated that about 450 million cases occur every year
with almost 3.5 million deaths yearly97. Together, pneumonia and influenza constitute the ninth
leading cause of death in the United States, resulting in approximately 50,000 estimated deaths in
201098. This data likely underestimates the incidence, since deaths from other conditions like
cancer, Alzheimer’s disease, in which pneumonia is the terminal event are coded separately92.
Similar to sepsis, incidence of pneumonia is the highest among very young and elderly.
Identification of the etiology of pneumonia/lower respiratory tract infection is challenging as few

28

develop bacteraemic illness and high prevalence of nasopharyngeal colonization by potentially
pathogenic bacteria, limits the use of respiratory samples for diagnosing bacterial pneumonia.
Sepsis and pneumonia has often been viewed to involve excessive immune inflammation that
leads to death, mostly due to multiple organ failure. Sepsis is initiated itself as a systemic
inflammatory response syndrome (SIRS) to an infection and proceeds to the development of the
compensatory anti-inflammatory response (hypoinflammatory status) syndrome (CARS)99,100.
Hyper-inflammation mediated multiple organ failure is a characteristic feature of SIRS, whereas
immunosuppression-mediated opportunistic/ nosocomial infection and the resultant multiple
organ failure is a characteristic feature of CARS (Figure 4). Development of multiple organ
failure, refractory hypotension and loss of homeostasis are the later events in sepsis contributing
towards the high fatality rates observed during septic syndrome. Current approach to treatment is
early goal directed therapy (EGDT). The plan of action upon detection of sepsis is: broadspectrum antibiotics for targeting infection-causing bacteria, fluid resuscitation to alleviate
hypotension and external life support to maintain homeostasis. Discussed below are the common
major events considered crucial to development and progression of both pneumonia and sepsis.

1.7.3: Inflammatory response and NFκB signaling during sepsis/pneumonia
During an infection, pathogen recognition receptors (PRRs) mediate recognition of
microbial components by immune cells leading to their activation and downstream production of
inflammatory mediators and recruitment of additional effector cells. Innate immune cells viz.,
neutrophils, macrophages and dendritic cells form the first line of defense against microbes and
aids in clearing infection by production of inflammatory mediators. Inflammatory mediators aid
in defense against infection via mediating up regulation of PRRs and potentiation of

29

phagocytosis101. Cytokines and secondary metabolites (NO, ROS) produced by immune cells
during sepsis can lead to vasodilatation, increased permeability and hypotension producing
collateral damage to the host102. On the contrary, defective inflammatory response leads to
uncontrolled growth of bacteria ultimately damaging the host103,104. Therefore, a regulated
inflammatory response is critical for resolution of infection with minimal damage to the host105.

Microbial recognition by Toll-like receptors (TLRs) activates kinases and transcriptional
factors, leading to the expression of immunoregulatory mediators. Among the transcription
factors activated during sepsis, NFκB plays a major role in immunoregulation. NFκB is critical
for the transcription of many molecules involved in inflammatory responses, including cytokines,
chemokines, adhesion molecules, nitric oxide synthase, and complement components. Although
the classic transcriptionally active form of NFκB hetero-dimer is p65 (RelA) and p50 subunits,
other compositional forms of NFκB exist, and utilize RelB, c-Rel, or p52 as hetero- or homodimers. Studies have shown that during sepsis, greater nuclear accumulation of NFκB in
peripheral blood mononuclear cells (PBMC) is accompanied by higher mortality and worse
clinical course in patients with sepsis106,107. Clinical studies have also demonstrated that there is
an early and persistent activation of NFκB in non-survivors compared to survivors. Furthermore,
in preclinical models of sepsis, it has been shown that inhibition of NFκB improves organ
function and survival, indicating that NFκB activation contributes to cellular and organ
dysfunction108,109.

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1.7.4: Cytokines and their cellular sources
Cytokines and other inflammatory mediators mediated by microbial sensing pathways
play a central role in the inflammatory process. Cytokines are soluble proteins, act as
intercellular messengers and regulate proliferation, differentiation and functional activity of
individual cells and hence have a profound effect on organ systems. Cytokines have variously
been called interleukins, chemokines and lymphokines based on their presumed origin, effects or
targets. Cytokine production is well regulated under homeostatic conditions; however, in sepsis
its production is often disregulated. Macrophages are often the cell type involved in initial
microbial recognition (Alveolar macrophages in lungs and peritoneal resident macrophages in
peritoneum). After being triggered by bacteria and bacterial components, macrophages induces
release of a number of early pro-inflammatory mediators, such as TNF-α, IL-1β, IL-6, IFN-γ, IL8, and MCP-1, as well as secondary mediators for tissue injury, e.g., NO and ROS.

TNF-α is mainly secreted by macrophages, but also by a range of other cell types. It is
rapidly released in animals and humans after bacterial infection or inoculation and has strong
pro-inflammatory properties, and is probably the major driving force in the early cytokine storm
of severe infections. IL-1β, secreted by monocytes and macrophages, appears shortly after TNFα and has similar pro-inflammatory properties. Endothelial activation with vasodilation and
increased capillary permeability is one example of IL-1 and TNF effects. IL-6 is secreted by Tcells and macrophages. It is one of the most important mediators of the acute phase response. IL8 is secreted by innate immune cells in response to TLR-signaling and attracts other immune
cells through chemotaxis.

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The anti-inflammatory cytokines, e.g., IL-10 and TGF-β, which are released in sepsis
from macrophages and PMNs, play immune regulatory functions. However, excessive
production of these anti-inflammatory mediators may later cause immune dysfunction against
pathogens.

IL-12 and IL-18 are also important cytokines in sepsis. They are released from
monocytes/macrophages and stimulate NK cells and T-cells to produce IFN-γ and various
cytokines. They also promote the development of cell-mediated immunity. Upon interaction with
APCs, the naïve T-helper (Th) cells proliferate and differentiate into Th1 cells that produce large
amounts of TNF-α, IL-2, IL-12, IFN-γ, and leukotrienes. IL-17A is a pro-inflammatory cytokine
that is mainly produced by Th17 cells. IL-17A is involved in mediating pro-inflammatory
responses by triggering the production of other cytokines (IL-1β, IL-6, and TNF-α) and provokes
cross-talk between lymphocytes and phagocytes.

On the other hand, IL-4-driven Th2 cells give rise to immunomodulatory cytokines, such
as IL-4, IL-5, IL-9, IL-10, and IL-13. A majority of the Th1 cytokines are produced at the early
phase of sepsis, followed by excessive production of Th2 cytokines. A shift in the balance from
Th1 to Th2 cytokines can cause immune suppression, which is evident at the late stage of sepsis,
rendering the host susceptibility to nosocomial infection and tertiary pneumonia.

1.7.5: Immunosuppression & Apoptosis
It is now appreciated that anti-inflammatory immunologic events develop concurrently
or subsequently during the time-course of sepsis. Although anti-inflammatory responses likely

32

are essential to the restoration of immune homeostasis following an inflammatory stimulus such
as infection, this anti-inflammatory state can result in immunosuppression and subsequent death
because of the inability to fight secondary infections in the post-septic period. Production of antiinflammatory cytokines and impairment of immune cell function are the two major events
leading to the immunosuppressive phase of sepsis. Anti-inflammatory cytokines including IL-4,
IL-10, IL-13, IL-1 receptor antagonist (IL-1ra), and transforming growth factor-beta (TGF-β) are
detected during the immunosuppressive phase and have the ability to inhibit synthesis of IL-1,
TNF-α and other pro-inflammatory cytokines. Also, IL-10 exerts a wide range of antiinflammatory effects including inhibition of cell surface expression of class II major
histocompatibility complex (MHC) expression by monocytes, down regulation of TNF receptors,
and inhibition of macrophage bactericidal activity. Septic milieu impairs the ability of the DCs to
initiate a TH-1 cytokine response and converts to TH-2 type cytokine response110 which is
observed during immunosuppression. It has been shown that during later stages of sepsis,
neutrophilic adherence and migration is impaired111,112. Furthermore, expression of neutrophil
activation markers is significantly lower in patients who do not survive sepsis113.

During sepsis, apoptosis is extensively observed in lymphoid organs (spleen and thymus)
and lymphoid tissues of large intestine, both in humans114 and in animal models115. Apoptosis of
lymphoid cells results in immunosuppression due to decrease in the number of effector cells and
decreased co-stimulatory potential of macrophages that phagocytose apoptotic cells. In addition
to lymphoid tissue, apoptosis is observed in gut epithelium, lung endothelium, kidney tubular
cells and skeletal muscles115. Apoptosis of non- lymphoid cells contributes to multi-organ
dysfunction, leading to fatality. Strategies to inhibit apoptosis, including bcl-2 overexpression

33

and caspase inhibition, have shown encouraging results in animal models116 and are promising
targets for future therapeutics.

1.7.6: Coagulation and Endothelial response
Pathogens, besides activating the innate immune cells can also trigger the coagulation
cascade. During sepsis, tissue factor (TF) expression is up regulated in monocytes and tissue
macrophages, resulting in the activation of the extrinsic coagulatory pathway. Fibrin formed
during this process immobilizes pathogens on the surface of the immune cells, facilitating their
phagocytosis and subsequent killing. Coagulation initiated through the extrinsic pathway is
amplified through the intrinsic pathway by mechanisms that involve cross-talk and feedback.
Nevertheless, in immunocompromised hosts or when the bacterial growth exceeds bacterial
killing, the coagulation is dysregulated and induces host tissue damage by disseminated
intravascular coagulopathy (DIC). The activated vascular endothelium is also capable of
modulating systemic inflammation and coagulation, thereby inducing DIC117. The endothelium
can undergo apoptosis and acquire a more pro-coagulant phenotype118. Apoptosis induced loss of
membrane integrity of endothelium is believed to induce organ dysfunction and global tissue
hypoxia119. Moreover, recent studies suggests that vascular endothelium mainly acts as a target
organ rather than a mediator of systemic inflammation and endothelial NFκB activation
contributes minimally to the systemic inflammation, but plays a pivotal role in contributing to
multiple organ damage120,121.

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1.7.7: Cardiac dysfunction
Cardiac dysfunction significantly contributes to mortality in septic patients. Early sepsis
and septic shock are characterized by circulatory abnormalities that usually relate to intravascular
volume depletion and vasodilation. Consequent underfilling of the heart leads to a reduced
cardiac output. Development of cardiac dysfunction is multifactorial and involves multiple
interrelated pathways, including circulating inflammatory mediators122, NO production123,
impaired mitochondrial function124,125, complement activation, and apoptosis126. Cellular hypoxia
mediated by systemic hypotension caused by cardiac dysfunction is one of the major reason for
cell death and eventual multiple organ failure (MOF)127.

1.7.8: CLP as a model for polymicrobial sepsis
Murine model of Cecal Ligation and Puncture (CLP) is a clinically relevant model that
emulates human sepsis induced as a result of perforation in the intestine (septic peritonitis, postoperative intestinal wound dehiscence, ruptured appendix etc.,). It closely mimics the clinical
parameters associated with sepsis including inflammatory response, cardiovascular dysfunction,
bacteremia, immunosuppression, multiple organ dysfunction and mortality128. The procedure
involves ligating and puncturing the cecum below the ileocecal valve in anaesthetized mice. The
patency of the puncture is maintained by extruding a small amount of cecal content through the
puncture. The severity of sepsis can be altered depending on the position of ligation, size of
needle used for puncture and the number of punctures11. The cecum is then returned to the
peritoneal cavity and is closed using a silk suture. This procedure introduces a mixture of live
and dead enteric bacteria, dead epithelial cells and digested food material into the sterile
peritoneal cavity leading to septic peritonitis. Control surgeries (Sham) involve exteriorizing and

35

manipulation of the cecum, without ligation and puncture. To understand the role of GRK5 in the
onset and progression of sepsis, we used CLP to induce poly microbial sepsis.

1.7.9: Per-oral inoculation of E. coli- as a model for pneumonia
Per-oral inoculation of bacteria into trachea is one of the many mouse models129 used to
mimic human pneumonia. Per oral tracheal instillations may be the preferred methodology, over
inhalation or trans tracheal protocols. The former method primarily targets the upper respiratory
tract and possibly exposes the investigator to potentially hazardous substances. On the other
hand, trans tracheal involves surgical manipulation and therefore increase surgical techniqueinduced modulation of inflammatory responses. In the per oral technique, the anaesthetized
mouse is placed on an angled wooden platform hanging by its incisors on the wire with the
mouth open. With the help of a laryngoscope or a light source, and a forceps, the tongue is pulled
gently out to visualize the tracheal opening. With the other hand, the inoculum is injected into
the trachea slowly. After injection the mouse is observed for signs of proper aspiration of the
inoculum (gasping and alteration in breathing pattern). The mouse is held upright for few
minutes and then carefully placed in a heating pad until anesthetic recovery. This method models
oropharyngeal aspiration and deliver a bolus of organisms to a localized area of lung
parenchyma, resulting in a higher ratio of infecting organisms to phagocytes and other local
defenses at the site of infection. Conversely, per-oral intubation is some associated with
contamination of the lower respiratory tract with oropharyngeal flora. Hence, this technique must
be perfected before using for experiments.

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To characterize the role of GRK5 in the onset and progression of pneumonia, we used
this method to induce pneumonia. Even though we cannot compare this model of pneumonia
directly with CLP, we can uncover certain additional roles of GRK5 in bacterial infections; since
the inoculum size and composition is characterized, the miniscule alteration in the
immunological events can be identified with ease, which is otherwise masked.

1.8: Significance
Treatment of sepsis involves an early-goal directed therapy that revolves around using
antibiotics and supportive care. Additionally, specific therapies have been developed during the
years to tackle derangements in coagulation and metabolism and hyper-inflammation observed in
sepsis using activated protein C, insulin therapy and low dose corticosteroids respectively.
Despite advances in the treatment and supportive care, sepsis still remains a persistent clinical
problem with reported mortality as high as 30-50%130. Sepsis is the number one cause of death
observed among ICU patients. In 2008, an estimated $14.6 billion was spent on hospitalization
and treatment of septic patients and the treatment cost is increasing at an average of 11.9%
annually131. Moreover, hospitalization rate has doubled from 2001 to 2008, implying that
incidence of sepsis and associated conditions are still on the rise. Current research on sepsis
focuses on understanding the pathophysiology of molecular events happening during sepsis and
eventually to target those events to improve the outcome of sepsis. According to recent report of
NIH GLUE grant132, GRK5 expression in immune cells correlates with the clinical attributes of
sepsis in human patients. Also, another independent study reported that GRK5 expression is
increased in blood neutrophils from septic patients47. Moreover, in animal models, GRK5 is an
important regulator of inflammation during endotoxic shock4. So, all these studies suggest that

37

GRK5 might play a crucial role in the development and progression of sepsis. Thus,
understanding GRK5's role in the pathogenesis of sepsis/pneumonia and deciphering the
involved mechanisms would provide clues for development of therapeutics for effective sepsis
management.

38

HYPOTHESIS AND SPECIFIC AIMS

The major aim of my thesis project is to understand the overall patho-physiological role
of GRK5 in acute inflammatory diseases mainly, sepsis. Based on the current knowledge on
GRK5, I propose the following overall hypothesis:

Hypothesis: GRK5 is an important regulator of NFB signaling pathway in vivo, and therefore
play an essential and critical role in the pathogenesis of sepsis.

Each of the chapter in this dissertation is dedicated to address the following specific aims:
Specific Aim 1: To determine the role of GRK5 deficiency in MyD88 dependent and TRIF
dependent cytokine production in vivo.

Specific Aim 2: To determine the role of GRK5 in a clinically relevant model of intraabdominal sepsis induced by cecal ligation puncture technique.

Specific Aim 3: To determine the role of GRK5 in acute lung injury/pneumonia induced by per
oral intra tracheal E. coli injection.

39

CHAPTER TWO

OVERLAPPING AND DISTINCT ROLES OF GRK5 IN TLR2-, AND TLR3-INDUCED
INFLAMMATORY RESPONSE IN VIVO
This chapter represents a manuscript that was published in Cellular Immunology.
2012;272(2):107-11.

Authors who contributed towards this study were: Nandakumar Packiriswamy, Sitaram
Parvataneni and Narayanan Parameswaran

40

2.1: Abstract
We recently demonstrated that G-protein coupled receptor kinase-5 (GRK-5) is an
important regulator of Toll-like-receptor-4 (TLR4) signaling in vivo and in vitro. It is not known
if role of GRK5 is selective for TLR4 or whether its role can be extended to other TLRs. In
addition, it is unclear if GRK5 mediates its effects via MyD88- or TRIF-dependent TLR
pathways. To address this, we tested the role of GRK5 in TLR2- (MyD88-dependent) and TLR3(TRIF-dependent) induced inflammatory response in vivo using GRK5 knockout and the
corresponding wild type mice. To activate TLR2, we injected Pam3CSK4 and to activate TLR3
we administered Poly(I:C). Both ligands induced a broad range of inflammatory cytokines in the
wild type mice. However, in the Pam3CSK4 treatment group plasma levels of IL-5, IL-12p40,
MCP-1, and G-CSF were significantly inhibited in the knockout mice. Whereas, in the Poly(I:C)
treatment group IL12p40, TNF, and G-CSF were inhibited in the GRK5 knockout mice
compared to the wild types. Together, these studies suggest that plasma IL-12p40 and G-CSF
levels are regulated by GRK5 at a level that is common to both MyD88 and TRIF. However, our
results also indicate that GRK5 regulation of IL-5, and MCP-1 are MyD88-dependent, while
regulation of TNF is TRIF-dependent. Together these results provide in vivo evidence that
GRK5 has overlapping as well as selective roles in TLR signaling pathways.

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2.2: Introduction
Toll-like receptors (TLRs) are a well-characterized family of receptors involved in the
recognition of pathogen-associated molecular patterns (PAMPs) derived from microbes. Eleven
TLRs in the human and 13 in the mice have been identified133. Activation of TLRs by PAMPs
leads to transcriptional regulation of many genes and the pattern of gene regulation and the
outcome is crucially dependent on the TLR that is activated and the specific cell type. In general,
TLR signaling can be broadly classified into MyD88-dependent and TRIF-dependent
pathways133. MyD88 signaling has generally been linked to NFB and MAPK signaling,
whereas TRIF-dependent signaling has been linked to activation of interferon response factors134.
Studies have also shown crosstalk and overlap between these two pathways depending on the
cell type involved. All TLRs except TLR3 utilize MyD88-dependent signaling, whereas TLR4
and TLR3 utilize TRIF-dependent signaling. TLR4 is unique in that it utilizes both MyD88- and
TRIF-dependent pathways135. The different TLRs are activated by different components of
various microbes. For example, TLR3 is activated by dsRNA from viruses, whereas, TLR2 is
activated by lipoproteins from bacteria.

TLRs are involved in a number of inflammatory diseases including non-infectious
diseases. Therefore, understanding the mechanistic basis of TLR signaling is a rapidly expanding
field. In this regard, we recently showed that TLR4 signaling is regulated by G-protein coupled
receptor kinases (GRKs) in vitro in macrophages and in vivo in mice40,41,136,137. GRKs are a
family of serine/threonine kinases discovered for their role in G-protein coupled receptor
desensitization. Of the seven GRKs, recent studies have suggested that GRK5 has a unique role
in regulating TLR4-stimulated NFB signaling in different cell types and in vivo in mice136. In

42

addition, studies have also shown important roles for GRK5 in signaling from microbes in
Drosophila and Zebra Fish models42. Even though initial studies suggest critical role for GRK5
in TLR4 signaling, it is not known whether the role of GRK5 is specific for TLR4 or whether it
regulates other TLRs. In addition, since TLR4 activates both MyD88- and TRIF-dependent
pathways, an important question still remains as to whether GRK5 is specific for one of these
pathways. To address these issues we utilized our previously described GRK5 knockout mice as
model and administered a TLR1/2 ligand (Pam3CSK4) that activates only the MyD88 pathway,
and a TLR3 ligand (Poly(I:C)) that activates only the TRIF pathway. Surprisingly, our results
suggest that GRK5 has unique and overlapping roles in inflammation induced by TLR2, and
TLR3.

2.3: Materials and Method
2.3.1: Animals and protocol
All animal procedures were approved by the Michigan State University Institutional
Animal Care and Use Committee and conformed to NIH guidelines. Generation and
characterization of GRK5 null mice has been described before136. Male mice, 6-8 weeks of age
were used for the experiments with appropriate littermate controls. Animals were housed 4–5
mice per cage at 22–24° C in rooms with 50% humidity and a 12-h light–dark cycle. All animals
were given mouse chow and water ad libitum. Wild type GRK5+/+ and Knockout GRK5-/- were
segregated into two different groups for intra-peritoneal injection of Pam3CSK4 (100 µg/mouse)
and Poly I:C (200 µg/mouse) ligands. Both ligands were obtained from Invivogen ( San Diego,
CA). The animals were euthanized and samples collected 9 hours post-injection.

43

2.3.2: Cytokine analysis
A mouse 23-plex multiplex based assay (from Biorad, Hercules, CA) was used to
determine the cytokine/chemokine concentrations according to manufacturer’s instructions via
Luminex 100 technology as described previously138. Plasma from mice treated with Pam3CSK4
and Poly(I:C) were used to assess the cytokine/chemokine levels.

2.3.3: Statistical methods
All values are represented as mean ± SEM. Data were analyzed and statistics performed
using GRAPHPAD PRISM software (La Jolla, California). The Student’s t-test was used to
compare mean values between two experimental groups. P value of less than 0.05 was
considered significant.

2.4: Results and Discussion
Although originally discovered as a regulator of G-protein coupled receptor
desensitization, GRK5 has now been shown to broadly regulate other aspects of cell signaling,
independent of GPCRs40,46,136,137. In this regard, recently we showed that GRK5 is an important
regulator of NFB signaling in mice and this was further confirmed in Drosophila, and Zebra
fish, suggesting an evolutionarily conserved role for GRK5 in NFB signaling. Using cells from
mice that are deficient in GRK5, we showed that lipopolysaccharide (TLR4 ligand)-induced
activation of NFB signaling and the consequent cytokine production are critically regulated by
GRK5. In addition, in the endotoxemia model, several cytokines/chemokines were significantly
attenuated in the GRK5 knockout mice136.

44

Figure 2.1: Plasma IL-12p40 and G-CSF are regulated by GRK5 in both Pam3CSK4 and
Poly (I:C) treatment groups: WT and GRK5 knockout mice were injected with Pam3CSK4
and Poly(I:C) and sacrificed 9 hours after administration. Plasma samples were collected and
cytokines measured as described in the methods. n=6 per group. *p<0.05

45

Figure 2.2: Plasma IL-5, MCP-1, MIP-1β and IL-6 production are regulated by GRK5 only
in the Pam3CSK4 treatment group: Mice were injected with respective ligands and samples
collected and analyzed as described in Fig 2.1. n=6 per group. *p<0.05.

46

To begin to understand the specificity of GRK5 in various TLR signaling pathways, we
used ligands for TLR2, and TLR3 to separately activate MyD88 and TRIF respectively. We
administered these ligands in wild type and GRK5 knockout mice. Nine hours after
administration, we examined the levels of various cytokine/chemokines using a 23-plex ELISA
136,138

. Pam3CSK4 activated all 23 cytokines/chemokines, whereas PolyI:C activated only 21 of

the 23 cytokines (data not shown). In the Pam3CSK4 injected group, IL12p40, IL-5, G-CSF and
MCP1 were significantly inhibited in the GRK5 knockout mice (Fig 2.1 and 2.2). IL-6 and
MIP1 levels showed similar results but were not statistically significant (Fig 5). Interestingly,
all the six cytokines/chemokines (IL12p40, IL5, IL6, GCSF, MCP1, MIP1) overlapped with
our previously reported findings using LPS136. Interestingly, when Poly(I:C) was administered,
only IL-12p40, TNF, and G-CSF were significantly inhibited in the GRK5 knockout mice
compared to the wild types (Fig 2.2 and 2.3). Poly (I:C)-induced RANTES was also inhibited in
the GRK5 knockout, but did not reach statistical significance. Unlike Pam3CSK4, IL1-, and
Eotaxin levels were not at detectable levels in the plasma after Poly(I:C) administration. There
was no significant difference in the levels of IL1, IL-13, IFN, IL1, IL-2, IL-3, IL-4, IL-9,
IL12p70 and GM-CSF when compared between the wild type and the GRK5 knockout mice in
either of the ligand groups (data not shown).

These results clearly indicate several outcomes as follows: 1. GRK5 regulation of IL12p40 and G-CSF production occurs at a level that is common to MyD88 and TRIF pathways. 2.
Regulation of IL-5, MCP-1 and possibly IL-6 and MIP1 production by GRK5 occurs at a level
that is specific to MyD88-dependent pathway. It is important to note that these cytokines/

47

Figure 2.3: Plasma TNF and RANTES are regulated by GRK5 only in the Poly (I:C)
treatment group: Mice were injected with ligands and samples collected and analyzed as
described before. n=6 per group. *p<0.05.

48

Figure 2.4: Cytokines/chemokines differentially regulated by GRK5 in response to
activation of TLR2 and TLR3 signaling pathway.

49

chemokines were also inhibited in the LPS-induced response, in our previous study. 3.
Regulation of TNF and possibly RANTES production by GRK5 occurs at a level that is likely
to be specific for the TRIF-dependent pathway. 4. In previous study, we reported that LPSinduced production of IFNγ, MCP-1, TNFα, IL-1β, IL-12(P40), IL-12(P70), IL-5, IL-17, IL-10,
EOTAXIN, IL-2, RANTES, IL-4, IL-9, IL-13 and RANTES are positively regulated by GRK5.
Of these IL-17, IL-10, Eotaxin are not inhibited in either TLR2 or TLR3 ligand groups
suggesting that regulation of these factors by GRK5 is likely to be unique to TLR4.

Previous studies have shown that GRK5 regulates NFB signaling and consistent with
that many cytokines/chemokines induced by LPS were inhibited in the GRK5 knockout mice.
Similarly, in this study many cytokines/chemokines induced by Pam3CSK4- and Poly(I:C)-were
inhibited in the knockout mice. While this regulation is likely due to GRK5’s role in NFB
pathway, it is possible that other signaling pathways are also being regulated by GRK5, given its
roles in many cellular processes40-42,47,137,139-142. The observation that GRK5 appears to regulate
various cytokines/chemokines at levels that are common to MyD88 and TRIF, as well as unique
to MyD88 or TRIF pathways (Fig 2.4) suggests that GRK5 regulates these inflammatory factors
via multiple pathways. Identifying these novel roles of GRK5 will be the focus of future studies.

50

CHAPTER THREE

GRK5 MEDIATES INFLAMMATION BUT DOES NOT REGULATE CELLULAR
INFILTRATION OR BACTERIAL LOAD IN POLYMICROBIAL SEPSIS MODEL IN
MICE

This chapter represents a manuscript that was published in Journal of Innate Immunity
2013;5(4):401-13

Authors who contributed towards this study were: Nandakumar Packiriswamy, Taehyung
Lee, Pongali B. Raghavendra, Haritha Durairaj, Hongbing Wang, and Narayanan Parameswaran

51

3.1: Abstract
NFκB-dependent signaling is an important modulator of inflammation in several diseases
including sepsis. G-protein coupled receptor kinase-5 (GRK5) is an evolutionarily conserved
regulator of NFB pathway. We hypothesized that GRK5 via NFB regulation plays an
important role in the pathogenesis of sepsis. To test this we utilized a clinically relevant
polymicrobial sepsis model in mice that were deficient in GRK5. We subjected wild type (WT)
and GRK5 knockout (KO) mice to cecal-ligation and puncture (CLP)-induced polymicrobial
sepsis and assessed the various events in sepsis pathogenesis. CLP induced a significant
inflammatory response in the WT and this was markedly attenuated in the KO mice. To
determine the signaling mechanisms and the role of NFκB activation in sepsis-induced
inflammation, we assessed the levels of IB phosphorylation and expression of NFBdependent genes in the liver in the two genotypes. Both IB phosphorylation and gene
expression were significantly inhibited in the GRK5 KO compared to the WT mice. Interestingly
however, GRK5 did not modulate either immune cell infiltration (to the primary site of infection)
or local/systemic bacterial load subsequent to sepsis induction. In contrast GRK5 deficiency
significantly inhibited sepsis-induced plasma corticosterone levels and the consequent thymocyte
apoptosis in vivo. Associated with these outcomes, CLP-induced mortality was significantly
prevented in the GRK5 KO mice in the presence of antibiotics. Together, our studies
demonstrate GRK5 as an important regulator of inflammation and thymic apoptosis in
polymicrobial sepsis and implicate GRK5 as a potential molecular target in sepsis.

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3.2: Introduction
G-protein coupled receptor kinases (GRKs) are serine/threonine kinases well known for
their role in phosphorylation of G-protein coupled receptors (GPCRs)143. Functionally GRKs
have been linked to a number of cell signaling processes, not only related to their role in GPCR
phosphorylation, but also in their ability to phosphorylate or scaffold a number of intracellular
signaling proteins17. GRKs are functionally grouped into three classes: GRK1-like (GRK1 and
GRK7, otherwise known as rhodopsin kinases), GRK2-like [GRK2 and GRK3, otherwise known
as ARK1 and 2 (-adrenergic receptor kinases 1 and 2)] and GRK4-like (GRK4, GRK5 and
GRK6). Even though there is some specificity in terms of their tissue distribution, GRKs
especially GRK2 and GRK5 are ubiquitously expressed in many cell types including immune
cells17.

Of the seven members of the GRK family, GRK5 was first identified as a kinase that
phosphorylates 2-adrenergic receptor (2AR), m2 muscarinic cholinergic receptor, and
rhodopsin144. Using GRK5 deficient mice, Gainetdinov et al29 further identified a critical role for
GRK5 in muscarinic receptor signaling in vivo. Subsequently, however, several studies have
demonstrated a broad role for this kinase in cell signaling. For example, GRK5 has been shown
to phosphorylate and/or interact with non-receptor substrates including arrestin-2140, F-actin67,
HDAC518, Hip1, IB43, p10540, Lrp6145, nucleophosmin146, and p5371. Based on its role in cell
signaling, GRK5 has been proposed to be a critical kinase in the pathogenesis of several diseases
including endotoxemia4, cancer147, Alzheimer’s148 and atherosclerosis57. In addition, GRK5
levels are modulated in a number of diseases including sepsis, heart failure, obesity, cystic
fibrosis, cancer and mental disorders17. We recently demonstrated that the GRK5 knockout mice

53

have attenuated ability to produce cytokines in vivo, in response to lipopolysaccharide (a Tolllike receptor-4, TLR4 ligand)4. We further showed GRK5 to be an important regulator of
signaling from multiple Toll-like receptor ligands including TLR2 and TLR3 in vivo77.
Interestingly, a recent study also demonstrated that GRK5 is a critical mediator of inflammation
in Drosophila and Zebra fish models42. However, role of GRK5 in the pathogenesis of a
clinically relevant model of polymicrobial sepsis is not known.

Sepsis is the leading cause of death among intensive care patients149. Dysregulated
inflammatory response is a prominent modulator of sepsis progression, causing coagulation
derangements, apoptosis of lymphoid and non-lymphoid tissues and organ dysfunction150.
Despite the improvements in resuscitation and antibiotic supportive care, the high incidence and
fatality in sepsis underscores the need for better understanding of the pathophysiology of sepsis
and to identify new molecular therapeutic targets. Using a clinically relevant polymicrobial
sepsis model151, we demonstrate here that GRK5 is an important modulator of sepsis
progression, inflammation, thymocyte apoptosis and mortality. We further demonstrate that
GRK5 is an important regulator of sepsis-induced NFB activation in the liver. Together, our
studies implicate GRK5 as an important molecular target in the pathogenesis of polymicrobial
sepsis.

3.3: Materials and Methods
3.3.1: Materials
Protease inhibitor cocktail tablets were from Roche Applied Science (Indianapolis, IN);
pIκBα, pERK1/2, pP38 and pJNK and tubulin antibodies were from Cell Signaling Technology,

54

Inc. (Danvers, MA) and Sigma (St. Louis, MO) respectively. Ultra-pure Escherichia coli
(0111:B4) LPS was from Invivogen (San Diego, CA) and dexamethasone was from Sigma (St.
Louis, MO).

3.3.2: Mice
GRK5 KO mice were obtained from Jackson labs and have been previously described4.
Animals used for experiments were 8-12 week old males. Animals were housed 4-5 mice per
cage at 22-24°C with 50% humidity and a 12 hour light-dark cycle. All animal procedures were
approved by Michigan State University Animal care and Use committee.

3.3.3: Sepsis model
Polymicrobial intra-abdominal sepsis was induced by cecal ligation puncture
technique152. Briefly, mice were anaesthetized by administering ketamine (80mg/kg) and
xylazine (5mg/kg) intraperitoneally. Cecum was exteriorized, ligated and punctured twice with
20 G needle. Sham surgeries were carried out to serve as control wherein the exteriorized cecum
was neither ligated nor punctured. All animals were administered with 1ml of warm saline postsurgery subcutaneously. In one set of survival experiments, antibiotics (ceftriaxone 25mg/kg and
metronidazole 15mg/kg) were administered (intraperitoneally) 1-hour post-sepsis and every 24
hours for 5 days.

3.3.4: Peritoneal Lavage fluid and blood collection
Peritoneal exudate fluid was collected at different time points post sepsis as described
before78. Briefly, peritoneal cavity was lavaged with 7 ml of RPMI media with 10% FBS and

55

peritoneal fluid collected, centrifuged to separate the cells and the supernatants stored at -80°C
until further analysis. Blood was collected by cardiac puncture and plasma was separated (by
centrifugation) and stored at -80°C until further analysis.

3.3.5: Cytokine/chemokine measurements
Cytokines and chemokines were measured from peritoneal exudate and plasma using
ELISA kits from eBiosciences, Inc. as described before153.

3.3.6: Bacterial counts
Bacterial load was determined in blood, and peritoneal fluid at different time points as
described before78. Briefly, blood, and peritoneal fluid were serially diluted and plated on
Trypticase™ Soy Agar with 5% Sheep Blood (BD Biosciences) and incubated at 37°C for 48
hours. Colony forming units were counted to determine bacterial load and expressed as colonyforming units per milliliter.

3.3.7: Determination of thymic cell numbers and apoptosis
Thymi were collected from septic and sham operated mice 20- and 36-hours post-sepsis
and single cell suspension prepared as described before154. For determining cell number changes,
cells were counted using hemocytometer. Cells were also labeled with Annexin V and propidium
iodide (following manufacturer’s instructions (eBiosciences, Inc)) to determine the apoptotic
cells by flow cytometry (LSRII, BD biosciences) as described before155. In addition, cells were
labeled with anti-CD4-PE-Cy7 and anti-CD8-PE for determining CD4+ and CD8+ cells in the

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thymus and data were acquired using LSRII (BD Biosciences) and analyzed using Flowjo
software (Tree StarInc., Ashland, Oregon).

3.3.8: Caspase activity assays
Thymocytes (obtained as described above) were lysed in buffer (50 mM HEPES, 0.1%
CHAPS, 1 mM DTT, 0.1 mM EDTA, 0.4% Triton-X100, pH 7.4) at 4°C for 15 min. The cell
lysate was collected and the protein content determined (BIO-RAD). 10 μg of the cell lysate was
incubated with the fluorescent substrates (Ac-DEVD-AFC, Z-IETD-AFC and Ac-LEHD-AFC)
to determine caspase-3, -8 and -9 activities, at 100 μM in the assay buffer (50 mM HEPES, 1%
Sucrose, 0.1% CHAPS, 10 mM DTT, pH 7.4) as described

156

. The fluorescence of the cleaved

substrates was determined spectro-fluorometrically (excitation of 400 nm and emission of
505 nm) in Tecan Spectra FluorPlus fluorescence plate reader. Data are presented as picogram of
cleaved AFC per mg protein per min calculated from a standard curve plot with free AFC.

3.3.9: In vitro stimulation of septic peritoneal cells
Peritoneal cells collected from wild type (WT) and GRK5 knockout (KO) mice 36 hourspost sepsis were washed with PBS and plated in 12-well plate at 1 million cells/well. Cells were
then stimulated (or not) with LPS (0.5 µg/ml) for 12 hours and supernatants collected and
assayed for the indicated cytokines by ELISA.

3.3.10: Restraint stress
Eight to 10 week old GRK5 WT and KO mice were subjected to stress with physical
restraint as previously described157. Briefly, mice were placed in a 50 ml centrifuge tube with

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multiple openings for ventilation and held horizontally for 30 minutes. After the stipulated time,
blood was collected for corticosterone measurement.

3.3.11: Corticosterone measurements
Plasma corticosterone levels were measured using a corticosterone EIA kit from Cayman
Chemical (Ann Arbor, MI) as per the manufacturer’s instructions.

3.3.12: RNA extraction and Real-time Q-PCR
Liver and lung samples from septic and sham operated mice were collected 12 hours
post-CLP, and total RNA was extracted using Qiagen’s RNeasy Mini kit. Reverse transcription
was carried out with 1 µg of RNA with promega cDNA synthesis kit. Real-time Q-PCR was
performed as described before for the expression of IкBα, IL-6, IL-1β, and HPRT43. Primers
were obtained from IDT DNA Technologies. Following primers were used: IB-Forward:
TGG CCA GTG TAG CAG TCT TG; Reverse: GAC ACG TGT GGC CAT TGT AG; IL-6Forward: ACA AGT CGG AGG CTT AAT TAC ACA T; Reverse: TTG CCA TTG CAC AAC
TCT TTT C; IL-1-Forward: TCG CTC AGG GTC ACA AGA AA; Reverse: CAT CAG AGG
CAA GGA GGA AAA C; HPRT-Forward: AAG CCT AAG ATG AGC GCA AG; Reverse:
TTA CTA GGC AGA TGG CCA CA. Real-time Q–PCR was performed using ABI fast 7500
(Applied Biosystems) and all the genes were normalized to HPRT.

3.3.13: Western blot analysis
Cytoplasmic extracts from frozen liver tissue samples were prepared by homogenizing
the tissue in lysis buffer (1M HEPES, 2M KCl, 0.5M EDTA, 0.1M EGTA along with protease

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and phosphatase inhibitors). The protein concentrations in the extracts were determined and
equivalent amounts of protein were loaded onto the gels for western blot analysis.
Immunoblotting was carried out for p-IкBα, p-ERK, p-JNK, p-p38 and tubulin as described
before4. The bands were quantified using image-J (for chemiluminescence) or Licor's Odyssey
program (for fluorescence).

3.3.14: Statistical analysis
All data are presented as the meanSEM. Two group comparisons were performed using
Student’s t-test and comparisons of more than two groups were done by ANOVA with postBonferroni test. Survival studies were analyzed by log rank test (Mantel-Cox) 158-160 as well as by
factorial analysis. All statistical analyses (except factorial analysis) were performed using
GraphPad Prism Software (San Diego, CA) and p<0.05 were considered statistically significant.
Factorial analysis for survival [Two genotypes (wild type and knockout) and two treatments
(without and with antibiotics)] was performed in consultation with MSU’s Center for Statistical
Training and Consulting and using the SPSS software program.

3.4: Results
In previous studies, we demonstrated that the GRK5 deficient mice have an attenuated
inflammatory response after in vivo stimulation with TLR ligands4,77. Given that the progression
of polymicrobial sepsis is in part dependent on stimulation of multiple TLRs, we examined the
role of GRK5 in a clinically relevant model of polymicrobial sepsis. For this, we subjected wild
type (WT) and GRK5 knockout (KO) mice to cecal ligation and puncture and determined the

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various pathogenic events including inflammatory response, immune cell infiltration, thymic
apoptosis, bacterial load and mortality.

3.4.1: GRK5 mediates sepsis-induced cytokine production
Cytokine and chemokine levels were examined in the peritoneal fluid and plasma from
the different groups of mice (sham and CLP in the WT and KO groups) at 12 hours after surgery.
As shown in Fig 3.1a, IL-6, IL-10, TNF and MCP-1 levels in the peritoneal fluid were
significantly decreased in KO septic compared to WT septic mice. Peritoneal IL-12/23 (total
p40) was also inhibited in KO mice but did not reach statistical significance. Similar to the
peritoneal fluid, plasma levels of IL-6 and IL-10 were significantly decreased in KO compared to
the WT septic mice (Fig 3.1b). Plasma levels of TNFα, IL-12/23 and MCP-1 did not
significantly differ between septic groups. Note that neither of the sham groups showed any
detectable levels or showed very low levels of cytokines/chemokines at the time points tested
(data not shown). Together these results suggest that GRK5 mediates sepsis-induced local and
systemic inflammation.

3.4.2: GRK5 mediates NFкB signaling in polymicrobial sepsis
Previous studies have shown that GRK5 is an important regulator of NFB signaling and
inflammatory gene expression in mouse, zebra fish and Drosophila models4,42. Our results in the
CLP model of polymicrobial sepsis suggest that GRK5 deficiency attenuates inflammatory
cytokine production both in the peritoneal cavity and in the plasma. To examine if GRK5
deficiency leads to attenuated NFB signaling in this sepsis model, we determined phosphoIB levels and mRNA expression of NFB-dependent genes in the liver from the two

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genotypes of mice subjected to sham or CLP surgery. As shown in Fig 3.2a, CLP-induced
significant phosphorylation of IB in the liver of WT mice and this was significantly inhibited
in the GRK5 knockout. To determine the expression of NFB-dependent genes, we examined
the mRNA levels of IкBα, IL-6 and IL-1 in the liver 12 hours after CLP. Consistent with the
role of GRK5 in IB phosphorylation, mRNA expression of IB, IL-1 and IL-6 was
significantly inhibited in the GRK5 KO mice (Fig 3.2b). Interestingly, this phenomenon was also
observed in the lungs (Fig 3.2b lower panel). To rule out other signaling pathways, we also
examined pERK, pJNK and pP38 and found no effect of GRK5 deficiency on these pathways
(Fig 3.2c). Together, these results demonstrate a crucial role for GRK5 in NFB activation in
vivo and the consequent inflammatory response in this polymicrobial sepsis model.

3.4.3: GRK5 does not regulate chemotaxis to the local site of injury/infection
Studies have shown that following CLP, peritoneal cell infiltration plays a critical role in
the progression of sepsis161 and that modulation of immune cell infiltration can have therapeutic
consequences162. GRKs have been shown to be important regulators of chemokine receptor
signaling and chemotaxis47. Therefore, to determine if GRK5 regulates immune cell infiltration
into the site of injury/infection (peritoneum), we examined the number of cells in the peritoneal
cavity following CLP at different time points in the two genotypes of mice. As shown in Fig 3.3,
GRK5 deficiency did not affect immune cell infiltration into the peritoneal cavity at any of the
time points tested. In addition there was no difference in infiltration of specific immune cell

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Figure 3.1: GRK5 mediates inflammatory response in cecal ligation and puncture model of
polymicrobial sepsis: GRK5 wild type and knockout mice were subjected to CLP surgery and
peritoneal (A) and plasma (B) fluids collected 12 hours after surgery as described in the methods.
IL-6, IL-10, TNF, IL12/23 (total p40), and MCP-1 were measured in these two fluid
samplesusing ELISA kits from eBiosciences. (n=12 for WT and n=9 for KO for the 12 hour
timepoint).*P<0.05, **P<0.01 and ***P<0.001 compared to the corresponding septic wild type
groups.
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populations (Fig 3.3). Together these results suggest that GRK5 is an unlikely regulator of
chemotaxis in this model of sepsis.

3.4.4: Deficiency of GRK5 does not affect bacterial load following sepsis
In order to examine if GRK5 is able to modulate bacterial load after CLP, we plated
peritoneal lavage fluid and blood samples from sham and septic mice onto 5% sheep blood agar
plates and determined the colony forming units (CFU). Interestingly, bacterial load was not any
different between the wild type and the KO mice at any of the time points tested (Fig 3.4a and
3.4b). Taken together, our data so far suggests that even though GRK5 mediates NFB signaling
and inflammation in sepsis, neither chemotaxis nor bacterial load is significantly affected.

3.4.5: Regulation of thymocyte numbers by GRK5 in sepsis
It is now well established that sepsis-induced thymocyte apoptosis contributes to the
pathogenic events and the consequent mortality in septic animals163 and human patients164.
Importantly, inhibiting thymic apoptosis has been shown to be beneficial in preventing sepsisinduced mortality in experimental models165. Previous studies have suggested a role for GRK5 in
irradiation-induced thymocyte apoptosis via a p53-dependent pathway71. Furthermore, NFB
signaling in thymocytes has been shown to be an important regulator of thymocyte
apoptosis166,167. Together, based on these rationale, we hypothesized that GRK5 could be an
important regulator of sepsis-induced thymocyte apoptosis. To test this, we first assessed the
number of thymocytes from WT and KO mice subjected to sham or CLP surgery. As predicted
from previous studies, in the WT septic mice thymocyte numbers were significantly decreased
compared to sham operated mice (Fig 3.5a). Interestingly however, septic KO mice had

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Figure 3.2: GRK5 mediates IB phosphorylation and expression of NF-κB dependent
genes in liver and lung: (a) Cytosolic extracts of the liver tissue from GRK5 wild type and
knockout mice 12 hours post-sepsis were subjected to Western blotting for phospho-IB and
tubulin (for normalization) as described before4. Representative blot is shown in the left and
quantitation on the right. n=11-12 each for wild type and KO mice CLP and n=3 for shams;
*P<0.05. (b) Liver and lung tissue samples from GRK5 wild type and knockout mice subjected

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to sham or CLP surgery were collected 12 hours post-sepsis and analyzed for the expression of
NF-κB-dependent genes as described in methods. (n= 8 for CLP and 4 for sham per genotype
*P<0.05, **P<0.01 compared to the corresponding septic wild type group. (c) Liver tissue
extracts described in (a) above were subjected to immunoblotting for pERK, pJNK and pP38 as
described in the methods. Western blot is shown on the left and quantitation on the right. n=5
each for wild type and KO mice CLP.

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significantly higher numbers of thymocytes compared to the corresponding WT mice at both
time points. In addition to the total cells, the frequency of double positive CD4+CD8+
lymphocytes (loss of these lymphocytes has been linked to poor survival168) was also markedly
decreased in the wild type septic mice compared to the sham (Fig 3.5b). Importantly, decrease in
these double positive CD4+CD8+ cells was significantly attenuated in the GRK5 deficient mice
(Fig 3.5b).

3.4.6: Regulation of thymocyte apoptosis by GRK5 in vivo
To further confirm the difference in thymocyte numbers is as a result of altered apoptosis
between wild type and GRK5 KO mice, we performed two separate assays: Flow cytometry
analysis of thymocytes stained with Annexin V and PI; and Caspase activity assays. Consistent
with the thymocyte numbers, increased frequency of Annexin V+PI- cells (early apoptotic cells)
was observed in wild type septic mice and this was significantly inhibited in the GRK5 KO mice
(Fig 3.5c). Furthermore, CLP significantly induced caspase-3 activity in the wild type
thymocytes and this was again markedly reduced in the GRK5 KO mice (Fig 3.5c). Unlike
caspase-3, activities of caspase-8 and -9 were not significantly induced in sepsis and did not
differ between the groups (Fig 3.5c). Together, these results implicate GRK5 in the regulation of
caspase-3-mediated thymocyte apoptosis following sepsis.

3.4.7: Mechanism of GRK5-mediated thymic apoptosis
Previous studies have shown that sepsis-induced corticosteroids induce thymic apoptosis
in the CLP model169. To determine whether GRK5 directly modulates corticosteroid-induced
thymocyte apoptosis, we stimulated thymocytes from wild type and GRK5 KO mice ex vivo

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Figure 3.3: Role of GRK5 in peritoneal cell infiltration following cecal ligation and
puncture: GRK5 wild type and knockout mice were subjected to CLP surgery and peritoneal
cells collected by lavaging peritoneal cavity and cells counted (a) as described in the methods.
The different immune populations (b) were assessed by flow cytometry as described in the
methods (n=9 for 4 and 12 hour time points and n=5-6 for 20 and 36 hour time points, per
genotype).
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Figure 3.4: Role of GRK5 on bacterial load following cecal ligation and puncture: Blood
and peritoneal samples from GRK5 wild type and knockout mice subjected to sham or CLP
surgery were assessed for bacterial load as described in the methods. CFU counts from peritoneal
(a) and blood (b) for different time points are shown from mice subjected to CLP. (n= 11-14 per
genotype at 12 hour time point; n= 5 per genotype at 20 hour time point; n= 9-12 for 36 hour
time point). Note that sham mice did not show any bacterial colonies (data not shown).

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Figure 3.5: GRK5 mediates thymocyte apoptosis in polymicrobial sepsis: Thymi from GRK5
wild type and knockout mice subjected to sham or CLP surgery were collected 20 and 36 hours
after surgery as described in the methods. Thymocytes were analyzed for total number (a),
CD4+, CD8+ (b), Annexin V+ and PI- staining and for caspase activity (c) as described in the
methods. (n=5-6 per genotype for assessing total thymus cell number and Annexin V+ and PIstaining; n=3-4 per genotype for the flow cytometry experiments). *P<0.05, **P<0.01 and
***P<0.001 compared to the corresponding septic wild type groups.

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Figure 3.6: Sepsis-induced plasma corticosterone levels are diminished in GRK5 knockout
mice: (a) Plasma samples collected 12 and 20 hours post-surgery (sham and CLP) from GRK5
wild type and knockout mice, were assessed for corticosterone levels using Cayman Chemical
EIA kit. (n=8 per genotype for both 12 and 20 hour time point for CLP; n=2-3 for sham at 12
hour time point). (b) Corticosterone levels in plasma samples obtained from GRK5 wild type and
knockout mice before and after restraint stress (n=7 per genotype). *P<0.05 compared to the
corresponding wild type groups.

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with dexamethasone (100 nM) and assessed apoptosis using flow cytometry (AnnexinV/PI).
Dexamethasone treatment induced significant apoptosis in both the genotypes and surprisingly
apoptosis was equivalent between the genotypes (data not shown). Because these results ruled
out any direct effect of GRK5 in thymic apoptosis (at least as induced by corticosteroids), we
next examined whether the levels of corticosterone are different between the two genotypes
during sepsis progression. Interestingly, plasma corticosterone levels were significantly higher in
the wild type septic mice (compared to sham) and the levels were markedly attenuated in the
GRK5 KO septic mice (Fig 3.6). This was more evident at the later time point (20 h). To
determine whether the difference in the corticosterone levels between the two genotypes is
specific to sepsis, we induced stress with physical restraint in both genotypes and measured
plasma corticosterone levels. We found that 30 minutes of physical restraint induced significant
increase in plasma corticosterone, but the levels were similar between the two genotypes (Fig
3.6). Together these results suggest that sepsis-induced corticosterone levels are attenuated in
GRK5 deficient mice and this might lead to enhanced thymocyte survival during later stages of
sepsis.

3.4.7: GRK5 inhibits immunoresponsiveness of peritoneal cells in sepsis
Decrease in lymphocytes due to apoptosis is thought to be an important pathogenic event
in the development of immunosuppression observed during sepsis progression. Because, thymic
apoptosis is reduced in the GRK5 deficient mice; we hypothesized that the consequent
development of immune suppression may be attenuated in GRK5 deficient mice. To test this in

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Figure 3.7: GRK5 suppresses immuno-responsiveness of peritoneal cells in sepsis:
Peritoneal cells from GRK5 wild type and knockout mice subjected to sham or CLP surgery
were collected 36 hours post-sepsis, stimulated with LPS and assessed for cytokine production as
described in methods. (n= 7-8 per genotype). *P<0.05, **P<0.01 compared to the corresponding
septic wild type peritoneal cells stimulated with LPS. #P<0.05 compared to the corresponding
septic wild type peritoneal cells without LPS stimulation.

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vitro, we obtained peritoneal cells from the two genotypes of mice subjected to sepsis (36 hours
post-CLP) and assessed their immunocompetency in response to in vitro lipopolysaccharide
(LPS) stimulation. Supernatants from these experiments were assayed for IL-6, IL-10, TNFα,
and IL-12/23 using ELISA. As expected, unstimulated GRK5 KO cells (from septic mice)
produced lower cytokine levels compared to the wild type septic mice (Fig 3.7). However, upon
stimulation with LPS, GRK5 KO cells produced significantly enhanced pro-inflammatory
cytokines (IL-6, TNF and IL-12/23) compared to the wild type cells. This effect was restricted
only to the typical pro-inflammatory group and not to IL-10. Basal IL10 level was much higher
in the wild type cells from septic mice and LPS stimulation did not further enhance IL10.
Together, these results show that even though the initial inflammatory response in the KO cells is
attenuated, cells from these septic mice respond better than the wild type cells to in vitro LPS
stimulation. This suggests that deficiency of GRK5 possibly renders the mice more immuneresponsive at later stages of sepsis.

3.4.8: Role of GRK5 in sepsis-induced mortality
Dysregulated inflammatory response, poor bacterial clearance and excessive loss of
lymphocytes have all been linked to poor survival in sepsis150. Our results using GRK5 deficient
mice indicate that GRK5 mediates inflammatory response and thymocyte apoptosis in sepsis.
Therefore, we predicted that septic GRK5 deficient mice might exhibit altered survival profile
compared to the corresponding wild type mice. Contrary to our expectations, mortality following
sepsis was similar between the two genotypes (Fig 3.8A). We then reasoned that because
bacterial load was similar between the two genotypes, GRK5 deficient mice might exhibit better
survival in the presence of antibiotics. To test this, we subjected wild type and GRK5 deficient

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mice to CLP and administered antibiotics (Ceftriaxone: 25µg/g and Metronidazole: 15µg/g body
weight) intra-peritoneally 1 hour post- CLP and every 24 hours thereafter for 5 days170.
Compared to wild type mice that did not receive any antibiotics (~70% mortality), mortality in
wild type mice that received antibiotics decreased to ~40%. Interestingly however, GRK5
deficient mice receiving antibiotics had only ~10% mortality (compared to ~90% mortality in
GRK5 deficient mice not receiving antibiotics) (Fig 3.8B). Together, these results demonstrate
that in the presence of antibiotics, GRK5 deficiency protects mice from sepsis-induced mortality.

3.5: Discussion
Given the high rate of mortality in sepsis, understanding the pathophysiologic events and
molecular mechanisms that mediate mortality in sepsis can help in developing new therapies and
better treatment strategies. In the CLP model of polymicrobial sepsis GRK5 deficiency inhibited
several but not all aspects of sepsis progression. Importantly, GRK5 deficiency significantly
enhanced survival only in the presence of antibiotics. Even though GRK5 deficiency attenuated
inflammatory response both at the systemic and organ levels, as well as significantly inhibited
thymocyte apoptosis, these changes were not sufficient to enhance survival. It is possible that
both the severity of sepsis and bacterial dissemination were too high, in spite of the favorable
effects on inflammation and thymocyte apoptosis in GRK5 deficiency. This is in part supported
by the observation that bacterial load per se, was not different between the two genotypes of
mice at early or later time points of sepsis. Thus it is possible that at this level of severity, both
antibiotics (to clear bacterial infection) and GRK5 deficiency yield better outcome. A similar
phenotype was reported in MyD88 knockout mice103 wherein deficiency of MyD88 led to a
diminished inflammatory response and attenuated lymphocyte apoptosis without any effect

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Figure 3.8: Role of GRK5 on survival following cecal ligation and puncture: GRK5 wild
type and knockout mice were subjected to sham or CLP surgery and survival was assessed for 7
days in the absence (a) or presence (b) of antibiotics as described in methods. (n=10 per
genotype in a; n=15-16 per genotype in b). *P<0.05 compared to the corresponding septic wild
type in the antibiotic treated group by log-rank test. Factorial design analysis of the data showed
the following: Between genotypes: p=0.204; between antibiotic and non-antibiotic groups:
p=0.024; Interaction between genotypes and with antibiotics: p=0.038.

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on bacterial load following polymicrobial sepsis. Importantly, similar to our studies, these effects
were not sufficient to prevent mortality following CLP. MyD88 is a critical adaptor molecule for
many TLRs including TLR4. Consistent with the phenotype of MyD88 knockout mice, blocking
TLR4 alone in a CLP model of sepsis did not prevent mortality because of persistent bacterial
load160. Similar to our model, TLR4 antagonist significantly improved survival only in the
presence of antibiotics160. Based on our previous studies4,77 showing that GRK5 is an important
regulator of TLR4 signaling, our results are consistent with these other studies in terms of the
outcome of sepsis.

Studies have consistently related cytokine response as playing a major role in resolution
of sepsis by activation of immune cells and subsequent clearance of microbes. However,
excessive production of cytokines with aberrant activation of immune cells can have ill effects
on the host. As shown in the endotoxemia model4, we demonstrate here that GRK5 deficiency
attenuates inflammatory cytokines following intra-abdominal polymicrobial sepsis. Consistent
with the systemic and peritoneal cytokines, mRNA expression of NFB-dependent genes in the
liver and lungs were also attenuated at 12 hours post-sepsis. In line with our observations in LPS
model as well as the demonstrated effects on bacterial infection in Drosophila and Zebra fish
models, polymicrobial sepsis-induced IB phosphorylation in the liver was significantly
inhibited in the GRK5 knockout mice. Together, these studies are consistent with previous
studies showing that GRK5 regulates NFB signaling and therefore is able to modulate NFBdependent gene expression.

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Contrary to our results in mice and that of Valanne et al’s42 results in Drosophila, Zebra
fish and human cells, other studies have also shown that GRK5 is a negative regulator of NFB
signaling in endothelial cells57 and vascular smooth muscle cells57. This effect of GRK5 has been
linked to its role in stabilizing nuclear IB levels whereas we demonstrated previously that
GRK5 is able to phosphorylate IB at the same sites as that of IKK and mediate
degradation43. Thus it is possible that GRK5 may have multiple roles in terms of NFB
regulation but the dominance of regulation might depend on the cell type and disease being
examined.

Lymphocyte apoptosis has increasingly been recognized as an important step in the
pathogenesis of sepsis, by inducing a state of ‘immune paralysis’ that renders the host vulnerable
to invading pathogens168. NFкB signaling plays a vital role in lymphocyte development, function
and apoptosis. In addition, NFкB signaling can either promote survival166 or apoptosis167 of
lymphocytes depending on the cell type involved. Even though we found GRK5 to be an
important regulator of NFB signaling in the liver, this phenomenon appeared to be tissue
specific since we did not observe any difference in NFB activity in the thymus from the two
genotypes subjected to CLP (data not shown). Since previous studies have shown that sepsisinduced thymocyte apoptosis is mediated by corticosterone169, we hypothesized that either
corticosteroid signaling or its plasma levels may be differentially regulated in the two genotypes.
Even though our in vitro results rule out any direct effect of GRK5 on corticosteroid-induced
thymocyte apoptosis, our in vivo results demonstrate that GRK5 is an important regulator of
sepsis-mediated increase in corticosterone levels. Since sepsis-induced thymocyte apoptosis has
been shown to be mediated by corticosterone, lower levels of plasma corticosterone may in part
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explain why GRK5 KO thymocytes are protected from apoptosis in vivo. In future studies, we
will determine whether the effect of GRK5 on thymocyte apoptosis in vivo is due to decreased
corticosterone levels or due to differential regulation of other signals that may be involved in
thymocyte apoptosis in sepsis.

Consistent with the better survival of thymocytes in the GRK5 deficient mice, septic
peritoneal cells from the GRK5 knockout were more immunocompetent than the cells from wild
type mice. While our studies don’t demonstrate a cause-effect relationship between thymocyte
apoptosis and peritoneal cell responsiveness in the septic mice, previous studies have suggested
that enhanced lymphocyte apoptosis leads to enhanced immunosuppression in animal models171.
In addition it is possible that enhanced plasma corticosterone levels in the wild type mice might
suppress LPS-induced cytokine production in the wild type cells compared to less suppression in
the knockout cells172. These mechanisms will be dissected out in future studies. Regardless of the
mechanism, our results suggest that under conditions where the innate cells are already exposed
to polymicrobial injury, GRK5 deficiency is able to modulate the cells to be more immuneresponsive compared to the wild type. The better immuno-responsiveness of innate cells during
the stage of sepsis when cells are likely “paralyzed” may lead to a beneficial outcome in
sepsis173. This has been shown before where innate immune paralysis observed later in sepsis has
been attributed to poor outcome174. Together, these results suggest that GRK5 deficiency could
modulate sepsis at several levels or stages.

In conclusion, we demonstrate here that GRK5 is an important regulator of sepsis
pathogenesis and that blocking GRK5 could result in a protective mechanism via modulating

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several aspects of sepsis. Even though bacterial load is not affected by GRK5, administration of
antibiotics in the presence of GRK5 inhibition might lead to a better outcome in sepsis-induced
mortality.

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CHAPTER FOUR

CRITICAL INVOLVEMENT OF G- PROTEIN COUPLED RECEPTOR KINASE 5
(GRK5) IN ESCHERICHIA COLI INDUCED PNEUMONIA

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4.1: Abstract
G-protein coupled receptor kinases are serine/threonine kinases involved in a range of
pathophysiological processes including inflammation. We recently showed that deficiency of Gprotein coupled receptor kinase-5 (GRK-5) improves sepsis outcome in a poly-microbial sepsis
model in mice. In this study, we examined the role of GRK5 in mono-microbial pulmonary
infection by injecting E. coli intra-tracheally at two different doses to induce a low- and highgrade inflammation. We observed distinct phenotypes in GRK5 deficient mice (KO) depending
on the dose of E. coli. With low-grade inflammation, GRK5 KO mice induced higher
CXCL1/KC production and recruited more neutrophils, which were associated with better E. coli
clearance compared to WT mice. Consistent with the increased clearance of bacteria, the
inflammatory response was diminished and resolution of inflammation (as determined by
increased number of efferocytosing macrophages) advanced in the KO mice. However, at higher
dose of E. coli, even though there was increased CXCL1 and enhanced neutrophil recruitment in
the KO mice, the bacterial burden remained high in the KO compared to wild type (WT) mice. In
addition, the neutrophils from KO mice from the high dose E. coli treatment group showed
decreased neutrophil activation as determined by CD11b expression. Decreased neutrophil
activation and increased bacterial burden in the KO at this dose suggest increased neutrophil
exhaustion in the KO mice. Consistent with the increased bacterial burden, the KO mice showed
decreased survival. Overall our data suggest GRK5 negatively regulates CXCL1/KC production
and modulates the outcome of pneumonia depending on the bacterial dose.

81

4.2: Introduction
Pneumonia is one of the leading causes of mortality in the United States and is a major
cause of severe sepsis175,176. Acute lung injury (ALI) is often caused by infection with bacteria,
viruses, and other organisms including fungi and chlamydia of which gram-negative bacterial
species are the dominating cause of pneumonia-induced ALI177. As in any bacterial infection,
neutrophil chemotaxis plays a vital role in clearing the infection. Neutrophil recruitment is
critical to clear bacteria, and neutrophil migration in the lung requires the production of
ELR+ (glutamic acid-leucine-arginine) CXC chemokines like CXCL1/KC, CXCL2/MIP2 and
CXCL5/LIX178-180. However, excess neutrophilic infiltration or defect in neutrophil removal after
clearing infection can propagate inflammatory process, which can be detrimental181. A key
strategy in reducing mortality is to modulate the innate immune system to enhance the host's
ability to combat microbial infection while limiting inflammation-induced tissue injury.

G-protein coupled receptor kinase 5 (GRK5) is a serine-threonine kinase, shown to
modulate inflammatory responses in vivo and in vitro by regulating inflammatory signaling
pathways40,42,43,77. Of the seven GRKs, GRK5 is highly expressed in normal12,182 and diseased
airways80. In addition, GRK5 expression level has been shown to be modulated in conditions
such as sepsis, cardiac failure, obesity, cystic fibrosis, cancer etc.

17

. Furthermore, our previous

studies revealed critical role of GRK5 in polymicrobial sepsis2. Even though GRK5 is expressed
in the airways, its role in modulating bacterial pneumonia is not known. Given its role in
inflammatory pathways and in polymicrobial sepsis and because GRK5 is highly expressed in
the airways, we hypothesized that GRK5 will play a critical role in modulating bacterial
pneumonia. We demonstrate here that GRK5 is a critical signaling molecule in the pathogenesis

82

of bacterial pneumonia by modulating CXCL1/KC levels and neutrophil recruitment; our results
however, highlight the complexities by demonstrating that the role of GRK5 is distinct
depending on the dose of E. coli.

4.3: Materials and methods
4.3.1: Materials
Protease inhibitor cocktail tablets were from Roche Applied Science (Indianapolis, Ind.,
USA); pIκBα, and tubulin antibodies were from Cell Signaling Technology, Inc. (Danvers,
Mass., USA) and Sigma (St. Louis, Mo., USA), respectively. Escherichia coli (O6:B1) was
obtained from ATCC (Manassas, VA, USA).

4.3.2: Experimental animals
GRK5 deficient mice previously described4 was used in the study. Healthy, male mice
belonging to the age of 8- to 12 weeks were used in the experiments. All mice were housed with
a 12-h alternating light-dark cycle at 25°C, with 50% humidity and free access to food and water.
All experiments performed were approved by the Institutional Animal Care and Use Committee
at the Michigan State University.

4.3.3: E. coli infection
A clinical isolate of E. coli (O6:B1, ATCC 25922) was used for all experiments. Bacteria
were grown in trypticase soy broth for 8 h at 37°C and bacterial colony forming units (CFU) of
each culture was determined by comparing the specific OD-600 value to a standard curve.
Bacteria were washed twice with sterile phosphate buffered saline (PBS) and adjusted to the

83

appropriate final concentration. For bacterial injection, mice were anaesthetized using xylazine
(5mg/Kg) and ketamine (80 mg/Kg), placed in an intubation stand and gently secured by the
incisors. Using a clean forceps tongue was pulled gently out to expose epiglottis and trachea.
With the tongue held in position 50 µl of bacterial inoculum was instilled. To avoid bias, WT
and KO mice were injected in a blinded fashion. Sham intra-tracheal inoculation were also
carried out with the use PBS and served as control. Mice were observed and euthanized at
various time points up to 48 hours for cell and tissue analysis.

4.3.4: Bacterial load determination
Mice infected with E. coli were euthanized 4, 12, or 24 h post infection. Broncho alveolar
lavage fluid (BAL), lung and blood were harvested aseptically. Post-caval lung lobe was
collected, weighed and homogenized in 1 ml of sterile PBS. All the samples were plated in
Trypticase Soy Agar (BD Biosciences) and incubated at 37 ° C for 48 h. Colony-forming units
(CFU) were counted to determine bacterial load and expressed as CFU per milliliter of blood or
50 µl of BAL or gram (g) of lung. In addition, bacterial presence in lungs was also measured
indirectly by measuring gene expression of bacterial 16S rRNA using Real-time Q-PCR (Primer
sequence for 16s rRNA forward: 5'-GGT GAA TAC GTT CCC GG-3'and reverse: 5'-TAC GGC
TAC CTT GTT ACG ACTT-3' HPRT used as housekeeping gene-Forward: 5’-AAG CCT AAG
ATG AGC GCA AG-3’; Reverse: 5’-TTA CTA GGC AGA TGG CCA CA-3’).

4.3.5: In vitro Bacterial killing assay
Neutrophils were collected from WT and KO mice by processing peritoneal lavage 4h
post injection of thioglycollate. A small aliquot was separated to assess the purity of neutrophils

84

by flow cytometry. Neutrophil numbers were enumerated and incubated with opsonized E. coli
for 30 minutes to enable bacterial uptake by neutrophils. Post incubation, the cells were treated
with gentamicin (200 µg) for 30 and 60 minutes to kill extracellular E. coli. The cells were then
washed twice with PBS and the pellet was lysed with 0.1% triton-X and plated onto Tryptic soy
agar. Plates were incubated at 37° C and Colony forming units were counted 24 hours post
plating.

4.3.6: Immune cell infiltration
Lung tissue, Broncho-alveolar lavage fluid cells and blood samples were collected at
specified time points post infection to measure the infiltration of immune cells to the lung. Left
lung lobe was teased and incubated in collagenase D (1 mg/ml) containing media at 37°C for an
hour with gentle shaking and then the tissue was disrupted to release cells. For determining cell
number changes, cells were counted using a hem cytometer. Cells were also labeled with cell
surface markers for various immune cells (Gr-1, CD11b, F4/80, CD11c, B220, CD3 and Ly6C)
and data acquired using LSRII (BD Biosciences) and analyzed using Flowjo software (Tree Star,
Inc., Ashland, Oreg., USA) as described before2.

4.3.7: Chemokine receptor expression
Bone marrow neutrophils were isolated from WT and KO mice using percoll gradient
centrifugation. Briefly, RBC lysed bone marrow cell suspension was gently layered on top 62%
percoll solution and centrifuged at 2200 rpm for 30 minutes. At the end of the gradient
centrifugation, the cloudy layer below the interface were collected and gently washed twice.
Neutrophil purity was assessed using flow cytometry. For the expression studies, neutrophils

85

were treated with controls or chemokine ligands CXCL1 (100ng) and CXCl2 (50ng) (obtained
from Peprotech, USA) for the specified time points. The samples were then fixed immediately,
processed and analyzed by flow cytometry.

4.3.8: Cytokine/Chemokine measurements
Plasma isolated from blood samples, tissue lysates from lung samples and bronchoalveolar lavage fluid samples were subjected to cytokines and chemokines analysis using ELISA
kits from eBiosciences, Inc. as described before2. CXCL1 and CXCL2 levels were determined
using ELISA kits from R&D system, Minneapolis, MN.

4.3.9: Immuno Blot Analysis
Lung tissue samples for immune blot analysis were prepared by homogenizing the tissue
in lysis buffer (1 M HEPES, 2 M KCl, 0.5 M EDTA and 0.1 M EDTA along with protease and
phosphatase inhibitors). The protein concentrations were determined and equivalent amounts of
protein were loaded onto the gels for Immuno blot analysis. Immunoblotting was carried out for
pIκBα and tubulin as described before2. The bands were quantified using the image-J (for
chemiluminescence) or Licor’s Odyssey program (for fluorescence).

4.3.10: Statistical Analysis
All data are presented as the mean ± SEM. Two group comparisons were performed
using Student’s t test and comparisons of more than two groups were done by ANOVA with the
post-Bonferroni Test. All statistical analyses were performed using GraphPad Prism Software
(San Diego, Calif., USA) and p < 0.05 were considered statistically significant.

86

4.4: Results
4.4.1: GRK5 modulates bacterial clearance but doesn’t mediate intracellular bacterial
killing
To determine the function of GRK5 in the host response and subsequent clearance of live
Gram-negative infection, we infected WT and KO mice intra-tracheally with log-phase growth
live E. coli at 1x106 CFU/mouse. After infection, lung homogenates, broncho-alveolar lavage
fluid and blood samples were prepared at specified time-points and plated onto tryptic soy agar
plates and CFUs determined 24 h later.

Infection of mice with E. coli at 1x106 CFU/mouse induced low grade acute
inflammation; bacteria were cleared from lungs by 24 h post infection and were not detected in
blood at any of the time-points tested. Mice of both genotypes had comparable bacterial load at
an earlier time-point (4 h) both in lungs and BAL fluid (Fig 4.1a and 4.1b). However, by 12
hours, bacterial counts were strikingly decreased in the GRK5 KO lung and BAL (Fig 4.1a, b, c).
These results suggest that GRK5 deficient mice are able to clear bacteria much better than the
WT mice, in this E. coli model.

Bacterial burden could be regulated at multiple levels including phagocytosis, bacterial
killing or by regulating infiltration of phagocytes. To determine if GRK5 directly regulates
phagocytosis or bacterial killing, we performed in vitro intracellular bacterial killing assay using
thioglycollate-induced peritoneal neutrophils. As shown in Fig 4.2, phagocytosis (as seen in 0
min time point) or intra cellular bacterial killing (as seen in 30 and 60 min time points) were
similar between the WT and GRK5 KO neutrophils. These data clearly suggest GRK5-regulated

87

2.0 10 0 6

50000

1.5 10 0 6
40000

CFU/50L of BAL

CFU/g of Lung

1000000
*

500000
50000
40000
30000

30000
20000
10000

20000

***

10000
0

4H

12H

0

24H

12H

24H

WT E.coli

5000

16S rRNA relative expression
compared to WT PBS control

4H

KO E.coli

4000
3000
2000
1000
0

***
4H

12H

24H

48H

Figure 4.1: Decreased bacterial burden in GRK5 deficient mice at low dose E. coli: GRK5
WT and KO mice were intra tracheally injected with 1x 106 CFUs of E. coli and after the time
points mentioned (a) lung and (b) Broncho alveolar lavage fluid were collected and plated for
bacterial growth as described in materials and methods. (c) Quantitative relative expression of
bacterial 16S rRNA in lung tissue of wild type and KO animals infected with E. coli. (n= 9 for
4hr; 12 for 12hr; 7 for 24h; 4 for 48h). * p < 0.05, * * * p < 0.001 compared to the corresponding
infected WT groups.
88

20000

WT
KO

CFU

16000
12000

4000
0

0min

30min

60min

Figure 4.2: GRK5 does not play a role in intra cellular killing of bacteria: Thioglycollate
induced neutrophils were collected from GRK5-/- and GRK5 +/+ as described in materials and
methods. 1x 106 neutrophils were incubated with 4x 106 CFUs of opsonized bacteria for 30
minutes to mediate phagocytosis of bacteria. The cells were then incubated with gentamicin for
specified time points to kill extra cellular bacteria and later washed twice with HBSS. The
washed pellet is then lysed with 0.1% tritonX to liberate intracellular bacteria and then plated
onto Tryptic soy agar to enumerate CFUs (n=6 ).

89

bacterial burden in vivo is unlikely through direct modulation of phagocytosis or intra cellular
killing by GRK5.

4.4.2: GRK5 mediates early neutrophil recruitment into lungs
GRK5 is known to regulate many GPCRs, including chemokine receptors. GRK5 was
shown to regulate CXCR4 in vitro by phosphorylating Hsp70 interacting protein (Hip) which in
turn aids in internalization of CXCR41. Since, our data ruled out a role for GRK5 in bacterial
killing, we shifted our focus to understand the dynamics of immune cell infiltration, which can
alter bacterial persistence during bacterial pneumonia in vivo. To study the dynamics of immune
cell infiltration, we induced lung infection with E. coli in both genotypes and examined the lung
tissue for immune cell infiltration using flow cytometry. When infected with E. coli, GRK5
deficient mice had a significantly increased recruitment of neutrophils into the lung at 4 h time
point (Fig 4.3a). However, at the later time points (12 to 48 h) the number of neutrophils in the
lung was similar between the genotypes, suggesting that GRK5 deficiency enhances early
neutrophil recruitment. Further to characterize the activation status of these neutrophils, we
examined CD11b expression. CD11b expression in neutrophils has been linked to increased
reactive oxygen species (ROS) production and increased respiratory burst, which have direct
consequence in bacterial killing183. Interestingly, 12 hours post-infection, CD11b expression in
GRK5 KO neutrophils were significantly higher compared to the WT cells (Fig 4.3d). This
coupled with early neutrophil infiltration likely cleared bacteria faster in the GRK5 KO mice.
In addition to these events, we also examined the percentage of CD11b+Gr1+F4/80+ (possibly
efferocytosing macrophages)184 cells in the lungs. Recently, these cells have also been described
to phenotype myeloid-derived suppressor cells (MDSCs), which function to restrict

90

(a)

(b)
10 6

5.0

WT E.coli

3.010 6

KO E.coli

3.010 6
2.010 6
1.010 6

*

5.010 5
0

B lymphocytes (counts)

(c)

4h

12h

2.010 6

1.010 6

0

24h 48h

(d)

2.010 5

1.510 5

1.010 5

10 4

5.0

0

4h

12h

24h 48h

4h

MFI of CD11b expression on neutrophils
(Fold change compared to WT)

Gr-1+F4/80+ (counts)

Neutrophil (counts)

4.010 6

12h

24h 48h

1.5
*
1.0

0.5

0.0

12H

Figure 4.3: GRK5 regulates early recruitment and activation of neutrophil upon E. coli
infection: Lung samples collected at different time points from E. coli infected mice were
analysed for cellular composition (a) Neutrophils, (b) Efferocytosing macrophages expressing
both Gr-1 and F4/80 and (c) B cells. GRK5 deficiency induced a small but significant increase in
neutrophil recruitment only at early time point (4H) and not at later time points.(d) Lung samples
collected 12 hours post infections were analyzed by flow cytometry for surface expression (mean
fluorescence intensity) of CD11b. (n= 8-10 for 4hr; 6-9 for 12hr; 5 for 24h; 5 for 48h)=* p <
0.05, compared to the corresponding WT groups.
91

Figure 4.4: Increased proportions of efferocytosing macrophages in GRK5 KO lungs at low
dose E. coli: (a) Lung samples analyzed by flow cytometry revealed increased percentage of
efferocytosing macrophages in GRK5 KO lungs. (b) Percentage of Gr-1+ cells and Gr-1+
F4/80+ cells from lung 12h post infection. (n=8-10). * p < 0.05, compared to the corresponding
WT groups.

92

ongoing inflammation and favor resolution184,185. Surprisingly, at 12 hours post-infection, this
resolution favoring cell type was significantly higher in the GRK5 KO mice compared to WT
(Fig.4.4). These data suggest that the enhanced neutrophil infiltration and activation together
with increase in efferocytosing macrophages/myeloid-derived suppressor cells could favor faster
resolution of inflammation observed in the GRK5 knockout mice.

4.4.3: GRK5 regulates CXCL1 but does not regulate CXCR2, CXCR4 and CCR1 cell
surface expression
Enhanced neutrophil infiltration in the GRK5 knockout could be due to regulation of
expression of chemokines or their respective receptors. Therefore, we examined the levels of
various chemokines (CXCL1, CXCL2 and MCP1) at different time points after E. coli infection.
Interestingly, CXCL1 levels were significantly increased in KO mice at 4 h time point coinciding
with the increased neutrophil recruitment (Fig. 4.5a). Expression of other chemokines, CXCL2
and MCP1 weren’t any different between WT and KO mice at 4 h time point. However, at later
time points, the levels of CXCL1, CXCL2 and MCP1 were decreased in KO mice compared to
WT mice. To examine if GRK5 also regulated chemokine receptor expression, we assessed cell
surface expression of CXCRs in neutrophils under basal and stimulated conditions in the two
genotypes. As shown in Fig 4.6, chemokine receptor cell surface expression decreased when the
neutrophils were treated with chemokines (likely due to receptor desensitization and
internalization). This however was similar between the genotypes under both basal and
stimulated conditions. Together these data suggest that in response to E. coli infection, GRK5
deficient mice were able to produce higher levels of chemokines early in the process that likely
led to enhanced neutrophil infiltration and therefore better clearance.

93

(a) Plasma

WT E.coli
25000

300000

40000

KO E.coli

*

200000

100000

30000

MCP 1 (pg/ml)

CXCL2 (pg/ml)

CXCL1 (pg/ml)

20000
15000
10000

10000

5000
0

4hr

0

12hr 24hr 48hr

20000

4hr

0

12hr 24hr 48hr

4hr

12hr 24hr 48hr

(b) BAL
150000

80000

60000
***

***

50000

MCP 1 (pg/ml)

**

CXCL2 (pg/ml)

CXCL1 (pg/ml)

60000
100000

40000

20000

40000
***
20000

0

4hr

12hr 24hr 48hr

0

4hr

12hr 24hr 48hr

0

4hr

12hr 24hr 48hr

Figure 4.5: GRK5 deficiency induced CXCL1 production early in low dose E. coli induced
pneumonia: (a) Plasma and (b) BAL samples were analyzed for expression of chemokines
CXCL1, CXCL2 and MCP1. (n= 7 for 4hr; 6 for 12hr; 5 for 24h; 4 for 48h).*p < 0.05, **p <0.01
and ***p<0.001 compared to the corresponding WT groups.

94

Figure 4.6: GRK5 does not regulate expression of neutrophil CXCR2, CXCR4 and CCR1 :
Bone marrow neutrophils were isolated using percoll density centrifugation from wild type and
knock out mice and were analyzed for expression of chemokine receptors with and without
stimulation using (a) CXCL1 and (b) CXCL2.( n= 5).

95

4.4.4: GRK5 modulates inflammatory status
The overall phenotype in this lower dose E. coli model suggests GRK5 deficiency
regulates neutrophil infiltration likely via CXCL1 expression and restricts bacterial presence and
as sequelae we expected inflammatory cytokine production during active pulmonary infection to
be decreased in KO mice. To assess the inflammatory status, we subjected lung, BAL fluid and
plasma from E. coli infected mice to cytokine analysis by ELISA.

Consistent with our

prediction, levels of pro-inflammatory cytokines including IL-6 and TNFα levels were
significantly decreased in the KO mice, in both plasma and lung (Fig.4.7a and 4.7b). These
effects however, were time-dependent. Also, consistent with protein levels, mRNA levels of
these cytokines were decreased in KO lung tissue (data not shown). Previous studies2,4,43 have
shown that GRK5 is a critical regulator of NFB pathway during acute inflammation. To assess
the role of GRK5 in NFB pathway in this model, we measured the levels of phospho-IκBα in
the lung tissue. Consistent with the decreased cytokine production, p-IκBα was significantly
decreased in the KO lung tissue (Fig.4.7c). These observations on inflammatory status in KO
mice are consistent with our previous studies on endotoxemia and CLP induced polymicrobial
sepsis2,4.

4.4.5: GRK5 confers survival advantage during E. coli induced pneumonia at higher
infection dose:
Our results so far suggest that the sub lethal dose of E. coli induces a sub-acute lung
inflammation and that the GRK5 knockout mice are protected from bacterial burden and
inflammation likely via multiple mechanisms. This however did not result in overt histo-

96

(a)

Figure 4.7: GRK5 deficiency induced decreased inflammatory response in low dose E. coli
induced pneumonia: (a) Immunoblot showing decreased pIĸBα levels in GRK5 deficient mice
after low dose E. coli injection. (n=4). (b) BAL and (c) Plasma samples were analyzed for
expression of cytokines IL-6, TNFα, IL-12p40 and IL-10. (n= 10-15 for 4hr; 9-15 for 12hr; 5 for
24h; 5 for 48h) *p < 0.05,**p <0.01 and ***p<0.001 compared to the corresponding WT groups.

97

Figure 4.3: (cont’d).

(b)

(c)

50000

*

IL-6 (pg/ml)

IL-6 (pg/ml)

60000

30000
20000

40000

20000

10000

4hr

12hr

0

24hr 48hr

25000

4hr

12hr

24hr 48hr

15000

20000

***

TNF (pg/ml)

TNF (pg/ml)

KO E.coli

***

40000

0

WT E.coli

80000

15000
10000

10000

5000

5000
0

4hr

12hr 24hr

0

48hr

15000

4hr

IL-12 (pg/ml)

IL-12 (pg/ml)

10000

5000

4hr

12hr

**

10000

5000

0

24hr 48hr

3000

4hr

***

IL-10 (pg/ml)

2000

1000

12hr

24hr 48hr

10000
*

8000

IL-10 (pg/ml)

48hr

**

***

0

12hr 24hr

15000

***

***
6000
4000
2000

0

4hr

12hr 24hr

0

48hr

98

4hr

12hr 24hr

48hr

pathological differences in resolution at 48 hours after infection (data not shown). Even though it
is possible that a difference in resolution may be evident at later time points, we wanted to
further examine if E. coli-induced mortality in this pulmonary model is different between the
genotypes. We hypothesized that since bacterial burden and inflammation are decreased in the
GRK5 knockout mice, this genotype would be protected from E. coli-induced mortality
compared to the wild type mice. This hypothesis was also based in part on our previous studies
in polymicrobial sepsis model, in which we showed that GRK5 deficiency enhances survival
especially in the presence of antibiotic therapy. Therefore, we tested the role of GRK5 with a
higher dose of E. coli that we predicted would cause ~50% mortality based on pilot studies and
literature. For this, we used E. coli dose of 5x106 CFUs/mouse and performed survival studies.
This dose resulted in approximately 50% mortality in wild type in 2 days. In contrast to our
prediction, GRK5 deficient mice succumbed to pneumonia much more readily than wild type. At
the end of 48 hours post infection GRK5 deficient mice had 90% mortality, whereas wild type
mice had only 58% mortality (Fig.4.8). Also, the median survival in WT mice was 33 hours,
whereas GRK5 deficient mice had a median survival of 18 hours. To begin to understand the
paradox, we first examined bacterial burden using the higher dose of E. coli. Surprisingly, and in
contrast to the low dose E. coli, with the higher dose of E. coli, GRK5 deficient mice showed
significantly increased bacterial load in the lung and blood (Fig. 4.9). This suggested that with
higher dose of E. coli, the GRK5 deficient mice are unable to clear bacterial infection. Some key
events that were observed with the low dose of E. coli were the enhanced infiltration of
neutrophils coupled with greater activation and increased CXCL1 production in the GRK5
knockout mice. To confirm if these were altered in the lethal dose model, we examined
neutrophil infiltration, CD11b expression and CXCL1 production in the two genotypes following

99

Percent survival

100

WT
KO

80
60
40
20
0

*p=0.0256
0

20

40

60

Hours

Figure 4.8: Increased mortality observed in GRK5 deficient mouse infected with E. coli at
5x106 CFU/mouse: GRK5 deficient mice had approximately 90% mortality as compared WT
mice having 58% mortality. (n= 11-12). ).*p < 0.05, compared to the corresponding WT groups.

100

2.010 8

10 7

1.0

5.010 6

1.510 8

20000

**
CFU/ml of Blood

CFU/g of Lung

CFU/50L of BAL

1.510 7

1.010 8

5.010 7

0

12H 15H

0

12H 15H

*

15000

10000

5000

0

12H 15H

Figure 4.9: Increased Bacterial burden in GRK5 deficient mice at high dose E. coli: GRK5
WT and KO mice were intra tracheally injected with 5x 106 CFUs of E. coli and after the time
points mentioned (a) lung, (b) Broncho alveolar lavage fluid and (c) Blood were collected and
plated for bacterial growth as described in materials and methods. (n= 3-4 for 12hr; 3 for
15hr).*p < 0.05, ** p < 0.01 compared to the corresponding infected WT groups.

101

(b)

4.0

2.010 6

0

4H

1.510 6

1.010 6

**

5.010 5

0

12H 15H

4H

3.010 5

2.010 5

1.010 5

0

12H 15H

(e)

1.5

0.5

12H

15H

**
400000

0

WT E.coli
KO E.coli

200000

200000

0.0

12H 15H

250000

600000

*

4H

(f)
800000

1.0

WT E.coli
KO E.coli

4.010 5

CXCL2 (pg/ml)

Mean CD11B on neutrophils
(Fold change compared to WT)

(d)

*

B lymphocytes (counts)

Gr-1+F4/80+ (counts)

10 6

(c)

2.010 6

6.010 6

CXCL1 (pg/ml)

Neutrophil (counts)

(a)

150000
100000
50000

4hr

12hr

0

4hr

12hr

Figure 4.10: GRK5 deficiency mediates early neutrophil recruitment into lungs in high
dose E. coli infected mice: Lung samples collected at different time points from E. coli infected
mice were analyzed for cellular composition (a) Neutrophils, (b) Efferocytosing macrophages
expressing both Gr-1 and F4/80 and (c) B cells. (d) Lung samples collected 12 and 15 hours post
infections were analyzed by flow cytometry for neutrophil surface expression (mean
fluorescence intensity) of CD11b, (e and f) Plasma samples from high dose E. coli were analyzed
for expression of chemokines CXCL1 and CXCL2. (n= 3-4 for 12hr; 3 for 15hr).*p < 0.05, ** p
< 0.01 compared to the corresponding infected WT groups.

102

infection with 5x106 CFUs/mouse. Interestingly, although neutrophil infiltration and CXCL1
production were enhanced in the GRK5 KO similar to the sub lethal dose, CD11b expression
was decreased in the GRK5 KO compared to the wild type mice (Fig.4.10). These data suggest
that neutrophils from GRK5 KO mice are likely “less activated” and therefore unable to clear
infection efficiently and therefore result in higher mortality. These data however, does not
completely rule out other aspects of regulation by GRK5 KO in terms of bacterial clearance,
especially under this high dose condition.

4.5: Discussion
Exaggerated inflammation is a characteristic feature of pneumonia and sepsis. Alteration
in the initial events of infection like microbe detection, inflammatory signaling, chemotaxis of
immune cells can alter the course of disease process. Studies have shown neutrophil recruitment
is essential in clearing bacteria from lungs and at the same time exaggerated neutrophil influx is
detrimental to the host.

In this study, we show GRK5 plays a paradoxical role in regulating E. coli induced
pneumonia depending on the dose of E. coli. At a sub lethal dose of E. coli, GRK5 deficiency
leads to efficient clearing of bacteria from lungs by increasing neutrophil recruitment. Being a
kinase regulating GPCRs, it is likely that GRK5 can regulate chemokine receptors and hence
modulate neutrophil chemotaxis. However, our data showed (Fig 4.8) GRK5 doesn't regulate
basal expression of CXCR2 and CXCR4, which are the major chemokine receptor responsible
for neutrophil migration. Instead, our data suggest, that the increased bacterial clearance was
probably a result of increased CXCL1 production (Fig.4.9a), which likely led to enhanced

103

neutrophil chemotaxis to the site of infection observed in the KO mice. In addition to the
increased bacterial clearance, GRK5 also mediated increased resolution of inflammation as
evidenced by increased proportion of efferocytosing macrophages (Fig.4.5), which are known to
clear dying and dead cells185. We also noticed decreased activation of NFκB (Fig. 4.10) in the
lungs of KO mice, which probably could be a result of faster bacterial clearance and therefore
less inflammatory signaling pathways being activated or due to previously demonstrated role of
GRK5 in NFB signaling.

Similar to the low dose model, E. coli at a lethal dose induced higher CXCL1 and
neutrophil recruitment in GRK5 KO mice compared to wild type. However in contrast to low
dose model, the increased neutrophil recruitment did not seem to clear bacteria effectively in the
KO mice (Fig. 4.2). In fact, high dose E. coli resulted in increased bacterial load in both lungs,
and blood and this data is consistent with increased mortality observed in KO mice. This data
suggests that neutrophils are probably altered in terms of their bacterial killing capacity or their
activation status with the high dose of E. coli. Our data clearly show marked differences in
CD11b expression (linked to neutrophil activation) between the high and low dose E. coli
infection. Even though at low dose infection, CD11b expression is significantly enhanced in the
KO mice neutrophils, this is not observed in the high dose model (which in fact is decreased in
the KO, in the high dose model), suggesting less activation GRK5 KO neutrophils in the high
dose model. CD11b is an integrin receptor and β2-Integrins (e.g., CD11a/CD18, CD11b/CD18)
are generally expressed at low levels basally in the neutrophils, but can be activated to a high
affinity state, which enables them to transmigrate from blood to tissues. Also, engagement of
CD11b/CD18 in neutrophils triggers generation of reactive oxygen species through NADPH

104

oxidase in close proximity to the target mediating bacterial killing186. Furthermore, CD11b being
a component of Complement receptor 3 can influence complement receptor pathways and
therefore regulate bacterial burden.

One of the key observations in this study is that deficiency of GRK5 significantly
enhances CXCL1 production. CXCL1/KC belongs to a class of ELR+ CXC chemokines, which
also includes other major chemokines like CXCL2/MIP2 and CXCL5/LIX. CXCL1 is produced
from hematopoietic and non-hematopoietic compartments and was shown to be essential for
neutrophil influx and clearing of Klebsiella pneumoniae from lungs187. Even though the
molecular mechanism by which GRK5 regulates CXCL1 production is not known, CXCL1 has
been shown to be regulated by TLR-mediated MAPK signaling. p38 MAPK and ERK1/2 were
known to regulate CXCL1 production in response to IL-1β188, FasL189 activation in a MyD88
dependent manner. Consistent with this, previous studies have shown that GRK5 negatively
regulates TLR4 induced ERK1/2 signaling via regulation of IKK-NFBp105 pathway40. Thus,
deficiency of GRK5 likely leads to over activation of ERK pathway thus leading to increased
CXCL1 production. This hypothesis will be tested in future studies.

GRK5 has been shown to positively43 (macrophages) or negatively41 (endothelial cells)
regulate NFκB signaling and modulate inflammatory response depending on the cell type
involved. In endotoxaemia4 and polymicrobial sepsis2, we showed GRK5 positively regulates
cytokine production and that GRK5 deficient mice exhibited attenuated inflammatory response
mostly in the later stages of sepsis. It is likely that, GRK5 can negatively regulate NFκB in
endothelial and epithelial cells, both of which can produce CXCL1 and therefore lead to

105

increased production of CXCL1 in GRK5 KO mice. Nevertheless, in this study we find that
IB phosphorylation was decreased in the KO lungs suggesting that NFB is unlikely to be the
regulator of CXCL1 production in the GRK5 KO mice

In conclusion, our current study demonstrates GRK5 negatively regulates CXCL1
production (an important chemokine responsible for neutrophil chemotaxis) and thereby
regulates neutrophil chemotaxis. In low dose E. coli model, this response seems to play a
beneficial role in the KO mice, as evidenced by decreased bacterial load and increased resolution
of inflammation. However, at the higher dose of E. coli, KO mice succumb to lethality due to
higher bacterial load in spite of enhanced neutrophil infiltration, likely as a result of decreased
neutrophil activation.

106

CHAPTER FIVE

SUMMARY AND CONCLUSION

107

More than two decades ago, GRKs were discovered in the context of GPCR
desensitization and mostly considered only to play a role in G- protein mediated functions.
However, recent studies show GRKs can interact with and phosphorylate non-GPCR targets.
However, the knowledge on their physiological and pathophysiological consequences of these
newly identified interactions of GRKs especially during inflammatory diseases is scarce. The
major focus of this dissertation research project was to understand the overall pathological role
of GRK5 in acute inflammatory diseases like sepsis and pneumonia. In this context, our lab
showed GRK5 to mediate NFĸB signaling by interacting with and phosphorylating IĸBα in vitro.
Furthermore, GRK5 role in TLR4 signaling had been previously identified, where in GRK5
acted as positive mediator of cytokine production. We further wanted to ascertain the role of
GRK5 in more clinically relevant models for acute inflammatory diseases like sepsis and
pneumonia. The individual aims and main findings from each project are summarized below.

5.1: Role of GRK5 in MyD88-dependent and -independent cytokine production in vivo
Toll like receptor signaling can be broadly classified into MyD88-dependent and TRIFdependent pathways. The activation of the MyD88-dependent pathway leads to the early phase
of NFκB activation and proinflammatory cytokine expression, while TRIF dependent pathway
mediates the late phase of NFκB activation and the induction of IFN-inducible genes in TLR4
signaling.

GRK5 regulates cytokine production in LPS induced TLR4 signaling pathway via
positively modulating NFĸB signaling. However, it is not known whether the role of GRK5 is

108

specific for TLR4 or whether it regulates other TLRs. Also, since both MyD88-dependent and
TRIF-dependent pathways converge onto NFĸB, it is important to know whether GRK5
regulates either one of them or both. To identify the role of GRK5 in these pathways, we injected
WT and KO mice with TLR2 ligand and TLR3 ligand separately and measured cytokine
production. TLR2 specifically activates MyD88 pathway whereas TLR3 specifically activates
TRIF pathway and by comparing the responses we can identify whether GRK5 has any role in
either of the pathways.

5.1.1: Results


GRK5 mediated cytokine production through both MyD88 and TRIF pathways.



GRK5 positively mediated IL-5, IL-6 and MCP-1 production via MyD88 pathway.



GRK5 positively mediated TNFα and RANTES production via TRIF pathway.



IL-12p40 and G-CSF levels are regulated by GRK5 at a level that is common to both
MyD88 and TRIF pathway

5.1.2: Conclusion
Our results show GRK5 has overlapping as well as selective roles in TLR signaling
pathways and can regulate multiple TLRs via both MyD88 and TRIF pathway. These findings
also suggest GRK5 can play a substantial role in acute inflammatory disease conditions like poly
microbial sepsis where in microbial TLR is the key players in mediating the disease.

109

5.1.3: Limitations and future directions
While we showed an evidence of GRK5 playing role in MyD88 and TRIF signaling, we
did not take into account the cross talk happening between the two pathways. It is likely that
MyD88 activation by itself can interact with partners of TRIF pathway and mediate TRIF
dependent responses and vice versa. Our future direction includes identifying role of GRK5 in
other pathways activated by the cross talk between MyD88 and TRIF signaling.

5.2: Role of GRK5 in Polymicrobial sepsis
The goal of this project was to understand the role of GRK5 in the pathophysiology of
sepsis. Sepsis is characterized by inflammatory cascade, in response to microbial infection or
extensive tissue damage. We induced polymicrobial sepsis by cecal ligation and puncture
technique in both WT and KO mice to study the role of GRK5 during sepsis.

5.2.1: Results


GRK5 KO mice had decreased NFĸB activation and diminished inflammatory responses
during sepsis.



GRK5 KO and WT mice showed comparable bacterial load.



GRK5 deficiency decreased sepsis-induced thymocyte apoptosis that correlated with
decrease plasma corticosterone production.



Peritoneal lavage cells from GRK5 KO mice during sepsis showed more immuneresponsive phenotype compared to the wild type cells.

110



GRK5 deficient mice were able to survive better than WT mice with administration of
antibiotics. However, there is no significant survival advantage for GRK5 KO mice in the
absence of antibiotics.

5.2.2: Conclusion
Our results demonstrate GRK5 as an important regulator of inflammation in
polymicrobial sepsis and implicate GRK5 as a potential molecular target in sepsis. Regulation of
inflammatory response, thymocyte apoptosis and immune suppression by GRK5 suggests that it
can play multiple roles during sepsis.

5.2.3: Limitations and future directions
Mortality in sepsis is multifaceted and results eventually from cardiovascular, renal and
pulmonary collapse. GRK5, being a GPCR regulating kinase could have potentially other
beneficial GPCR dependent roles to play during sepsis like regulation of β-adrenergic system
and preventing cardio vascular system collapse, regulation of coagulation and preventing
disseminated intravascular coagulation (DIC), etc. Role of GRK5 in these processes is unknown
and will be the subject of future studies. Non-hematopoietic cells also play an important role in
the pathogenesis of polymicrobial sepsis by modulating neutrophil migration and phagocytosis.
GRK5 is a ubiquitously expressed protein with a role in cell signaling in both the hematopoietic
and non-hematopoietic compartments. Hence, our future direction is to assess the role of GRK5
in non-hematopoietic compartments using bone marrow chimeric mice or cell-type targeted
knockout mice.

111

5.3: Role of GRK5 in E. coli induced pneumonia
GRK5 is highly expressed in normal and diseased airways. GRK5 regulates Toll like
receptor signaling and airways are often exposed to multiple pathogens. So, the goal of this
project is to discern the role of GRK5 in Gram negative bacteria induced pneumonia. We
induced pneumonia by per oral tracheal injection of live E. coli into WT and KO mice at two
different doses to characterize the role played by GRK5 in low grade and high grade airway
inflammation.

5.3.1: Results


At low dose E. coli, GRK5 KO mice had low bacterial burden, whereas at five-fold
higher dose GRK5 KO mice had increased bacterial load and suffered from increased
mortality compared to WT mice.



GRK5 KO mice recruited more neutrophils to the site of infection in both high and low
dose models.



Increased neutrophil recruitment into lungs at low dose proved beneficial in the GRK5
KO mice, as the KO mice cleared bacteria sooner and mediated early resolution of
inflammation.



Increased neutrophil recruitment into lungs at high dose in the GRK5 KO mice was not
effective at clearing bacteria and the mice harbored more bacteria causing early death.



Increased neutrophil recruitment in both models in KO mice was associated with
increased CXCL1/KC and/or CXCL2/MIP2 production.

112



Neutrophils activation assessed by CD11b expression showed increased activation of
neutrophils in KO mice at low dose E. coli but decreased activation of neutrophils in KO
mice at high dose E. coli.

5.3.2: Conclusion
Our results show GRK5 plays a critical role in bacterial clearance and neutrophil
chemotaxis irrespective of low or high doses of E. coli. At low doses, GRK5 deficiency induces
appropriate neutrophil infiltration to enhance clearance of bacteria, whereas at high dose of E.
coli, GRK5 deficiency induces exaggerated neutrophilic infiltration leading to neutrophil
exhaustion causing bacterial overgrowth and quicker death. Our data exemplify the complexity
in the mechanism of progression of pneumonia and suggests modulating GRK5 levels can be
beneficial depending on the patient inflammatory status to achieve patient recovery.

5.3.3: Limitations and future directions
Although increased CXCL1 production from GRK5 deficient mice is contrary to the
GRK5’s positive regulatory role on cytokine production, the overall phenotype in high dose is
comparable to essential role of NFĸB in pneumonia and suggests GRK5 likely mediates NFĸB
pathway. Furthermore GRK5 has been shown to regulate NFĸB signaling either positively or
negatively depending on the cell type. It is likely that GRK5 might negatively regulate NFĸB and
CXCL1 production from epithelial or endothelial cells of lung, thus altering neutrophil
chemotaxis. To answer these questions, GRK5 role in CXCL1 production from hematopoietic
and non-hematopoietic compartments must be ascertained.

113

Together, this dissertation research uncovers novel roles of GRK5 during acute
inflammatory diseases. Through this project, GRK5 was shown to regulate both MyD88 and
TRIF pathway, modulate immune cell apoptosis and development of immune suppression during
poly microbial sepsis, and regulate CXCL1 production thereby enabling neutrophil recruitment
and bacterial clearance. These roles are very critical in the progression of acute inflammatory
diseases and further work on discerning the involved molecular mechanisms will likely lead to
therapeutic strategies to target GRK5 in inflammation.

114

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