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The role of ErbB signaling in dendritic cells during inflammatory bowel disease

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The role of ErbB signaling in dendritic cells during inflammatory bowel disease

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The role of ErbB signaling in Dendritic Cells during Inflammatory Bowel Disease

by

Natalie Pilikian

A Thesis Presented to the
FACULTY OF THE USC KECK SCHOOL OF MEDICINE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)

May 2021

Copyright 2021 Natalie Pilikian
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TABLE OF CONTENTS

List of Tables………………………………………………………..……………………..…....iii
List of Figures………………………………………………………………………..………….iv
Abstract…………………………………………………………………………..…………...….v
Introduction…………………………………………………………………..…….…………….1
Methods………………………………………………………………………….....................20
Results………………………………………………………………………………………….26
Discussion………………………………………………………………………………..…….41
References…………………………………………………………………………………..…46

iii

List of Tables

Table 1. qPCR probes……………………………………………………..…….……………26

iv

List of Figures

Figure 1. Primary and accessory organs of the human gastrointestinal system…….…17
Figure 2. Intestinal tissue………………………….……………………..…….…………….18
Figure 3. Gut-Associated Lymphoid Tissue (GALT) ………………………..…………….18
Figure 4. Interaction between CD80 and CD86 costimulatory molecules on dendritic
cells with their receptors CD152 and CD28on T lymphocytes………..…….…………….19

Figure 5. ErbB family of RTKs and their effects in the cell……………………………….19
Figure 6. IFN-ɣ added to dendritic cells……………………………………….………...….25
Figure 7. Dendritic cells were stimulated with agonists and LPS……………………..…25
Figure 8. EGFR is regulated by DC activation……………………………………...……..29
Figure 9. ErbB2 is regulated by DC activation…………………………………...…….….30
Figure 10. ErbB3 is regulated by DC activation…………………………………….....…..31
Figure 11. ErbB4 is regulated by DC activation……………………………………...……32
Figure 12. TNF is regulated by DC activation……………………………………...………33
Figure 13. IL-12 is regulated by DC activation………………………………...……..……34
Figure 14. EGFR is not differentially regulated by ErbB ligand treatment………………35
Figure 15. ErbB2 is not differentially regulated by ErbB ligand treatment………………36
Figure 16. ErbB3 is not differentially regulated by ErbB ligand treatment………………37
Figure 17. ErbB4 is not differentially regulated by ErbB ligand treatment………………38
Figure 18. Activation-induced Tnf expression by DCs was not affected by ErbB
ligands…………………………………………………………………………………...……...39

Figure 19. Activation-induced IL-12 expression by DCs was not affected by ErbB
ligands………………………………………………………………………………….……….40

v

ABSTRACT

Inflammatory Bowel Disease (IBD) is characterized by chronic inflammation of
the gastrointestinal tract, and it can be caused by a combination of factors including
overaggressive immune cell responses. Disruption of intestinal homeostasis by aberrant
inflammation plays a major role in the pathology of this disease. The intestinal
environment is complex and comprised of interactions between cells of the immune
system, gut microbiota, and intestinal epithelial cells. Therefore, it is crucial to
understand the complexity of the intestinal inflammation that occurs in IBD. In this study,
I sought to determine if a family of growth factor receptors recently identified in other
immune cell types (ErbB family:), EGFR, ErbB2, ErbB3, and ErbB4, may play a role in
the regulation of dendritic cell (DC) function and activation. I also investigated how ErbB
stimulation affects expression of DC-associated cytokines tumor necrosis factor (TNF)
and interleukin (IL)-12. ErbB receptors have many specific functions in various tissue
types, such as cell survival, proliferation, apoptosis, and differentiation of cells.
However, their role in DCs is unknown. In this study, I generated bone-marrow derived
DCs from mice and performed in vitro experiments looking at cytokine production in
activated DCs, a major effector response of these cells that is important for amplifying
the innate immune response to limit pathogens. The data indicates that expression of
the ErbB family is regulated by activation of DCs (with interferon-ɣ and
lipopolysaccharide). Upon activation, EGFR and ErbB2 show elevated expression levels
suggesting they are potentially involved in regulating DC activation or functions. In
contrast, stimulation of DCs with ErbB-specific ligands showed no effect on receptor
expression, suggesting that ligand-dependent feedback mechanisms do not provide
substantial feedback inhibition on regulating ErbB receptor expression. With regard to
DC activation, both of the cytokines, TNF and IL-12 were not significantly altered in
naive or activated DCs, or those treated with ErbB-specific ligands. This study serves as
a first step in understanding the regulation of ErbB signaling in DCs. Future work will be
necessary to understand the role that DC-specific ErbB signaling plays in the chronic
inflammatory response of IBD.
1

INTRODUCTION

BACKGROUND:
The human gastrointestinal (GI) tract is a complex system consisting of distinct
regions, cell types, and microorganisms. It is made up of primary and secondary
organs. The primary organs include the mouth, esophagus, stomach, small intestine,
large intestine, rectum, and anus. The accessory organs are the salivary glands, liver,
gallbladder, and pancreas, which support the digestive process (Figure 1). Within each
of these organs, there are specific functions that support the role of the tissue. The main
functions of the gastrointestinal tract as a whole are digestion, nutrient absorption,
waste excretion, and protection against pathogens (Cheng, 2010). The focus of this
study is the intestine, the predominant interface between your body and the food you
digest. As this area is exposed to external factors such as a rich microbial ecosystem
(your GI microbiota), it also plays a major role in immunity. The large intestine alone
contains approximately 400-500 microbial species, the highest number out of the rest of
the gastrointestinal organs. Many of these microbes play beneficial roles in your body
and typically exist in an equilibrium, but a well-regulated inflammatory response guided
by your intestinal tissue is important for ensuring pathogenic microbes that cause
disease cannot do so, while at the same time not mounting reactions to beneficial,
commensal microbes (Berg, 1996).

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GI TRACT ANATOMY:
Anatomically, the intestine is made up of muscle, mesenchyme, sub-mucosa,
and epithelium. At the microscopic level, these tissues are folded into crypt-villus
structures (Figure 2) that increase absorptive surface area. The intestinal submucosa is
a thin layer of loose connective tissue that surrounds the mucosa. The submucosa is
surrounded by a muscularis layer that has many muscle cells that contract for the
movement of the intestines. There are two layers of muscles, the inner circular and
outer longitudinal muscle layers. There are bands of smooth muscle on the large
intestine that create haustra, which look like pouches.
The intestinal epithelium is a single cell layer that serves as the interface
between the intestinal contents and the body. The structural and immune development
of the intestinal mucosal epithelium of the gut is very particular due to the functions it
serves. This is a simple polarized epithelium, consisting of several cell types with
specialized functions. Some of these cells are enterocytes (absorptive cells), Paneth
cells (source of host defense peptides), goblet cells (source of mucus), and M cells
(provide immune sampling functions). These cells interact with one another to protect
the gut barrier and act as the first line of defense during an immune response. The gut
barrier also includes junctions such as tight junctions, adherens junctions, and
desmosome, which each regulate the passage of molecules and proteins across cells
(McElroy et al., 2018). The transmembrane proteins that make up these junctions cause
intracellular scaffolding and act as sensors and signaling proteins. During inflammation,
these proteins can be disrupted, for example, by tumor necrosis factor (TNF) and
interferon-gamma (IFN-ɣ) which are secreted by immune cells and can disrupt the
3

intestinal barrier. One specialized cell known as a goblet cell produces and secretes
mucus, which helps the protective intestinal barrier between the microbes in the lumen
and the mucosa. They can also present small antigens to dendritic cells (DCs) in the
intestinal lamina propria. Another specialized cell, Paneth cells, are found and remain in
the crypts of the intestinal epithelium near the stem cells in order to protect these
undifferentiated cells from microbial exposure. Disruption of Paneth cells is associated
with inflammatory bowel disease, since they contribute to intestinal homeostasis.
Microfold cells (M cells) are very important specialized cells that are within the intestinal
epithelium covering the lymphoid tissue there. M cells present luminal antigens to
immune cells, mainly the dendritic cells.

GI IMMUNITY:
The intestinal immune system is composed of cells of both the innate immune
response and the adaptive immune response. The innate immune cells, which are
nonspecific and act as fast first-responders, are dendritic cells, macrophages,
neutrophils, NK cells, mast cells, basophils, and eosinophils. Epithelial cells, especially
the M cells and Paneth cells, are sometimes considered part of this response as well.
Macrophages and dendritic cells act as antigen-presenting cells to activate the adaptive
immune cells, which are the B lymphocytes and T lymphocytes. These specific
lymphocytes then undergo stimulation and proliferation in order to fight off pathogens as
part of the adaptive immune response (Yamauchi et al., 2019). The dendritic cells,
which are the focus of this study, are key antigen-presenting cells of the immune
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system. They sense and sample luminal contents. They have long finger-like
projections, which can reach and take up antigens in the intestine in order to present
them to T lymphocytes that are found in the lymphoid organs. Dendritic cells internalize
antigens from M cells through different pathways such as by using toll-like receptors
(TLRs), receptor-mediated endocytosis, or nucleotide-binding oligomerization domain
(NOD) receptors. M cells, which are found within the epithelial layer of the intestine,
sample antigens and passage them to the submucosa to be presented to dendritic cells.
Then, the dendritic cells present these antigens to the lymphocytes, initiating the
activation of B cells and T cells (Figure 3). DCs mediate immunoglobulin isotype
switching to IgA in B cells in the intestinal mucosa, and can also stimulate T cell
differentiation to either cytotoxic or regulatory subtypes (Rescigno, 2010).
The lymphoid tissue of the intestine is known as Gut-Associated Lymphoid
Tissue (GALT). There are two parts of GALT, the effectors and the inductors. The sites
of the inductors are where immune responses are induced, which are known as the
mesenteric lymph nodes, isolated lymphoid follicles, and the Peyer’s patches. This is
where antigen-presenting cells such as dendritic cells activate T cells and B cells. The
effector sites are the lamina propria and the intestinal epithelium, which are where the
lymphocytes reside (McElroy et al., 2018).
Dendritic cells have pattern recognition receptors (PRRs), the most commonly
known ones being toll-like receptors (TLRs). TLRs, which are transmembrane proteins,
recognize and bind to pathogen-associated molecular patterns (PAMPs) that are found
on the surface of foreign microorganisms. One example of a PAMP is bacterial
lipopolysaccharide (LPS). LPS endotoxin has previously been detected in the plasma of
5

inflammatory bowel disease patients, due to the permeability of the gut barrier and
abnormal gut flora. Some TLRs are more specific than others. Their extracellular
component binds to the particular PAMP, and its intracellular component does
downstream intracellular signaling (Caradonna et al., 2000). Some important signaling
pathways that the bound PRR-PAMP complex induce are the nuclear factor κB (NF- κB)
and the mitogen-activated protein kinase (MAPK) pathways. Both of these pathways
can induce the inflammatory response. The NF- κB pathway has been found to regulate
the activation of certain chemokine genes during bacterial infection (McElroy et al.,
2018).
The gut continuously protects the body by crosstalk between all of the species of
microbiota, epithelial cells, and all the other specialized cells found there. The mucus
layer and epithelial cells separate the microbiota from the body. This contributes to the
regulation and homeostasis in the gut, as in the development of the immune cells to
recognize foreign versus indigenous cells. Colonization resistance is a major factor in
the gut (McElroy et al., 2018).

INFLAMMATORY BOWEL DISEASE:
Due to the complexity of the intestinal environment and its many factors, there
can be numerous disruptions to the intestinal homeostasis at many different levels.
When the balance is dysregulated, this may lead to chronic inflammation of the
intestinal tract, known as inflammatory bowel disease (IBD). Though there are many
contributing factors to the development of IBD, an overaggressive immune response is
6

central to the pathology of the disease, and thus the focus of my project. IBD is
subdivided by symptoms and location into Crohn’s disease and ulcerative colitis.
Crohn’s disease is inflammation throughout the colon and terminal ileum, consists of
patchy damaged areas with non-caseating granulomas, and is characterized by
transmural lesions, while ulcerative colitis is inflammation only of the large intestine and
presents as continuous diffuse damage and mucosal ulceration and abscesses in the
intestinal crypts (Xavier et al., 2007). IBD affects one in every 250 Americans and
lowers their quality of life. Its symptoms include persistent diarrhea, weight loss, bloody
stools, abdominal pain, and fatigue. The number of people diagnosed with IBD
continues to increase every year in the United States by about 70,000 cases. Though
the likely causes of IBD are numerous and probably different in each patient, there are
several common factors that have been found to contribute to disease development.
One major contribution is genetic susceptibility. There have been many genes found to
be associated with IBD, such as the NOD2 gene, and the ErbB family of proteins, which
will be discussed later (Xavier et al., 2007). Another factor contributing to IBD is the gut
microbiota. The balance between the commensal intestinal flora and the host immune
response is very important, and if deregulated or if microbes invade through the barrier
epithelium through the M cells, can lead to pathogenesis. Commensal microorganisms
are in continuous crosstalk with the host cells, and they affect the certain cytokines and
chemokines being released at any given time. These may be pro-inflammatory or anti-
inflammatory. Examples of anti-inflammatory cytokines include interleukin (IL)-1 and IL-
10. If these are not balanced or there is an overproduction of pro-inflammatory
cytokines, it can cause prolonged inflammation. Also, T cells have been previously
7

found to be infiltrated in the intestinal mucosa of Crohn’s disease patients (Rogler et al.,
1998). This introduces the next factor, which is the immune response. As mentioned
earlier, the developing immunity in the gut environment is crucial in training the body’s
defense system to identify what is foreign or not, to activate the correct cells, and
respond appropriately. An overactive immune response in the GALT that is imbalanced
may lead to IBD. Lastly, environmental factors, such as smoking, diet, and antibiotic
use, may contribute to IBD.

THE IMMUNE RESPONSE IN IBD:
As DCs play a key role in the initiation of inflammation in the intestine, this study
centers specifically around the dendritic cells’ role in chronic inflammation. While
several factors play a role in IBD development and persistence, dendritic cells are one
of the main players in this disease process. Dendritic cells consist of different subtypes
with a variety of functions. They are formed in the bone marrow from hematopoietic
progenitor cells, and they rely on granulocyte macrophage-colony stimulating factor
(GM-CSF) for proliferation. Dendritic cells are part of the innate immune system, which
is the body’s first line of defense against any inflammation and infection. Being part of
innate immunity, dendritic cells act immediately, and they are non-specific. But the most
important aspect about them is that they are the connection between innate immunity
and adaptive immunity. Adaptive immunity is specific and provides long-lasting
protection. It includes memory T lymphocytes and antibodies. Dendritic cells help relay
a response from innate to adaptive immunity in order to resolve infections and
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inflammation. One dendritic cell is capable of activating about 100-300 T lymphocytes,
making it the most effective of all the antigen-presenting cells. They have PRRs on their
surface, which include TLRs, in order for them to recognize and respond to infection.
They activate naïve CD8 and CD4 T cells (den Hartog et al., 2018). In the chronic
inflammation and microbial imbalance that occurs in IBD, dendritic cells become
activated, these receptors are upregulated, and they produce the appropriate cytokines,
which include IL-12 and TNF. TNF is heavily involved in the intestinal inflammatory
response. One mechanism that TNF is involved in is inducing intracellular pathways
leading to cell proliferation, survival, and cell death. It also affects wound healing in the
gut by its induction of the ErbB-dependent pathway. Most important, however, it is a
regulator in the interaction between dendritic cells and epithelial cells in the intestine.
Anti-TNF antibodies have been used to treat the inflammation in IBD patients (Leppkes
et al., 2014). IL-12 is a key pro-inflammatory cytokine released by dendritic cells that
activates naive T lymphocytes. It has been previously known to mediate intestinal
inflammation in colitis (Monteleone et al., 1997). These cytokines are essential in the
activation of the adaptive immune response. As dendritic cells recognize foreign
structures in the gut using their TLRs during persistent inflammation in IBD, DCs
become activated, and there is upregulation of these receptors. Cytokines IL-12 and
TNF are also produced and secreted during this time (Sun et al., 2015). Intestinal DCs
seem to be the main initiators of this inflammatory response that occurs in IBD.
Dendritic cells are very unique to their environment. They are found in almost
every organ in the body, and this determines their function and phenotype. Furthermore,
they maintain the immune tolerance in the gut. DCs have two functional states:
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immature DCs and mature DCs. The main function of immature DCs is phagocytosis
and macropinocytosis. Immature dendritic cells have a few cytoplasmic projections.
Mature DCs function in antigen presentation and are present in lymphoid organs and
have several distinct long cytoplasmic processes. They also have a very high
expression of costimulatory molecules on their surface. While immature DCs can take
up antigens, they cannot present them on their surface. Only a mature DC can make an
MHC Class II complex with the antigen peptide and present it on its plasma membrane.
For a DC to convert from an immature cell to a mature one, pathogenic ligands or
proinflammatory molecules are sensed. The DCs are then upregulated for a short
period of time. They then go on to upregulate chemokine receptors, an important one
being CCR7, which will be described later. A subset of DCs has also been found to
support the growth of regulatory T cells that perform immune tolerance as part of the
anti-inflammatory response. There have also been DCs found to exhibit mucosal
functions, such as the converting of Foxp3+ regulatory T cells, Th17 cell differentiation,
and promoting IgA class switching. This shows the importance of the function of DCs,
because they are involved in both immune tolerance and the pro-inflammatory immune
response (McNamee et al., 2015). This shows their weight in maintaining and regulating
homeostasis in the intestine. DC function is regulated by its local environment, which
consists of both immune and nonimmune cells, as well as the microbiome. They all
contribute to the intestinal balance, so if any of these are deregulated, the function of
DCs may be affected and this can lead to intestinal disease such as IBD. When DCs
cannot function correctly, they may cause abnormal T cell responses, for example
against the normal intestinal flora. T cells can remain active for too long and contribute
10

to inflammation in the gut, or proinflammatory cytokines can be secreted also
contributing to the inflammation. This could lead to a positive feedback loop in which gut
inflammation becomes chronic and pathological.
A unique aspect of intestinal DCs is that the PRRs on these DCs recognize and
bind to PAMPs on microbes in order to remove them via phagocytosis as the first line of
defense of the innate immune response. This inflammation anergy is important because
the intestinal tissue is very different than other tissues of the body. It holds residence to
a variety of microbiota that it should not react to. Without this immunological non-
responsiveness, chronic intestinal inflammation can occur.
DCs are found in many parts in the intestinal tissue. One is a secondary
lymphoid organ in the intestinal tissue known as Peyer’s patches. Other regions are the
lamina propria of the small and large intestines, isolated lymphoid follicles, and
mesenteric lymph nodes. The secondary lymphoid organs are where mature DCs reside
and express their costimulatory molecules and instruct T cells when appropriate. In the
lamina propria, DCs are able to constantly surveil the contents of the intestinal lumen.
They are part of a group of cells known as lamina propria mononuclear cells, and they
act as the first guards of the gut-associated lymphoid tissues. They dispose of microbes
and can stimulate the adaptive immune response, while simultaneously maintaining a
tolerant gastrointestinal environment. Dendritic cells in the lamina propria act directly in
the maintenance of both tolerance to self and tolerance to foreign antigens in the
intestinal lumen. Lamina propria dendritic cells (LPDCs) express high amounts of MHC
Class II on their surface, which shows their role in the communication with and influence
on the adaptive immune response.
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In order for a T cell to be stimulated and activated to begin the adaptive immune
response, two signals are necessary. The first signal is the presentation of an antigen
by the dendritic cells, with the antigen being presented on an MHC on the surface of the
DCs. The second signal, which is just as important, is the costimulatory signal. These
two steps must occur for the adaptive immune response to be initiated. One signal
alone is not sufficient. These costimulatory molecules, such as CD86 and CD80, are
essential for activating the T lymphocytes during an immune response. Both of the
costimulatory molecules CD80 and CD86 amplify the interaction between dendritic cells
and T cells. These molecules are found on the surface of DCs. The point of contact
between a T cell and a dendritic cell is known as an immunological synapse (IS). While
other molecules, such as B cells, can also activate T cells during an immune response,
these molecules usually have a single immunological synapse with the T cell, whereas
the IS between a dendritic cell and T cell has been found to be multifocal, thus showing
a stronger relationship. A previous study has found that when CD80 and CD86 are
blocked by antibodies, T cell activation by DCs is attenuated, which shows how
important these costimulatory molecules are in the strength of the interaction between T
cells and DCs. Furthermore, cytokine secretion (both TNF and IL-12) was also reduced
when CD80 and CD86 were simultaneously inhibited by antibodies, further confirming
how important the role of these costimulatory molecules is in T cell activation by DCs
(Lim et al., 2012).
CD80 (B7-1) and CD86 (B7-2) molecules act as ligands for CD28 and CD152 on
T cells. CD28 and CD152 have opposing functions in T cell activation. CD152 is for
immune tolerance. Both ligands can bind to either receptor. CD28 receptor has a
12

stimulatory function on T cells, while CD152 has an inhibitory function on T cells. These
two receptors’ structures are different. CD28 is a constitutive membrane receptor, while
CD152 is a more complex receptor that is only expressed when a T cell is active. It is
not expressed in resting T lymphocytes. It also translocates inside the cell and is then
recycled back to the cell surface. CD80 is a dimer and it has two binding sites, one for
CD28 and another for CD152. On the other hand, CD86 is a monomer with a single
binding site. CD28 is monovalent, while CD152 is a bivalent dimer. CD86 binds more
effectively to the CD28 receptor, and CD80 has higher affinity to CD152 (Figure 4).
CD152 has been previously found to have an inhibitory effect on T cells, therefore
reducing immune hyper-responsiveness. When there is no inflammatory stimulus, CD80
prefers to interact with CD152 to prevent T cell interaction. However, after inflammatory
stimuli, CD86 expression is upregulated to activate T-cells and overrides the inhibitory
CD152-CD80 complex effects. DCs predominantly express CD80 during no
inflammation. During inflammation, such as in the presence of LPS, the dendritic cells
mature and express CD86 at high amounts (Sansom et al., 2003).

DENDRITIC CELLS IN IBD:
Migration of DCs:
When DCs respond to infection, they are primarily in an immature state, in which
they internalize antigens. In order for them to present to and stimulate the T cells, they
must mature as well as migrate to secondary lymphoid organs where T cells reside.
Therefore, when DCs are switching from an immature state to a mature state, they
downregulate their phagocytosis and increase their expression of costimulatory
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molecules and MHC. The type of chemokine receptors present and expressed by the
DCs determines their maturation state. C-C motif chemokine receptor 7 (CCR7) has
previously been found to be expressed on the surface of a mature DC for the purpose of
migration (Al-Hassi et al., 2013).
Intestinal lamina propria DCs (LPDCs) require the chemokine CCR7 to migrate
from the lamina propria to the mesenteric lymph nodes. Peyer’s Patches’ DCs are the
most known, and they are the location where DCs train specific T cells to make and
release cytokines IL-4 and IL-10 and specify the T cell to the gut. Much less is known
about the LP-DCs in which the projections of DCs reach into the lumen and sample the
antigens there (Jang et al., 2006). CCR7 is a chemotactic receptor, but it also has other
functions. The ligands of CCR7 are CCL21 and CCL19. The ligands CCL19 and CCL21
have been found to have increased expression in intestinal inflammation. The
significance of CCR7 has previously been shown through CCR7-deficient mice whose
immune responses were very delayed. Besides migration and maturation, CCR7 can
also alter the morphology of DCs, increasing the dendritic cell protrusions during an
immune response. CCR7 has also shown to somewhat lengthen the life span of
dendritic cells that are on their way to the T cells to increase the efficiency of the
immune response (Sánchez-Sánchez et al., 2006). Studies have shown that in IBD,
more specifically Crohn’s disease, there is an increased number of mature DCs with
CCR7 expression, causing high T cell proliferation (Middel et al., 2006). Also, there is
upregulation of CCR7’s ligands, CCL19 and CCL21 (McNamee et al., 2015). CCR7 acts
in an autocrine and paracrine fashion in the bowel wall (Middel et al., 2006). With its
ligands present, there is upregulation of CCR7 on dendritic cells, and mature DCs
14

become trapped in the bowel wall, and the inflammatory response with the T cell
expansion continues during IBD (von der Weid et al., 2011).

THE ROLE OF ERBBs in IBD:
A major class of molecules, the ErbB family of receptor tyrosine kinases (RTKs)
and their ligands are involved in wound healing, intestinal epithelial cell growth, and
survival. The family includes epidermal growth factor receptor (EGFR, aka ErbB1),
ErbB2, ErbB3, and ErbB4. As cell surface receptors, they can be targeted and used for
therapeutic intervention in IBD. Many biochemical pathways are activated during
inflammation of the gut. ErbBs have been previously found to have a major role in
intestinal homeostasis, so their deregulation may cause intestinal pathology.
There are various growth factors acting in the GI tract, which contribute to
processes such as wound repair, immune cell regulation and growth, and maintenance.
Growth factor signaling consists of a series of steps, beginning with ligand release,
extracellular binding of the ligand to a corresponding receptor, and signal transmission.
The ErbBs, since they are RTKs, are the receptors that take signals from their ligands
outside of a cell based on their specificity and affinity and help transmit these changes
into the cell. Then, intracellular signaling occurs through a variety of pathways and
activation of transcription factors which result in transcriptional regulation (Figure 5)
(Frey and Polk, 2014).
Each member of the ErbB family of receptors is an integral membrane protein,
consisting of a unique extracellular domain, a transmembrane domain, and a
cytoplasmic domain, as well as proteolytic cleavage sites. Upon ligand binding, these
15

RTKs either homodimerize or heterodimerize and are activated through increased
kinase activity. Ligands bind directly to EGFR/ErbB1, ErbB3, and ErbB4, but ErbB2
cannot directly bind to ligands. Therefore, ErbB2 must form a heterodimer in order to be
fully active. ErbB3 can bind ligand but has low kinase activity, so it also needs to form
heterodimers to signal. After forming homodimers and heterodimers with one another,
this causes transphosphorylation of the receptors’ tyrosine residues on their cytoplasmic
domains. Then, these act as docking sites for a vast array of other proteins, and
downstream signaling occurs. An example ligand for EGFR is EGF, a key ligand for
ErbB4 is NRG4 (Neuregulin-4), and there are several NRGs like NRG1 that bind both
ErbB4 and ErbB3. Previous studies show that EGF, NRG1, and NRG4 have anti-
inflammatory actions during colitis and reduce the severity of disease in experimental
models (Schumacher et al., 2017). There are many combinations of ligands and
receptor pathways, as well as intracellular pathways that overlap and are connected,
making signaling very complex, which allows for control and regulation of actions in the
intestinal tissue. So, overexpression or loss of expression can disturb the balance of the
GI tract, thus promoting disease.
In previous studies, in cultured intestinal cells, activation of EGF has had a
protective action during an inflammatory response, binds to EGFR homodimers and
EGFR/ErbB2 heterodimers. Upon binding, a variety of pathways are activated, including
PI3-kinase, ERK/MAPK, JNK, and more, leading to many outcomes (Frey et al., 2013).
EGFR has been found to reduce apoptosis and stimulate proliferation of intestinal
epithelial cells. It may also cause wound healing and migration. EGFR is expressed on
the basolateral surface of the epithelium in the intestine, so EGF must cross this barrier
16

from the lumen to assist with wound repair and healing and immune surveillance during
injury. EGF expression increases in response to insult to the GI mucosa in order to
restore the homeostasis of the gut. In chronic inflammation, EGF expression increases
and consequently EGFR activation increases. Furthermore, NRG4 binding to ErbB4
contributes to colon cell survival (Bernard et al., 2012) but not migration or proliferation
as EGFR does (Almohazey et al., 2014). This further illustrates the complexity and
selectivity of these different combinations of ligands, RTKs, growth factors, and cellular
pathways. The better these are understood, the more specific and effective IBD therapy
will be.
Previous findings have shown that ErbB4, ErbB3, and ErbB2 knockout mice have
impaired recovery from colitis (Ni et al., 2019). Furthermore, when ErbB4 is activated by
NRG4, injury from colitis is reduced, and this involves the PI3-kinase/AKT pathway.
These findings show the different roles that ErbB signaling plays and how they work
together to protect the intestinal environment. Previous studies have shown that ErbB
signaling is interrupted in IBD, partly due to the reduction in the ErbB ligands (Frey and
Polk, 2014). However, because of the complexity of the ligand and receptor
combinations, contradictory results have been found.
Currently, dendritic cells are difficult to identify, since they don’t have a lineage-
specific marker (Dilioglou et al., 2003). Furthermore, studies on the role of biophysical
interactions between T-cells and APCs, particularly dendritic cells, remain limited at this
point. Since the ErbB family of receptors has been found to be involved in intestinal
inflammation, such as in wound healing, migration, proliferation, and immune
maintenance, this study investigates the role of ErbB signaling in dendritic cells during
17

IBD. The hypothesis of this study is that ErbB receptors regulate dendritic cells activity,
and the questions to test this hypothesis are: what do the ErbB ligands do to receptor
expression, and how does ErbB stimulation affect expression of DC-associated
cytokines TNF and IL-12?

Figure 1. Primary and accessory organs of the human gastrointestinal system. Primary:
mouth, esophagus, stomach, small intestine, large intestine, rectum, and anus.
Accessory: salivary glands, liver, gallbladder, and pancreas. (Adapted from
https://smart.servier.com/)

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Figure 2. Intestinal tissue: The submucosa, a thin layer of loose connective tissue,
surrounds the mucosa. There are two layers of muscles, the inner circular layer and the
outer longitudinal layer. The villi, outward projections, point towards the intestinal lumen.

Figure 3. Gut-Associated Lymphoid Tissue (GALT).

19

Figure 4. Interaction between CD80 and CD86 costimulatory molecules on dendritic
cells with their receptors CD152 and CD28 on T lymphocytes.

Figure 5. ErbB family of RTKs and their effects in the cell. Upon ligand binding, homo-
or heterodimerization, and transphosphorylation of the tyrosine kinases on the
cytoplasmic domain of the receptors, various downstream signaling pathways are
activated which are involved in proliferation, migration, cell survival, wound healing, and
apoptosis.

20

METHODS

Animals:
Animal use was approved by the Children's Hospital Los Angeles Institutional
Animal Care and Use Committee (Animal Welfare Assurance #A3276-01). In this
protocol, mice are housed with free access to water and chow at all times in the
AAALAC-accredited animal care facility. C57Bl/6J background mice obtained from
Jackson Laboratory aged 12 weeks were used for experiments.

Bone-Marrow Derived Dendritic Cells:
Circulating dendritic cells in vivo are derived from bone marrow. I performed an
experimental technique where bone marrow was isolated from mice, then treated with
specific growth factors to differentiate the stem cells in bone marrow to dendritic cells.
For this protocol, mice were sacrificed by humane isoflurane methods whereby
isoflurane was added by dropper to a chamber. The mouse was placed in chamber for
five minutes until it was euthanized, followed by cervical dislocation. The femur and tibia
were then removed and cleaned, and their epiphyses were cut. Dendritic cell media
(DCM) was prepared containing 500mL DMEM (Dulbecco’s Modified Eagle Medium),
10% (55mL) fetal bovine serum, and 1% (5.5mL) penicillin-streptomycin solution. This
media is commonly used to grow and culture mammalian cells. Next, a 23-gauge
needle with a 10mL syringe filled with this DMEM media was used to flush the bone
marrow from the bones through a 40 micrometer Nytex mesh filter into a 50mL tube.
Then the needle was removed from the syringe, and any remaining cells were pushed
down through the filter using the plastic stopper. A small amount of additional medium
21

was added to filter through the remaining cells. These bone marrow cells were then
centrifuged at 300 RCF (Relative Centrifuge Force) for 5 minutes, and then the
supernatant was removed. Next, 5 mL 10X red blood cell (RBC) lysis buffer was diluted
by adding 45mL of water to it in a 50mL tube to prepare a 1X solution. 25 mL of 1X RBC
lysis buffer was added to the pellet, and the pellet was resuspended. This solution was
left to sit for 5 minutes to lyse RBCs from the collected bone marrow. Next, the cells
were again centrifuged at 300 RCF for 5 min and the supernatant was removed. In this
50mL tube, 30 mL of DCM was added to the bone marrow cell pellet. After re-
suspension, 15mL was added to 2 culture dishes. Then, 20 ng/mL of GM-CSF
(granulocyte-macrophage colony-stimulating factor) was added to induce the
differentiation of dendritic cells. The plates were then placed in an incubator set to 37
degrees Celsius at 5% CO2 for 4 days.
On day four, 20 ng/mL of GM-CSF was again added to each of the plates to
continue differentiation of the bone marrow cells. On day 7, loosely adherent cells were
removed, washed, counted, and placed into wells for experimentation. First the DCM
media was warmed up for 20 minutes in a warm water bath, which is 37 degrees
Celsius. Next, loosely adherent dendritic cells were removed by pipetting the fluid up
and down on the cells, then transferred to separate 15mL tubes. These tubes were then
centrifuged at 300 RCF for 5 minutes and the supernatant was removed. To ensure
similar levels of cells in each experiment, the cells were counted. This was performed
by first adding 40 microliters of the cell solution to a microcentrifuge tube, and then
adding 40 microliters of Trypan blue dye (this dye is taken up by dead cells and allows
us to exclude them from the count). The cell solution and the dye have a 1:1 ratio. This
22

mixture was then put onto an appropriate chamber slide and the slide was inserted into
a cell counter. After counting, an appropriate volume of DCM containing 200,000 cells
was transferred to each well of a 12-well plate. There was a total volume of 2mL in each
well (Figure 6).
For experiments, one set of wells were labeled as unstimulated dendritic cells,
and the other set were stimulated dendritic cells. To elicit activation of these cells, I
used a 2-step process where cells were given signal 1 (interferon-ɣ) overnight, followed
by treatment with signal 2 (lipopolysaccharide/LPS) the following day (Figure 6). IFN-ɣ
is a cytokine of both innate and adaptive immunity that activates dendritic cells. LPS is a
bacterial endotoxin that stimulates dendritic cells as a PAMP during the innate immune
response.
To test the impact of ErbB receptor tyrosine kinase signaling in these cells, I
treated them with agonists stimulating activation of ErbBs: epidermal growth factor
(EGF), neuregulin-1 (NRG1), and neureglin-4 (NRG4) at the same time as LPS (100
ng/ml) treatment (Figure 7). As a control for vehicle delivery, Phosphate-Buffered Saline
(PBS) was added to unstimulated and stimulated control wells. EGF (100 ng/ml), NRG1
(100 ng/ml), and NRG4 (100 ng/ml) were added to respective wells. The wells were
then placed into the incubator at 37 degrees Celsius for 6 hours before collection. (See
Figure 7 for complete final content in each well.)

23

RNA extraction and isolation:
To determine the effect of growth factors on ErbB receptor tyrosine kinase
expression and activation of dendritic cells, I isolated cells and extracted the RNA using
the Omega Bio-Tek EZNA Total RNA Kit 1 (#R6834). For this, the cells from each well
were collected into corresponding labeled microcentrifuge tubes. The tubes were
centrifuged at 13,000 RCF for 2 minutes, then supernatant was aspirated. Then 350
microliters of cell lysis buffer was added to each well, and the liquid in each well was
individually pipetted up and down to mix. The liquid of each well was placed into
corresponding labeled “homogenizer” tubes (Omega homogenizer columns HCR003),
which were placed on top of white/clear sterile tubes. These were centrifuged for 2
minutes at a speed of 20,000 RCF to complete cell lysis and release of cell contents
including RNA. The flow-through was mixed 1:1 with 350mL of RNase-free 70%
ethanol-water mixture to precipitate the RNA. This RNA-containing mixture was then
transferred to corresponding labeled binding mini column. These mixtures were then
centrifuged for 1 minute at 10,000 RCF to bind the RNA to the column and the flow-
through was discarded. Then 500 microliters of RNA Wash Buffer I was added, followed
by centrifuging again for 1 minute at 10,000 RCF and the flow-through was discarded.
Next, 500 microliters of RNA Wash Buffer II was added to the tubes, then the tubes
were centrifuged once again for 1 minute at 10,000 and the flow-through was discarded.
This last step with the RNA Wash Buffer II and centrifuge was repeated one more time.
Then these bind mini columns were placed into new sterile microcentrifuge tubes. Next,
50 microliters of nuclease-free water were added to each tube, and then the tubes were
24

centrifuged at maximum RCF 20,000 for 2 minutes. This last step re-suspended the
RNA from the columns into water.

cDNA conversion:
Isolated RNA was converted into cDNA in preparation for expression level
analysis, using the Applied Biosystems™ High-Capacity cDNA Reverse Transcription
Kit (4368814). For this step, a master mix was prepared, consisting of 10x Reverse
Transcriptase Buffer, 10x random oligonucleotide primers, DNTP mix, and Reverse
Transcriptase (the enzyme which converts RNA to cDNA). Then, to individual 0.2
microliter PCR tubes, 10 microliters of master mix were added along with 10 microliters
of each individual corresponding RNA sample. These tubes were then placed in a
thermal cycler set to an appropriate program to enable generation of the cDNA over 40
cycles.

Real-time quantitative polymerase chain reaction (qPCR):
To determine the expression level of the cytokines and ErbB receptors, real-time
quantitative polymerase chain reaction (qPCR) was performed on both the stimulated
and unstimulated samples. The gene expression was being evaluated relative to mouse
HPRT (Hypoxanthine phosphoribosyltransferase), which served as the endogenous
control (Table 1). The samples were then run on a OneStep ThermoCycler by Applied
Biosystems. Fold change was calculated using the 2−ΔΔCt method.
25

Figure 6. IFN-ɣ added to dendritic cells. Each labeled well with an appropriate volume
of DCM containing 200,000 cells. There was a total volume of 2mL in each well. IFN-ɣ
was added to the stimulated dendritic cells.

Figure 7. Dendritic cells were stimulated with agonists and LPS. To test the impact of
ErbB receptor tyrosine kinase signaling in these cells, the cells were treated with
agonists stimulating activation of ErbBs (epidermal growth factor (EGF), neuregulin-1
(NRG1), and neureglin-4 (NRG4) at the same time as LPS treatment.

26

Gene: Product code:
mHPRT Mm03024075_m1
Mouse IL-12 Mm00434169_m1
Mouse TNF Mm00443258_m1
mEGFR Mm01187858_m1
mErbB2 Mm00658541_m1
mErbB3 Mm01159999_m1
mErbB4 Mm01256793_m1
Table 1. qPCR probes. To determine the expression level of the cytokines and ErbB
receptors, real-time quantitative polymerase chain reaction (qPCR) was performed on
both the stimulated and unstimulated samples. The gene expression was evaluated
relative to mouse HPRT, which served as the loading control gene.

RESULTS

Dendritic cells express ErbB receptor tyrosine kinases:
ErbB family members have previously been detected in immune cells, however
their expression and role in dendritic cells are not well known. As a first step to
understand their role in these cells, I measured expression of the four ErbB family
receptor tyrosine kinases in unactivated naïve dendritic cells and in cells activated with
IFN- ɣ and LPS. This was performed by RNA expression analysis by qPCR as
described in the methods. DC activation induced EGFR expression and ErbB2
expression, but repressed ErbB3 expression. It had no effect on ErbB4 expression. The
27

qPCR results suggest that EGFR may regulate dendritic cell activity, due to its spike in
expression (Figure 8) (p=0.07). ErbB2, which also increased in stimulated DCs, may
also be involved in regulating DCs (Figure 9) (p=0.21). Although the induction of EGFR
and Erbb2 were did not achieve significance, they did trend toward a significant
increase. However, ErbB3 expression significantly decreased in activated DCs
compared to unactivated DCs, suggesting that repression of this receptor may be
involved in DC activation (Figure 10) (p<0.0001). ErbB4 showed no difference in its
expression between stimulated and unstimulated DCs (Figure 11). The cytokines TNF
and IL-12 were induced by DC activation. TNF had a significant increase in expression
(Figure 12) (p<0.0001). IL12, although insignificant, trended towards a significant
increase in expression (Figure 13) (p=0.07).

ErbB-family ligand treatment do not alter ErbB receptor expression:
As expression of these receptors is sometimes regulated by feedback loops, I
looked at what the ligands for these ErbB receptors did to receptor expression.
Epidermal growth factor (EGF), neuregulin-1 (NRG1), and neuregulin-4 (NRG4) were
then tested as these ligands can activate signaling through EGFR, ErbB2, ErbB3, and
ErbB4. In particular, EGF is known to activate EGFR, NRG1 activates ErbB3 and
ErbB4, and NRG4 activates ErbB4 (Schumacher et al., 2017). Though trends were
detected for induction of ErbB2 by NRG1 (Figure 15), and for induction of ErbB4 by
NRG4 treatment (Figure 17), neither of these results were significant. This suggests that
ligand-dependent feedback mechanisms do not provide substantial feedback inhibition
on regulating ErbB receptor expression (Figures 14-17).
28

ErbB ligand treatment do not alter IL-12 and TNF levels in activated dendritic
cells, one marker of dendritic cell activation:
Lastly, I looked at what these same ligands did to cytokine expression. When
DCs are activated in the body, they release cytokines. Here I tested the cytokines TNF
and interleukin (IL)-12, as a measure of dendritic cell activation (Neurath, 2014).
Surprisingly, neither TNF nor IL-12 levels were altered by ErbB ligand treatment, either
in unstimulated DCs or DCs that were stimulated with IFN-ɣ and LPS (Figures 18 and
19).

29

Figure 8. EGFR is regulated by DC activation. Bone marrow-derived dendritic cells
were activated by IFN-gamma for 24 hours and LPS for 6 hours. EGFR levels were
determined by qPCR analysis. n=5 independent experiments. P-value did not achieve
significance but trends towards a significant increase of EGFR.

30

Figure 9. ErbB2 is regulated by DC activation. Bone marrow-derived dendritic cells
were activated by IFN-gamma for 24 hours and LPS for 6 hours. ErbB2 levels were
determined by qPCR analysis. n=5 independent experiments. P-value did not achieve
significance but trends towards a significant increase of ErbB2.

31

Figure 10. ErbB3 is regulated by DC activation. Bone marrow-derived dendritic cells
were activated by IFN-gamma for 24 hours and LPS for 6 hours. ErbB3 levels were
determined by qPCR analysis. n=5 independent experiments. DC activation significantly
repressed ErbB3 expression.

32

Figure 11. ErbB4 is regulated by DC activation. Bone marrow-derived dendritic cells
were activated by IFN-gamma for 24 hours and LPS for 6 hours. ErbB4 levels were
determined by qPCR analysis. n=5 independent experiments. DC activation had no
effect on ErbB4 expression (N.D=Not Detected).

33

Figure 12. TNF is regulated by DC activation. Bone marrow-derived dendritic cells
were activated by IFN-gamma for 24 hours and LPS for 6 hours. TNF levels were
determined by qPCR analysis. n=5 independent experiments. DC activation significantly
increased TNF expression.

34

Figure 13. IL-12 is regulated by DC activation. Bone marrow-derived dendritic cells
were activated by IFN-gamma for 24 hours and LPS for 6 hours. IL-12 levels were
determined by qPCR analysis. n=5 independent experiments. P-value did not achieve
significance but trends towards a significant increase of IL-12.

35

Figure 14. EGFR is not differentially regulated by ErbB ligand treatment. Bone
marrow-derived dendritic cells were activated by IFN-gamma for 24 hours and LPS for 6
hours, +/- ErbB ligands as indicated. EGFR levels were determined by qPCR analysis.
n=5 independent experiments.

36

Figure 15. ErbB2 is not differentially regulated by ErbB ligand treatment. Bone
marrow-derived dendritic cells were activated by IFN-gamma for 24 hours and LPS for 6
hours, +/- ErbB ligands as indicated. ErbB2 levels were determined by qPCR analysis.
n=5 independent experiments. Trends were detected for induction of
ErbB2 by NRG1 although not significant.

37

Figure 16. ErbB3 is not differentially regulated by ErbB ligand treatment. Bone
marrow-derived dendritic cells were activated by IFN-gamma for 24 hours and LPS for 6
hours, +/- ErbB ligands as indicated. ErbB3 levels were determined by qPCR analysis.
n=5 independent experiments.

38

Figure 17. ErbB4 is not differentially regulated by ErbB ligand treatment. Bone
marrow-derived dendritic cells were activated by IFN-gamma for 24 hours and LPS for 6
hours, +/- ErbB ligands as indicated. ErbB4 levels were determined by qPCR analysis.
n=5 independent experiments. Trends were detected for induction of
ErbB4 by NRG4 treatment, but these results were not significant.

39

Figure 18. Activation-induced Tnf expression by DCs was not affected by ErbB
ligands. Bone marrow-derived dendritic cells were activated by IFN-gamma for 24
hours and LPS for 6 hours, +/- ErbB ligands as indicated. Tnf levels were determined by
qPCR analysis. n=5 independent experiments.

40

Figure 19. Activation-induced IL-12 expression by DCs was not affected by ErbB
ligands. Bone marrow-derived dendritic cells were activated by IFN-gamma for 24
hours and LPS for 6 hours, +/- ErbB ligands as indicated. Il-12 levels were determined
by qPCR analysis. n=5 independent experiments.

41

DISCUSSION

Dendritic cells are key immune cells in mediating inflammation that play a
significant role as the bridge between innate and adaptive immunity. In particular, they
have identified roles in amplifying the innate immune response (e.g. recruitment of
neutrophils, macrophages) as well as activating adaptive immune responses (i.e.
stimulating cytotoxic T cells) important for competent reaction to infection or injury
(Rescigno, 2010). DCs perform these functions through a variety of processes including
amplified cytokine release, antigen capture and presentation, migration to lymph nodes,
and other means (Middel et al., 2006, Rescigno, 2010). In this study, I sought to
determine if a family of growth factor receptors recently identified in other immune cell
types (ErbB family), may play a role in the regulation of DC function and activation. To
do this, I generated bone-marrow derived DCs from mice and performed several in vitro
analyses.
The data suggests that the ErbB RTKs potentially have a role in the regulation of
DCs upon stimulation by IFN-ɣ and LPS. Clear changes in expression of the panel of
ErbBs after activation suggest a functional role, though this will have to be confirmed.
While the data illustrate regulation of ErbB receptors in DCs during activation, further
research needs to be done to learn what these receptors are actually doing in DCs
during the inflammatory response. Also, ErbBs have various functions in the tissue,
ranging from wound repair, proliferation and migration of epithelial cells (Frey and Polk,
2014), and death of proinflammatory macrophages (Schumacher et al., 2017). Further
research is necessary to understand the downstream actions during different phases of
the inflammatory response.
42

Dendritic cell activation induced EGFR and ErbB2 expression. EGFR drives
proliferation, wound repair, apoptosis, and migration of epithelial cells, and is increased
during inflammation to restore intestinal homeostasis. Its role in immune cells, however,
is poorly understood. Similarly, ErbB2 is involved in the recovery of intestinal injury
(Zhang et al., 2012), but its function in immune cells is much more obscure than its
effects on epithelial cells which generally trend towards survival and growth.
In contrast to EGFR and ErbB2, ErbB3 levels were suppressed by dendritic cell
activation. Erbb3 is involved in the recovery of intestinal injury (Zhang et al., 2012), but
like EGFR and ErbB2, its function in immune cells is largely unexplored. The coordinate
and inverse regulation of EGFR and ErbB2 versus ErbB3 suggests an important role in
DC function, which has yet to be identified.
In terms of DC activation, I tested two cytokines. TNF and IL-12 are well-known
to be induced in DCs following stimulation (Neurath, 2014). TNF may be mediating
crosstalk between the dendritic cells of the innate immune system and the epithelial
cells in the intestinal tissue, and therefore participating in the wound healing, cell death,
and permeability during intestinal inflammation (Leppkes et al., 2014). IL-12 secretion
from intestinal dendritic cells may be mediating the proinflammatory interaction between
the DCs and the T cells in order to stimulate T cells to proliferate and begin a cytotoxic
response in the tissue (Neurath, 2014). Therefore, TNF and IL-12 are regulating the
chronic inflammatory response that contributes to IBD. However, ligands for ErbB
receptors did not alter TNF and IL-12 levels in DCs, regardless of cell activation state.
This suggests that either the basic process of DC activation is not substantially affected
by ErbBs, or that the EGFR and ErbB2 expressed are already maximally activated by
43

endogenous ligand and thus additional ligand has no effect. Alternatively, it is possible
that there are differences in other cytokines known to be released by activated DCs.
These include IL-10, IL-6, and IL-1 (Rogler et al., 1998). Future work will identify
whether these additional cytokines are altered in DCs by ErbB ligand, and whether
timing and concentration of DC activation or ErbB ligand treatment have an effect on
these outcomes. An additional question raised by my work is whether ErbB3
downregulation is necessary for DC activation, which would not be determined by ligand
treatments in these experiments but could be tested by ectopic overexpression of the
receptor in future studies.
Now that I know that ErbB receptors are regulated in DCs, but I don’t know what
they are doing yet, further research is needed to gain a deeper understanding of their
role in these cells. Going forward, it could be more effective to test the cytokines at
different time points. Other future aims are to test amount of protein expression for
correlation with the amount of RNA expression to confirm the results of this study, since
RNA expression does not always equal protein expression, especially in living cells in
the human body. Then, testing these responses using bone marrow-derived DCs which
are deficient in EGFR, ErbB2, ErbB3, or ErbB4 will indicate which receptors are
required.
Additional functional assays for dendritic cell activation with ligand treated
cells can be done with testing the CD86 and CD80 costimulatory molecules, and then
investigating the influence of these costimulatory molecules on the strength of the
interaction between DCs and T cells. Presentation of an antigen on the MHC II on the
surface of DCs is a crucial step in the initiation of the adaptive immune response. An
44

assay testing for MHC II expression on activated and naïve dendritic cells and further
evaluation of consequent T cell activation can be performed. Since CCR7 is a major
chemotactic receptor involved in the migration of intestinal DCs in the lamina propria to
the mesenteric lymph nodes in order to activate T cells, a functional assay on mature
DCs could be performed to test the migration of DCs, with and without the presence of
the ligands of CCR7, which are CCL21 and CCL19. Additionally, as DCs present
antigen to B-cells and influence the production of antibodies (Rescigno, 2010), further
experiments on DC-B-cell proliferation could be performed in cells in which ErbB
signaling is experimentally targeted.
Dendritic cells not only interact with other immune cells, but also can
influence the function of the intestinal epithelium, the lining of the intestinal tract. An
overabundance of DC-released cytokines may promote or inhibit epithelial function. In
particular, since EGFR and its corresponding ligand EGF have been previously found to
be involved in wound repair, testing DCs with an EGFR inhibitor and seeing the effects
this has on wound healing in co-culture experiments would be effective in further
exploring the role of ErbBs in DCs and their potential involvement in IBD.
This study lays the foundation for the beginning of the understanding of the role
of ErbB receptors and cytokines in the regulation of intestinal dendritic cell activation
during the chronic inflammatory response of IBD. Knowing that there is involvement of
these RTKs is a crucial first step in the exploration towards learning what the
homodimers and heterodimers of these receptors and their ligands are doing
downstream, the many diverse pathways they are activating or inactivating, and how
this is specifically contributing to the development and continuous inflammation
45

characterized by IBD. This allows for more opportunities for more effective therapeutic
treatments for this chronic pathological condition.

46

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mouse colon epithelial cell survival. Laboratory investigation, 92(3), pp.437-450.

Abstract (if available)

Abstract Inflammatory Bowel Disease (IBD) is characterized by chronic inflammation of the gastrointestinal tract, and it can be caused by a combination of factors including overaggressive immune cell responses. Disruption of intestinal homeostasis by aberrant inflammation plays a major role in the pathology of this disease. The intestinal environment is complex and comprised of interactions between cells of the immune system, gut microbiota, and intestinal epithelial cells. Therefore, it is crucial to understand the complexity of the intestinal inflammation that occurs in IBD. In this study, I sought to determine if a family of growth factor receptors recently identified in other immune cell types (ErbB family:), EGFR, ErbB2, ErbB3, and ErbB4, may play a role in the regulation of dendritic cell (DC) function and activation. I also investigated how ErbB stimulation affects expression of DC-associated cytokines tumor necrosis factor (TNF) and interleukin (IL)-12. ErbB receptors have many specific functions in various tissue types, such as cell survival, proliferation, apoptosis, and differentiation of cells. However, their role in DCs is unknown. In this study, I generated bone-marrow derived DCs from mice and performed in vitro experiments looking at cytokine production in activated DCs, a major effector response of these cells that is important for amplifying the innate immune response to limit pathogens. The data indicates that expression of the ErbB family is regulated by activation of DCs (with interferon-ɣ and lipopolysaccharide). Upon activation, EGFR and ErbB2 show elevated expression levels suggesting they are potentially involved in regulating DC activation or functions. In contrast, stimulation of DCs with ErbB-specific ligands showed no effect on receptor expression, suggesting that ligand-dependent feedback mechanisms do not provide substantial feedback inhibition on regulating ErbB receptor expression. With regard to DC activation, both of the cytokines, TNF and IL-12 were not significantly altered in naive or activated DCs, or those treated with ErbB-specific ligands. This study serves as a first step in understanding the regulation of ErbB signaling in DCs. Future work will be necessary to understand the role that DC-specific ErbB signaling plays in the chronic inflammatory response of IBD.

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Asset Metadata

Creator Pilikian, Natalie (author)

Core Title The role of ErbB signaling in dendritic cells during inflammatory bowel disease

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Publication Date 04/15/2021

Defense Date 03/15/2021

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag Dendritic cells,ErbB,IL-12,inflammatory bowel disease,OAI-PMH Harvest,TNF

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Zandi, Ebrahim (committee chair), Frey, Mark (committee member), Li, Jie (committee member)

Creator Email nataliepilikian@gmail.com,pilikian@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-443054

Unique identifier UC11667441

Identifier etd-PilikianNa-9463.pdf (filename),usctheses-c89-443054 (legacy record id)

Legacy Identifier etd-PilikianNa-9463.pdf

Dmrecord 443054

Document Type Thesis

Rights Pilikian, Natalie

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA