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Negative Regulation of Inflammation: Implications for Inflammatory Bowel Disease and Colitis Associated Cancer
Daniel E. Rothschild
Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
In Biomedical and Veterinary Sciences
Irving C. Allen, Chair Nick Dervisis
Tanya LeRoith Liwu Li
Kenneth Oestreich
September 21, 2018 Blacksburg, Virginia
Keywords: IRAK-M, inflammation, organoid, IBD, cancer
Negative Regulation of Inflammation: Implications for Inflammatory Bowel Disease and Colitis Associated Cancer
Daniel E. Rothschild
ABSTRACT
The ability to sense and respond to external environmental signals is closely regulated
by a plurality of cell signaling pathways, thereby maintaining homeostasis. In particular,
the inflammatory signaling cascade contributes to cellular homeostasis and regulates
responses prompted by external stimuli. Such responses are diverse and range from a
variety of processes, including tissue repair, cell fate decisions, and even immune-cell
signaling. As with any signaling cascade, strict regulation is required for proper
functioning, as abnormalities within the pathway are often associated with pathologic
outcomes. A hyperactive inflammatory response within the gastrointestinal tract, for
example, contributes to inflammatory bowel disease (IBD), presenting as Crohn’s
disease or ulcerative colitis. Furthermore, as a chronic condition, IBD is associated with
an increased risk for the development of colitis-associated cancer.
In order to resolve inflammation and thus restore homeostasis, negative regulation may
be utilized to mediate the activity of inflammatory molecules. The mechanistic action of
a specific negative regulator of interest, interleukin receptor associated kinase M (IRAK-
M), is explored in detail within the present dissertation. Investigation of IRAK-M in
mouse models of colitis, which mimics human IBD, and in mouse models of
inflammation-driven tumorigenesis, which models colitis associated cancer,
demonstrated that loss of this molecule contributes to host protection. Therefore, IRAK-
M may be a suitable target for inhibition in order to advance therapeutic options for
human patients afflicted with a GI-related inflammatory disease, such as IBD and colitis
associated cancer.
Furthermore, an ex vivo method that models the interaction of intestinal epithelial cells
with microbes present in the GI tract was optimized and is described in the present
dissertation. This method takes advantage of primary intestinal derived organoids, also
termed “mini-guts”, which display similar features corresponding to intestinal tissue in
vivo. For this reason, the use of “mini-guts” has several advantages, particularly for the
enhancement of personalized medicine. The method discussed herein aims to
normalize experimental conditions in order to enhance reproducibility, which can further
be used to uncover microbial-epithelial interactions that contribute to a pathological
state, such as IBD. Finally, this method of intestinal epithelial cell culture was utilized to
evaluate the role of a protein, termed NF-B inducing kinase (NIK), in intestinal
epithelial cell growth and proliferation. Ultimately, ex vivo organoid culture can serve as
an important model system to study the contribution of NIK in intestinal stem cell
renewal, cancer progression, as well as in maintenance of the integrity of the
gastrointestinal barrier.
Negative Regulation of Inflammation: Implications for Inflammatory Bowel Disease and Colitis Associated Cancer
Daniel E. Rothschild
GENERAL AUDIENCE ABSTRACT
Inflammation is a tightly regulated physiologic process that is employed by body
systems such as the gastrointestinal (GI) tract to handle pathogenic insult, aid in wound
healing, and help prevent infections. When abnormal inflammatory responses occur,
this can lead to the progression of severe diseases such as ulcerative colitis and
Crohn's disease. When inflammation persists in the GI tract, such as in inflammatory
bowel disease, this can predispose patients to the development of inflammation-
associated colorectal cancer. In order to improve the treatment options for patients
afflicted with these maladies, this dissertation is aimed at studying the signaling
pathways of the innate immune system that regulate such inflammatory responses.
Furthermore, this body of work encompasses a detailed method for isolating and
culturing intestinal stem cells, which can be applied in personalized medicine for
patients with intestinal diseases. This method was utilized in this dissertation to study
genetically modified intestinal stem cells, and can further be used to investigate the
interactions of intestinal epithelial cells with pyogenic bacteria that contribute to
inflammatory maladies in the GI tract.
Acknowledgements
First and foremost I would like to thank Dr. Irving Coy Allen for giving me the opportunity
to be a part of his lab. I am immensely grateful for the mentorship he has provided, and
for his open door policy and willingness to always discuss science. I have learned many
lessons, but most importantly how to think critically, and how to grow from failure. I will
take many of the lessons I learned as your protégé with me. Your mentorship has made
me a more critical and fundamentally better scientist.
I would like to thank my committee members for the helpful guidance, critiques, and
their time. Your support has helped me grow as a person and a scientist. For this, I am
immensely grateful.
I would also like to thank the other graduate students and the undergraduates in the
Allen lab; specifically, Dylan, Sheryl, Kristin, and Veronica. Your help, advice, company
and friendship has helped make the lab a positive and cohesive environment. I wish
everyone the best of luck in their future endeavors. I would also like to thank Becky
Jones for all her help and support.
I often question if I could have made it through this endeavor without the help of Dr.
Tara Srinivasan. I am forever indebted for your help throughout my career, and grateful
for your friendship. I will always cherish our conversations and it is safe to say that I
would not be where I am today without you.
I would also like to thank Dr. Renata Goncalves. Your critical suggestions always left
me in awe of your brilliance as a scientist; you are one of the most impressive I've come
across. You embody why we need more women in science.
Lastly, I would like to thank my extended family, my siblings and my parents. Staying
true to my personality, no words. I hope to see you out on the lake, cheers.
Attribution
Chapter 1 includes portions of previously published work that was co-first authored by
myself, Dylan McDaniel and Veronica Ringel-Scaia. The portions that have been
included in this dissertation from that publication were sections that I had written.
Footnotes that reference the original publication have been included in Chapter 1.
Chapter 2 includes work that was a collaborative effort. Figures 2.1, 2.2, 2.4-2.10
contains data that was generated in Dr. Irving Allen's lab by myself and Dr. Allen, while
figure 2.3 contains data contributed by Dr. Liwu Li's lab including: Yao Zang, Na Diao,
Christina K. Lee, and Keqiang Chen. I wrote the original draft of the manuscript,
including descriptions of the figures, with edits contributed by Dr. Liwu Li and Dr. Irving
C. Allen. Yao Zang provided the data and figure legend for figure 2.3. I solely generated
the data for figures: 2.7-2.10. Methodology as well as formal data analysis for the
remaining figures contained contributions from myself, Yao Zang, Na Dial, Christina K.
Lee, Keqiang Chen, Tanya LeRoith, Clayton C. Caswell, Daniel J. Slade, Richard Helm,
Liwu Li, and Irving C. Allen.
Chapter 3 includes a step-by-step method with contributions from myself and Tara
Srinivasan. I wrote the entire manuscript, and generated all of the figures. The
remaining authors provided described in the method. Tara Srinivasan provided technical
assistance, as well as scientific advice to help frame the method described.
Chapter 4 contains contributions from both myself and Kristin Eden. While working in
collaboration with Kristin Eden on this project, I conducted the experiments for and
generated figures 4.1 and 4.2, and wrote the entirety of chapter 4. Kristin Eden
contributed to the methodology as well as formal data analysis for figure 4.1 and 4.2.
The experiment that was conducted to generate Figure 4.3 contains equal contribution
from myself and Kristin Eden.
ix
Table of Contents
Acknowledgements v
Attribution vii
Table of Contents ix
List of Figures xi
Chapter 1: Introduction 1
Chapter 2: Enhanced Mucosal Defense and Reduced Tumor Burden in Mice with the
Compromised Negative Regulator IRAK-M
A. Abstract 67
B. Introduction 68
C. Materials and Methods 71
D. Results 79
E. Discussion 100
F. References 107
Chapter 3: The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of
Mouse Intestinal Organoids
A. Abstract 115
B. Introduction 116
C. Materials and Methods 117
D. Results 132
x
E. Discussion 138
F. References 139
Chapter 4: NF-B inducing kinase (NIK) is essential for adequate expression of the
stem cell marker LGR5 and results in enhanced proliferation of intestinal epithelial cells.
A. Abstract 142
B. Introduction 143
C. Materials and Methods 144
D. Results 147
E. Discussion 152
F. References 154
Chapter 5: Discussion and Future Directions 158
Appendix
A. Works Completed 181
B. Copyright Permissions 183
xi
Figures
Figure 1.1. Intestinal epithelial cells of only a single cell layer line the GI tract. 8
Figure 1.2. Three dimensional graphic of the small intestine depicting the lumen
lined with intestinal villi. 11
Figure 1.3. Wnt signaling drives intestinal stem cell niche. 12
Figure 1.4. MyD88 Dependent Signaling Pathway 19
Figure 2.1. IRAK-M Expression is Increased During Inflammatory Bowel
Disease Exacerbation and in Advanced Colorectal Cancer Patients. 81
Figure 2.2. Attenuated Experimental Colitis Pathogenesis in Irak-m-/- Mice. 84
Figure 2.3. Increased Neutrophil and T-cell Function in Irak-m-/- Mice 87
Figure 2.4. Increased Morbidity and Mortality in Antibiotic + DSS Treated Mice 89
Figure 2.5. Colitis Associated Tumorigenesis Progression is Significantly 91
Reduced in Irak-m-/- Mice
Figure 2.6. Irak-m-/- Mice Display Attenuated Polyp Formation in the Colitis
Associated Tumorigenesis Model. 93
Figure 2.7. Disruption of the Murine Irak-m Locus Results in the Formation 95
of an Irak-mrΔ9-11 Splice Variant.
Figure 2.8. Differences in TLR-2, TLR-7 and TLR-9 Stimulation in Irak-m 97
Mutant Mice.
Figure 2.9. Comparison of Male and Female Bone Marrow Derived Macrophages. 98
Figure 2.10. Western Blot Evaluation of Protein Expression of IRAK-M. 100
Figure 3.1. Small Intestinal Organoid Growth 133
Figure 3.2. mRNA Expression of Inflammatory Cytokines Following 24 hr 134
xii
PAMP Challenge
Figure 3.3. Relative Fluorescent Staining of Organoids for Normalization 135
Figure 3.4. Standard Curve generated from Caco-2 Cells 136
Figure 3.5. Graphic Representation of the Technique 137
Figure 4.1. Growth tracking of organoids from single cell suspensions 148
Figure 4.2. Relative expression of Lgr5 from Wild-type and Nik-/- colonic crypts 149
Figure 4.3. Organoid colonies and FACS analysis of single cell suspensions 151
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Chapter 1:
Introduction
The gastrointestinal (GI) system, similar to the integumentary system, has an important
immunological role, and acts as a physical barrier that helps to prevent the access of
microbes within the body. The cells of the GI system are renewed on a daily basis,
which maintains proper barrier and physiologic function. Several cells of the immune
system are located just beneath the epithelial cells of the GI system, mainly for
protection due to the high probability of encountering a foreign pathogen if this site is
breached. When a pathogen manages to invade host tissues, an immune response
ensues to handle the invading microbes. Many immune cells will then produce
inflammatory molecules that relay information to surrounding cells that directs a proper
cellular defense reaction in response to the foreign microbes. Resolution of these
inflammatory molecules must occur; otherwise continued production might lead to
catastrophic tissue damage. As such, several regulatory molecules are produced by the
cell to prevent hyperactive inflammation. The research in the present dissertation
focuses on these molecules, specifically on a positive and a negative regulator of NF-B
signaling.
As a primary focus of this dissertation, a molecule induced by the cell to limit the
inflammatory cascade, termed interleukin receptor associated kinase-M (IRAK-M), is
evaluated with respect to its function and contribution to inflammatory related diseases
in the GI system. Both epithelial and immune cells contribute to host defenses, but due
to complexities that exist with in vivo animal models, the elucidation of precise
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molecular mechanisms can be complicated. Therefore, in order to simplify and study the
epithelial contribution to disorders such as inflammatory bowel disease (IBD), a method
is described herein and is optimized utilizing intestinal derived organoids, or “mini-guts”,
aimed to simplify conditions through ex vivo techniques. Further, the method described
is integrated for experiments conducted in Chapter 4 and is used to help elucidate the
role of NF-B inducing kinase (NIK), a protein that participates in the induction of the
alternative NF-B pathway. As such, this dissertation collectively investigated the role of
a negative and positive regulator of the NF-B pathway, being IRAK-M and NIK,
respectively. Further, the method utilizing intestinal organoids was applied to study the
epithelial cell specific contribution of these molecules to intestinal epithelial cell biology.
1. Introduction to Innate immunity and adaptive immunity
From the perspective of a pathogenic microorganism, metazoans (i.e. multicellular
organisms) represent a well-suited environment to infect and reside. In particular,
mammalian physiology affords conditions that are favorable to foreign microbes, which
include a stable temperature, abundant energy source, and interaction with species
within a population to facilitate pathogenic transmission (Sund-Levander et al., 2002),
(Speakman, 2005), (Engering et al., 2013). Therefore, in order to prevent such
infections from occurring, evolution has selected elegant mechanisms for the host to
prevent and limit the spread of disease through the action of the immune system
(Schultz and Grieder, 1987). The mammalian immune system is a complex network of
cells that orchestrate diverse functions, most of which are attributed to protecting the
host from disease causing pathogens (Muller et al., 2008). This is accomplished by
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what evolution has afforded, and what immunologists have classified as the two main
branches of the immune system: the innate and adaptive immune system (Powers and
Dean, 2016). Simplistically, innate immunity is known for its ability to act as a barrier
from the external environment with internal tissues. Furthermore, innate immunity also
involves the recognition of broad components of pathogens, and can rapidly induce an
effector response once a pathogen is recognized (Akira et al., 2006). Adaptive immunity
is known for being acquired following pathogenic insult, is highly specific to previously
encountered pathogens through the action of T and B lymphocytes, and is long lasting
(Bonilla and Oettgen, 2010). Tight regulation of both branches of the immune system
are required for normal physiological process to occur, and pathologies ensue when
signaling aberrations deviate from homeostatic norms. Hyperactive aberrations in both
the innate and adaptive immune responses can result in severe tissue damage;
whereas hypoactive aberrations can result from improper recognition of a pathogen,
leading to opportunistic infection (Blach-Olszewska and Leszek, 2007), (Al Anazi,
2009).
1.1 Cells of the Innate Immune System
The importance of innate immunity cannot be understated. All metazoans have cells
that compose an innate immune system, which protects from pathogens; whereas, the
adaptive immune system is more specialized, and it is believed to have evolved
stemming from a common ancestor with an innate immune system (Kimbrell and
Beutler, 2001), (Beutler, 2004). The innate immune system is composed of a diverse
range of cell types, which are categorically assigned based on the criteria of being a
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foremost line of host defense (Alberts et al., 2008). It should be noted that clear cut
definitions of innate immune cells are not always possible. Recently, cell types with both
innate and adaptive immune cell characteristics have been discovered, and fittingly
termed innate lymphoid cells. Innate immune cells include cell types that compose and
maintain the anatomical barriers to the external environment; however, the main cell
types that are pertinent to the innate immune response usually refer to the white blood
cells including both mononuclear and polymorpho-nuclear phagocytes. Mononuclear
phagocytes include the monocytes, macrophages, and dendritic cells, whereas
polymorpho-nuclear phagocytes include neutrophils, basophils and eosinophils (Beutler,
2004). Monocytes are the circulating precursors to macrophages, and mature into
macrophages once they have migrated from the circulation into tissues. It should be
noted that not all macrophages are created equal. In particular, there are groups of
tissue resident macrophages that are present in all organs and they display alternate
epigenetic gene signatures. Currently, it is believed that the surrounding
microenvironment is in constant communication with, and contributes to the diverse
function of tissue resident macrophages (Lavin et al., 2014). Macrophages and dendritic
cells are well known for their ability to engulf foreign microbes, as well as cellular debris,
through a cellular process called phagocytosis, followed by presentation of foreign
antigens to the adaptive immune system on class II MHC molecules (Metchnikov,
1884), (Banchereau and Steinman, 1998). Neutrophils are polymorphonuclear cells that
are likely the first-responders to a pathogen. They undergo maturation in the bone
marrow and enter into the circulation following their differentiation (Bainton et al., 1971).
Ultimately, neutrophils have a limited lifespan following maturation, as they undergo
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apoptosis as shortly as 6 hours after their entry into circulation (Cronkite and Fliedner,
1964), (Brinkmann and Zychlinsky, 2007). Neutrophils have the ability, similar to
macrophages, to engulf foreign substances via phagocytosis (Cohn and Hirsch, 1960),
and have recently attracted attention for the finding pertaining to their ability to exude
their cellular contents, termed neutrophil extracellular traps (NETs), which combats
pathogenic bacteria (Brinkmann et al., 2004). Basophils and eosinophils compose a
small percentage of circulating leukocytes in the human body (Stone et al., 2010). There
are aspects of basophil function that are unknown; however, basophils are currently
understood to contribute to host defense against parasites (Min, 2008). They store
preformed granules that contain histamine, and thus contribute to the allergic responses
(Stone et al., 2010). Eosinophils are known for their response, mainly to helminth
infection and allergies, but can also play a role in rare diseases (Stone et al., 2010).
Once matured into fully differentiated eosinophils, they migrate into circulation, and will
increase in abundance and locality due to the presence of Th2 associated cytokines
(Rosenberg et al., 2007).
All of the cell types described above share a common feature, in that they all share a
common lineage ancestor. All of these cells described above differentiate from a
hematopoietic stem cell that normally resides in the bone marrow. These phenomena
are highly relevant, because depending on which type of cell immunologists wish to
investigate, isolated stem cells from the bone marrow can be differentiated ex vivo when
the proper signals are provided that drive differentiation to a specific lineage. This
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allows researchers to simplify complex conditions to solve specific questions, pertaining
to a specific cell.
1.2 Interplay of innate immune system with GI system
Barrier surfaces, such as the GI tract, are a highly susceptible point of entry for
pathogenic microorganisms when breached. Therefore, an adequate epithelial barrier,
and immune response is necessary to prevent and contain any pathogenic insult. The
GI system of humans, as well as other mammals, harbors a diverse array of microbes
that share a commensal relationship with the host (Human Microbiome Project, 2012).
This phenomenon poses a really fascinating question, one that currently perplexes
researchers, and is related to immune activation versus immune tolerance. How is the
body able to recognize and respond to a pathogen emanating from the GI system, while
also subverting an active immune response against the commensal microbes that can
share many similarities with the pathogen? The answer to this question is obviously not
a simple one, but the answer partially lies in the several molecular mechanisms that
regulate the interaction between the GI tract and immune system that allow
homeostasis with the microbiota. This collectively starts with the physical barrier of
epithelial cells that prevent translocation of bacteria from the intestinal lumen to the
lamina propria. This intestinal epithelium is composed of polarized columnar epithelial
cells that are remarkably only a single layer in thickness. (Abreu, 2010), (Mowat and
Agace, 2014), (Williams et al., 2015). The turnover rate of epithelial cells in the GI tract
is one of the fastest in the human body, occurring every 3-4 days, and is possibly
related to the exposure of this tissue to hostile substances (e.g. bacterial products,
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dietary components) (Creamer et al., 1961), (Cheng and Leblond, 1974). In the
unfortunate event that a pathogen breaches the epithelial barrier, immune cells of both
the innate and adaptive immune system strategically located beneath the layer of
epithelial cells within the lamina propria will recognize and engage the pathogen (Figure
1.1). The lamina propria of the GI tract contains the highest abundance of
macrophages, T-cells, and IgA secreting plasma cells; additionally, under normal
conditions, macrophages constitute the most abundant leukocyte in the lamina propria
of the GI tract (Mowat and Agace, 2014). Such a high abundance of phagocytic cells in
the lamina propria is likely due to the high occurrence, or probability of foreign microbial
encounter. Under homeostatic conditions, tissue resident macrophages found in the
lamina propria are under consistent turnover, renewed by circulating monocytes that
travel into the tissue from the blood stream (Bain et al., 2014). Once these cells migrate
into the lamina propria, they can be distinguished by three major factors. These include
the expression of the fractalkine receptor CX3CR1, the secretion of high amounts of IL-
10 and TNF, and by being relatively inert to the presence of LPS (Bain et al., 2013). The
low responsiveness of these macrophages to LPS suggests an important regulatory
function, one that likely includes phagocytosis of debris without inducing an immune
response. These cells contribute to several pleiotropic responses due to the secretion
of the cytokines IL-10 and TNF. IL-10 is important for the regulation of immune cell
function, and TNF can govern epithelial cell turnover (Shouval et al., 2014), Proper
tissue homeostasis is partially maintained by constant crosstalk between the epithelium,
immune cells, and the microbiota. It is fascinating that all the physiological processes
work, in most cases, without any insult or abnormalities. As can be expected, these
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interactions between tissue and commensal microbes, as well as the molecular
mechanisms that regulate immune tolerance versus immune activation are at the
forefront for many scientific investigators. Aberrations in any of these interactions can tip
Inte
stin
al lu
men
Lamina Propria
Intestinal bacteria
IgA
antibodies
Intestinal Epithelial cells
Intestinal Immune
cells
Figure 1.1. Intestinal epithelial cells of only a single cell layer line the GI tract, and physically separate intestinal microbes from entering the body. Beneath the epithelial cells are immune cells within the lamina propria, many of which are macrophages. Figure from: (Mowat, A. M. & Agace, W. W. 2014). See copyright permissions.
9
the homeostatic balance, and ultimately have the potential to contribute to tissue
pathologies.
2. Introduction to the GI system
The GI system collectively refers to the hollow tube that begins at the mouth and
terminates at the anus. The main functions of the GI system involve obtaining nutrients
via digestion and absorption followed by the excretion of waste, while also protecting
the host during these processes by forming a physical barrier with the external
environment (Cheng et al., 2010). As chemoorganoheterotrophs, humans require
energy sources from complex organic forms of carbon (i.e. carbohydrates, proteins and
lipids) and must break these macromolecules down to simpler subunits in order for
proper metabolism to occur. The GI system has evolved to mechanically and chemically
break down ingested food, beginning in the mouth and ending at the colon, while also
providing a large surface area of epithelial cells to maximize the absorption process,
occurring from small intestine to colon. The colon aids in absorption of nutrients and
water, and also harbors trillions of bacteria that form a commensal relationship with the
host, and is referred to as the microbiome.
2.1 Anatomy of Small intestine and Colon
The GI tract is a hollow tube that is derived from the endoderm during gastrulation
(Lewis and Tam, 2006). The small intestine is the segment of GI tissue that begins after
the stomach, and ends at the cecum, which is followed by the large bowel (i.e. the
colon). From its proximal to distal ends, the small intestine is further subdivided into
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three segments: duodenum, jejunum, and ileum, respectively. The function of the
duodenum pertains to acid neutralization, whereas the jejunum and ileum involve
nutrient absorption. The major functions of the small intestine are to aid in digestion of
ingested nutrients, and absorption of water, electrolytes and nutrients. The anatomical
structures of the small intestine are quite striking; the surface epithelium is lined with
fingerlike projections, termed villi (Figure 1.2 A, B). These fingerlike projections
maximize the intestinal surface area, which enhances the efficiency in which nutrients
can be absorbed into the blood stream. The intestinal epithelium is one of the most
rapidly dividing tissues in the human body, with epithelial turnover being every 3-5 days
(Cheng and Leblond, 1974), (Mayhew et al., 1999). Cell division is driven by intestinal
stem cells that reside in the base of the intestinal crypts, which in a conveyor belt
fashion, push the epithelial cells up towards the tip of the intestinal villus, where they
eventually undergo apoptosis (Williams et al., 2015) (Figure 1.2 C). This results in a
constant shedding of epithelial cells into the intestinal lumen to be excreted with the
feces (Schuijers and Clevers, 2012). The colon shares similarities with the small
intestine; however, there are distinct differences between the two tissues. Regarding
epithelial anatomy, the colon does not contain villi, but rather has a flat surface
epithelium (Colony, 1996), (Schuijers and Clevers, 2012). Similar to the small intestine,
the colon contains crypts that harbor stem cells, and the epithelial cells of the colon are
under constant renewal. The main epithelial cell types of the colon are columnar
epithelial cells, goblet cells, and enteroendocrine cells (Colony, 1996). One key feature
of the colon is that it is in constant contact with millions of bacteria, and thus contains
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several goblet cells to produce mucus, which provides another physical barrier to GI
bacteria.
2.2 Stem cells as contributors of epithelial cell fates and the Wnt signaling
pathway
Intestinal stem cells reside at the crypt base, and in the case of the small intestine,
reside next to the Paneth cells, which produce antimicrobial peptides and contribute to
stem cell maintenance (Cheng and Leblond, 1974), (Sato et al., 2011). Intestinal stem
cells give rise to all of the differentiated epithelial cells that compose the intestinal
epithelium (Cheng and Leblond, 1974), (Bjerknes and Cheng, 2006). The past two
decades of research has demonstrated the importance of the Wnt signaling pathway in
the maintenance of the intestinal epithelium (Figure 1.3). WNT is a ligand that binds to
Fizzled receptors that ultimately culminates in the stabilization and nuclear localization
Figure 1.2. A. Three dimensional graphic of the small intestine depicting the lumen lined with intestinal villi. B. Birdseye view of the fingerlike projections (villi). Intestinal crypts are at the base of the villi. C. Transverse section of intestinal villi. Intestinal crypts are the U-shaped structures at the base of the intestinal villi. Figure from: (Schuijers and Clevers, 2012). See copyright permissions.
A. B. C.
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of the transcription factor β-catenin. The importance of canonical Wnt signaling was first
demonstrated with transgenic mice lacking Tcf4, a key transcription factor that is
activated in response Wnt
ligands. Loss of Tcf4 in mice resulted in premature death, and interestingly, the
intestinal epithelium of neonatal mice was entirely differentiated (Korinek et al., 1998).
This provided the first clue that Wnt signaling contributed to the intestinal stem cell
niche. A major breakthrough came recently, when it was discovered that both small
intestine and colon intestinal stem cells express the cell surface receptor leucine-rich
repeat-containing G-protein coupled receptor 5 (LGR5) (Barker et al., 2007). LGR5 was
identified as a Wnt target gene, and further identified as the receptor for R-spondin. In
the presence of WNT ligands, R-spondin amplifies and sustains Wnt signaling that is
necessary to retain the stem cell niche (Kazanskaya et al., 2004), (Kim et al., 2008), (de
Lau et al., 2011). ). With this knowledge, isolated intestinal crypts or single Lgr5+/GFP
stem cells can be cultured ex vivo with the addition of several niche factors that
Figure 1.3. Wnt signaling drives intestinal stem cell niche. (left) intestinal stem cells divide and differentiate into all the epithelial cell types found in the GI system. (Right) Wnt signaling is amplified in intestinal stem cells by R-spondins that bind to LGR5. Figure from: (Koo and Clevers, 2014). See copyright permissions.
13
stimulate the Wnt signaling pathway (Sato et al., 2009). These “mini guts”, also termed
organoids possess the characteristics of intestinal epithelium, and are proving to be a
vital tool for GI research. Organoids are proving to be excellent tools to study GI
epithelial cell proliferation, because the confounding factors of the immune system have
been removed.
3. Pattern Recognition Receptors of the Innate Immune System
3.1 The Families of Pattern Recognition Receptors
Following pathogenic insult, the normal series of innate immune responses are to
recognize, respond, and resolve the insult from the foreign invader (Beutler, 2004), but
what mechanisms govern the ability of host cells to recognize a pathogen? It was
Charles Janeway that first proposed the existence of so called “pattern recognition
receptors” by innate immune cells, and rationalized that host innate immune cells likely
recognize conserved molecules (i.e. patterns) that are unique to foreign microbes
(Janeway, 1989). Time later proved that Janeway was ultimately correct in his
prediction. The major breakthrough came when it was demonstrated that Toll-like
receptor 4 (TLR4) was the bona fide receptor for the conserved bacterial molecule
lipopolysaccharide (LPS) (Poltorak et al., 1998). This discovery, along with work
conduced in the fruit fly by Jules Hoffman and works pertaining to dendritic cells linking
innate and adaptive immunity by Ralph Steinman, was ultimately awarded the Nobel
Prize in physiology and medicine in 2011. These findings were the first to demonstrate
the importance of pattern recognition receptors (PRRs) to innate immunity. Broadly, it is
now known that PRRs are located based on topology relative to the cell. Soluble
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extracellular PRRs include members of the complement system, as well as the
pentraxins, which bind to phosphocholine in a calcium dependent manner (Pepys and
Hirschfield, 2003); cell surface receptors include the Toll-like receptors (TLRs) and C-
type lectin receptors (CLRs); and finally, Nod-like receptors (NLRs), Rig-I-like helicases
(RLRs) and AIM2 receptors, which are located intracellularly in the cell cytosol.
PRRs recognize conserved molecules residing on or contained within foreign microbes
that are fundamentally distinct from healthy host cells. These foreign patterns (i.e.
molecules) can be of bacterial, viral, fungal, or protozoan origin; and certain PRRs can
even recognize self-damage patterns (Thompson et al., 2011). The molecules
recognized by PRRs are termed pathogen associated molecular patterns (PAMPs) and
self-patterns are termed damage/danger associated molecular patterns (DAMPs)
(Janeway, 1989), Following recognition of a PAMP/DAMP by a particular PRR leads to
a rapid cellular response. This ensues via multiple coordinated signal transduction
cascades that culminate in either the release of inflammatory molecules, an increase in
the transcription of genes involved in cellular migration of immune cells, and when the
signal is robust, activation of the adaptive immune system (Steinman and Witmer,
1978), (Medzhitov and Janeway, 1997), (Kawai et al., 2001), (Rahman et al., 2009).
Lastly, resolution of the immune response must ensue in order to prevent excessive
inflammation that can result in destructive tissue damage (Beutler, 2004). The resolution
phase occurs through negative feedback loops, which are a mechanism to restore
homeostasis, and are paramount to proper physiological function. This can occur
through a variety of means, for example, intracellular molecules that down-regulate the
15
signaling cascades that culminate in the activation of the inflammatory response (PTEN,
SOCS-1, SHIP1, IRAK-M, CYLD, A20). Because both arms of the immune system are
highly involved in the production of inflammatory mediators, many inflammatory related
diseases can ensue when proper function is not maintained. Other mechanism to
restore homeostasis can include the production of anti-inflammatory cytokines, such as
interleukin-10 (IL-10), as well as the activation of regulatory cells that blunt the immune
system, i.e. T-regulatory cells (Tregs) and myeloid derived suppressor cells (MDSCs).
In summary, there are many ways the cell can restore balance once a response is
generated.
3.2 Cell types of both GI and innate immune systems that express PRRs
The expression of PRRs on both intestinal epithelial cells (IEC) and innate immune cells
has been shown to be crucial for proper intestinal and immune physiology (Vijay-Kumar
et al., 2007). The cells that line the GI tract are composed of polarized epithelial cells,
meaning they have an apical surface that borders the intestinal lumen, and a
basolateral surface that borders the lamina propria (Abreu, 2010). The families of PRRs
that have been most extensively studied pertaining to IEC are the TLRs, and their
expression varies regarding localization on either the apical or basolateral membrane of
the IEC. In the human colon, TLR5, which senses bacterial flagella, is expressed on the
basolateral membrane of IECs and not the apical membrane on IECs (Gewirtz et al.,
2001), (Rhee et al., 2005). This suggests that spatial separation of TLR5 from the
intestinal lumen is important in preventing constitutive interaction of this receptor with
the intestinal microbiota, and likely recognizes bacterial components once they have
16
breached beyond the apical membrane. Further, TLR1, TLR2, TLR4 and TLR9 are
expressed in human small intestinal epithelial cells (Otte et al., 2004). The expression of
these TLRs also display spatial polarization, with the highest expression on the
basolateral surface of IECs; however, their expression is not limited to the basolateral
membrane as they are found on the apical surface as well (Cario et al., 2002), (Lee et
al., 2006). This begets the question, if TLRs are expressed on the apical membrane of
IEC that are in contact with the luminal microbiota, why are these cells not constantly
driving inflammation? This is likely due to the importance of proper TLR expression, and
signaling for cellular maintenance of IECs. Indeed, TLR engagement of commensal
PAMPs results in the production of protective factors, and ablation of the GI microbiota
with antibiotics limits GI TLR activation, and results in profound susceptibility to dextran
sulfate sodium (DSS), which is a chemical that leads to colitis in mice (Rakoff-Nahoum
et al., 2004).
3.3 Inflammation and the NF-κB signaling pathway1 (Rothschild et al., 2018)
Inflammation, in certain contexts harbors a negative umbrella of pathologies; however,
under proper physiological conditions, it serves a very important purpose. Broadly,
inflammation functions to coordinate the repair of damaged tissue by informing the
immune system that tissue damage is taking place. Inflammation results in cellular
activation, increased blood flow to the affected area, and an influx of cells associated
with the immune system. This coordinated effort from cells of the host immune system
1 Published in: ROTHSCHILD, D. E., MCDANIEL, D. K., RINGEL-SCAIA, V. M. & ALLEN, I. C. 2018. Modulating
inflammation through the negative regulation of NF-kappaB signaling. J Leukoc Biol.
17
functions to clear the pathogenic insult, repair tissue damage and curb the inflammatory
response in order to restore tissue homeostasis. At the cellular level, this is coordinated
via an important regulator of transcription: the nuclear factor kappa light chain enhancer
binding protein (NF-κB) transcription factor.
NF-κB is an evolutionarily conserved transcription factor found in species ranging from
Drosophilia to humans, which underscores its critical role in the host immune response.
(Ghosh et al., 1998). The last 3 decades of research have contributed greatly to our
understanding of NF-κB. NF-κB functions as a prominent inducible transcription factor
that regulates the immune system Briefly, NF-κB it is known to regulate a vast array of
genes ranging from the development of the embryo, to cell fate decisions, and is well
known for its role as a prominent transcription factor that regulates the immune system
(Beg et al., 1995), (Alcamo et al., 2001), (Boersma et al., 2011), (Zhang et al., 2017).
Due to its diverse and broad biological functions, strict regulation of NF-κB signaling is
paramount to proper tissue allostasis. When NF-κB signaling is aberrant, several
maladies can ensue, such as susceptibility to infections, autoimmunity and cancer
(Oeckinghaus and Ghosh, 2009). (Sun et al., 2013), (Greten et al., 2004). Thus, as with
many major signal transduction pathways, several mechanisms tightly regulate NF-κB
signaling and maintain the proper balance of activation and repression.
NF-κB in mammals consists of a total of five proteins that are predominantly present in
an inactivated state in the cytosol as either homo- or hetero-dimers that include: RelA
(p65), RelB, c-Rel, p105 and p100. The p105 and p100 proteins are unique because
18
they must undergo post-translational processing through the proteasome to form the
active subunits, p50 and p52, respectively (Amir et al., 2004), (Ghosh et al., 1998). All
isoforms contain a common Rel homology domain, which is responsible for DNA
binding, as well as binding to the cytosolic inhibitory proteins, termed Inhibitor of –κB
(IκB) (Ghosh et al., 1998). Several families of cellular receptors signal through NF-κB to
initiate gene transcription and are known to coordinate signaling through either the
canonical NF-κB pathway or the non-canonical NF-κB pathway (which is also termed
the “alternative” NF-κB pathway). Molecules that signal via the canonical NF-κB
pathway include cytokines, such as tumor necrosis factor (TNF), interleukin-1 beta (IL-
1β), and the majority of PRRs (Ghosh et al., 1998). The non-canonical pathway is
initiated by a much smaller repertoire of molecules that are members of TNF family,
such as CD40, B-cell activating factor (BAFF), and lymphotoxin beta (LT-β) (Sun et al.,
2013).
3.4 Canonical NF-κB signaling pathway1
Convergence on the canonical NF-κB signaling pathway occurs through the activation
of several different families of receptors (i.e. TNFR, TLRs, NLRs, IL-1R) (Ghosh and
Hayden, 2012). As a classical example of canonical activation, we will focus on MyD88-
dependent TLR signaling (Figure 1.4), a process that occurs for the interleukin 1
receptor (IL-1R) and all TLRs with the exception of TLR3 (Yamamoto et al., 2003). IL-
1/TLR signaling commences via binding of their respective ligands. Engagement of a
PAMP to an extracellular TLR (i.e. TLR1, TLR2, TLR4, TLR5, TLR6) induces a
conformational change and hetero- or homo-dimerization of the TLR. Dimerization leads
19
to a change in conformation of the receptor, followed by recruitment of adaptor proteins
to the toll/interleukin receptor (TIR) domain of the TLR. The adaptor molecule MyD88 is
recruited to extracellular dimers composed of TLR5 receptors, whereas Mal followed by
MyD88 are recruited by TLR1/TLR2, TLR2/TLR6, TLR4 dimers (Kawai et al., 1999),
(Fitzgerald et al., 2001), (Yamamoto et al., 2002),(Horng et al., 2002),(Didierlaurent et
al., 2004), (Nishiya and DeFranco, 2004). MyD88 acts as a protein scaffold, as it
contains both a TIR domain and a death domain (DD) in its protein structure. The DD of
MyD88 recruits interleukin receptor associated kinase (IRAK) IRAK-4, followed by
IRAK-1, all in a helical assembly to form a Myddosome complex (Wesche et al., 1997),
(Lin et al., 2010). The close spatial proximity of IRAK-4 to IRAK-1 allows for IRAK-1 to
Lys63 Ubiquitination
Phosphorylation
Ubiquitin
Lys48 Ubiquitination
MyD88
IRAK-4
IRAK-1
TRAF6
TAK-1
IKK Complex
NF-! B
I! B
" TrCP
NF-! B Signaling (MyD88 Dependent )
26s Proteosome
Inflammation Cell Migration
Cell Survival
Negative Regulators
Figure 1.4. MyD88 Dependent Signaling Pathway. Engagement of a Toll-like receptor (TLR), with the exception of TLR3, induces a signal transduction cascade via the Myeloid Differentiation Factor 88 (MyD88) dependent pathway. MyD88 dependent signaling results in the activation of the canonical NF-κB pathway. Activation of NF-κB results in the transcription of multiple genes involved in inflammation, cell migration, cell survival, and negative feedback proteins to eventually restore homeostatic norms of the cell.
20
become rapidly phosphorylated by IRAK-4, and then undergo auto-phosphorylation
(Wesche et al., 1997), (Li et al., 2002). Once phosphorylated, IRAK-1 leaves the
receptor complex and interacts with TRAF-6. Upon activation, TRAF6 associates with
Uev1A and Ubc13 and results in TRAF6 lysine-63 (Lys-63)-mediated poly-ubiquitination
(Takaesu et al., 2000), (Deng et al., 2000). Lys63-mediated poly-ubiquitination of
TRAF6 acts as an important scaffold resulting in the recruitment and docking of TAB2/3
and TGF-β activated kinase-1 (TAK1) (Wang et al., 2001), (Kanayama et al., 2004).
Close association of the TAB2/3/TAK1 complex with poly-ubiquitinated TRAF-6 allows
TAK1 to undergo auto-phosphorylation, resulting in TAK1 activation (Wang et al., 2001).
Once TAK1 becomes phosphorylated, it can mediate downstream signaling by
phosphorylating the inhibitor of κB kinase (IKK) complex on IKKβ subunits (Ninomiya-
Tsuji et al., 1999), (Wang et al., 2001). IKKβ phosphorylation results in the IKK complex
activation, composed of NEMO/IKKα/ IKKβ subunits, to phosphorylate IκB (Mercurio et
al., 1997). Phosphorylation of IκB results in its subsequent recognition by the SCF-
βTrCP ubiquitin (Ub) ligase complex that covalently modifies IκB with Lys-48 poly-Ub
chains, which ultimately leads to the degradation of IκB by the 26s proteasome (Chen et
al., 1995). Once IκB is degraded, NF-κB dimers are liberated and predominantly shuttle
into the nucleus and bind to -κB promoter and enhancer elements involved in the
regulation of immunity, cell migration, cell adhesion, cell death and inflammation.
3.5 The Non-Canonical NF-κB Signaling Cascade1
The activation of the non-canonical or alternative NF-κB signaling cascade is tightly
regulated and has a much smaller group of ligands and receptors that can induce its
21
activation (Sun, 2012). Members of the TNF/TNFR superfamily, which include the
lymphotoxin-β receptor (LTβR), CD40, B-cell activating factor receptor (BAFFR),
receptor activator of NF-κB (RANK), TNFR2 and CD27 have all been shown to signal
through the non-canonical pathway (Sun, 2012). Upon ligation of TNF family members
to their receptors, the TRAF2-TRAF3-cIAP1/2 E3 complex is recruited to the receptor
(Sun, 2012). This recruitment leads to accumulation of TRAF2 molecules, allowing
TRAF2 to shift cIAP1/2’s Lys-48 Ub ligase activity towards TRAF3 via Lys-63 poly-
ubiquitination of cIAP1/2 (Sun, 2012), (Hostager et al., 2003). When TRAF3 is modified
via this mechanism, it ultimately leads to proteasomal degradation of TRAF3, allowing
for accumulation of MAP3K14 (also commonly known as NF-κB Inducing Kinase (NIK)),
resulting in increasing the levels of cytosolic NIK (Sun, 2012). Mechanistically, in an
unstimulated state, TRAF3 acts as a binding partner for NIK bringing it close to the
cIAP1/2 E3 ligase complex, which in this case leads to its degradation (Zarnegar et al.,
2008, Liao et al., 2004). NIK is essential for the processing of p100 into active p52(Xiao
et al., 2001), (Sun, 2012), which is a defining feature of the non-canonical NF-κB
signaling pathway. However, NIK has also been shown to phosphorylate IKKα, which
can act as a secondary kinase that mediates the activation of p100 (Senftleben et al.,
2001). For this to occur, NIK must be present in relatively high concentrations inside the
cell before interacting with IKKα. Thus, stabilization of NIK results in its accumulation
and interactions with IKKα, culminating in phosphorylation and activation (Sun,
2012),(Senftleben et al., 2001).
22
It is thought that IKKα-mediated phosphorylation of p100 occurs on Ser-872 in vitro and
Ser-866 and 870 in vivo(Sun, 2012),. While the cause of this discrepancy is currently
unclear, presumably, IKKα acts as a site-specific kinase for p100. This phosphorylation
step targets the C-terminal inhibitory domains (the PID and the ankyrin repeat domain)
of p100 for degradation via the proteasome(Sun, 2012),. Ankyrin repeat domains
(ARDs) have been shown to mask the nuclear localization sequence (NLS) of IκBα and
interestingly, p100’s phosphorylation site contains a sequence similar to IκBα (Sun,
2012),(Oeckinghaus and Ghosh, 2009). Therefore, disruption of the ARD of p100 via
proteasomal degradation may lead to unmasking of the NLS and subsequent
translocation of p52/RelB to the nucleus (Oeckinghaus and Ghosh, 2009). Once in the
nucleus, p52/RelB drives the transcription of a limited repertoire of genes, including
CCL19, CCL21, CXCL12, and CXCL13. In contrast, when the TNFR family members
are unstimulated, NIK is instead degraded via Lys-48 poly-ubiquitination(Sun, 2012).
Consequently, IKKα will not become activated and phosphorylate p100. Thus, in this
scenario, the NF-κB dimer cannot enter the nucleus and initiate transcription of target
genes. Due to its essential function, NIK represents a “bottleneck” in the non-canonical
NF-κB signaling cascade and is frequently targeted by mechanisms that have evolved
to negatively regulate this alternative pathway.
4 Negative Regulation of Inflammation
4.1 Inducible Negative regulators of Inflammation
Well-controlled mechanisms to resolve NF-κB activation are required to prevent its
potential destructive activities if this transcription factor if left unencumbered. These
23
include mechanisms that inhibit the action of NF-κB following its nuclear translocation.
There are over 785 different molecules that counteract the activity of NF-κB that are
either constitutively expressed in the cell, or induced by NF-κB following its activation;
there are even exogenous molecules that inhibit its activity (i.e. from plants) (Gilmore
and Herscovitch, 2006). The molecules (i.e. microRNA, proteins) that are induced are
often referred to as negative regulators, because they act to restore homeostasis via
negative feedback loops (Renner and Schmitz, 2009). Several, if not all of these
molecules serve a very important function that act as the “brakes” to halt NF-κB activity.
Certainly, these are not the only means utilized by the cell to reduce the activity of NF-
κB, but proteins expressed following NF-κB activation will be the focus here. Analogous
to an automobile losing its breaks, catastrophic outcomes can ensue if the cell loses
these abilities to halt the actions of NF-κB. These include diseases resulting from
abnormalities of the immune system, as well as certain types of cancers, mainly
lymphomas as well as GI cancers (Clevers, 2004), (Karin, 2009). One can anticipate
that loss of key components of a signal transduction cascade will result in severe
maladies, if not lethality. The same is true for cellular systems; in fact, mutations in
several of these proteins result in severe inflammatory disorders in both humans and
mice, if and when such mutations are actually viable.
4.2 Inhibitor of κB (IκB)1
There are 8 IκB proteins, known to negatively regulate NF-κB dimers, two of which are
the inhibitory regions on both p100 and p105 prior to proteosomal processing. The
mechanism of regulation functions, in the case of the canonical NF-κB pathway, by
24
binding to the Rel homology domain of NF-κB subunits via ankyrin repeats on the IκB
proteins. IκBα is induced by NF-kB, and this mechanism of induction demonstrates an
auto-regulatory feedback loop (Sun et al., 1993). The binding of IκBα to NF-κB results in
the concealment of the nuclear localization sequence on NF-κB dimers, thus keeping
NF-κB mainly contained in the cytosol under normal conditions (Ghosh and Hayden,
2012). This parallels the nuclear export signal on IκBα, which renders the flux of NF-κB
dimer mainly in the cytosol. Once IκBα is degraded by the proteasome, the nuclear
import signal on NF-κB is no longer blocked, and this allows for translocation to the
nucleus to regulate gene transcription (Sun et al., 1993). One question arises regarding
this feedback loop: if NF-κB upregulates IκBα, which contains a nuclear export signal,
and NF-κB is in the nucleus with a nuclear import signal, how does IκB-α access NF-κB
to halt its function? It turns out that both NF-κB and IκBα can shuttle into and out of the
nucleus; thus, they are in constant flux between these cellular compartments. When the
majority of IκBα is bound to NF-κB, the cytosolyic flux predominates and NF-κB is
limited to the nucleus. When NF-κB is free of IκBα, the nuclear flux predominates and
allows for NF-κB to bind enhancer regions on target DNA (Ghosh and Hayden, 2012).
4.3 Negative Regulation From The Interleukin Receptor Associated Kinase
Family1
There are four interleukin receptor associated kinases (IRAK) in mammals: three of
which are known as IRAK-1, IRAK-2 and IRAK-4. These three family members act as
positive regulators of NF-κB signal transduction (Thomas et al., 1999), (Muzio et al.,
1997), (Suzuki et al., 2002), (Kawagoe et al., 2008). One family member, IRAK-3 or
25
IRAK-M, is unique from the other family members, because it acts as an inducible
negative regulator of the canonical NF-κB signaling pathway (Wesche et al., 1999),
(Kobayashi et al., 2002). All four family members share a N-terminal death domain that
is important for homotypic protein-protein interactions with either MyD88, or with other
IRAK members for signal transduction. More central in the protein sequence for all
members is the kinase domain (Flannery and Bowie, 2010). IRAK-1 and IRAK-4 were
first described as being the only functional kinases in the family, with IRAK-2 and IRAK-
M being inactive or pseudo-kinases (Thomas et al., 1999), (Muzio et al., 1997), (Suzuki
et al., 2002), (Kobayashi et al., 2002). This was determined by both biochemical and
analytical methods. Analytically, the functional activity of a protein kinase can be
predicted by its primary amino acid sequence (Hanks and Hunter, 1995). There are
specific residues in the sequence of the kinase domain that are invariant for kinase
function, and these include residues in the kinase subdomain VIb known as the HRD
and DFG motifs (Hanks and Hunter, 1995), (Meylan and Tschopp, 2008). The invariant
aspartic acid residues, that are essential for proper function, are mutated in IRAK-2 and
IRAK-M to an asparagine and serine, respectively. This provides the rationale for the
nonfunctional activity of these IRAK family members (Meylan and Tschopp, 2008). All
IRAK proteins, apart from IRAK-4, contain a unique C-terminal domain (Flannery and
Bowie, 2010). This domain contains TRAF6 binding motifs that, as the name implies,
are important for interaction with TRAF6, and aid to propagate downstream signaling.
4.4 Molecular Mechanism of IRAK-M Function as a Negative Regulator1
26
Early functional studies suggested that IRAK-M shared a redundant function with IRAK-
1 and IRAK-2 (Wesche et al., 1999), despite the later prediction that IRAK-M was
inactive or a pseudo-kinase. In overexpression systems and luciferase reporter assays,
IRAK-1, IRAK-2, and IRAK-M were shown to function as positive regulators of NF-κB
signaling. However, with in vitro kinase assays, human IRAK-M was demonstrated to
have very weak intrinsic kinase activity, especially compared to IRAK-1 (Wesche et al.,
1999). Further, murine IRAK-M was shown to contain detectable kinase activity; albeit,
at a lower level when compared to the robust auto-phosphorylation of human IRAK-1
(Rosati and Martin, 2002). These data suggest that the auto-phosphorylation, and
therefore the kinase activity of IRAK-M is negligible for its function; however, it is
tempting to speculate that IRAK-M may indeed function as an active kinase on a
currently unknown cellular substrate following its induction. Despite these initial findings
and speculation, the prevailing literature suggests that IRAK-M functions as a negative
regulator of NF-κB signaling following TLR activation. These data are based on findings
utilizing genetically modified mice with targeted deletions of the Irak-m gene (Kobayashi
et al., 2002). The Irak-m gene contains 12 total exons and in order to define the role of
IRAK-M, exons 9-11 were targeted for deletion (Kobayashi et al., 2002). These exons
encode the amino acids predicted to constitute the putative kinase domain. Full length
IRAK-M was not detected when a western blot was performed with an antibody specific
for the C-terminus of IRAK-M (Kobayashi et al., 2002). Using these genetically modified
animals, Irak-m was found to be induced by NF-κB, along with IκB and A20 following
TLR stimulation, suggesting a negative feedback mechanism. Subsequent co-
immunoprecipitation experiments demonstrated that IRAK-M has the capacity to bind to
27
TRAF6, leading to the current model that IRAK-M functions to inhibit IRAK-1 and
TRAF6 interactions, which in turn inhibits downstream NF-κB activity (Kobayashi et al.,
2002). The expression of IRAK-M can be induced by other transcription factors, such as
C/EBP, Smad4, AP-1 and CREB (Lyroni et al., 2017), suggesting additional functions
beyond the feedback mechanism currently described. Phenotypically, Irak-m-/- mice do
not display any abnormal fetal or post-natal development, but do develop osteoporosis
later in life due to hyperactive osteoclast activity (Kobayashi et al., 2002), (Li et al.,
2005).
These Irak-m-/- mice were instrumental in defining IRAK-M as a negative regulator of
NF-κB signaling and have been widely utilized to define its function. For example,
consistent with increased NF-κB signaling, Irak-m-/- bone marrow derived macrophages
(BMDMs) display impaired endotoxin tolerance and hyper-production of inflammatory
cytokines (i.e. IL-6, TNF and IL-12p40) following TLR stimulation with specific PAMPS,
as well as, L. monocytogenes and S. typhimurium (Kobayashi et al., 2002). Consistent
with the ex vivo BMDM studies, Irak-m-/- mice were found to display enhanced small
intestinal inflammation following in vivo exposure to S. typhimurium (Kobayashi et al.,
2002). Interestingly, in these initial studies, Irak-m-/- mice do not display enhanced
morbidity or mortality following infection, despite the increased inflammation (Kobayashi
et al., 2002). These findings are consistent with a more recent study that showed the
Irak-m-/- mice are protected in models of experimental colitis and colitis associated
tumorigenesis (Rothschild et al., 2017). Mice lacking IRAK-M were found to have a
robust immune response to bacteria translocating from the lumen following chemical
28
induced damage to the intestinal epithelial cell barrier (Rothschild et al., 2017). The
attenuation in pathogenesis was associated with large expansions of gastrointestinal
associated lymphoid tissue (GALT), increased neutrophil function, and enhanced T-cell
recruitment (Rothschild et al., 2017). Complementary data revealed that the
gastrointestinal (GI) tract of the Irak-m-/- mice had a lower total colonic bacterial load
compared to wild type counterparts, which could also contribute to attenuation of
disease pathogenesis (Kesselring et al., 2016). Mechanistically, these data suggest the
immune system in Irak-m-/- animals is primed and more prone to a robust inflammatory
response. In the GI tract, this improves the efficiency of the host response to pathogenic
and commensal components of the host microbiome that drive disease processes.
It should be noted that while the consensus data identifies IRAK-M as a negative
regulator of NF-κB signaling, contrary data suggests a possible alternative mechanism.
Recently, it was demonstrated that IRAK-M participates in TAK-1 independent NF-κB
activation downstream of TLR7 through MEKK3 (Zhou et al., 2013). With the use of
IRAK-1/IRAK-2 double deficient mice and IRAK-1/-2/-M triple deficient mice, this study
demonstrated that under highly specific conditions, IRAK-M functions in the absence of
IRAK-1 and IRAK-2 and can actually activate NF-κB signaling (Zhou et al., 2013).
Mechanistically, IRAK-M interacts with IRAK-4 to form an IRAK-M myddosome complex
in the absence of both IRAK-1 and IRAK-2 that modulates NF-κB signaling through
TAK-1 independent mechanisms (Zhou et al., 2013). While these data are certainly
intriguing, the study ultimately concluded that IRAK-M exerts an inhibitory effect under
normal conditions by indirectly inducing inhibitory proteins (A20, SHIP-1, SOCS1 and
29
IκB-α) (Zhou et al., 2013). Further clouding mechanistic insight, it has recently been
revealed that the original Irak-m-/- mice commonly used to characterize this protein may
actually contain a truncated version of IRAK-M (Rothschild et al., 2017). Detailed
sequencing analysis of the Irak-m gene product was conducted following TLR
stimulation of BMDM from genotype confirmed Irak-m-/- mice (Rothschild et al., 2017).
Under these conditions, a splice variant of the Irak-m gene was identified that resulted
from the splicing of exon 8 with exon 12, in essence splicing around the neo cassette
(Rothschild et al., 2017). This type of splice variant is a common occurrence in
genetically modified animals where functional domains are targeted, as opposed to the
gene’s start site. Typically, these truncated proteins are dysfunctional and degraded by
the cell, which preserves the knockout status of the animals. It is also important to note
that the truncated IRAK-M protein has not yet been detected in situ and may not exist in
vivo (Rothschild et al., 2017). However, overexpression of Irak-mrΔ9-11 and functional
studies using the recombinant protein revealed that this truncation mutant is significantly
more potent at activating NF-κB signaling then the wild type version (Rothschild et al.,
2017).
Considering these data, we agree with the consensus findings that IRAK-M functions to
negatively regulate NF-κB signaling under normal conditions. However, the possibility
remains that IRAK-M may actually have a dual role under certain cell type or temporal
specific conditions to also activate NF-κB signaling. This could be directly correlated
with the cellular concentration of other IRAK family members or other critical cellular
substrates. Resolution of the crystal structure of IRAK-M would provide valuable insight
30
into these questions. It should also be pointed out that the history of IRAK-M is similar to
that of IRAK-2, which was first believed to function as an inactive pseudo-kinase
(Meylan and Tschopp, 2008). However, it was later determined that IRAK-2 function
was independent of IRAK-1 and plays a critical role in sustaining the late phase of NF-
κB signaling through potentially functioning as an active kinase (Kawagoe et al., 2008).
Future studies will indicate whether our interpretation into the function of IRAK-M will be
modified, as we have previously seen with IRAK-2.
4.5 IRAK-M and mucosal tissues
IRAK-M was appropriately named upon its discovery in mammals based on its
homology to the other IRAK family members and its tissue specific expression IRAK-M
mRNA expression was first found to be limited to cells of the myeloid lineage, hence the
“M” in IRAK-M. However, as scientific efforts intensified regarding the functional role of
IRAK-M, evidence emerged that suggested IRAK-M expression is not limited to cells of
myeloid origin. In fact, several reports have demonstrated the expression of IRAK-M in
a wide array of tissues comprising the bone, liver, and cells being osteoclasts, epithelial
cells, and neutrophils (Li et al., 2005), (Sumpter et al., 2011), (Kesselring et al., 2016),
(Rothschild et al., 2017). The Irak-m -/- mouse has greatly enhanced the current
understanding regarding the functional role of IRAK-M. Originally, Irak-m -/- mice
displayed enhanced inflammation in the small intestine when challenged with S.
typhimurium, and due to the revelation that these mice did not succumb to potentially
lethal effects due to endotoxic shock, it was concluded that these mice have enhanced
innate immunity compared to wild-type mice (Kobayashi et al., 2002). The lung and GI
31
tract are mucosal surfaces that are constantly interacting with foreign substances, often
described as luminal antigens, which include commensal microorganisms. Thus, these
mucosal tissues represent a first line defense against exposure to foreign substances,
and serve, therefore, as a strategic defense site for cells of the innate immune system.
For this reason, many studies have focused on the role of IRAK-M in the GI tract, as
well as other mucosal surfaces including the lung (Berglund et al., 2010), (Biswas et al.,
2011),(Deng et al., 2006). It is interesting to note that innate immune cells, particularly
monocytes isolated from septic patients display so called enhanced endotoxin
tolerance, and reduced response to antimicrobial peptides (Deng et al., 2006).
Interestingly, in peritonitis-induced sepsis models, Irak-m -/- mice display enhanced
bacterial clearance, and had higher survival rates compared to wild-type mice following
secondary challenge with the bacteria S. arengosa (Deng et al., 2006). This enhanced
bacterial clearance, and ultimate protection from endotoxin tolerance was attributed to
increased MIP-2 chemokine production, resulting in an influx of neutrophils to the lung
to handle secondary pulmonary bacterial challenge (Deng et al., 2006).
Several mouse models exist that are utilized to mimic GI inflammatory diseases, such
as ulcerative colitis and colitis-associated cancer (Okayasu et al., 1990), (Neufert et al.,
2007). These include both chemically induced models of colitis, as well as genetic
models utilizing mice that are predisposed to developing colitis (Wirtz et al., 2007),
(MacDonald, 1994). In one chemically induced model conducted with the agent dextran
sulfate sodium (DSS), Irak-m -/- mice displayed robust GI inflammation, with an
increase in plasma concentrations of inflammatory cytokines, namely IL-6 and TNF
32
(Berglund et al., 2010). Furthermore, Irak-m -/- mice displayed reduced splenic and
thymic weight, which normally increases in weight, and as these weights increase, a
correlation is observed with respect to increased disease severity of colitis (Berglund et
al., 2010). A genetic model of colitis is one that utilizes mice with a targeted disruption
for the gene that encodes the cytokine IL-10 (Kuhn et al., 1993). Regarding IRAK-M,
mice that are double deficient for IL-10/IRAK-M display increased inflammation in the
colon leading to exacerbated colitis, as well as increased pro-inflammatory cytokine
expression (Biswas et al., 2011). Evidence supports the claim that Irak-m-/- have an
enhanced inflammatory phenotype. This phenotype, however, appears uniquely titrated
at mucosal surfaces. Though these mice display robust inflammation following
pathogenic encounter, they also display enhanced innate immunity to these pathogenic
encounters, and mice are uniquely protected when monitoring their survival (Deng et al.,
2006), (Rothschild et al., 2017).
Regarding GI colitis-associated tumorigenesis, Irak-m -/- mice were first characterized
by robust tumorigenesis induction following completion of the AOM/DSS model
(Klimesova et al., 2013). Klimesova et al. attributed this finding to an altered microbiota
composition in Irak-m -/- mice, because wild-type mice were rescued from tumor
formation when supplemented with antibiotics in their drinking water. Conversely, Irak-m
-/- mice developed tumors both with and without antibiotics, and tumor formation in the
GI tract of Irak-m -/- mice was attributed to increased inflammatory cytokine production
(Klimesova et al., 2013). It is well recognized that inflammation is a growth promoting
condition that contributes to the progression of cancer (Greten et al., 2004); therefore,
33
the authors concluded that this was the main mechanism attributed to tumorigenesis in
the Irak-m -/- mice (Klimesova et al., 2013). It should be noted that microorganisms in
the GI tract are important contributing factors in models that use DSS as an inducing
agent for colitis. Laboratories often use different combinations of antibiotics to clear the
microbiota, which should minimize the catastrophic inflammatory effects of DSS;
however, this is not always the case. When a combination of ampicillin, vancomycin,
neomycin and mitronidizole are used in combination with DSS, mice often display
extreme morbidity and mortality, which is counter to what one would expect (Rakoff-
Nahoum et al., 2004),(Hernandez-Chirlaque et al., 2016). Results with a combination of
antibiotics should be interpreted with caution, because of heightened the variability of
due to fluctuations in the GI microbiota.
Reports from other groups have demonstrated Irak-m -/- mice with decreased tumor
progression in both xenograft models and AOM/DSS models (Xie et al., 2007),
(Standiford et al., 2011), (Kesselring et al., 2016), (Rothschild et al., 2017). This has
been attributed to enhanced innate immune functions in Irak-m -/- mice. Recently,
Kesselring et al. demonstrated reduced tumor progression in Irak-m -/- mice following
AOM/DSS colitis-associated tumorigensis (Kesselring et al., 2016). The authors of this
study reported increased GI inflammation in Irak-m -/- mice during early phase colitis
(i.e. acute colitis models); however, decreased inflammation driven tumorigenesis
following AOM/DSS. This decrease in tumor burden was attributed to multiple factors,
which include the absence of epithelial expressed IRAK-M, as demonstrated with bone
marrow chimeric mice, and substantiated by a reduced overall absolute bacterial load in
34
the colon of Irak-m -/- mice (Kesselring et al., 2016). Interestingly, co-housing studies,
which aim to transfer GI bacteria of mice, due to their coprophagic nature, did not
recapitulate tumor progression in wild-type mice with an inherited IRAK-M microbiome.
Therefore, the mechanism attributed to decreased carcinogenesis was the induced
expression of IRAK-M, leading to the stabilization of the transcription factor STAT-3,
which has a prominent oncogenic role in colon cancer (Kesselring et al., 2016),
(Jenkins, 2016), (Yu et al., 2014).
Evolution has provided unique and interesting mechanisms to tolerate commensal
microbes, while also maintaining an adequate immune response following pathogenic
encounter. There are multiple mechanisms, as well as negative regulatory proteins that
restore NF-κB transcriptional activity to homeostatic norms. It is clear that IRAK-M
participates in this process, and it is interesting that, at least in mice, tumor initiation and
progression appears to be reduced. This suggests that IRAK-M represents a novel
target for inhibition as a cancer therapeutic, with the intention of enhancing innate
immunity to limit the progression of cancer.
4.6 A20: An inducible negative regulator of inflammation that works via post-
translational modifications1
A20 or Tumor necrosis factor alpha-induced protein 3 (TNFAIP3) is a protein that is
rapidly induced by NF-κB, and functions through a feedback mechanism to halt the
canonical signaling cascade. A20 is one of the most studied negative regulatory
proteins targeting components of the NF-κB pathway and functions primarily by
35
modifying positive regulatory proteins in the cascade (Lee et al., 2000). The two
mechanisms in which A20 functions is by modifying the posttranslational status of target
proteins directly as a ubiquitin modifying enzyme (i.e. as both a ubiquitin ligase, and as
a protein deubiquitinase (Wertz et al., 2004). How can a single protein blunt NF-κB, and
other signaling pathways, by acting as both a ubiquitin ligase and de-ubiquitinase
(DUB)? The answer lies in the type of linkage of poly-ubiquitin chains to proteins.
Ubiquitin is a small 8.5 kDa peptide found in eukaryotic cells that can be covalently
attached to proteins to regulate their function post-translationally. This is accomplished
via the addition of poly-ubiquitin (poly-Ub) chains to amino acid residues on target
proteins. Ubiquitin itself contains seven different lysine residues, and the particular
lysine chain that is utilized to make the ubiquitin chain determines the regulatory status
of the protein. The seven-lysine residues found on ubiquitin are K6, K11, K27, K29,
K33, K48, and K63, with K48 and K63 linkages being the best studied, with currently
known regulatory function (Tenno et al., 2004), (Varadan et al., 2004). Generally, when
a target protein is modified by Lys-48-linked poly-ubiquitination, this serves as a
molecular tag on the protein for recognition, and further destruction by the 26S
proteasome (Wilkinson et al., 1980), (Voges et al., 1999). Additionally, Lys-63 linked
poly-Ub is a posttranslational modification that generally results in the activation of
tagged proteins (Tenno et al., 2004). In terms of A20, this describes the mechanism for
its ubiquitin ligase domain; removing Lys-63 linked poly-Ub chains from a protein results
in the attenuation of activity of the protein. By also functioning as a ubiquitin ligase, A20
has been shown to inhibit the canonical NF-κB pathway by the addition of Lys-48 linked
poly-Ub chains to receptor interacting protein 1 (RIP1) (Wertz et al., 2004), (Bertrand et
36
al., 2008). RIP1 is an essential molecule that activates canonical NF-κB pathway
downstream of the TNF receptor. Addition of Lys-48 linked poly-Ub chains to RIP1
targets this protein for degradation, thus removing a positive signal with the intent of
restoring cellular homeostasis.
The importance of A20 as a key negative regulator of inflammation was demonstrated
with the use of A20 knockout mice (Lee et al., 2000). In the absence of A20, mice
display a robust inflammatory phenotype in the liver, intestine, bone joints, skin and
kidney (Lee et al., 2000). Furthermore, severe cachexia occurs in several organs, and
mortality commences postnatally within a few weeks of age; this can be perpetuated
further when mice are given low doses of TNF or LPS (Lee et al., 2000). A20 does not
appear to be crucial for fetal development, because mice display an appropriate
Mendelian ratio when born. It was demonstrated in mouse embryonic fibroblasts (MEFs)
that in the absence of A20, NF-κB transcriptional activity is sustained, and IκB-α mRNA
is transcribed with similar, even possibly increased levels when compared to wild type
mice. However, IκB-α protein levels do not rebound following TNF treatment. This was
attributed to the continued activity of the IKK complex, because in the presence of a
proteasome inhibitor named MG-132, IκB-α levels were restored. Furthermore, it was
demonstrated that IκB-α was continually phosphorylated by the IKK complex following
translation, which resulted in its constant degradation in A20 null cells (Lee et al., 2000).
A20-Tnf and A20-Tnfr1 deficient mice also develop severe spontaneous inflammation,
indicating a role for A20 in modulating either alternative innate immune pathways, or
adaptive immune pathways (Boone et al., 2004). LPS stimulated A20-Tnf double
37
deficient BMDMs display a robust production of IL-6 and nitric oxide (NO) compared to
WT, which was slightly attenuated compared to A20 deficiency alone. This, along with
robust inflammation in A20-Rag1 double knockouts, suggested that A20 played a
prominent role in negatively regulating innate immune pathways beyond those regulated
by TNF (Boone et al., 2004).
Indeed, A20 was demonstrated to robustly regulate TLR signaling, as MyD88-A20
double deficient mice do not develop severe multi-organ inflammation and cachexia as
seen in A20 knockout mice alone Furthermore, components of the microbiota were
found to drive spontaneous inflammation, as chimeric mice reconstituted with A20-/-
bone marrow are rescued from severe spontaneous inflammation when treated orally
with an antibiotic cocktail that diminishes the intestinal microbiota (Turer et al., 2008).
4.7 CYLD1
CYLD is a constitutively expressed DUB that was originally described as a tumor
suppressor gene (Bignell et al., 2000). Loss of function of CYLD in humans is
associated with familial cylindromatosis, which are multiple benign skin cancers usually
localized to the face and neck (Bignell et al., 2000). Additional mechanistic studies
revealed that loss of CYLD contributes to an overall cellular resistance in apoptosis
(Brummelkamp et al., 2003), which likely contributes to cancer pathogenesis. CYLD is a
member of the Ub-specific protease family (USP), which represents the largest family of
DUBs in the human genome (Nijman et al., 2005). As a bona fide cysteine protease,
CYLD cleaves Lys-63 linked chains of poly-Ub on target proteins (Komander et al.,
38
2008). This serves as a repressive function because addition of poly-Ub on Lys-63 is
generally a post-translational modification that leads to increased activity of modified
proteins. Interestingly, in addition to Lys-63 mediated de-ubiquitination, CYLD has the
ability to de-ubiquitinate via linear Ub chains (Sato et al., 2015). It is interesting to note
that CYLD and A20 cleave Ub chains on several of the same substrates that participate
in the signal transduction cascade in the NF-κB pathway, including but not limited to
RIP1, IKKγ, TRAF2 and TRAF6 (Kovalenko et al., 2003, McDaniel et al., 2016,
Trompouki et al., 2003, Brummelkamp et al., 2003). This overlap in negative regulatory
function demonstrates the importance of signaling molecules, such as TRAF6, as
primary nodes in the induction of the inflammatory cascade. The post-translational
modification of several of the same molecular targets between A20 and CYLD could be
due to steady-state and inducible negative regulation. Further kinetics and substrate
avidity studies are necessary to explain the similar molecular targets between these two
molecules.
Studies utilizing independently generated Cyld-/- mice have yielded highly insightful
findings, albeit somewhat conflicting. In one study, the Cyld-/- animals demonstrated
significant defects in NF-κB and JNK signaling in macrophages following TLR and CD40
stimulation (Zhang et al., 2006). However, this effect appeared to be stimuli specific, as
TNF stimulation did not appear to be impacted by CYLD deficiency (Zhang et al., 2006).
Importantly, this study observed normal B and T-cell development and function (Zhang
et al., 2006). While there are several differences in the proposed mechanistic actions of
CYLD, these findings are in general agreement with other groups using these and other
39
independently generated Cyld-/- animals (Massoumi et al., 2006), (Lim et al., 2007).
However, at least one additional group has not observed these phenotypes using
another independently generated Clyd-/- mouse line (Reiley et al., 2004). These animals
did not recapitulate the defects in NF-κB, JNK, ERK, or p38 signaling (Reiley et al.,
2004). Rather, these animals showed significant attenuation of T cell receptor (TCR)
signaling in the absence of CYLD (Reiley et al., 2004).
5. Inflammatory maladies of the GI tract
5.1 Inflammatory Bowel Diseases
Inflammatory bowel disease (IBD) is an inclusive term for two similar, yet distinct
diseases, being ulcerative colitis (UC) and Crohn’s disease (CD); both diseases are
characterized by aberrant inflammation resulting in the pathogenesis of tissues localized
to the gastrointestinal (GI) system. The distinguishing features between these two
diseases are based on the localization of the GI inflammation, gross pathology, and
microscopic pathological characteristics (Ahmad et al., 2002), (Satsangi et al., 2006).
Based on locality, ulcerative colitis is generally limited to inflammation of the large
bowel, also termed the colon; whereas Crohn’s disease can cause inflammation-driven
pathologies, usually in an intermittent pattern, affecting any portion of the GI tract, even
the mouth. The most common GI area affected in Crohn’s disease is the colon and
terminal ileum of the small intestine (Abraham and Cho, 2009). Crohn’s disease patients
can have inflammatory lesions that affect all GI tissue layers, defined as transmural
inflammation; whereas, inflammation in ulcerative colitis usually only affects the
mucosal layer of the colon (Abraham and Cho, 2009). Ulcerative colitis, as the name
40
implies, display distinct ulcers present in the colon that may contribute to blood in the
stool. Crohn’s disease patients may have similar ulcerative lesions, but a diagnosis can
be distinguished over ulcerative colitis if these lesions are found in other interspersed
portions of the GI tract.
Gross pathologies are a method utilized to confirm a diagnosis between ulcerative
colitis and Crohn’s disease; however, because IBD is a multifactorial disease of differing
severities in the human population, diagnosis is not always simple (Yantiss and Odze,
2006). Gastroenterologists have built upon two models, aimed to guide clinicians to
determine if a patient suffers from ulcerative colitis or Crohn’s disease, which include
methods of identification termed the Vienna and Montreal models, which were
conveniently named after the cities where gastroenterologists convened when the
guidelines were established(Satsangi et al., 2006). The Vienna model, first proposed in
1998, described features to distinguish Crohn’s disease based on three predominant
elements. These include A, B and L classifications, which designated for age of onset
(A), disease behavior, and location of the disease (L), respectively (Satsangi et al.,
2006). In 2005, gastroenterologists reassessed the Vienna model and came up with
new guidelines that describe the Montreal model, which expanded on the guidelines to
identify Crohn’s disease, as well as the distinguishing features of ulcerative colitis
(Satsangi et al., 2006). Thus, the more comprehensive Montreal model is the preferred
system used among clinicians (Spekhorst et al., 2014).
5.2 Factors that influence IBD pathogenesis
41
There are several genetic mutations that may predispose individuals to IBD. Genome
wide association studies (GWAS) have demonstrated that NOD2 and IL23r mutations
are associated with the predisposition of individuals to Crohn’s disease; however, there
is currently no single gene mutation solely responsible for IBD pathogenesis (Ma et al.,
1999), (Hugot et al., 2001), (Satsangi et al., 1996), (Kanaan et al., 2012). Interestingly,
more genetic mutations have been found that are associated with predisposing
individuals to Crohn’s disease compared to ulcerative colitis, as demonstrated via
monozygotic twins, but different genetic alterations are seen in both diseases
(Thompson et al., 1996), (Orholm et al., 2000). A definitive cause of IBD onset and
pathogenesis is currently unknown, yet environmental and genetic components have
been described to play a significant role (Podolsky, 1991). Patients that have the
highest predisposition for IBD are Caucasians, usually of European decent, and disease
onset usually takes place in the fertile years of a patient’s life (Probert et al., 1993).
Interestingly, males and females are equally likely to develop IBD, thus, there are no
gender biases seen in human patients (Rosenblatt and Kane, 2015). Furthermore,
because there are several components that contribute to the development of IBD,
treatment regimens are generally aimed to improve quality of life for patients, as no cure
currently exists for both Crohn’s disease and ulcerative colitis (Braus and Elliott, 2009).
5.3 The Interrelationship Between Inflammation and Cancer
Chronic inflammation, which is common in IBD patients, is an enabling characteristic of
cancer (Hanahan and Weinberg, 2011). Notably, IBD patients, especially ulcerative
colitis patients, are at a heightened risk for the development of GI related cancers,
42
termed colitis-associated cancer (Thorsteinsdottir et al., 2011). Empirical evidence was
provided in mice that ultimately linked inflammation and cancer when components of the
NF-κB signaling cascade were mutated (Greten et al., 2004). Components of the
immune system are important players in this context, because they normally contribute
to the inflammatory milieu, especially in response to pathogens. As was seen earlier,
aberrations in NF-κB signaling can result in hyperactive inflammatory signaling, and
production of these cytokines can have a profound impact on the surrounding tissue. In
terms of chronic inflammation, this can result in a pro-growth environment that leads to
cancer progression. The revelation that inflammation is highly regulated by components
of the immune system, it is vital to understand the contribution of the immune system to
inflammatory related diseases, such as IBD.
Concluding Remarks
Several factors contribute to proper physiology and maintenance of the GI system.
Having a comprehensive understanding at the cellular level is a good starting point for
advancing therapeutic potential to treat maladies, particularly IBD and colon cancer.
For this reason, it is important to investigate specific components of cellular signaling
pathways, such as negative regulatory proteins, to determine their contribution
pertaining to these particular diseases. Further, broadening our perspectives to
encompass instances that alter the interactions of host cells and endogenous microbes
shows great potential to uncover unknown aspects that contribute to disease. There will
always be a need to understand these complex phenomena, and hopefully recent
43
advances, as well as ones to come, will not only impact our understanding, but also
strengthen treatment options for patients in the near future.
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Chapter 2:
Enhanced Mucosal Defense and Reduced Tumor Burden in Mice with the
Compromised Negative Regulator IRAK-M
Published as:
Rothschild, D. E., Zhang, Y., Diao, N., Lee, C. K., Chen, K., Caswell, C. C., Slade, D. J.,
Helm, R. F., Leroith, T., Li, L. & Allen, I. C. 2017. Enhanced Mucosal Defense and
Reduced Tumor Burden in Mice with the Compromised Negative Regulator IRAK-M.
EBioMedicine, 15, 36-47.
A. Abstract
Aberrant inflammation is a hallmark of Inflammatory Bowel Disease (IBD) and colorectal
cancer. As discussed in the previous chapter, the inflammatory signaling pathway has
many regulatory mechanisms, both through positive and negative regulation. IRAK-M is
a critical negative regulator of TLR signaling and overzealous inflammation. Here we
utilize data from human studies and Irak-m-/- mice to elucidate the role of IRAK-M in the
modulation of gastrointestinal immune system homeostasis. In human patients, IRAK-M
expression is up-regulated during IBD and colorectal cancer. Further functional studies
in mice revealed that Irak-m-/- animals are protected against colitis and colitis associated
tumorigenesis. Mechanistically, our data revealed that the gastrointestinal immune
system of Irak-m-/- mice is highly efficient at eliminating microbial translocation following
epithelial barrier damage. This attenuation of pathogenesis is associated with expanded
areas of gastrointestinal associated lymphoid tissue (GALT), increased neutrophil
migration, and enhanced T-cell recruitment. Further evaluation of Irak-m-/- mice revealed
68
a splice variant that robustly activates NF-κB signaling. Together, these data identify
IRAK-M as a potential target for future therapeutic intervention.
B. Introduction
Crohn’s disease (CD) and ulcerative colitis (UC) represent the clinical manifestations of
inflammatory bowel disease (IBD). In humans, there is a strong association in patients
afflicted with IBD to further develop colitis associated cancer (CAC) (Greten et al.,
2004). Gastrointestinal (GI) immune homeostasis is, in part, maintained by pattern
recognition receptors (PRRs); including, but not limited to the Toll-like receptors (TLRs).
There are 10 known TLRs in humans that recognize pathogen associated molecular
patterns (PAMPs) associated with invading microbes. (Kawai and Akira, 2011). In the
gut, TLRs sense and respond to commensal flora translocation, resulting from damage
to the epithelial cell barrier during the course of disease progression (Saleh and Elson,
2011). Following TLR engagement to its pathogenic ligand, a highly coordinated
signaling cascade leads to the activation of specific transcription factors including
nuclear factor-κB (NF-κB), activating protein-1 (AP-1), or in the case of viral recognition,
interferon regulatory factor-3 (IRF-3). The dominant signaling pathway utilized depends
upon which TLR is stimulated in response to the pathogenic ligand; however, all TLRs,
with the exception of TLR-3, signal through the myeloid differentiation factor 88 (MyD88)
adaptor molecule (Kawai et al., 1999), (Takeuchi et al., 2000), (Jiang et al., 2006). A
MyD88 independent pathway exists for both TLR-3 and TLR-4 that has been shown to
signal through the adaptor molecule TIR-domain-containing-adapter-inducing interferon-
β (TRIF) (Yamamoto et al., 2003), (Hoebe et al., 2003), (Kawai et al., 2001). In healthy
69
individuals, these TLR signaling cascades are responsible for rapid activation of innate
host defenses and further activation of the adaptive immune response (Medzhitov and
Janeway, 1997). However, uncontrolled activation of TLR signaling promotes chronic
inflammation and is associated with a variety of autoimmune diseases, including IBD
(Cook et al., 2004).
The TLRs that utilize the MyD88 dependent pathway have been reported to signal
through a family of Interleukin Receptor Associated Kinases (IRAKs), which contain four
known family members being: IRAK-1, IRAK-2, IRAK-M, and IRAK-4 (Wesche et al.,
1997), (Muzio et al., 1997), (Wesche et al., 1999), (Li et al., 2002). IRAK-1, IRAK-2, and
IRAK-4 have been described to play a role in the transduction of downstream signaling
from MyD88; conversely, IRAK-M is rather unique and has been shown to be a negative
regulator of TLR signaling (Kobayashi et al., 2002). The initial reports pertaining to
IRAK-M described it as a member of the Pelle/IRAK family, and when overexpressed in
mammalian cells, had the capacity to activate NF-κB through interactions with TNF
receptor associated factor-6 (TRAF-6) (Wesche et al., 1999). Following the generation
of an Irak-m-/- mouse, IRAK-M was confirmed to function as a negative regulator of TLR
signaling (Kobayashi et al., 2002).
Here we report that IRAK-M is significantly up-regulated in human patients with IBD and
colitis associated neoplasia. We also show that IRAK-M is up-regulated in advanced
stages of colorectal cancer (CRC). Prior studies have evaluated IRAK-M in models of
experimental colitis and colitis associated tumorigenesis utilizing Irak-m-/- mice and
70
shown that these animals are highly sensitive to both inflammation and neoplasia.
However, contrary to these prior observations, more recent studies have revealed that
Irak-m-/- mice are actually protected from inflammation driven tumorigenesis in the colon
(Kesselring et al., 2016). IRAK-M was found to support colorectal cancer progression
through the reduction of antimicrobial defenses and the stabilization of STAT-3
(Kesselring et al., 2016). Due to these conflicting reports, we sought to better elucidate
the contribution of IRAK-M during IBD and colitis associated tumorigenesis. Our studies
are, in general, complementary to the more recent findings that support a role for IRAK-
M in disease pathogenesis. Here we report that Irak-m-/- mice were significantly
protected against dextran sulfate sodium (DSS)-mediated GI inflammation and
azoxymethane (AOM)/DSS mediated tumor formation. We further show that the
immune system in the GI tract of Irak-m-/- mice is primed and highly efficient at
eliminating components of the microbiome translocating from the GI lumen following
damage to the epithelial barrier. This priming and attenuation of disease appears to be
associated with expanded areas of gastrointestinal associated lymphoid tissue (GALT),
increased and efficient neutrophil migration, and enhanced T-cell recruitment.
Upon further evaluation of the Irak-m-/- mice, we discovered the formation of a Irak-m
splice variant. The splicing event joins exon 8 with exon 12, splicing around the
neomycin resistance cassette (Kobayashi et al., 2002), forming a Irak-mrΔ9-11 truncation
mutant. This truncation mutant functions to robustly induce NF-κB signaling when
overexpressed in HEK293T cells. Together, our data confirms previous findings that
71
IRAK-M functions as a critical mediator of inflammatory signaling pathways and is
essential in maintaining immune system homeostasis in the GI system.
C. Materials and Methods:
Human Metadata Analysis
IRAK-M expression was assessed using a publicly accessible microarray metadata
analysis search engine (http://www.nextbio.com/b/nextbioCorp.nb), as previously
described (Kupershmidt et al., 2010). The following array data series were analyzed to
generate the human patient and cell expression data: GSE10714; GSE59071;
GSE9686; GSE13367; GSE36807; GSE16879; GSE10191; GSE52746; GSE9452;
GSE38713; GSE4183; GSE37283; GSE37364; GSE10715.
Reagents
Ultra-pure LPS from E.coli, PGN-SA, Pam3CSK4, Poly (I:C) H.M.W, Flagellin-BS, ODN-
1668, Imiquimod were purchased from Invivogen. Mouse TNF-α was purchased from
Biolegend. Mouse IL-6, IL-10, TNF-α, IL-1β and IFNγ ELISA kits were purchased from
BD Biosciences. Rabbit polyclonal IRAK-M antibody was purchased from Millipore
(Millipore 07-1481). Rabbit monoclonal β-Actin (Cell Signaling 13E5), Rabbit
monoclonal-HA (Cell Signaling C29F4) and anti-rabbit-HRP were from Cell Signaling.
Antibodies for Ly6G, CD11b, CXCR2, CD14, Ly6C, IAE, and CD62L were from
Biolegend. PCR primers were generated against exons of murine Irak-m utilizing
Primer3 software and purchased from Integrated DNA Technologies. Dextran sulfate
sodium DSS salt (36,000-50,000 M.Wt.) was purchased from MP Biomedicals.
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Azoxymethane (AOM) was from Sigma. RNA isolation Miniprep kit was from Zymo
Research. Espresso mammalian expression CMV cloning kit was purchased from
Lucigen. Plasmid pGL4.32[luc2P/NF-ĸB-RE/Hygro] and pRL-null were from Promega.
Cell Culture
HEK293T cells (ATCC) were grown in Eagle’s Minimum Essential Medium (EMEM) +
10% Fetal bovine serum (FBS) (Atlanta Biologicals). THP-1 cells (ATCC) were grown in
RPMI + 10% FBS + 0.05 mM 2-mercaptoethanol.
Bone Marrow Derived Macrophage Assessments
Mouse bone marrow was harvested from both tibias and femurs for individual mice and
plated in non-tissue culture treated sterile petri dishes containing 1x DMEM
supplemented with 10% FBS + 20% L929 conditioned media for 7 days. Bone marrow
derived macrophages (BMDMs) were re-plated on day 7 by aspirating L929 growth
media and incubated with 10 ml ice cold sterile 1x PBS containing 5mM EDTA. Cells
were incubated at 4 oC for 30 minutes and gently scraped following incubation. Cell
containing supernatants were transferred to 50 ml conical tubes and centrifuged for 10
minutes at 300 x g. BMDMs were resuspended in 20 ml warm growth media and
replated in tissue culture treated sterile plastic ware at: 2x105 cells per well in triplicate
for 24 well plates; 2x106 cells per 60mm2 dish; 5x106 cells per 10mm2 dish. Wild-type
and Irak-m-/- BMDMs were re-plated in separate 24 well plates and allowed to adhere
overnight. Cell culture media was aspirated the following day and BMDMs were washed
with 1 ml 1x PBS. Following PBS aspiration the cells were treated with 500 μl total
73
volume per well of 1x DMEM +10% FBS containing specific pathogen associated
molecular patterns (PAMPs) for 24 hours at a concentration of: Poly I:C (6.25 μg/ml),
PGN-SA (10 μg/ml), LPS (10 ng/ml), Pam3CSK4 (300 ng/ml), TNF-α (75 ng/ml).
Colon organ culture supernatants
Following the acute colitis model or AOM/DSS model, colon sections were also
collected to establish organ cultures to determine local cytokine levels as previously
described (Greten et al., 2004), (Allen et al., 2010), (Williams et al., 2015). Briefly,
approximately 1-cm2 strip of the distal colon was removed and washed with 200 U/ml
Penicillin and 200 μg/ml streptomycin in 1x PBS. Following the wash, each respective
colon section had the total mass measured and recorded for downstream normalization.
Each individual distal colon section was then transferred to an individual well of a 24
well plate and supplemented with 1ml of DMEM media containing 200 U/ml Penicillin
and 200 μg/ml streptomycin with no additional supplements. Following overnight
incubation, the supernatants were collected and centrifuged at 300 x g for 10 minutes to
collect cell free supernatants. ELISAs were then conducted on the supernatants to
determine the respective cytokine level and each sample was normalized to their
respective colon section mass.
Bacterial Counts.
Bacterial counts were determined from mouse whole blood collected via cardiac
puncture. 25 μl of whole blood was plated, as well as two 10 fold serial dilutions were
74
prepared in sterile 1x PBS and 25 μl aliquots were plated on LB agar plates. Following
overnight incubation at 37 oC, bacterial colonies were counted.
Western Blot
Experiments utilizing WT and Irak-m-/- BMDMs were seeded in 60mm2 dishes and
allowed to adhere overnight. Experiments with HEK293T cells were seeded in 6 well
plates at 3x105 cells per well. BMDMs were treated with control media or stimulated with
10 ng/ml LPS, 10 μg/ml PGN-SA or 300 ng/ml Pam3CSK4 for 24 hr. Cells and BMDMs
were washed with ice cold 1x PBS and were subsequently lysed in cell lysis buffer
containing: 2% SDS (Fisher Sci), 100 mM NaCl (Fisher Sci), 10 mM Tris-HCl pH 7.4
(Fisher Sci), 1x Halt Protease inhibitor (Thermo). Whole cell lysates were sonicated to
shear endogenous nucleic acids. Protein concentration was quantified by BCA (Pierce)
and 15 - 20 μg total protein was boiled in 1x Blue Loading Buffer + 1x DTT (Cell
Signaling) at 95 oC for 5 min and electrophoresed with either Bolt 4-12% Bis-Tris gels
(Thermo) or Biorad 4-12% Criterion XT Bis-Tris Gel in 1x MOPs buffer (Thermo). Gels
were transferred onto 0.2 μm PVDF membranes (Millipore) in 1x Tris-glycine + 20%
methanol. Membranes were blocked for 30 minutes with 5% non-fat dry milk (Carnation
Brand) dissolved in TBS+0.05% Tween 20 (TBST). Primary antibodies were used
1:1000 and incubated at 4 oC overnight with gentle shaking. Primary antibodies were
either: anti-IRAK-M (Millipore 07-1481), anti-β-Actin (Cell Signaling 13E5) and anti-HA
(Cell Signaling C29F4). IRAK-M, β-Actin and HA were visualized using anti-rabbit IgG
linked to HRP, detected with ECL western blotting substrate Pico (Pierce) or Dura
(Pierce) by x-ray radiography. Densitometry was analyzed using ImageJ software.
75
RT-PCR
BMDM were seeded in 10mm2 dishes and allowed to adhere overnight. Cells were then
treated with 1x DMEM +10% FBS without PAMP stimulation or 300 ng/ml Pam3CSK4
(Invivogen) for 24 hr. RNA was isolated using Zymo Research miniprep kit. Total RNA
was quantified and 2 μg was converted to cDNA with High Capacity cDNA reverse
transcription kit (Applied Biosystems) according to the manufacture’s instructions. 5-10
ng of cDNA was amplified for different exons of murine Irak-m by RT-PCR. The primer
sequences used for Irak-m exons are:
Irak-m Exon Primers
Exon: Forward Primer (5’3’) Reverse Primer (5’3’)
5-8 GGACATTCGAAACCAAGCAT TGTGCCATTTGTGCACTGTA
9-10 CCAGCTCCAACCCAAACTAA TGTTTCGGGTCATCCAGCAC
10-11 GGACCTCCTCATGGAACTGA CCAGAGAGGACAGGACTTCG
12 TCCTTCAGGTGTCCTTCTCCACTG CCTCTTCTCCATTGGCTTGCTC
5-12 GTGCAGAGAAAACGACCCTG TGGGAGGGTCTTCTGCAAAA
RT-PCR products were electrophoresed in 1x TBE and visualized on 1% agarose gel
stained with ethidium bromide.
Molecular Cloning, Overexpression and Dual Luciferase Assay.
76
Molecular cloning of the Irak-m gene of both wild-type (Irak-m) and mutant (Irak-mrΔ9-11)
was performed from BMDMs stimulated for 24 hr with 300 ng/ml Pam3CSK4. RT-PCR
was conducted utilizing Phusion DNA high fidelity polymerase (Thermo) with primer
sequences generated with homology to either the WT Irak-m gene, or the Irak-mrΔ9-11
gene, and the espresso CMV cloning and expression system (Lucigen) according to the
manufacture’s instructions. Primer sequences are:
CMV-Irak-m-HA Cloning Primers
WT Irak-m-HA
(Forward) 5’3’
GAAGGAGATACCACCATGGCCGGCCGGTGCGGGGCCCGT
WT Irak-m-HA
(Reverse) 5’3’
GGGCACGTCATACGGATACTGCTTTTTGGACTGTTCATG
Irak-mrΔ9-11-HA
(Forward) 5’3’
GAAGGAGATACCACCATGGCCGGCCGGTGCGGGGCCCGT
Irak-mrΔ9-11-HA
(Reverse) 5’3’
GGGCACGTCATACGGATAAGGACGTGGGAGGGTCTT
6-well plates (Corning) were seeded with 3x105 HEK293T cells (ATCC) per well and
allowed to adhere overnight. Cells were transfected with a total of 2.7 μg of DNA using
Lipofectamine P3000 reagent (Thermo) according to the manufacturer’s instructions.
Transfection included: 100 ng pGL4.32[luc2P/NF-ĸB-RE/Hygro] (promega), 100 ng
pRL-null (promega), and decreasing amounts of either pME-CMV-Irak-m-HA tagged
(625ng-10ng); pME-CMV-Irak-mrΔ9-11-HA tagged (2500ng-40ng); 500 ng pME-CMV-β
77
galactosidase-HA; or 2500 ng pME-CMV-Empty Vector. The total amount of transfected
plasmid DNA remained constant for each treatment by co-transfecting with the proper
amounts pME-CMV-Empty Vector. 500 ng pME-CMV- -HA served as a
positive control for transfection efficiency. Following 18 hr of transfection, a pME-CMV-
galactosidase-HA transfected well was treated with 10 ng/mL human IL-
for 6 hr as a positive control for the dual luciferase assay. Following 24 hr transfection,
luminescence catalyzed by firefly luciferase and renilla luciferase of the cell lysates was
determined using Dual-Luciferase Reporter Assay System (promega) according to the
manufacturer’s instructions.
Experimental Animals
All mouse studies were approved by the Institute for Animal Care and Use Committee
(IACUC) at Virginia Tech and in accordance with the Federal NIH Guide for the Care
and Use of Laboratory Animals. The Irak-m-/- mice were generated as previously
described (Kobayashi et al., 2002) and purchased from The Jackson Laboratory. All
studies were controlled with either littermate and/or co-housed WT animals that were
maintained under specific pathogen-free conditions and received standard chow
(LabDiet) and water ad libitum. All experiments described utilized age matched male
mice.
Experimental Colitis and Colitis Associated Tumorigenesis
In order to assess acute experimental colitis, mice were given either 3 or 5% DSS
dissolved in drinking water available ad libitum for 5 days as previously described
78
(Williams et al., 2015b), (Schneider, 2013). On day 5, mice were withdrawn from DSS
and given regular drinking water ad libitum until euthanasia was performed on day 7.
Cumulative semi quantitative clinical scores for acute experimental colitis were
assessed as previously described (Williams et al., 2015b), (Schneider, 2013).
Tumorigenesis was induced via a single intraperitoneal (i.p.) injection of AOM (10 mg/kg
of total body weight) and supplemented with three cycles of 2.5% DSS in drinking water
available ad libitum for 5 days with 2 weeks of recovery between cycles, as previously
described (Neufert et al., 2007), (Allen et al., 2010). While subjected to DSS, mice were
monitored for weight loss, physical body condition, stool consistency, and rectal
bleeding. Upon completion of each model, whole blood was collected by cardiac
puncture for bacterial counts, flow cytometry assessments of leukocyte populations, and
serum isolation. Colon sections were collected for H&E staining. Blinded to treatment
and mouse genotype, examination of histopathology was conducted by a board-certified
veterinary pathologist (T.L.). Colon H&E sections were evaluated and scored as
previously described (Williams et al., 2015b). Additional colon sections were further
prepared for immunohistochemistry and stained with anti-β-catenin and DAPI to
determine β-catenin levels in the AOM+DSS studies.
FACS
Leukocytes and lymphocytes were evaluated from either: bone marrow, spleen, or
whole blood utilizing flow cytometry. Cells were immunostained with antibodies for the
respective cell surface markers prior to FACS analysis. Sorted cells were further
evaluated for CXCR2, CD14, CD11b+, Ly6G+, CD4+, CD8+, Ly6C+, IAE, and CD62L.
79
ELISA
Cell culture supernatants or colon organ culture supernatants were collected from each
individual well in 1.7 ml tubes and centrifuged at 300 x g for 10 minutes to remove
residual cells. Cell-free supernatants were then assayed for mouse IL-6 and/ or IL-10
(BD Biosciences) according to the manufacturer’s instructions.
Statistical Analysis
Data are represented as mean ± standard error of mean (S.E.M.) unless otherwise
indicated. Graphs and statistical analysis were conducted via GraphPad PRISM
software. Complex data sets were analyzed by 1 way analysis of variance (ANOVA)
and followed by either Tukey-Kramer HSD or Newman-Keuls method. The Kaplan-
Meier test was conducted to determine group survival. A value of p<0.05 were
considered statistically significant.
D. Results
IRAK-M Expression is Significantly Increased in Human Patients with IBD and
CRC
Previous studies have shown that IRAK-M expression is increased in IBD patients
(Fernandes et al., 2016), (Gunaltay et al., 2014). Thus, we initially sought to evaluate
these findings and expand our analysis to further evaluate IRAK-M expression in the
context of colitis associated neoplasia and CRC using a retrospective metadata analysis
of publicly available gene expression data (Kupershmidt et al., 2010). Our analysis
revealed that the relative expression of IRAK-M is significantly increased in human
80
patients with active forms of IBD (Figure 2.1A). Patients that suffer from IBD have a
higher predisposition to CAC (Karlen et al., 1999); thus, we also included CAC patients
in our data analysis and found that IRAK-M is also significantly increased in patients
with active UC with inclusive areas of neoplasia (Figure 2.1A). Independent of CAC, we
were also interested in evaluating IRAK-M expression in the context of CRC. Thus, we
also analyzed expression levels of IRAK-M in patients diagnosed with both low and
high-grade CRC. CRC patients were stratified based on the Dukes’ staging system
(A/B) and (C/D). From these data, it is apparent that IRAK-M expression is significantly
increased in patients with more advanced CRC (grades C/D) compared to the patients
with less advanced CRC (grades A/B) and patients not diagnosed with CRC (Figure
2.1B). To gain greater insight into IRAK-M function, we next sought to evaluate IRAK-M
expression in different human cell types of relevance to IBD, CAC, and CRC, with
particular emphasis on specific immune cell populations and colon epithelial cells
Figure 2.1. IRAK-M Expression is Increased During Inflammatory Bowel Disease
Exacerbation and in Advanced Colorectal Cancer Patients.
81
Figure 1. IRAK-M Expression is Increased During Inflammatory Bowel Disease Exacerbation and in Advanced Colorectal Cancer Patients
A. B.
C.
* †
*
†
*
†
¶
#
§
*
†
‡
‡ ¶ #
§
Healthy Inactive CD UC UC+Neo
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n
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2
3
4
5
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1000
1500
2000
2500
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82
(Figure 2.1C). Prior literature documents the expression of IRAK-M in cells of the
myeloid linage (Wesche et al., 1999). This is supported by our metadata analysis
(Figure 2.1C). However, our findings further revealed that IRAK-M is also highly
expressed in eosinophils and neutrophils (Figure 2.1C). We also found IRAK-M
expressed in all of the other cell types assessed, albeit at lower levels than the
macrophages, myeloid progenitor cells and granulocytes (Figure 2.1C). Together, these
data suggest that IRAK-M plays a significant role in modulating the immune response in
the GI system and is up-regulated in the context of IBD and CRC. This up-regulation is
likely in response to increased TLR and interleukin 1 receptor (IL-1R1) signaling in
these disease states.
Irak-m-/- Mice Are Protected Against DSS Induced Colitis
Figure 2.1. IRAK-M Expression is Increased During Inflammatory Bowel Disease Exacerbation and in Advanced Colorectal Cancer Patients. A. Retrospective analysis of metadata from colonic biopsies revealed that IRAK-M expression was significantly increased in Crohn’s Disease (CD) and ulcerative colitis (UC) patients during exacerbation, compared to biopsies from currently diagnosed IBD patients during inactive disease (inactive) and patients not diagnosed with IBD (healthy). IRAK-M was also significantly up-regulated in UC patients with neoplasia (UC + Neo). The fold-change values were extracted and averaged from 7 separate datasets and reflect the change in IRAK-M expression between each patient population and the respective healthy controls. *, †, ‡, §, ¶, #p<0.05. B. Colorectal cancer (CRC) patients were stratified based on the Dukes’ staging system (A/B) and (C/D). IRAK-M expression was significantly increased in patients with more advanced disease (CRC C/D) compared to the less advanced (CRC A/B) patients and patients not diagnosed with CRC (healthy). The fold-change values were extracted and averaged from 2 separate datasets and reflect the change in IRAK-M expression between each patient population and the respective healthy controls. *, † p<0.05. C. IRAK-M is differentially expressed in multiple human cell types of relevance to IBD and CRC, with the highest levels of expression in cells of the myeloid lineage. The mean expression of IRAK-M in all human cell types is identified by the dotted line (218).
83
We next sought to better characterize the contribution of IRAK-M in the context of IBD.
Prior studies have evaluated Irak-m-/- mice in both dextran sulfate sodium (DSS) and
Il10-/- colitis models (Berglund et al., 2010), (Biswas et al., 2011). These prior studies
revealed that IRAK-M functions to attenuate the progression of experimental colitis
(Berglund et al., 2010), (Biswas et al., 2011). Here we utilized Irak-m-/- mice in an acute
colitis model induced with 5% DSS (Okayasu et al., 1990). This is a standard model
utilized previously by our laboratory in similar studies evaluating mediators of innate
immunity in IBD (Williams et al., 2015a), (Williams et al., 2015b), (Allen et al., 2012),
(Allen et al., 2010). We found that Irak-m-/- mice are protected in this experimental colitis
model (Figure 2.2). Clinically, the Irak-m-/- mice demonstrate significant improvements
in weight change and clinical parameters associated with disease progression
compared to the wild-type (WT) animals (Figure 2.2A and 2.2B). Consistent with our
clinical observations, Irak-m-/- mice also displayed significantly increased colon length
and less severe tissue damage as evident by histopathology evaluation of H&E stained
colon sections compared to the WT counterparts (Figure 2.2C and 2.2D). Interestingly,
when viewing the histopathology of the colon from Irak-m-/- mice we observed localized
and highly structured areas of lymphoid cells throughout the colon. These structures
were identified and confirmed to be expanded areas gut associated lymphoid tissue
(GALT) by a board certified veterinary pathologist (T.L.) (Figure 2.2E). We
hypothesized that the protective phenotype observed in the Irak-m-/- mice following
acute DSS exposure was due, in part, to the significant increase in GALT in the colon of
the Irak-m-/- mice, which were present irrespective of DSS treatment (Figure 2.2E).
Figure 2.2. Attenuated Experimental Colitis Pathogenesis in Irak-m-/- Mice.
84
0 2 4 6 8-15
-10
-5
0
5
Figure 2. Attenuated Experimental Colitis Pathogenesis in Irak-m-/- Mice
D. Mock Irak-m-/- +DSS Wild Type +DSS
*
B.
0
2
4
6
8
10
Mock
Irakm
r!9-
11
Wi ld
Type
*
Clin
ica
l Sc
ore
Cli
nic
al S
co
re
F.
0
100
200
300
Ba
cte
ria
l Co
un
ts in
Blo
od
*
Wi ld
Type
Irakm
r! 9
-11
Ba
cte
ria C
ou
nts
in
Blo
od
C.
0
2
4
6
8
10
Mock
Irakm
r!9-
11
Wi ld
Type
**
Co
lon
Le
ng
th (c
m)
Co
lon
Len
gth
(c
m)
G.
0
500
1000
1500
Co
lon
IL
-6 (p
g/m
l)
DSSMock
Wi ld
Type
Wi ld
Type
Irakm
r!9-
11
Irakm
r!9-
11
*
**
Co
lon
IL
-6 (
pg
/ml/
mg
)
Mock DSS
A.
*
†
* *
* †
† †
Day
% W
eig
ht
Ch
an
ge
Mock
Wild Type
Irak-m-/-
E. Irak-m-/- Mock GALT
* †
* †
DSS
85
To test this hypothesis, we measured the systemic bacteremia in whole blood of both
WT and Irak-m-/- mice following the acute DSS model. Our data lend support to this
hypothesis as bacterial counts were significantly reduced in Irak-m-/- mice (Figure 2.2F).
Further, we observed increased levels of IL-6 from Irak-m-/- colon organ culture
supernatant following DSS treatment, which is consistent with the increased GALT
(Figure 2.2G). While increased IL-6 is typically associated with detrimental
inflammation, our histopathology assessments revealed that the inflammation was
highly localized to these areas of GALT, with minimal damage to the epithelial cell
barrier (Figure 2.2D). Collectively, our data suggest that Irak-m-/- mice are protected
against DSS induced colitis by displaying increased resistance to bacterial translocation
and bacteremia.
Attenuation of Experimental Colitis in the Irak-m-/- Mice is Associated with
Enhanced Neutrophil and T-Cell Responses.
Figure 2.2. Attenuated Experimental Colitis Pathogenesis in Irak-m-/- Mice. A. Irak-m-/- mice demonstrated significantly improved weight change following DSS exposure compared to similarly treated WT mice. Irak-m-/- mock weight is not included for simplicity. B. Clinical features associated with disease progression were significantly attenuated in Irak-m-/- mice. C. Irak-m-/- mice had significantly increased colon length compared to WT animals. D. Representative H&E stained histopathology images of colon sections from WT and Irak-m-/- mice following the acute colitis model. Scale bars = 100 μm E. Irak-m-/- mice displayed large areas of Gut Associated Lymphoid Tissue (GALT), regardless of exposure to DSS. Scale bars = 100 μm. F. Bacteria count in 25μl of blood from WT (n=5) and Irak-m-/- mice (n=5) with DSS induced colitis. G. IL-6 levels were significantly increased in colon sections harvested from Irak-m-/- mice following DSS exposure. Data are represented as mean ± SEM. *p<0.05; †p<0.05 by 1 way ANOVA, Newman-Keuls post-test. WT mock, n=3; WT DSS, n=6; Irak-m-/- mock, n=3 (not shown); Irak-m-/-
DSS, n=5. Data are representative of 3 independent studies.
86
We hypothesized that the increased GALT observed in the Irak-m-/- mice was a
significant contributing factor in protecting these animals during experimental colitis. To
test whether Irak-m-/- protection from acute DSS was associated with increased
leukocyte recruitment and function, we evaluated the leukocyte composition in whole
blood using flow cytometry (Figure 2.3A). Under basal conditions, we found the
neutrophil count was significantly higher in the blood from naïve Irak-m-/- mice compared
to WT animals (Figure 2.3A). Interestingly, following exposure to DSS the number of
neutrophils significantly increased in WT mice; however, the number of neutrophils in
the Irak-m-/- mice maintained elevated levels with or without exposure to DSS (Figure
2.3A). We further measured the levels of CXCR2 and CD14 from both naïve animals
and mice in the experimental colitis model (Figure 2.3B and 2.3C). Under both naïve
and DSS treated conditions we observed increased levels of CXCR2 and CD14 from
Irak-m-/- Ly6G+/CD11b+ leukocytes, suggesting Irak-m-/- neutrophils are primed prior to
tissue insult, which likely improves the efficiency in recruitment to a site of infection,
such as the colon when bacterial translocation occurs. To further support this
hypothesis we performed a neutrophil chemotaxis assay in response to macrophage
inflammatory protein 2 (MIP-2). When bone marrow from WT and Irak-m-/- mice was
primed with doses of LPS and subjected to the chemotaxis assay, we observed
increases in neutrophils in a dose dependent manner for Irak-m-/- mice (Figure 2.3D).
Further, we also observed increased numbers of CD4+, CD8+ T-cells and monocytes
Figure 2.3. Increased Neutrophil and T-cell Function in Irak-m-/- Mice.
87
WT Irak-m-/- L
y6
G
CD11b
Na
ïve
D
SS
A.
C. D.
W T IR AK M - /-
2 0
4 0
6 0
8 0
Ly
6G
+C
D1
1b
+%
in
Blo
od
* *
Ly
6G
+C
D11b
+%
20
40
60
80
**
25
Ly
6G
+C
D1
1b
+%
in
Blo
od
W T IR A K M - /-
0
5
1 0
1 5
2 0
2 5 * *
0
5
10
15
20
Ly
6G
+C
D11b
+%
**
W T IR AK M - /-
0
2 0
4 0
6 0
8 0
1 0 0
CD
14
MF
I
* * *
CD
14
MF
I
20
0
40
60
80
100 ***
W T IR AK M - /-
4 0
6 0
8 0
1 0 0
1 2 0
CD
14
MF
I
* * *
CD
14
MF
I
40
60
80
100
120
***
CD
4+
%
W T IR AK M -/-
0
5
1 0
1 5
2 0
2 5
*
CD
8+
%
W T IR AK M -/-
0
5
1 0
1 5
*
Ly
6G
-Ly
6C
+C
D1
1b
+%
W T IR AK M -/-
0 .0
0 .5
1 .0
1 .5
2 .0
*
E.
0
5
10
15
20
25
CD
4+
%
0
5
10
15
CD
8+
%
0.0
0.5
1.0
1.5
Ly
6G
-LY
6C
+C
D11
b+
%
2.0 * *
*
WT
IRAKM-/-
IAE CD62L
WT Irak-m-/-
IAE
MF
I
W T IR AK M -/-
0
2 0
4 0
6 0
8 0
* *
CD
62
L M
FI
W T IR AK M -/-
0
2 0
4 0
6 0
8 0
1 0 0 * *
F.
0
20
40
60
IAE
MF
I
80
0
20
40
60
CD
62
L M
FI
80
100 ** **
WT Irak-m-/-
Naïve DSS
CD14 CD14
WT Irak-m-/- Irak-m-/-
WT
Figure 3. Increased Neutrophil and T-cell Function in Irak-m-/- Mice
B.
W T IR AK M -/-
2 0 0
2 5 0
3 0 0
3 5 0
4 0 0
CX
CR
2 M
FI
* * *
200
250
300
350
400
CX
CR
2 M
FI
***
W T IR AK M- /-
3 0 0
3 2 0
3 4 0
3 6 0
3 8 0
4 0 0
CX
CR
2 M
FI
*
CX
CR
2 M
FI
300
320
340
360
380
400 *
CXCR2
Naïve
CXCR2
DSS
WT Irak-m-/-
WT
Irak-m-/-
Irak
-m-/
-
Me
dia
M
IP-2
PBS 100pg/ml LPS
10ng/ml LPS
1µg/ml LPS
Wil
d T
yp
e
Me
dia
M
IP-2
Fo
ld I
ncre
ase
F
old
In
cre
ase
F
old
In
cre
as
e
0.5
1.0
1.5
2.0
0.5
1.0
1.5
2.0
*** **
* *
88
isolated from the spleen of Irak-m-/- mice following exposure to DSS (Figure 2.3E). This
finding is consistent with prior studies that indicated increased T-cell recruitment in Irak-
m-/- mice in the experimental colitis model (Berglund et al., 2010), (Klimesova et al.,
2013). Monocytes isolated from the spleen of Irak-m-/- mice following DSS exposure
displayed increased cell surface expression of IAE and decreased expression of CD62L
(Figure 2.3F). Further, we hypothesized that the differences in neutrophil and T-cell
recruitment in Irak-m-/- mice was the result of differences associated with the GI
microbiota composition. When mice were treated with antibiotics for two weeks prior to
DSS administration, similar susceptibilities to pathogenesis were observed between WT
and Irak-m-/- mice (Figure 2.4A-C). Collectively, our data suggests that Irak-m-/- mice
are protected from experimental colitis due to efficient recruitment of neutrophils and T-
cells following microbial translocation. These data provide a mechanism of acute colitis
protection and lend support to the phenotype that GALT in Irak-m-/- mice contributes to
the overall protection observed in the experimental colitis model.
Irak-m-/- Mice Are Protected Against Inflammation Driven Colon Tumorigenesis.
Figure 2.3. Increased Neutrophil and T-cell Function in Irak-m-/- Mice. A. Representative FACS plots of neutrophils and the percentage of neutrophils (Ly6G+CD11b+) in the blood of naïve mice and mice with DSS-induced ulcerative colitis. B. The expression of CXCR2 on the Ly6G+CD11b+ cells in the blood. C. The expression of CD14 on the Ly6G+CD11b+ cells in the blood. *p<0.05, **p<0.01, ***p<0.001, n=5. D. Chemotaxis of neutrophils to MIP-2. Neutrophils purified from the bone marrow were primed with different doses of LPS, then subjected to chemotaxis assay. PBS is used as control. *p<0.05, **p<0.01, ***p<0.001, n=5. Scale bars ≈ 10 μm. E. Percentages of CD4+, CD8+ T cells and monocytes in the spleen from mice with DSS-induced ulcerative colitis. F. The expression of IAE and CD62L on Ly6G-Ly6C+CD11b+ monocytes in the spleen from mice with DSS-induced ulcerative colitis. *p<0.05, **p<0.01, n=4.
89
Our retrospective metadata analysis revealed that IRAK-M likely modulates IBD, CAC,
and CRC in human patients (Figure 1A and 1B). Based on our findings in the IBD
model, we next sought to evaluate the Irak-m-/- mice in a model of colitis associated
tumorigenesis. Here we utilized the AOM+DSS model (Williams et al., 2015a),
Figure 2.4. Increased Morbidity and Mortality in Antibiotic + DSS Treated Mice
C
0 2 4 6 80
25
50
75
100
125
Irakm-/- + DSS + Antibiotic
Irakm-/- + Antibiotic
Wild Type+DSS+ Antibiotic
Wild Type + Antibiotic
Days
Pe
rce
nt s
urv
iva
l
A. B.
80
90
100
110
120
Irakm-/- + DSS + Antibiotic
Irakm-/- + Antibiotic
Wild Type + DSS + Antibiotic
Wild Type + Antibiotic
5% DSS
10 2 3 4 5 6 7
Days
Bo
dy
We
igh
t E
vo
lutio
n (
%)
4
5
6
7
8 N.S.
N.S. N.S.
N.S.
Co
lon
Le
ng
th (c
m)
90
(Allen et al., 2012), (Allen et al., 2010). Irak-m-/- mice displayed improved morbidity and
mortality throughout the course of the model compared to the WT animals, suggesting
attenuation of disease progression compared to the WT counterparts (Figure 2.5A-C).
When colon organ culture supernatants were assayed for IL-6 and IL-10, Irak-m-/- mice
displayed significant increases in both cytokines (Figure 2.5D-E). Beyond the significant
attenuation of clinical features and enhanced cytokine responses, the Irak-m-/- mice
displayed dramatic resistance to tumorigenesis (Figure 2.6). The overall gross polyp
formation was significantly higher in WT mice; conversely, no polyps were detected in
Irak-m-/- mice over the course of the study (Figure 2.6A-C). Decreased tumor burden
was further supported by histopathology assessments of H&E stained colon sections
(Figure 2.6D). As described for the experimental colitis model, GALT formation was
prominent in Irak-m-/- mice (Figure 2.6E). Consistent with the macroscopic colon
evaluation, histopathology scoring revealed significant reductions in areas of
hyperplasia and dysplasia in Irak-m-/- mice (Figure 2.6F-G). Likewise, we found that
Figure 2.4. Increased Morbidity and Mortality in Antibiotic + DSS Treated Mice Wild type and Irak-m mutant animals were subjected to two weeks of antibiotic (Ab) treatment prior to DSS administration available ad libitium consisting of 0.5mg/ml Ampicillin, 0.5mg/ml Metronidazole, 0.5mg/ml Neomycin, 0.25mg/ml Vancomycin and 12mg/ml splenda (Rutkowski et al., 2015), (Hernandez-Chirlaque et al., 2016). Wild type and Irak-m mutant animals were then given either Ab water or Ab + 5% DSS for 5 days, followed by Ab water alone for two days. A. Body weight evolution of mice once supplemented with DSS. B. Colon length of mice following completion of the study. C. Survival curve of mice throughout the study. n=5 Wild type + Ab; n=5 Irak-m-/- +Ab; n=5 Wild type Ab+DSS; n=5 Irak-m-/- + Ab+DSS.
91
Figure 2.5. Colitis Associated Tumorigenesis Progression is Significantly
Reduced in Irak-m-/- Mice
†
D a y s
Pe
rc
en
t s
urv
iva
l
0 2 0 4 0 6 00
2 0
4 0
6 0
8 0
1 0 0
Ira k m- /-
A O M + D S S
W ild T y p e A O M + D S S
M o c k + A O MMock AOM
Irak-m-/- AOM+DSS
WT AOM+DSS
0 20 40 60
Days
0
100
80
60
40
20
% S
urv
iva
l
*
*
†
A.
Figure 4. Colitis Associated Tumorigenesis Progression is Significantly Reduced in Irak-m-/- Mice
B.
D. E. C.
0 10 20 30 40 50 60
Days %
We
igh
t C
han
ge
-20
-15
-10
-5
0
5
10
15
20
25 Mock AOM
Irak-m-/- AOM+DSS
WT AOM+DSS
Clin
ica
l S
co
re
0.0
0.5
1.0
1.5
2.0
2.5
AOM+DSS
**
0
0.5
1.0
1.5
2.0
2.5
AOM + DSS AOM + DSS
0
100
200
300
400
500
AOM+DSS
**
IL-6
(p
g/m
l/mg
)
0
100
200
300
400
500
IL-6
(p
g/m
l/m
g)
0
10
20
30
40
50
IL-1
0 (
pg
/ml/m
g)
AOM+DSS
*
*
0
10
20
30
40
50
AOM + DSS
IL-1
0 (
pg
/ml/
mg
)
DSS DSS DSS
*
*
*
92
the levels of β-catenin are also markedly reduced in Irak-m-/- mice following AOM+DSS
(Figure 2.6H). Collectively, our data indicates that the Irak-m-/- mice are resistant to
both experimental colitis and inflammation driven tumorigenesis.
Identification of a Splice Variant of the Irak-m Gene in Irak-m-/- Mice
Expression levels of IRAK-M are highest in macrophages and previous studies have
utilized both human and mouse macrophages to study IRAK-M function (Wesche et al.,
1999), (Kobayashi et al., 2002). Thus, we were interested in determining the response
of IRAK-M in murine bone marrow derived macrophages (BMDM) when challenged with
diverse PAMPs. In our hands, BMDM from our Irak-m-/- mice display significantly
increased levels of IL-6 compared to WT BMDMs when stimulated for 24 hours with
different TLR ligands, specifically the TLR2 ligand Pam3CSK4 (Figure 2.7A). Further,
we assessed a broad panel of secreted cytokines following 24 hour treatment with TLR
1-9 agonists. Specifically, Irak-m-/- BMDMs display increased levels of TNF with TLR1/2
Figure 2.5. Colitis Associated Tumorigenesis Progression is Significantly Reduced in Irak-m-/- Mice. A. Kaplan-Meier plot of Irak-m-/- and WT survival. *p<0.05; †p<0.05. B. WT mice demonstrated significant weight loss throughout the AOM/DSS model, whereas weight loss in the Irak-m-/- mice was minimal. All mock and mock+AOM mice gained weight throughout the duration of the model, regardless of genotype. The WT mock+AOM are shown for comparison. C. Clinical features associated with disease progression were significantly improved in Irak-m-/-
mice. Data shown reflect the clinical scores on the final day of the model (Day 65). D-E. Both IL-6 and IL-10 levels were significantly increased in colons harvested from Irak-m-/- mice compared to mock, mock+AOM, and WT AOM+DSS treated mice. Data are represented as mean ± SEM. *p<0.05; †p<0.05; p<0.05 by 1 way ANOVA, Newman-Keuls post-test. Mock WT, n=5 (not shown); mock Irak-m-/-, n=5 (not shown); AOM+mock WT, n=5; AOM+mock Irak-m-/-, n=5 (not shown); AOM+DSS WT, n=18; AOM+DSS Irak-m-/-, n=5. Data are representative of 3 independent studies.
93
Figure 2.6. Irak-m-/- Mice Display Attenuated Polyp Formation in the Colitis
Associated Tumorigenesis Model.
Figure 5. Irak-m-/- Mice Display Attenuated Polyp Formation in the Colitis Associated Tumorigenesis Model
A. B.
F.
D. E.
Wild Type Irak-m-/-
Dis
tal
Pro
xim
al
C.
G.
Irak-m-/- AOM+DSS Wild Type AOM+DSS
GALT Irak-m-/-
Mock
*
*
ND ND
AOM + DSS
0
1
2
3
Nu
mb
er
of
Ma
cro
sc
op
ic P
oly
ps
/Co
lon
AOM+DSS
**
Nu
mb
er
of
Ma
cro
sc
op
ic P
oly
ps/C
olo
n
0
1
2
3
0
1
2
3
4
AOM+DSS
Po
lyp
Siz
e (m
m2)
**
ND ND
AOM + DSS
Po
lyp
Siz
e (
mm
2)
0
1
2
3
4
0
5
10
15
20
25
HA
I S
co
re
* *
AOM+DSSAOM + DSS
HA
I S
co
re
0
5
10
15
20
25
0
1
2
3
Hy
pe
rpla
sia
an
d D
ys
pla
sia
AOM+DSS
* *
AOM + DSS
Hyp
erp
lasia
& D
ys
pla
sia
0
1
2
3 DAPI Merge ! -catenin
Wil
d T
yp
e
Ira
k-m
-/-
H.
94
agonists and decreased IL-10 with TLR 1/2, 7, and 9 agonists (Figure 2.8A-E). This is
consistent with prior reports pertaining to IRAK-M being a negative regulator of TLR
signaling (Kobayashi et al., 2002). We also observed differences in IL-6 secretion
between male and female Irak-m-/- BMDMs when stimulated with Pam3CSK4 (Figure
2.9). However, during routine follow-up studies, we observed IRAK-M protein induction
in both the WT and Irak-m-/- strains when stimulated with various TLR ligands using an
antibody specific for the C-terminus of IRAK-M (Figure 2.10). This was unexpected, as
our Irak-m-/- mice were acquired from a commercial vendor, albeit reconstituted from
frozen embryos, and previously characterized (Kobayashi et al., 2002). It was previously
reported that generation of the Irak-m-/- mouse targeted exons 9-11 for deletion by
homologous recombination inserting the neomycin resistance cassette in place of these
Figure 2.6. Irak-m-/- Mice Display Attenuated Polyp Formation in the Colitis Associated Tumorigenesis Model. A. Representative image of macroscopic polyp formation in WT and Irak-m-/- mice. Black arrows indicate macroscopic polyps in WT mice, with none found in Irak-m-/- animals. Scale = mm. B. Average number of macroscopic polyps per colon of WT and Irak-m-/- mice following the AOM/DSS model. ND = none detected. C. Average macroscopic polyp size between WT and Irak-m-/- mice. D. Representative H&E stained histopathology images of colon sections from WT and Irak-m-/- mice following the AOM/DSS model. Scale bars = 100μm. E. Representative H&E staining of Gut Associated Lymphoid Tissue (GALT) from the colon of Irak-m-/-mice following the AOM/DSS model. Areas of expanded GALT were detected in the Irak-m-/- mice irrespective of exposure to AOM/DSS. Scale bar = 100μm F. Irak-m-/- mice demonstrated a significant attenuation in histopathological features associated with the AOM/DSS model, as indicated by the histopathological activity index (HAI) score. G. Histopathology assessments also revealed that Irak-m-/- mice had significantly attenuated development of hyperplasia and dysplasia compared to the WT animals. H. Representative images of immunofluorescence staining of the colon sections from WT and Irak-m-/- mice following the AOM/DSS treatment. The tissue sections were fixed and stained with anti–β-catenin (red) and DAPI (blue). Scale bars = 30 μm. Data are represented as mean ± SEM. *p<0.05; p<0.05 by 1 way ANOVA, Newman-Keuls post-test. Mock WT, n=5 (not shown); mock Irak-m-/-, n=5 (not shown); AOM+mock WT, n=5; AOM+mock Irak-m-/-, n=5 (not shown); AOM+DSS WT, n=18; AOM+DSS Irak-m-/-, n=5. Data are representative of 3 independent studies.
95
Figure 2.7. Disruption of the Murine Irak-m Locus Results in the Formation of an
Irak-mrΔ9-11 Splice Variant.
! "
#"
Figure 6. Disruption of the Murine Irak-m Locus Results in the Formation of an Irak-mr! 9-11 Splice variant
C.
800
400
600
1000
1500
Exon: 5-12
500/517
700
2000
(bp) 2531
! 400bp
WT Irak-m-/-
C Pam3 C Pam3 !
Neg.
A.
D.
Neo
WT Irak-m
Irak-mr! 9-11
1 2 3,4,5,6,7,8 9,10,11 12
1 2 34 5 6 7 8 9-11 12
1 2 34 5 6 7 8 12
1 2 3,4,5,6,7,8 12
B.
5-8, 9-10, 10-11, 12
!
Exon: 5-8 9-10 10-11 12
300
600
100
200
400 500/517
700
WT Irak-m-/- Neg. Control
C Pam3 C Pam3!
WT Irak-m-/- WT Irak-m-/- WT Irak-m-/-
C Pam3 C Pam3! C Pam3 C Pam3! C Pam3 C Pam3!
E.
102kDa
! actin (42 kDa)
HA (short exposure)
Em
pty
Ve
cto
r !
Ga
l!
Ga
l+IL
-1!
WT Irak-m Irak-mr! 9-11
150kDa
76kDa
52kDa
38kDa
HA (long exposure)
150kDa
102kDa
76kDa
52kDa
38kDa
Empty
Vecto
r
500n
g Beta
Gal
500n
g Beta
Gal +IL
-1beta
Wt 625
Wt 320
Wt 160
WT 80
Wt 40Wt 2
0Wt 1
0
Irakm
2500
Irakm
1250
Irakm
625
Irakm
320
Irakm
160
irakm
80
Irakm
40
0
20
40
60
80
*
*
†
†
WT Irak-m Irak-mr! 9-11
Re
lati
ve
Lu
min
es
cen
ce
Un
its
0
20
40
60
80
Mock
poly IC
PG
N-S
ALPS
Pam
3CSK4
TNFa
0
2000
4000
6000
80009000
14000
19000
24000
29000
WT
Irakm-/-
IL-6
Co
nc
en
tra
tio
n (
pg
/ml)
WT Irak-m-/-
*
96
three exons (Kobayashi et al., 2002). In order to test the hypothesis that IRAK-M was
present in our mutant mice, we proceeded by further investigating the phenomena at
the
Figure 2.7. Disruption of the Murine Irak-m Locus Results in the Formation of an Irak-mrΔ9-11 Splice Variant. A. Bone marrow derived macrophages (BMDM) from Irak-m-/- mice demonstrate increased production of IL-6 following 24 hr stimulation with specific PAMPs. Data shown are pooled from 3 separate and independent experiments. n=3 mice per genotype. B. Disruption of the Irak-m locus resulted in the loss of Exons 9-11. BMDM from WT and Irak-m-/- littermates were un-stimulated or stimulated for 24 hr with Pam3CSK4. After 24 hr, cells were harvested for RNA and RT-PCR was utilized to amplify Irak-m corresponding to exons: 5-8, 9-10, 10-11 and 12, respectively. C. A size difference was detected in the RT-PCR product corresponding to Exons 5-12 between WT and Irak-m-/- BMDMs treated for 24 hr with Pam3CSK4. The shift of approximately 400 nucleotides in the Irak-m-/- BMDMs corresponds to the deleted exons 9, 10 and 11. D. Sequencing of the exon 5-12 RT-PCR product revealed a splice immediately after exon 8 together with exon 12. Normal splicing occurs in the WT BMDM, whereas Irak-m-/- BMDM splice around the inserted Neo-cassette after exon 8 and generates a splice variant connecting exon 8 with exon 12. E. (Left) Dual-Luciferase assay of HEK293T cells transfected for 24 hr with equal amounts of NF-κB-firefly luciferase reporter plasmid and decreasing amounts of either CMV-WT Irak-m-HA plasmid (625ng-10ng) or CMV-Irak-mrΔ9-11-HA plasmid (2500ng-40ng) from three independent and pooled experiments. Empty Vector is shown as a negative control. β-Galactosidase-HA is shown as a positive control for transfection. A separate β-Galactosidase-HA transfection was treated with 10 ng/mL human IL-1β for 6hr following 18hr of transfection, and serves as a positive control for NF-kB-firefly luciferase activation. All treatment groups received a constant amount of plasmid DNA described in the Experimental Methods and are normalized to the Renilla luciferase activity. (Right) Representative western blot of WT IRAK-M and IRAK-MrΔ9-11 protein expression. Overexpression inserts are HA-tagged at the C-
-actin is shown as a loading control. The WT IRAK-M expresses readily as seen in the short exposure and migrates at ~ 68kDa. The IRAK-MrΔ9-11 protein is present in the long exposure migrating at ~ 38kDa. Data in panel A. is represented as mean ± SEM. *p<0.05 by 1 way ANOVA, Tukey-Kramer post-test. Data in panel E. is represented as mean ± SEM. *p<0.001, †p<0.01 by 1 way ANOVA, Tukey-Kramer post-test. Statistical significance between all treatment groups in E is not shown for simplicity. A minimum of 3 independent experiments was conducted for all assays.
97
2.8 Differences in TLR-2, TLR-7 and TLR-9 Stimulation in Irak-m Mutant Mice.
Con
torl
PolyIC
LPS
PGN-S
A
Pam
3CSK4
Flage
llin
CpG
DNA
Imiquim
od
IL-1
b
TNFa
0
1000
2000
3000WT
Irak-m-/-
***
**
TN
F C
oncentr
ation (
pg/m
l)
Con
torl
PolyIC
LPS
PGN-S
A
Pam
3CSK4
Flage
llin
CpG
DNA
Imiquim
od
IL-1
b
TNFa
0
WT
Irak-m-/-
No Detection
IFN
-g C
oncentr
ation (
pg/m
L)
Con
torl
PolyIC
LPS
PGN-S
A
Pam
3CSK4
Flage
llin
CpG
DNA
Imiquim
od
IL-1
b
TNFa
0
10
20
30
40
8000
10000
12000
14000
WT
Irak-m-/-
IL-1
b C
oncentr
ation (
pg/m
l)
Con
torl
PolyIC
LPS
PGN-S
A
Pam
3CSK4
Flage
llin
CpG
DNA
Imiquim
od
IL-1
b
TNFa
0
500
1000
1500
2000
2500
WT
Irak-m-/-
***
***
**
IL-1
0 C
oncentr
ation (
pg/m
L)
Con
trol
PolyIC
LPS
PGN-S
A
Pam
3CSK4
Flage
llin
CpG
DNA
Imiquim
od
IL-1
b
TNFa
0
5000
10000
15000
20000
25000
WT
Irak-m-/-
***
***
**
IL-6
Concentr
ation (
pg/m
l)
A.
B.
D.
E.
C.
98
Figure 2.9. Comparison of Male and Female Bone Marrow Derived Macrophages
Figure 2.8. Differences in TLR-2, TLR-7 and TLR-9 Stimulation in Irak-m Mutant Mice. Bone marrow was harvested from 8-week age matched males. BMDMs were derived and seeded in 24 well plates as described in the Materials and Methods. Cells were stimulated with either: Control media, 6.25μg/ml Poly(I:C), 10 ng/ml LPS, 10 μg/ml PGN-SA, 300 ng/ml Pam3CSK4, 50 ng/ml standard Flagellin-BS, 5 μM CpG DNA ODN1668, 1 μg/ml Imiquimod (R837), 20 ng/ml m-IL-1β, 75 ng/ml m-TNFα. Cell free supernatants were collected and assayed by ELISA for extracellular: A. IL-6. B. IL-10. C. TNF (75ng/ml TNFα treatment indicates the upper limit/saturation range of the ELISA). D. IL-1β (Data plotted is extrapolated, as the values were below the detection limit of the ELISA). E. IFN-γ. Data indicates the mean from n=3 WT, n=3 Irak-m-/- mice. Error bars indicate SEM. Data was analyzed by 1-way ANOVA followed by Tukey’s HSD. *p<0.05, **p<0.01,***p<0.001.
99
mRNA level. When BMDMs were stimulated for 24 hours with the TLR1/2 ligand
Pam3CSK4 to induce Irak-m transcription, we observed an amplification band
corresponding specifically to exon 12 of Irak-m by RT-PCR (Figure 2.7B). PCR primers
were further designed to amplify the region spanning the length of exons 5 and 12
(Figure 2.7C). Interestingly, a shift of approximately 400bp was observed in the BMDMs
from our Irak-m-/- animals, which suggested a splice variant had been generated. This
was indeed confirmed by Sanger sequencing, which revealed a splice occurred after
exon 8 and connecting it with exon 12 in the amplification band (Figure 2.7D). Loss of
the neo cassette only occurred at the mRNA level, likely after exon splicing, and not at
the DNA level. This is evident because the genotyping primers are targeted against a
portion of the neo cassette, which is present in the genotyping for the Irak-m-/- mice.
Based on this revelation, we have defined this splice variant as Irak-mrΔ9-11. In order to
test the functionality of this truncated transcript, we cloned both the WT and mutant Irak-
mrΔ9-11 transcripts. Overexpression in HEK293T cells of both the WT and truncated Irak-
mrΔ9-11 transcripts both had the capacity to activate an NF-
reporter. However, NF-κB activation was robustly increased following overexpression of
Irak-mrΔ9-11 compared to the WT (Figure 2.7E), suggesting a functional and potent role
for this mutant protein. We further validated the specificity of the commercially available
Figure 2.9. Comparison of Male and Female Bone Marrow Derived Macrophages. IL-6 ELISA of BMDM stimulated for 24 hrs with respective Pamps. Concentrations of Pamps are described in the Materials and Methods. n=3 WT males, n=3 Irak-m-/- males, n=3 WT females, n=3 Irak-m-/- females. Data was analyzed by 1-way ANOVA followed by Tukey’s HSD. ***p<0.001.
100
Figure 2.10. Western Blot Evaluation of Protein Expression of IRAK-M.
IRAK-M antibody used in Figure S1, which displayed no specificity for the WT-IRAK-M
or Irak-mrΔ9-11 overexpression constructs (data not shown).
E. Discussion
Overzealous inflammation associated with dysregulated innate immune signaling is a
significant component of IBD pathogenesis. This hyper-inflammation is often associated
with aberrant TLR signaling. There is significant interest in better characterizing the
contribution of proteins, like IRAK-M, that regulate PRR signaling in IBD, CAC, and
CRC. This interest is due to the revelation that these regulatory proteins significantly
IRAK-M
(68 kDa)
β-actin
(42 kDa)
(4.2) (0.08) (0.4) (0.9) (0.7) (0.08) (0.58) (0.9) (0.6)
Figure 2.10. Western Blot Evaluation of Protein Expression of IRAK-M. Western blot evaluation revealed that IRAK-M protein is present in both the WT and Irak-m-/- treatment groups following 24hr stimulation with specific PAMPs. Western blot is normalized to β-actin and human THP-1 cells are used as a positive control. Densitometry is quantified with numerical values below β-actin using ImageJ. Further, independent validation of the IRAK-M antibody used displayed no specificity for the murine WT-IRAK-M-HA or Irak-mrΔ9-11-HA overexpression constructs (data not shown).
101
attenuate disease pathogenesis in IBD mouse models. For example, prior studies by
our group and others have shown that a diverse group of negative regulatory proteins,
including NLRP12, NLRX1, TOLLIP, A20, and GIT2, also function to maintain immune
system homeostasis in the gut through the attenuation of hyper-responsive
inflammation (Mukherjee and Biswas, 2014), (Vereecke et al., 2014), (Hammer et al.,
2011), (Boj et al., 2015), (Allen et al., 2012), (Zaki et al., 2011), (Soares et al., 2014),
(Singh et al., 2015). Many of these negative regulatory proteins have been found
dysregulated in IBD and CRC patients. For example, previous reports have indicated
that IRAK-M expression is significantly increased in IBD patients with both UC and CD
(Fernandes et al., 2016), (Gunaltay et al., 2014). These findings are consistent with our
analysis of retrospective metadata that revealed increased IRAK-M expression, not only
in active UC and CD patients, but also in the context of colitis associated neoplasia
(Figure 2.1). Our retrospective metadata analysis revealed that IRAK-M expression
increases with severity of CRC progression (Figure 2.1). Together these data likely
reflect an increase in the innate immune response during both IBD and CRC that is
driven by PRR activation. These data are consistent with recent findings that revealed
increased IRAK-M induction in colon tumor cells associated with the combined effects of
Wnt and TLR activation (Kesselring et al., 2016). The increase in IRAK-M expression,
as well as the expression of other genes that encode negative regulators of PRRs, is
likely an attempt to reign in overzealous inflammation and maintain some level of
immune system homeostasis during IBD and CRC.
102
The Irak-m-/- mouse model has been an essential tool to determine the negative
regulatory mechanisms underlying TLR signaling. Irak-m-/- mice have proven to be more
sensitive to TLR stimulation and display impaired endotoxin tolerance, likely due to the
hyper-activation of NF-κB signaling (Kobayashi et al., 2002). In addition to negatively
regulating canonical NF-κB signaling, IRAK-M inhibits the non-canonical NF-κB
cascade, in part, through modulating the degradation of NF-ĸB inducing kinase (NIK)
(Su et al., 2009). In prior tumor injection models, loss of Irak-m-/- has been shown to
result in enhanced innate immune responses and the attenuation of tumor growth (Xie
et al., 2007), (Standiford et al., 2011). These studies were based on tumor injection
models with Irak-m-/- mice. However, the overall conclusions of each study are
consistent with the observations reported here. In both prior studies, the Irak-m-/-animals
demonstrated significant resistance to tumor growth and pathogenesis following
inoculation. Mechanistically, the attenuation of tumorigenesis was associated with
increased activation and proliferation of B cells and T cells, specifically CD4+ and CD8+
T cells (Xie et al., 2007). Our group has previously shown that Irak-m-/- macrophages
display enhanced uptake of acLDL, as well as increased percentages of macrophages
that uptake apoptotic thymocytes (Xie et al., 2007). Reports have demonstrated IL-10R
signaling plays a dramatic role in macrophage function (Shouval et al., 2014);
additionally, IL-10 is produced from macrophages following the uptake of apoptotic cells
(Chung et al., 2007), (Zhang et al., 2010). Therefore, though we have not explicitly
tested, we speculate that increased phagocytosis of apoptotic cells by Irak-m mutant
macrophages results in increased IL-10 leading towards a tolerant, and potentially
protective phenotype.
103
Our findings in the experimental colitis and colitis associated tumorigenesis models
were initially surprising, as Irak-m-/- mice appear to have increased inflammation.
However, upon further investigation we discovered that the inflammation is actually
highly compartmentalized and isolated to regions of enhanced GALT, with little damage
or effect to the epithelial cell barrier. As we detail in the current manuscript, we believe
that these expanded areas of GALT are actually beneficial to these mice and function to
improve the efficiency of the immune response to translocating microbes from the GI
lumen. This is further supported as (Kesselring et al., 2016) described increased GI
epithelial barrier permeability in Irak-m-/- mice using FITC labeled dextran.
IRAK-M modulation of experimental colitis and colitis associated tumorigenesis has
been previously evaluated in Irak-m-/- mice (Berglund et al., 2010), (Biswas et al., 2011,
(Klimesova et al., 2013). In the initial study that evaluated IRAK-M function during DSS-
induced colitis, Irak-m-/- mice were treated with 3% DSS for 5 days and allowed 2 days
to recover prior to harvest (Berglund et al., 2010). In this model, Irak-m-/- mice were
found to be sensitive to DSS and presented with significantly increased clinical and
histopathological features associated with disease progression (Berglund et al., 2010).
This study also found elevated cytokine, chemokine, and T-cell transcription factor
mRNA expression in Irak-m-/- colon tissue and increased systemic IL-6 and TNF levels
in the plasma following DSS administration (Berglund et al., 2010). Subsequent studies
by a different group utilizing the AOM+DSS colitis associated tumorigenesis model in
both conventional and germfree conditions also reported increased sensitivity and
104
tumorigenesis in the Irak-m-/- mice (Klimesova et al., 2013). Similar to the study by
Berglund et al, the Irak-m-/- mice were shown to have enhanced pro-inflammatory
responses and increased T-cell accumulation in the tumor tissue and local lymph nodes
(Klimesova et al., 2013). The mechanism associated with the increased sensitivity was
correlated with altered commensal microbe metabolic activity in Irak-m-/- mice,
suggesting a difference in the microbiome between the WT and Irak-m-/- animals
(Klimesova et al., 2013). Beyond DSS based models, IRAK-M has also been evaluated
in the Il-10-/- model of spontaneous colitis (Biswas et al., 2011). Similar to the findings
from the DSS models, loss of IRAK-M resulted in increased TLR signaling, resulting in
increased inflammation and expression of pro-inflammatory signaling pathways (Biswas
et al., 2011). The Il-10-/- model is highly dependent on the intestinal commensal flora
and subsequent germfree studies evaluating Irak-m expression further suggest that
Irak-m-/- sensitivity in this experimental colitis model is dependent on the composition of
the resident GI microbiota (Biswas et al., 2011). The microbiota composition cannot be
underestimated for DSS based models as the severity to DSS colitis can drastically
change based on the microbiota composition (Hernandez-Chirlaque et al., 2016). We
postulate that differences observed between labs utilizing Irak-m-/- mice in DSS models
could be due to altered microbiome compositions or differing percentages and lots of
DSS used among labs.
Though our data provides contradictory evidence compared to the previous findings
pertaining to IRAK-M, (Berglund et al., 2010), (Biswas et al., 2011), (Klimesova et al.,
2013) our data is consistent with a more recent study that also showed Irak-m-/- mice
105
display reduced tumor burden compared to their WT counterparts in the AOM/DSS
model (Kesselring et al., 2016). This was attributed to enhanced epithelial cell barrier
function, specifically localized to tumor sites in the GI tract of Irak-m-/- mice (Kesselring
et al., 2016). This was shown to be associated with reduced activity of the oncogene
STAT-3. Our findings described in the current manuscript lend support to this
conclusion pertaining to Irak-m-/- mice challenged in the AOM/DSS model (Figure 2.5
and Figure 2.6). We further confirm the findings pertaining to decreased Wnt signaling
(Kesselring et al., 2016) as demonstrated by the reduced intracellular β-catenin in our
study (Figure 2.6H). Finally, the analysis of human biopsies from this prior study
revealed that human patients with increased IRAK-M expression have worse cancer
survival (Kesselring et al., 2016), which corroborates our metadata analysis described
(Figure 2.1). Collectively, our data pertaining to IRAK-M is complementary to the major
findings by Kesserlring et al. Together, these studies extend the mechanistic insight
associated with IRAK-M modulation of IBD and colitis associated tumorigenesis.
IRAK-M functions as a negative regulator of TLR and IL-1R1 signaling by either
attenuating the IRAK-1/IRAK-4 phosphorylation event or stabilizing the
TLR/MyD88/IRAK-4 complex (Kobayashi et al., 2002). Originally, we sought to better
define the mechanisms associated with IRAK-M attenuation of inflammation in the
context of experimental colitis and colitis associated tumorigenesis. Through the course
of our experiments, our in vitro data suggested that BMDMs from Irak-m-/- mice
displayed an augmented inflammatory response (Figure 2.7A), which is consistent with
the initial reports characterizing IRAK-M and the Irak-m-/- mice (Kobayashi et al., 2002).
106
However, we were intrigued after discovering a protein band pertaining to IRAK-M in
BMDMs from the Irak-m-/- mice after treating with specific pathogenic ligands (Figure
2.4). As previously described in the original manuscript reporting the generation of Irak-
m-/- mice, exons 9-11 were targeted for deletion by homologous recombination and the
insertion of a neo cassette (Kobayashi et al., 2002). Our genotyping confirms the
successful targeting of Irak-m. However, as we show in our current studies, a splice
variant of the Irak-m gene is formed following BMDM stimulation in the targeted Irak-m-/-
animals. The splicing event circumvents the neo cassette and joins exon 8 with exon 12,
defined here as Irak-mrΔ9-11. Together, our data suggests that the inclusion of exon 12 in
the mRNA effectively stabilizes the transcript. Further functional studies using
overexpression systems revealed that this truncation has the potential to robustly
activate NF-κB signaling (Figure 2.7E). If this splice variant is present in the Irak-m-/-
mice, then it is possible that the Irak-mrΔ9-11 variant could result in potential phenotypes
not characteristic of a true knockout mouse. These could include reduced or hyperactive
activity for IRAK-M functioning as either a dominant negative or potentially even a
dominant positive.
In conclusion, our data strongly suggests that IRAK-M functions to modulate
inflammatory signaling pathways and is critical in maintaining immune system
homeostasis in the gut. However, increased IRAK-M is associated with increased
disease pathogenesis and increased cancer severity in human patients. Our findings in
mice revealed that the immune system in Irak-m-/- animals is primed and highly efficient
at eliminating microbes translocating from the GI lumen. This increased microbial
107
clearance is associated with reduced experimental colitis and colitis associated
tumorigenesis. Together, our data identify IRAK-M as an essential regulator of
inflammation and is critical in the maintenance of mucosal immune system homeostasis
in health and disease.
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Chapter 3:
The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of
Mouse Intestinal Organoids
Published as:
Rothschild, D. E., Srinivasan, T., Aponte-Santiago, L. A., Shen, X. & Allen, I. C. 2016.
The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal
Organoids. J Vis Exp. May 18; (111).
A. Abstract:
While the previous chapter employed conventional in vivo and in vitro techniques to
study the role of IRAK-M, the present chapter discusses the utility and potential of
optimizing primary intestinal organoids as a model system in mucosal immunology.
The complexities of the organoid growth characteristics carry significant caveats for
the investigator. Specifically, the growth patterns of each individual organoid are
highly variable and create a heterogeneous population of epithelial cells in culture.
With such caveats, common tissue culture practices cannot be simply applied to the
organoid system due to the complexity of the cellular structure. Counting and plating
based solely on cell number, which is common for individually separated cells, such
as cell lines, is not a reliable method for organoids unless some normalization
technique is applied. Normalizing to total protein content is made complex due to the
resident protein matrix. These characteristics in terms of cell number, shape and cell
type should be taken into consideration when evaluating secreted contents from the
organoid mass. This protocol has been generated to outline a simple procedure to
culture and treat small intestinal organoids with microbial pathogens and pathogen
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associated molecular patterns (PAMPs). It also emphasizes the normalization
techniques that should be applied when gene expression and protein analysis are
conducted after such a challenge.
B. Introduction:
The ability to harvest and culture primary organoids have been described for small
intestine, colon, pancreas, liver and brain and are exciting advances germane to
understanding a more physiologically representative phenomena for tissue biology(Sato
et al., 2009)-(Lancaster et al., 2013). The first methods describing the culture and
maintenance of small intestinal organoids was reported by Sato et al. out of the lab of
Hans Clevers(Sato et al., 2009). Prior to this method, harvesting and culture of primary
intestinal epithelial cells proved to be limited and ineffective in sustaining epithelial cell
growth. Methods included dissociation of tissue via incubation with enzymes, such as
collagenase and dispase, which would ultimately lead to the outgrowth of intermixed
primary fibroblast cells(Freshney and Freshney, 2002). These conditions would also be
time restricted in sustaining the epithelial cell culture. Minimal to no epithelial cell niche
would form, as the epithelial cells would enter apoptosis due to the lack of appropriate
growth factors or loss of contact integrity, termed anokis(Vachon et al., 2002). The
advent of the 3D-organoid culture system has provided a method to culture primary
intestinal cells containing a spectrum of intestinal cell types in sustained culture(Sato et
al., 2009). These epithelial organoids have advantages over cell lines being that they
are composed of several differentiated cells, and better mimic the organ they are
derived from in vivo(Hynds and Giangreco, 2013). The process to ultimately “grow a
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mini gut in a dish” has proven to be a valuable tool for assessing the response of
intestinal epithelium under different stimuli. Investigating the interaction of primary
intestinal cells with microbial PAMPs is relevant to the field of immunology as these
molecular patterns can regulate diverse responses from both host and microbe(Kaiko
and Stappenbeck, 2014). Not only can investigators now explore these interactions with
mouse organoids, but they can be cultured from humans as well(Sato et al., 2011). This
technology has the potential to dramatically alter personalized medicine and it is
tempting to speculate about advances that this technique will make possible in the near
future.
The overall goal of this method is to provide a protocol for the culture, expansion,
and treatment of intestinal organoids with a variety of stimuli. Such stimuli can
ultimately range from vaccines, bacterial pathogen associated molecular patterns
(PAMPs), live pathogens, gastrointestinal (GI) and cancer therapeutics. This
method is focused on describing an adequate technique for proper normalization
when working with non-homogenous cell structures, which must be taken into
consideration when conducting an assay based on cell number.
C. Materials and Methods:
All research was approved and conducted under Virginia Tech IACUC guidelines
1. Prepare R-Spondin1 Conditioned Media From HEK293T-Rspo1 Cell Line
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1.1) Seed HEK293T-Rspondin1 secreting cells at 5-10% confluency in a T-175 flask
with 40 ml of growth media and incubate at 37 oC + 5% CO2. R-spondin1 can be
alternatively purchased as a recombinant growth factor.
1.2) Grow the HEK293T-Rspondin1 cells for seven days or until reaching 95%
confluency determined by bright-field microscopy. Harvest the 40 ml of conditioned
media and transfer to a 50 ml conical tube. The T-175 flask of HEK293T-Rspondin1
secreting cells can be discarded.
1.3) Centrifuge at 300 x g for 10 min at 4 oC to pellet cellular debris. Collect the
supernatant, termed R-spondin1 conditioned media, and aliquot into 15 ml tubes. Store
aliquots at -20 oC for short term or -80 oC for long term storage.
2. Preparation of Organoid Growth Media and Reagents for Harvesting Small
Intestinal Crypts
2.1) One-day prior to harvesting, place the frozen protein matrix on ice and thaw
overnight at 4 oC. Autoclave dissecting scissors, forceps and glass slides that will be
used for crypt harvest.
2.2) The following day, begin by preparing 40 ml of organoid growth media containing
supplements and growth factors by adding stock concentrations to 40 ml of 1x
Advanced DMEM/F-12. Media supplements contain: 10 mM HEPES - , 1x
glutamine supplement - , 1x Vitamin B27 without vitamin A - , 1x N2 - 200
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, 1 mM N-acetyl-cysteine - , 100 ng/ml m-Noggin - , 50 ng/ml m-EGF -
and place in a 37 oC water bath until usage. Warm a 15 ml aliquot of R-spondin1 to 37
oC in a water bath.
2.3) Warm three sterile 24 well plates to 37 oC by placing in an incubator. Prepare 50 ml
per mouse 1x PBS + 2 mM EDTA and cool to 4 oC. Prepare 100 ml per mouse 1x PBS
containing 10% FBS and cool to 4 oC.
3. Harvesting Mus musculus Small Intestinal Crypts for Organoid Culture(Sato et
al., 2009)
3.1) Sacrifice a male C57B6/J mouse age 6-12 weeks of age raised on standard rodent
chow and water available ad libitum according to institutional guidelines. Euthanize via
CO2 asphyxiation followed by cervical dislocation. Note: Keep carcass on ice until
tissue harvest and perform the tissue harvest in a biological safety cabinet to minimize
the risk of contamination.
3.2) Note: It is optional shave the mouse to remove the fur before submersing in 70%
ethanol.
Submerse euthanized mouse in 70% ethanol for 2 min and pin limbs to pinning board
with the dorsal side of the mouse touching the board.
3.3) Use pre-sterilized dissecting scissors and forceps to make a mid-line incision on
the ventral portion of the mouse. Make the incision at the genitalia proceeding cranial to
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the base of the neck. Open the skin incision laterally with tweezers and pin the skin to
the pinning board to expose the peritoneum.
3.4) Make a mid-line incision in the peritoneum of the abdomen with dissecting scissors
and fold the peritoneum with forceps laterally and pin to the pinning board in order to
expose the abdominal organs. Note: take care to avoid cutting abdominal organs.
3.5) Remove the small intestine from the abdominal cavity with dissecting forceps and
scissors by cutting the proximal junction of the small intestine connected to the stomach
and the distal junction connected to the caecum. Place the small intestine a sterile petri
dish containing 10 ml of ice cold 1x PBS.
3.6) Flush the contents contained within the lumen of the small intestine with ice cold 1x
PBS using a 1 ml pipette. Repeat this step until the debris within the lumen of the small
intestine is removed.
3.7) Cut the small intestine with dissecting scissors into 3-4 approximately equal length
strips and lay the strips longitudinally on a new sterile petri dish. For each strip, insert
the cutting edge of the dissecting scissors inside the lumen of the small intestine to
make an incision the entire length of the strip. Use forceps to laterally fold open the
incision in order to expose the lumen of the small intestine.
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3.8) Use a sterile glass slide to gently scrape away the villi on the luminal surface of the
small intestine. Cut the small intestine into 1-2 cm length strips with dissecting scissors
and transfer these strips to a 50 ml conical tube containing 10 ml ice cold 1x PBS.
3.9) Mix the contents gently and allow the tissue contents to settle to the bottom of the
50 ml conical tube. Aspirate the 1x PBS and repeat this three times by washing the
tissue with 10 ml of ice cold 1x PBS. On the final wash leave the conical tube on ice for
10 min containing 10 ml 1x PBS and the small intestine tissue segments.
3.10) Aspirate the 1x PBS and add 25 ml of 1x PBS + 2 mM EDTA. Place on a
rocking platform at 4 oC or in an ice bucket for 45 min. While the tissue is incubating,
label six 15 ml conical tubes “fraction #1-6.”
3.11) After incubation, allow the tissue contents to settle to the bottom of the tube
and aspirate 1x PBS + 2 mM EDTA. Add 10 ml of 1x PBS +10% FBS and shake the
tube vigorously by hand 10 times.
3.12) Allow the tissue contents to settle to the bottom of the tube. Remove and
transfer the supernatant to the tube labeled “fraction 1”. Repeat the 1x PBS + 10%
FBS shaking step five times and transfer the supernatant contents in order to the
appropriately labeled fraction tube.
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3.13) Centrifuge at 125 x g for 5 min. Aspirate the supernatant and resuspend
pellets in 5 ml pre-warmed 1x Advanced DMEM/F-12, with no growth factors added.
3.14) Centrifuge at 78 x g for 2 min. Aspirate 4 ml of the supernatant and resuspend
each pellet in 1 ml of remaining media.
3.15) Remove 0 μl from each fraction and add to a glass slide. Visualize crypts
and debris under a light microscope and determine which fractions to combine and
pool accordingly to achieve the greatest percentage of crypts to debris ratio. The
authors find that fractions 2-6 yield the greatest percentage of crypts to be plated.
The fractions to pool are most often fraction 3 with fraction 4, and fraction 5 with
fraction 6.
3.16) Centrifuge fractions to be plated at 125 x g for 5 min. Aspirate supernatant
leaving 50-100 remaining above the pellet.
3.17) Keep tubes on ice and add 1 ml of protein matrix to the pooled fractions. Pipet
up and down slowly to prevent addition of air bubbles.
3.18) Remove pre-warmed 24 well plates from 37 oC incubator and add 50 μl of
protein matrix/crypt suspension in the middle of each well. Transfer the seeded plate
to a 37 oC incubator for 10 min to allow the protein matrix drop to solidify.
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3.19) Add 450 μl of previously prepared organoid growth media + 50 μl of R-
spondin1 conditioned media per well. Incubate plates at 37 oC + 5% CO2 overnight.
3.20) Add 50-100 μl of R-spondin1 conditioned media per well daily. Every 3rd day
replace entire organoid growth media. The protein matrix drop maintains its integrity
for one week, and after 7 days proceed to passaging organoids.
4. Passaging Organoids Every 7th Day
4.1) Thaw the protein matrix on ice overnight at 4 oC the day before passaging.
Warm one sterile 24 well plate to 37 oC for at least 30 min. Prepare organoid growth
media described in step 2.2 and keep at 37 oC until usage.
4.2) Remove organoid plate from incubator and commence passaging of plate on
ice.
Aspirate growth media with a 10 ml pipette from 3-4 wells to start.
4.3) In the aspirated wells, pipette up and down in order to dislodge the protein
matrix drop from the plate. Gentle scraping with the tip of a 10 ml pipette is helpful in
dislodging the protein matrix drop. Transfer the contents to a 15 ml conical tube.
4.4) Incubate the 15 ml conical tubes on ice for 10 min and centrifuge at 125 x g for
5 min at 4 oC. Observe a white pellet at the bottom of the conical tube. Gently
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aspirate the majority of the supernatant above the organoid pellet leaving 100-200 μl
above the pellet.
4.5) Break up the organoid crypts by using a 23 gauge needle attached to a 1 ml
syringe. Aspirate and eject 10-15 times in order to dissociate the crypts. Minimize air
bubble formation due to excessive pipetting.
4.6) Resuspend the organoids in 500 μl of protein matrix and add 50 μl per well in a
new pre-warmed 24 well plate. Add 450 of previously prepared organoid growth
media + 50 μl of R-spondin1 conditioned media per well. Incubate plates at 37 oC
overnight.
5. Plating Organoids on Day 14 for Pattern Recognition Receptor Stimulation
with PAMPs and Listeria monocytogenes for Gene Expression Analysis
5.1) Two days prior to challenge, streak out L. monocytogenes on BHI agar plate.
5.2) The following day pick a L. monocytogenes colony and grow in 10 ml of BHI
broth at 37 oC overnight shaking at 200 rpm.
5.3) Thaw the protein matrix on ice overnight at 4 oC the day before organoid
challenge. Warm one sterile 24 well plate to 37 oC for at least 30 min. Prepare
organoid growth media described in step 2.2 and keep at 37 oC until usage.
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5.4) Remove organoid culture from incubator and commence passaging of
organoids.
5.5) Aspirate media from 3-4 wells and pipette up and down in order to dislodge the
protein matrix drop from the plate. Gentle scraping with the tip of a 10 ml pipette is
helpful in dislodging the protein matrix drop. Transfer the contents to a 15 ml conical
tube.
5.6) Incubate the 15 ml conical tubes on ice for 10 min. Centrifuge at 125 x g for 10
min at 4 oC. Observe a white pellet at the bottom of the conical tube. Gently aspirate
the supernatant leaving approximately 100 μl of residual supernatant behind.
5.7) Resuspend the organoid pellet in 5 ml of ice cold 1x PBS and remove a 10 μl
aliquot for counting. Add the 10 μl aliquot to the middle of a hemocytometer without
the glass cover slip. Gently overlay the glass coverslip on top of the drop. The
authors find that the hemocytometer will clog with organoids if the glass slide is not
first removed.
5.8) Count the total number of organoids via a light microscope in every grid/ the
entire chamber of the hemocytometer and determine the organoid concentration:
number of total organoids counted/10 μl.
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5.9) Remove a appropriate volume from the 15 ml conical tube that will allow
adequate numbers of organoids to be seeded for the assay and centrifuge this
suspension at 125 x g for 10 min. Note: The authors find that a range between
40-100 organoids per well of a 24 well plate is sufficient for RNA analysis.
5.10) Aspirate the supernatant and resuspend the pellet in 500 μl of protein matrix
without generating air bubbles. Add 50 μl of organoid/protein matrix suspension per
well in a 24 well plate. Note: The amount of protein matrix used to resuspend
organoids will vary for the number of treatment conditions and technical
replicates.
5.11) Add 500 μl of organoid growth media (without N-acetyl-cysteine) to each well
and incubate at 37 oC + 5% CO2 for 1 hr.
5.12) Prepare 2x concentrations of PAMPs and live L. monocytogenes. Measure the
OD of live L. monocytogenes and steak on a BHI plate for counting. Treat organoids
at 1 x 106 CFU/ml. For example, the authors find that an OD of 0.6 ≈ 1 x 109 CFU/ml
under typical conditions in their laboratory. The OD to CFU determination will vary
by bacteria type and should be assessed prior to organoid stimulation.
5.13) Streak out a serial dilution from the treatment stock of L. monocytogenes to
accurately determine the treatment CFU/ml. The authors find that a 1 x 108 dilution
127
of 100 μl on a BHI agar plate is sufficient to count colonies to determine an accurate
CFU/ml.
5.14) Prepare a heat killed solution of L. monocytogenes by taking a 1 ml aliquot of
the live L. monocytogenes solution and centrifuge at 5,000 x g for 10 min. Aspirate
the supernatant and wash the bacterial pellet with 1 ml of 1x PBS.
5.15) Centrifuge the L. monocytogenes at 5,000 x g for 10 min and resuspend the
pellet in 1 ml of 1x PBS. Heat kill the L. monocytogenes in a 1.7 ml polypropylene
tube by heating at 80-90 oC for 1hr. Culture a 100 μl aliquot of the heat killed L.
monocytogenes on a BHI agar plate to confirm no live bacteria remain.
5.16) Add 500 μl of the appropriate 2x PAMP and/or microbe treatment to 500 μl
resident media in designated wells determined by the user. Adding a 500 μl aliquot
of a 2x PAMP/microbe treatment to 500 μl of resident media will bring the
treatment concentration to 1x. Incubate at 37 oC + 5% CO2 for 24 hr.
5.17) The following day count L. monocytogenes colonies for an accurate
determination of treatment CFU/ml.
5.18) Aspirate media from the plate of treated organoids and wash wells three times
with 1x PBS.
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5.19) Proceed to RNA extraction via phenol/chloroform(Chomczynski and Sacchi,
2006) or a RNA extraction kit according to manufacturers instructions. Evaluate
gene expression using standard qRT-PCR(Allen et al., 2011).
6. Plating Organoids on day 14 for PAMP and L. monocytogenes Challenge for
Protein Analysis in Supernatant
6.1) Two days prior to challenge start a cell culture of Caco-2 cells in a T-75 flask
with 1x DMEM + 10% FBS and incubate the Caco-2 cells at 37 oC + 5% CO2. Begin
a bacterial culture of L. monocytogenes on BHI plate and incubate plate in a
bacterial incubator at 37 oC.
6.2) The following day pick a L. monocytogenes colony and grow in 10 ml of BHI
broth at 37 oC overnight. Thaw the protein matrix on ice overnight at 4 oC the day
before organoid challenge.
6.3) The following day warm one sterile 96 well black-walled plate to 37 oC for at
least 30 min. Prepare organoid growth media without N-acetyl-cysteine described in
step 2.2 and keep at 37 oC until usage. Remove organoid culture from incubator and
commence passaging of organoids.
6.4) Aspirate media from 3-4 wells and resuspend with a pipette in order to dislodge
the protein matrix drop from plate. Gentle scraping with a 10 ml pipette is helpful in
dislodging the protein matrix drop. Transfer the contents to a 15 ml conical tube.
129
6.5) Incubate the 15 ml conical tubes on ice for 10 min. Centrifuge at 125 x g for 10
min at 4 oC. Observe a white pellet at the bottom of the conical tube. Gently aspirate
the supernatant leaving 100 μl of residual supernatant behind.
6.6) Resuspend the organoid pellet in 5 ml of ice cold 1x PBS and remove a 10 μl
aliquot for counting. Add the 10 μl aliquot to the middle of a hemocytometer and
gently overlay the glass coverslip. The authors find that the hemocytometer will clog
with organoids if the glass slide is not first removed.
6.7) Count the total number of organoids via a light microscope in every grid of the
hemocytometer/ the entire chamber and determine the organoid concentration:
number of total organoids counted/10 μl.
6.8) Remove a appropriate volume from the 15 ml conical tube that will allow
adequate numbers of organoids to be seeded for the assay and centrifuge this
suspension at 125 x g for 10 min. Note: The amount of protein matrix used to
resuspend organoids will vary for the amount of treatment conditions and
technical replicates. The authors find that 40 - 100 organoids is sufficient for
secreted protein analysis.
6.9) Aspirate the supernatant and resuspend the pellet in 500 μl of protein matrix
/well without generating air bubbles. The amount of protein matrix used to
130
resuspend organoids will vary for the amount of treatment conditions and
technical replicates.
6.10) Leave at least two columns empty on the 96 well black-walled tissue culture
plate as these will serve as wells seeded with Caco-2 cells for the generation of a
standard curve. Add 50 μl of protein matrix + organoid suspension per well to a pre-
warmed 96 well black-walled tissue culture plate. Store the plate at 37 oC for 10 min
to allow protein matrix to solidify.
6.11) Add 100 μl of organoid growth media (without N-acetyl-cysteine) to each well
and incubate at 37 oC + 5% CO2 for 1 hr.
6.12) Prepare 2x concentrations of PAMPs and live L. monocytogenes. Add 100 μl of
each given treatment condition per well and incubate for 24 hr. Incubation and
Treatment times to be determined by the investigator.
6.13) The following day plate Caco-2 cells in the standard curve wells. Begin by
warming sterile 1x PBS, 0.25% trypsin and 1x DMEM + 10% FBS to 37 oC.
6.14) Remove the T-75 flask containing Caco-2 cells and aspirate the cell culture
media. Wash the cells with 10 ml 1x PBS. Aspirate the 1x PBS, add 2 ml 0.25%
Trypsin and place T-75 flask in 37 oC incubator for 15 min.
131
6.15) Visualize the flask under a light microscope to ensure the cells have detached
then add 8 ml of 1x DMEM + 10% FBS to the detached Caco-2 cells and transfer
into a 15 ml conical tube. Remove a 10 μl aliquot and count the cells by light
microscopy using a hemocytometer. Note: The authors find that a upper cell
number of 60,000 cells/well work well and dilutions can be conducted for generation
of the standard curve down to 2500 cells/well.
6.16) Resuspend the Caco-2 cells in protein matrix and add 50 μl per well to
appropriate wells. Allow the protein matrix to solidify at 37 oC for Caco-2 cells.
6.17) Following the treatment time for the organoid assay, aspirate and save the
organoid cellular supernatant media and keep on ice. Wash the wells three times
with 1x PBS. Add 100 µl of 100% methanol and incubate at 4 oC or on ice for 20-30
min to fix the organoids.
6.18) Following the methanol incubation, wash the wells three times with ice cold 1x
PBS. The plate can now be stored at 4 oC for 1-2 weeks.
6.19) Add nuclear staining dye at a concentration of 1 µg/ml per well, cover the plate
with aluminum foil and incubate at 4 oC overnight.
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6.20) Use a 96 well plate reader to measure nuclear staining dye excitation/emission
(350nm/461nm respectively) and normalize each organoid well to the standard
curve of Caco-2 cells.
6.21) Proceed to downstream assays, such as ELISA(Allen et al., 2011), of cell
culture supernatant normalizing to cell number.
D. Results:
When following this protocol to cultivate intestinal organoids, characteristic sphere
shaped organoids will be present after harvesting. The addition of R-spondin1
conditioned media daily will initiate the growth and budding of the organoids. The
growth of organoids is shown in Figure 1 (A-F), and is representative of intestinal
organoids on days 1, 2, 4, 5, 6 and day 14. Figure 1F represents the non-
homogeneous growth characteristics of organoids on day 14 of PAMP challenge.
Once the organoids are grown to an adequate number they can be replated and
challenged with various PAMPs and/or microbes. Expression analysis can be
performed via standard techniques. This is represented in Figure 2 (A-C) which
shows the mRNA expression of inflammatory cytokines IL-18, IL-6, and
a 24 hr challenge of organoids with heat
killed L. monocytogenes and the PAMP flagellin. Figure 3 (A-C) demonstrates the
relative nuclear staining of intestinal organoids with nuclear staining dye following
fixation with minimal background staining of debris in the resident protein matrix.
133
This method of fixation and staining can be applied to normalize assays, which will
account for differing cell numbers in each well. This is apparent in Figure 4 when
generating a standard curve of serial dilutions of Caco-2 cells plated in protein
matrix, then fixed and stained with nuclear staining dye. The R2 value of 0.89
indicates a linear relationship between cell number and mean fluorescent intensity,
and the linear equation can be used to normalize organoids to cell number.
Figure 3.1
134
Figure 3.1. Small Intestinal Organoid Growth
Time course of growth for murine small intestine derived organoids following isolation.
A) Day 1. B) Day 2. C) Day 4. D) Day 5. E) Day 6. F) Day 14. Images are
representative of organoid growth for each given day.
Figure 3.2
135
Figure 3.2. mRNA Expression of Inflammatory Cytokines Following 24 hr PAMP
Challenge
Relative mRNA expression of wild-type intestinal organoids challenged for 24 hr with
PAMPs and heat killed Listeria monocytogenes. A-C) represent fold change of the
inflammatory cytokines IL-18, IL-
Figure 3.3
136
Figure 3.3. Relative Fluorescent Staining of Organoids for Normalization
Nuclear staining of organoids with following fixation. A) Bright field of organoids. B)
Fluorescent staining of organoids with nuclear staining dye. C) Merged image of
bright field and fluorescent staining.
Figure 3.4
Figure 3.4. Standard Curve generated from Caco-2 Cells
Standard curve generated from triplicate wells. Cell seeding had a range starting at
60,000 cells per well with dilutions down to 2,500 cells per well.
137
Figure 3.5
Figure 3.5. Graphic Representation of the Technique
1. Harvesting R-spondin conditioned media. 2. Harvesting small intestinal crypts,
and growing organoids. 3. Plating organoids, followed by PAMP treatment, and
normalization.
Cell Number
Em
issi
on
Stomach {
Small intestine {
Cecum/ Large intestine {
} Villus
} Crypts
2 mM EDTA
Fractionate and Plate
2
3 4 5 6
14 Days
A. B. C.
D.
Caco-2
6 5 4 3 2 1
1
2
3
4
5
6
Figure 5.
1.
2.
3.
138
E. Discussion:
The culture and maintenance of intestinal organoids is a procedure that can be
mastered by any individual with adequate tissue culture technique. There are subtleties
in passaging when compared to growing cells in a more conventional monolayer, but
these subtleties are not difficult to overcome. The critical steps of this method involve
being able to grow the organoids to a high enough density for optimal seeding.
Experiments must be scaled down with organoids as large seeding densities that can
commonly be achieved with cell lines are not practical. This becomes especially
apparent when there are multiple treatment groups.
This protocol is intended to provide a step-by-step method to study host-pathogen
interactions of the intestinal epithelium with a variety of different bacterial, viral, and
fungal pathogens, as well as address difficulties using this system with respect to
normalization. Reports are available that describe the interactions of intestinal
organoids with the bacterial pathogen Salmonella, yet do not address normalization
methods when measuring secreted cytokines(Zhang et al., 2014). There are several
difficulties that are encountered when normalizing organoid cultures by different
methods. Normalizing secreted protein via BCA is an option; however, the growth
components required for organoid culture (N2 and/or Vitamin B27) interfere with the
BCA assay (data not shown). Normalizing via cellular viability, such as a modified MTT
assay has been described(Grabinger et al., 2014); however, a treatment that will alter
the mitochondrial metabolic activity of the organoids will introduce an inaccurate method
for normalization via this technique as MTT is based on reduction by the action of
139
mitochondrial dehydrogenases(Slater et al., 1963). It is also necessary to remove N-
acetyl-cysteine from the media if normalization via the MTT technique is desired.
The authors find that fixing with methanol and staining nuclei with nuclear staining dye
is an effective normalization technique against a standard curve of Caco-2 cells.
Although the growth characteristics of this colon cancer cell line do not exactly mimic
the growth characteristics of intestinal organoids, using a nuclear stain and measuring
mean fluorescence intensity is a good strategy to normalize against total cell number.
Taken together, the technique described here provides a good starting point to mimic
host-pathogen interactions with adequate normalization that is essential for making
accurate interpretations using this model system. The significance of this technique with
respect to alternative methods is that proper normalization must be performed when
conducting any evaluation of secreted protein, such as ELISA based assays.
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GRIS, D., RONEY, K. E., ZIMMERMANN, A. G., BOWZARD, J. B., RANJAN, P.,
MONROE, K. M., PICKLES, R. J., SAMBHARA, S. & TING, J. P. 2011. NLRX1
protein attenuates inflammatory responses to infection by interfering with the
RIG-I-MAVS and TRAF6-NF-kappaB signaling pathways. Immunity, 34, 854-65.
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acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something
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FRESHNEY, R. I. & FRESHNEY, M. G. 2002. Culture of epithelial cells, New York,
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GRABINGER, T., LUKS, L., KOSTADINOVA, F., ZIMBERLIN, C., MEDEMA, J. P.,
LEIST, M. & BRUNNER, T. 2014. Ex vivo culture of intestinal crypt organoids as
a model system for assessing cell death induction in intestinal epithelial cells and
enteropathy. Cell Death Dis, 5, e1228.
HYNDS, R. E. & GIANGRECO, A. 2013. Concise review: the relevance of human stem
cell-derived organoid models for epithelial translational medicine. Stem Cells, 31,
417-22.
KAIKO, G. E. & STAPPENBECK, T. S. 2014. Host-microbe interactions shaping the
gastrointestinal environment. Trends Immunol, 35, 538-48.
LANCASTER, M. A., RENNER, M., MARTIN, C. A., WENZEL, D., BICKNELL, L. S.,
HURLES, M. E., HOMFRAY, T., PENNINGER, J. M., JACKSON, A. P. &
KNOBLICH, J. A. 2013. Cerebral organoids model human brain development
and microcephaly. Nature, 501, 373-9.
SATO, T., STANGE, D. E., FERRANTE, M., VRIES, R. G., VAN ES, J. H., VAN DEN
BRINK, S., VAN HOUDT, W. J., PRONK, A., VAN GORP, J., SIERSEMA, P. D.
& CLEVERS, H. 2011. Long-term expansion of epithelial organoids from human
colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology,
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SATO, T., VRIES, R. G., SNIPPERT, H. J., VAN DE WETERING, M., BARKER, N.,
STANGE, D. E., VAN ES, J. H., ABO, A., KUJALA, P., PETERS, P. J. &
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CLEVERS, H. 2009. Single Lgr5 stem cells build crypt-villus structures in vitro
without a mesenchymal niche. Nature, 459, 262-5.
SLATER, T. F., SAWYER, B. & STRAEULI, U. 1963. Studies on Succinate-Tetrazolium
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VACHON, P. H., HARNOIS, C., GRENIER, A., DUFOUR, G., BOUCHARD, V., HAN, J.,
LANDRY, J., BEAULIEU, J. F., VEZINA, A., DYDENSBORG, A. B., GAUTHIER,
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142
Chapter 4:
NF-B inducing kinase (NIK) is essential for adequate expression of the stem cell
marker LGR5 and results in enhanced proliferation of intestinal epithelial cells.
A. Abstract
In the previous chapter, we discussed an optimized method for the culture and
stimulation of intestinal organoids. Here, we utilized this method further to study
intestinal epithelial cell proliferation. Intestinal epithelial cell (IEC) proliferation is
required to maintain homeostasis throughout the gastrointestinal (GI) tract. IECs act as
a protective barrier from the external environment within the GI lumen, exhibiting rapid
turnover and thus maintaining the integrity of the barrier. Cellular renewal relies on
several signal transduction pathways working in synchronization; and aberrancies in this
regenerative process contribute to cancer initiation within the intestine. The non-
canonical NF-B signaling pathway is emerging as an important pathway that aids in
adequate proliferation of intestinal epithelial cells. Here, we investigated the contribution
of the non-canonical NF-B pathway to IEC proliferation in a mouse model deficient for
NF-B inducing kinase (NIK). In the absence of NIK, IEC organoids demonstrated
defects in proliferation, consistent with limited expression of the intestinal stem cell
marker, LGR5. Our results demonstrate the essential role of NIK to proper IEC
proliferation, and ultimately GI maintenance.
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B. Introduction
Epithelial cells lining the small intestine and colon within the GI tract undergo rapid
turnover compared to those residing in other tissues. For example, epithelial cells
composing the skin turnover on a 47-48 day basis while, in contrast, intestinal epithelial
cells turnover every 4-5 days (Iizuka, 1994), (Creamer et al., 1961). This rapid renewal
process is associated with protection from various diseases, such as colon cancer,
since increased turnover on a bi-weekly basis prevents cells from accumulating
mutations due to toxic substances encountered within the GI tract. Intestinal epithelial
cells differentiate from intestinal stem cells (ISC) at the base of the intestinal crypt and
migrate up the villus in single-file manner. Upon reaching the apex of the villus, IECs
undergo apoptosis and are subsequently shed into the intestinal lumen to be excreted.
This consistent and rapid renewal is important in protecting the GI tract from the hostile
environment within the lumen of the intestine. In the absence of constant IEC renewal,
any breach in the intestinal epithelial barrier may lead to deleterious effects. Such
consequences may include endotoxic shock due to the plethora of bacterial pathogen
associated molecular patterns (PAMPS) that stimulate surrounding immune cells.
Furthermore, neutrophils responding to local invaders produce vast quantities of
reactive oxygen species (ROS), which can be genotoxic to surrounding cells.
The non-canonical, or alternative NF-B signaling pathway has been shown to be vital
for the proper development of secondary lymphoid organs, as well as proper B-cell
maturation (Claudio et al., 2002). This alternative NF-B pathway is activated
144
downstream of TNF-family receptors, including BAFF, LTR, and CD40; thus, absence
of this pathway results in immunodeficiency of the adaptive immune system. One crucial
molecule that propagates downstream signaling when this pathway is stimulated is NF-
B inducing kinase (NIK). In quiescent cells, NIK is constantly poly-ubiquinated and
degraded, thus maintaining the level of NIK within the cell at low levels. Following a
stimulus, NIK is stabilized, and levels rise within this cell, allowing NIK to act as a
functional kinase on its substrate IKK, which ultimately leads to NF-B activation,
primarily through RelB-p52 heterodimers. Recently, it has been demonstrated that the
silencing of NIK in the surrounding stroma of the intestine results in increased
metastasis of colon cancer cells to distant sites in the body (Maracle et al., 2018). The
purpose of this study was to investigate the role of NIK in relation to the GI system. In
vivo, NIK maintains GI homeostasis by preventing aberrant cellular hyperplasia and
inflammation. When cultured ex vivo, NIK is required for proper IEC proliferation. Our
results demonstrate that loss of Nik results in decreased IEC turnover due to reduced
expression of the stem cell receptor LGR5.
C. Materials and Methods
Reagents
Advanced DMEM F12 media, N2 supplement, B27 without Vitamin A, HEPES,
Glutamax supplement were purchased from ThermoFisher Scientific. HEK-293T-
Rspondin1 cells were cultured as previously described (Kim et al., 2005), and utilized to
generated R-spondin 1 conditioned media previously described (Rothschild et al.,
2016). L- cells were purchased from ATCC and utilized to generate Wnt3a conditioned
145
media as described by ATCC. qRT-PCR primers for mouse LGR5 and KRT20 were
purchased from TheromFisher Scientific. Antibodies for EphB2, EpCAM4, anti-mouse
APC, anti-donkey Alexaflur488 were purchased from ThermoFisher.
Colonic Organoid Isolation and Culture.
Colons from age matched 6-12 week old C57BL6J and Nik-/- mice were dissected
separately and colonic crypts were isolated as previously described (Sato et al., 2011).
For each genotype, approximately 1000 crypts were plated in each well of a 6-well
tissue culture (TC)-treated plate, containing in 100ul matrigel per well. Matrigel was
allowed to solidify for 10 minutes in a 37oC TC incubator. Organoids were cultured
900ul basal media containing: 1x N2, 1x B27without vitamin A, 10mM HEPES, 1x
Glutamax supplement, 100ng/ml m-Noggin, 50ng/ml m-EGF, 1mM N-acetyl cysteine,
25nM Y27632, and 1x Penicillin/Streptomycin. An additional 100 ul R-spondin1
conditioned media and 100 ul Wnt-3a media was added to each well during seeding
(day 0) for initial culturing. After 24 hours, the culture media was aspirated, each well
was washed with warm 1x PBS without Calcium/Magnesium; fresh medium without
Y27632 was then added to each well. Following day 2, 100ul R-spondin 1 and 100ul
Wnt-3a media was added to each well daily. The medium was completely replaced
every 3 days.
Growth tracking of organoids
Following 7 days of culture, organoids for each respective genotype were passaged and
single cell suspensions were made. This was accomplished by scraping and
146
transferring matrigel drops to a 50ml conical tube. Tubes were allowed to incubate on
ice for 15 min, and were subsequently centrifuged at 300g for 10 min. PBS was
aspirated and matrigel pellets were manually disrupted using a syringe with a 22G
needle. Organoids were centrifuged again and pellets were incubated for 10 min at
37oC with 1x TrypLE, centrifuged and TrypLE was aspirated. Cells were then passed
through a 70 um cell strainer to make single cell suspensions. Cells were counted,
assessed for viability with Trypan blue, and plated at 20,000 cells per well in a 6 well
dish. Single cells were tracked for growth over the next 6 days. Media was changed
each day, and each genotype received the same media, media volume, and
concentration of organoid growth factors.
qRT-PCR
Colon crypts were isolated as previously described (Sato et al., 2011). Crypts were
pelleted by centrifugation and harvested for RNA using Zymogen quick RNA isolation
kit. 2g RNA was converted to cDNA using high-capacity cDNA reverse transcription kit
according to the manufacturer’s instructions. 50ng cDNA was plated in each well as a
template, and amplified using TaqMan gene expression master mix according to the
manufacturer’s instructions.
FACS
Single cell suspensions of colonic crypts were made by first isolating colonic crypts.
Crypts were incubated for 20 min at 37oC in TrypLE (ThermoFisher). Crypt digests were
centrifuged and resuspended in 1x DMEM + 10 % FBS and passed through a 70 m
147
cell strainer. Single cell suspensions were stained with respective antibodies for 30 min
at 4oC, and washed with 1x PBS before FACS sorting.
Statistical Analysis
Graphs are represented as the mean ± standard error of mean (S.E.M.). Graphs and
statistical analysis were conducted via GraphPad PRISM software. Data sets were
analyzed by Mann-Whitney U test, and statistical significance was determined using a P
value < 0.01 unless otherwise indicated.
D. Results
NIK is needed for proper proliferation of colonic organoids.
Previous studies have demonstrated that the Wnt signaling pathway is critical when
culturing intestinal organoids ex vivo (Barker et al., 2007). Furthermore, a correlation
has been observed between inflammation driven by the classical NF-B pathway and
colon cancer (Greten et al., 2004). Here we tested the alternative NF-B pathway in
relation to colonic organoid proliferation by utilizing Nik-/- mice. In order to assess
whether NIK influences colonic proliferation, we cultured colonic crypts from Nik-/- mice
ex vivo for one week using both wild-type and ApcMin crypts as negative and positive
controls, respectively (Figure 4.1A). As anticipated, ApcMin crypts proliferated at an
accelerated rate compared to those derived from wild-type mice. To our surprise, Nik-/-
crypts proliferated, however the proliferation rate was drastically reduced when
compared to both wild-type and ApcMin crypts. This was observed both qualitatively
148
using microscopy (Figure 4.1A), and quantitatively when organoid diameter was
measured after one week (Figure 4.1B).
Colonic organoids from Nik-/- mice have impaired proliferation due to reduced
Lgr5 expression and enhance Krt20 expression.
In order to determine the cause of the reduced proliferation of Nik-/- colonic organoids,
stem cells derived from each of the different strains were characterized. This was
accomplished by evaluating the expression of Lgr5, a crucial receptor and marker of
intestinal stem cells (ISC), from freshly isolated colonic crypts, and evaluating gene
Figure 4.1 A. Growth tracking of organoids from single cell suspensions of wild-type,
ApcMin, and Nik-/-. Images are representative for day 1-6. Scale bar = 100m B. Quantification of organoid diameter following one week of growth. 30 organoids were measured in a similar manner for both wild-type and Nik-/- from randomly selected tissue culture wells. ApcMin quantification was omitted to not obscure the graph. Experiments were repeated a minimum of two times with similar results.
149
expression by qRT-PCR. Interestingly, we found Lgr5 levels of Nik-/- crypts to be
significantly reduced compared to the wild-type crypts (Figure 4.2A).
A reduction in the expression of Lgr5 suggests either that the number of stems cells
from Nik-/- is lower compared to the wild-type, or the amount of receptor on a per stem
cell basis is drastically reduced. We also analyzed the expression of Krt20, which has
been shown to increase in expression to correlate with differentiated IEC, as opposed to
ISC (Merlos-Suarez et al., 2011). A greater expression of Krt20 was observed from
Nik-/- crypts (Figure 4.2B). Collectively, these expression results suggest that in the
absence of NIK, not only are stem cell pools reduced, but the differentiated intestinal
Figure 4.2 A. Relative expression of Lgr5 from wild-type and Nik-/- colonic crypts. B. Relative expression of Krt20 from wild-type and Nik-/- colonic crypts. For both figures: wild-type n=3 mice, Nik-/- n=3 mice. Gapdh was used as the housekeeping gene and experiments were repeated a minimum of two times with similar results.
150
epithelial cell lineages are greatly increased. These data provide a possible mechanistic
explanation for the reduced proliferation of the Nik-/- colonic organoids.
Single cell suspensions of individual ISC from Nik-/- crypts yields reduced
organoid colonies
In order to determine whether NIK plays a role in not only stem cell number, but in
proliferation as well, we tested single cell suspensions from harvested colonic crypts. As
was consistent with our pervious findings, this experiment yielded a reduction in the
total number of colonic organoid colonies from Nik-/- crypts (Figure 4.3A). This
suggests that the stem cell pool from Nik-/- crypts are reduced, thus leading to a
reduction in the number of individual organoid colonies that were derived from single
stem cells. To further evaluate the difference in stem cell number between wild-type and
Nik-/- stem cells, we performed FACS analysis on the ISC surface marker EphB2 and
the IEC marker EpCAM1 as shown by Merlos-Suárez et al. (Figure 4.3B). The results,
though subtle, do display altered populations in the EphB2-high pool and EpCAM1-high
pool between wild-type and Nik-/- crypts. Separate gating strategies are indicated to
assess the differences between these two groups.
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Figure 4.3 A. Total organoid colonies from single cell suspension made after one week of culturing colonic organoids. B. FACS analysis of single cell suspensions made from both wild-type and Nik-/- colonic crypts. Cells were stained for EphB2 and EpCAM. A separate gating strategy is depicted as previously described by Merlos-Suárez et al. to emphasize the ISC pools between the genotypes. Experiments were performed once.
152
E. Discussion
One major role of the NF-B pathway is to enhance cellular survival by driving anti-
apoptotic mechanisms (Karin, 2009). The ability of the NF-B pathway to act as an
oncogene is possible, yet rare, and limited to certain types of leukemia (Karin, 2009).
Evidence suggests that the classical and alternative NF-B pathways are capable of
crosstalk between each other, with NIK having the ability to stimulate the classical
pathway (O'Mahony et al., 2000), (Ramakrishnan et al., 2004). Based on our ex vivo
culture of Nik-deficient colonic organoids, reduced growth and proliferation may result
from globally reduced stimulation of the classical NF-B pathway as a direct
consequence of the loss of NIK. Reduced cellular survival, and increased apoptosis
would be expected with reduced classical NF-B activation, similar to the phenotype
observed in Nik-deficient organoids. However, reduced Lgr5 expression in Nik-deficient
organoids suggests a mechanism that may involve the collaboration between the
alternative NF-B pathway and the Wnt signaling pathway, or some component of the
alternative pathway that directly contributes to the reduced expression of LGR5.
The discovery of LGR5 as the receptor responsible for contributing to ISC maintenance
has greatly advanced our understanding of intestinal stem cell biology (Barker et al.,
2007). Not only is Lgr5 a Wnt responsive gene, but changes in Lgr5 expression suggest
the Wnt signaling pathway has been altered in the intestine (Huels and Sansom, 2017).
It was recently shown that both R-spondins and Wnt ligands are both necessary to
maintain ISCs and allow them to differentiate into IECs that compose the crypt-villus
153
architecture (Yan et al., 2017). Yan et al. demonstrated that when Wnt ligands are
absent, R-spondins are unable compensate for the loss of Wnt signaling, resulting in
crypt death. Conversely, if R-spondins are lost, Wnt signaling is temporarily sufficient for
crypt maintenance; however, the LGR5 stem cell pool is lost, and the crypt eventually
dies. A similar phenomenon may explain the results observed in Nik deficient organoids;
one possible explanation is that there may be alternative sources of secreted R-
spondins other than epithelial cells in vivo, thereby promoting crypt survival.
Previous studies have shown NIK to be an essential protein kinase that participates in
the activation of NF-B downstream of TNF, CD95, and IL-1 receptors in the alternative
NF-B pathway, (Malinin et al., 1997). Furthermore, Nik deficiency in mice leads to
reduced B-cells numbers in the lymph nodes and spleen, which also contributes to
impaired IgA production (Brightbill et al., 2015). Since IgA is the predominant class of
antibody to be produced in mucosal tissues, one possibility for the alternative responses
observed in vivo and ex vivo could be explained by reduced IgA production in NIK
deficient mice. An impaired IgA response may ultimately contribute to a disrupted
intestinal microbiome, which may perpetuate an enhanced inflammatory state within the
intestinal system. Since IgA coating has been shown to predict the bacterial populations
that contribute to colitis in mice (Palm et al., 2014), it is possible that loss of IgA coating,
and ultimately immune defenses, may perpetuate a hypersensitive state in the GI tract.
Our studies demonstrate NIK to be a pleiotropic molecule that is of critical importance to
IEC homeostasis; therapeutic targeting of NIK may be eventually used for treatment of
GI-related maladies.
154
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Chapter 5
Discussion and Future Directions
5.1 The Contribution of IRAK-M to GI Immunology
Our findings pertaining to the contribution of IRAK-M were slightly unexpected. One
would anticipate that absence of a negative regulatory protein of the inflammatory
cascade would lead to a runaway inflammatory effect in the host, as well as contribute
to increased carcinogenesis, because increased inflammation can lead to increased
cancer, especially in the colon (Greten et al., 2004). However, in DSS-induced colitis
and AOM/DSS-induced colitis-associated tumorigenesis studies, it was apparent that
absence of IRAK-M was actually protective in both of these respective models. In our
hands, Irak-m-/- mice display decreased morbidity and mortality when challenged in the
acute DSS colitis model. Our mechanistic data explaining this discrepancy suggests
that Irak-m-/- mice have increased inflammation following DSS challenge, but also
enhanced innate immune functions, particularly with neutrophils. Furthermore, the
innate immune function of these neutrophils greatly contributes to and counteracts the
destructive tissue damage that occurs from bacteria, which breach the intestinal barrier
following DSS challenge. We propose that this enhanced state of immunity present in
Irak-m-/- mice also signals the adaptive immune system, mainly the T-lymphocytes, to
be in a heightened activation state. Following this logic, it is likely that cells of the
adaptive immune system from Irak-m-/- mice contribute to the anti-tumorigenic state
displayed in these animals following AOM/DSS challenge, as T cells are known to
destroy cancerous cells (Martinez-Lostao et al., 2015). An alternative explanation is that
it is likely that difference in the microbiome in the GI tract between Irak-m-/- mice and
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the wild-type mice plays a role in the differences observed in the acute colitis model. In
fact, when an antibiotic cocktail of ampicillin, vancomycin, neomycin, and metronidazole
were given to mice for two weeks prior to DSS challenge, both wild-type and Irak-m-/-
mice displayed similar morbidity and mortality following DSS administration. This
suggests that specific components of the microbiota contribute to the protective
phenotype displayed in Irak-m-/- animals. Metabolomics of specific bacterial products
produced in the GI tract could help explain the discrepancies observed between our
laboratory and others that have utilized these mice.
5.1.1 The Significance of the Irak-mrΔ9-11 Truncation Mutant
The central dogma of biology is that cells contain DNA, and the genes that are
expressed from DNA are transcribed into mRNA; further, mRNA is then translated into
proteins that carry out the functional roles in the cell. At the transcription level,
specifically in eukaryotes, mRNA is processed in a “cut and paste” fashion in the
nucleus by a small nuclear RNA (snRNA)-protein complex called the spliceosome
(Berget et al., 1977). The major function of the spliceosome is to process newly
transcribed mRNA into fully mature mRNA that then leaves the nucleus and encodes for
proteins in the cytosol. In eukaryotes, not all of the nascent mRNA that is transcribed in
the nucleus is encoded into proteins. Exons of the mRNA template, which only make up
a small fraction of the encoded mRNA, are spliced together to form much smaller
mature mRNA, while introns are removed from the mRNA template and degraded
(Berget et al., 1977). As such, a single gene can encode for many altered versions of a
protein based upon which exons are spliced together, and are termed splice variants.
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One unexpected and interesting finding was that the commercially available Irak-m-/-
animals form a spliced Irak-m mRNA that joins exons 8 with exon 12. Murine Irak-m is
located on chromosome 10, and contains 12 total exons that form the coding region for
the Irak-m gene. There are 5 naturally occurring Irak-m splice variants, and only two
form fully encoded proteins, using 1-12 and exons 2-12 to encode for splice variant
proteins, respectively (ensemble.org). The truncation mutant that is present in Irak-m
mutant animals is strictly a result of the knockout strategy used by the scientists that
generated these animals, and is not a normal splice variant that is known to occur in
wild-type mice. This was unexpected, because the first reports describing this knockout
mouse did not describe exon 12 being present in the mRNA transcript that was seen via
northern blot (Kobayashi et al., 2002). The relevance to having exon 12 present in this
transcript lies in the potential to encode a possibly stable truncated IRAK-M protein.
There are several factors that contribute to the stability of mRNA inside the cell, and one
factor for stability that contributes to mRNA translation is the addition of a
polyadenylation (poly-A) tail to the 3’ end of the mRNA. Since exon 12 is the last
naturally occurring exon for the Irak-m gene, the poly-A tail is likely contained in the
mRNA transcript, and it is therefore equally likely that the addition of this exon
contributes to the stability of this spliced transcript for protein translation. Therein lies
the problem for a truncated protein to be encoded from this transcript, which may
function in an unknown mechanism inside the cells. As it happens, the exons that were
targeted for deletion, being exons 9 through 11, encode a portion of the kinase domain
of IRAK-M. Interestingly, the current understanding for the function of IRAK-M describes
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this molecule as a non-functional kinase, so a mutation was targeted in this gene in an
already non-functional domain. Further, even more relevance is apparent because the
mechanism for IRAK-M has been described that demonstrates this protein’s function is
mediated through protein-protein interactions with other molecules, namely IRAK-1 and
IRAK-4 (Wesche et al., 1999), (Zhou et al., 2013). This mainly occurs through death
domain- death domain interactions with other proteins, and the death domain for IRAK-
M is encoded at the 5’end (N-terminal) region of this protein, which would be present in
a protein encoded from the resulting Irak-mrΔ9-11 splice variant.
Though this finding is novel and poses interesting questions into the functional role for
IRAK-M, it still remains highly speculative as to whether a protein is present and
functional. The fact that these Irak-m mutant mice display an alternative phenotype
compared to wild-type animals demonstrates the successful mutation of this gene. It is
highly likely that the mutation generated to make this mouse was successful in creating
a null Irak-m allele; however, research is still required to fully validate whether or not this
is the case. A strategy that could be used to validate whether a truncated IRAK-M
protein is present in these animals, would be to generate an antibody that is specific for
the N-terminal domain of IRAK-M, as most of the current commercially available
antibodies target the C-terminal region of this protein. Then, a simple western blot could
be conducted to validate if a protein is or is not present in these mice. Of the five
commercially available antibodies specific for murine IRAK-M, only one of these
antibodies were successfully validated in our laboratory. This was conducted by cloning
wild-type IRAK-M, along with a C-terminal HA tag, into a plasmid containing the strong
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mammalian CMV promoter. In this fashion, wild-type IRAK-M will be highly expressed
when transfected into HEK293T cells. Unfortunately, the antibody that was successfully
validated will not provide answers as to whether a IRAK-MrΔ9-11 protein is present in the
mutant mouse strain, because the epitope that this antibody recognizes is encoded in
exons 9 and 10.
5.1.2 Studies to Overcome the Truncation Mutant Conundrum
An alternative strategy, without the use of antibodies and western blot would be a
genetic approach. An option would be to test whether mice that are heterozygous for
this mutation have similar inflammatory phenotype as wild-type mice or the Irak-m-/-
mice. Assuming a truncated protein is present, the heterozygous mouse will display
similar responses to the knockout mouse if the mutation created is in fact a dominant
mutation. Alternatively, if the mutation generated is a null mutation, then the resulting
wild-type allele from the heterozygous mice will function in a similar manner to the
homozygous wild-type mouse.
Overall, our results, along with other laboratories demonstrate that IRAK-M is an
important intracellular signaling molecule with complex functions. Our research appears
to demonstrate that the function of IRAK-M following TLR ligation is TLR specific. Our
results suggest that some, but not all of the murine TLRs, when stimulated with
respective ligands displayed significant differences in IL-6, TNF, and IL-10. These
included TLR-2, TLR-7 and TLR-9, and all of these respective TLRs utilize the MyD88
dependent pathway exclusively. One possibility is that that these particular TLRs signal
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through similar NF- κB dimers, recall that NF-κB can form up to 15 combinations of
dimers, and each dimer can have slightly alternating functions (Zhang et al., 2017). This
also could be due to the concentration and treatment time of the particular PAMPs
utilized to stimulate the cells. This poses an interesting question related to dose
response and receptor threshold limits. The complexity of cellular signaling is
highlighted in many aspects of dosage, timing and kinetics, because stimulation of a cell
can result in early activation, late activation, and repressive responses at the gene level
(Wang et al., 2012). An interesting avenue to potentially pursue would be to determine
the precise mechanistic role for IRAK-M when specific TLRs are activated. This has
already been done in the case of TLR-7(Zhou et al., 2013); however, investigation of
TLR-9 and TLR-2 would provide additional interesting insight into the mechanistic
functions of IRAK-M.
5.1.3 Therapeutic Targeting of IRAK-M and the Clinical Implications
Our findings implicate IRAK-M to be an candidate molecular target for patients afflicted
with inflammatory maladies, such as IBD and inflammation driven colorectal cancer.
IRAK-M appears to be a molecule that shifts the balance toward host protection in both
acute colitis and inflammation driven tumorigenesis models. Though more mechanistic
research is required to fully elucidate the functions of IRAK-M, it is tempting to envision
a therapeutic strategy targeting IRAK-M in patients that currently suffer from, or are
predisposed to IBD and inflammation driven colorectal cancer. Based on our findings,
acute inhibition of IRAK-M would be an optimal therapeutic approach to treat patients
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with such maladies. Though no molecular inhibitors for IRAK-M currently exist,
compounds that inhibit the interaction of IRAK-M with other death domain containing
proteins would be a potential strategy. Another interesting approach could be to inhibit
IRAK-M and the mRNA level with a DNAzyme approach. DNAzymes are catalytically
active antisense strands of DNA that will bind to sense mRNA targets, and cleave them
for degradation (Burgess, 2012). Designing a DNAzyme specific for IRAK-M mRNA
could be utilized as a strategy to block the function of IRAK-M with minimal off target
effects, due to the rationale that IRAK-M is expressed in a cell type specific manner,
and is induced following cellular activation by transcription factors like NF-κB and AP-1,
as opposed to being constitutively transcribed (Lyroni et al., 2017). From our results,
one would anticipate that utilizing this approach would lead to a reduction in colitis, and
even prevention of colitis associated tumorigenesis in patients with active tumors.
IRAK-M is an interesting target for modulating the immune system, specifically via
inhibition, because it does not appear to be as essential of an inhibitory molecule when
compared to other negative regulatory molecules of inflammation like A20. Absence of
IRAK-M, at least acutely, does not result in severe multi-organ inflammation and tissue
damage, as is the case in mice with the loss of A20 (Lee et al., 2000). Therefore, if this
molecule is nonfunctional in mutant Irak-m-/- animals, complete inhibition of IRAK-M
may result in similar therapeutic benefits, as our results have demonstrated in the Irak-
m mutant mice. Further, IRAK-M may be a key molecular target for patients suffering
from life-threating infections. One can envision a therapeutic strategy that would aim to
inhibit the function of IRAK-M, which would result in heightened immunological functions
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of the immune system. This could be a novel strategy to treat patients with a
suppressed immune system, as is common under conditions of sepsis, as well as
elderly patients with infectious pneumonia. IRAK-M appears to be a novel molecule for
this type of approach, because its inhibition results in enhanced bacterial clearance,
without negative consequences, such as destructive tissue damage.
5.1.4 IRAK-M Targeted Therapies
Recent technologies have emerged that are very promising for gene therapy;
specifically, with enzymes that can be manipulated to efficiently edit DNA, via the use of
CRISPR-Cas9 technology (Brouns et al., 2008). Utilizing a gene editing approach
would be an option for creating a similar IRAK-M mutant in humans. One would
anticipate that targeting exons 9 through 11 of human IRAK-M for deletion, with
CRISPR-Cas9, would result in a similar outcome that has been seen in the mutant Irak-
m-/- mice. Further, it would be expected that this type of edit of IRAK-M would lead to
diminished colitis, and resistance to colorectal cancer in patients that carried a mutation
in the kinase domains of IRAK-M. This type of application could be conducted on the
precursor cells to macrophages, many of which are the hematopoietic stem cells found
in the bone marrow. In a manner that similar to generating chimeric antigen receptor
(CAR) T-cells, bone marrow could be extracted from human patients, hematopoietic
stem cells could then be individually isolated and transfected in the laboratory with the
specific CRISPR-Cas9 guide RNAs targeting exons 9-11 of IRAK-M for deletion. The
successfully transfected stem cells could then be returned to the patient that carry the
desired IRAK-M mutation, and would ideally result in a permanent therapeutic benefit
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for the patient. One of the promising options of this technique is that the therapy is
essentially permanent; therefore, the need for additional medications in the future
should be reduced. Additionally, one can envision an option in which expression or
repression of a gene, following the CRISPR-Cas9 strategy, is induced with a chemical
compound; analogous to the inducible mouse models that utilize estrogen receptor-
CRE-lox system. In this way, a mutation could be induced, or a gene repressed when
an exogenous molecule, such as tamoxifen is administered.
5.1.5 Future Directions with IRAK-M
In terms of future research regarding IRAK-M, it is necessary to develop and validate a
functional antibody to the murine protein, preferably a monoclonal antibody that
recognizes the N-terminus of IRAK-M. This would help solve the question of what is
occurring in the knockout mouse. In addition to this, one would anticipate the actual
mechanistic role of human IRAK-M to be reexamined. Though this molecule is currently
believed to act as a non-functional kinase, intensive biochemical analysis would help
validate whether IRAK-M is completely devoid of kinase activity. First and foremost,
currently IRAK-M has not been crystalized, and it would be highly relevant to determine
the full 3-D structure of this molecule. In addition, when IRAK-M was first discovered, it
was shown to have weak autophosphorylation capacity (Wesche et al., 1999), yet it is
believed that this molecule is completely devoid of kinase activity. One hypothesis is
that this molecule actually behaves as a functional kinase, but doesn’t have as strong
phosphorylation capacity when compared to other IRAK family members. It would be
very interesting to determine if IRAK-M acts on an unknown substrate that it modulates
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via post-translational modification to affect cellular signaling. An approach to solve this
perplexing question would be to make an overactive kinase mutant in kinase subdomain
VIb. Would changing the endogenous serine in the HRD motif to an aspartic acid
restore kinase function, and if so, what would be the substrate that IRAK-M
phosphorylates? If indeed IRAK-M acts as a functional kinase on a currently unknown
substrate, it is essential to determine what this substrate would be, as it is difficult to
decipher the exact cellular signaling events that IRAK-M would modulate without fully
understanding the kinase-substrate relationship (Berwick and Tavare, 2004).
If indeed IRAK-M phosphorylates a particular substrate, a “chemical genetic” approach
could be used experimentally to answer this question (Shah and Shokat, 2003). This
would involve making a mutation in the ATP binding pocket of the kinase of interest, in
this case IRAK-M, and converting a hydrophobic amino acid to one with a small side
chain. In this manner, the kinase would still be catalytically active, but has now gained
the capacity to use a form of ATP that can be labeled at the γ-phosphate with a
covalently attached traceable substituent, like biotin (Shah and Shokat, 2003), (Berwick
and Tavare, 2004). Even cell permeable ATP analogs can be used, so this technique
would not be limited to use with only cell lysates (Fouda and Pflum, 2015). Furthermore,
because this mutated kinase is the only kinase able to use this analog of ATP as a
substrate, the phosphorylated species, i.e. the substrate of the kinase, will become
uniquely labeled. Regarding IRAK-M, this could entail making a mutation that converts
the tyrosine gatekeeper of the ATP binding pocket of IRAK-M to a glycine or alanine.
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Then, this type of assay could be conducted to determine whether IRAK-M functions as
an active kinase on a particular substrate.
Another interesting method would be to use a Reverse In Gel Kinase Assay (RIKA) (Li
et al., 2007). This method is optimal because of its ingenuity and the relative ease in its
practical application; it requires common sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) reagents to perform this experimental procedure. To
summarize, a catalytically active kinase of interest is mixed with the reagents to make a
SDS-PAGE gel, and in this fashion, the kinase will be embedded into the gel matrix
once the gel has polymerized. Then, cell lysates can be run by electrophoresis, and
following its completion, a series of buffer exchange steps can be performed that cause
the proteins within the gel to refold back into their native conformations. Further,
radiolabeled ATP at the γ-phosphate can be incubated with this gel, and because of the
close proximity of the embedded kinase with potential substrates, the kinase will add
radioactive or “hot” ATP to the potential substrates. Now, any radioactive molecules can
be visualized with radiographic methods, and excised from the gel to be processed via
mass spectrometry. Thus, this provides a relatively inexpensive method to determine
previously unidentified substrates of the protein kinase.
4.2 Impact of Recent Methodologies to Study GI Immunology.
4.2.1 Elucidation of the GI Microbiota
Significant progress within the last decade has been made with respect to GI research.
The use of transgenic technology, particularly the use of genetically modified mice, has
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been at the forefront of many of these advances (Thomas and Capecchi, 1987), and
additional creativity from the scientific community continues to fuel progress within the
field. With respect to GI immunology and pathology, momentum has been building with
research pertaining to the symbiotic interactions between the GI systems and the
intestinal microbiota, and how the composition of the microbiota can influence health
and disease of the host. Studying these interactions are complex due to several factors,
but are partially due to the overwhelming diversity of bacterial genera present in the GI
tract, particularly in the colon (Human Microbiome Project, 2012). In fact, the
reproducibility between laboratories with respect to DSS colitis studies can be
confounded, and attributed in part, to the variability in the microbiota composition within
mice from lab to lab (Hufeldt et al., 2010). In order to minimize confounding factors
associated with the GI microbiota, researchers should make every effort to understand
and validate the composition of the microbiome present in the GI tract of mice, which
will help limit discrepancies among investigators. In addition, many laboratories have
adopted the practice of utilizing germ-free animal facilities, in which animals are derived
under sterile conditions, and are thus unable to be colonized by the microorganisms that
constitute the microbiome. This provides an excellent tool for researchers as specific
bacterial species can be added back on an individual microbe basis to these sterile
mice, allowing their microbiome to be colonized by a single microbial species. This
allows the interaction between host and microbe to be studied without other
confounding microbial communities. Of course, this system is not perfect: because
humans have many diverse types of microbes in the GI tract, it is likely that a shift in
composition of the whole microbial community contributes to health and disease.
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Though germ-free animals are a very useful and powerful scientific tool, interpretations
should be made with this caveat in mind.
4.2.2 Bioinformatics and the Impact of Intestinal Organoids on Future Research
A strategy to accomplish such a thorough undertaking for determining the microbiome
associated between laboratories would be to obtain microbiome profiles of the mouse
colony; specifically, with 16s ribosomal DNA sequencing technology, as well as
conducting metabolomics of bacterial products produced in the GI tract. This would
greatly advance our understanding of specific bacterial communities’ contribution
regarding colitis and cancer models, as well as how the composition may vary between
differing genotypes of mice. Additionally, ex vivo tissue culture, specifically with
intestinal organoids derived from the intestinal stem cells is an excellent strategy to
reduce complex confounding effects that occur when using live animal models. This is
one significant advantage of culturing ex vivo organoids from specific tissues, such as
the gut. Specifically, gastric, small intestinal and colonic organoids can be cultured ex
vivo and screened with substances such as specific drugs, microbial species, and
PAMPs to assess, for example, proliferative and anti-proliferative effects on epithelial
cells. A great example that has demonstrated the usefulness of this strategy was
conducted by Kaiko et. al.,2016. With the use of a Cdc25a-luciferase reporter mouse,
Kaiko et. al. screened colonic organoids with a broad panel of microbiome derived
metabolites with the aim of determining if any metabolites enhanced or inhibited cellular
proliferation. Kaiko et. al. determined that the microbial metabolite butyrate inhibited
stem cell proliferation under physiologic concentrations that are normally present in the
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GI tract. Further, convincing evidence supported their hypothesis that the production of
butyrate contributed to the invaginations that form the intestinal crypt (Kaiko et al.,
2016). The rationale was that differentiated colonic epithelial cells toward the top of the
crypt metabolize butyrate rapidly, which prevents its availability and interaction with the
colonic stem cells at the base of the crypt. Therefore, it is likely that the relationship of
microbial metabolites, namely butyrate, contributes to the U-shaped invaginations that
form the colonic crypt. The work by Kaiko et. al. highlight the powerful importance of the
ex vivo organoid culture, along with screening technology to the scientific method to
advance our current understanding of intestinal anatomy and physiology.
4.2.3 Research Applications Utilizing Intestinal Organoids
One advantage of the organoid system over traditional immortalized cell lines is due to
the inclusion of many intestinal cell types in the organoid (Sato et al., 2009). Mimicking
the intestinal stem cell niche allows for maintenance of the intestinal stem cells that give
rise to the fully differentiated epithelial cells present in the GI system; thus, this closely
models the physiology of the GI tract without the complex confounding interactions with
other organs that are present in a live animal, such as the immune system and others.
The importance of this methodology is demonstrated by the ability to culture intestinal
organoids from human patients with a minimally invasive biopsy taken from a patient.
For example, ex vivo organoids derived from patients with genetic mutations that cause
cystic fibrosis have been useful for devising and evaluating therapeutic strategies.
Patients with cystic fibrosis have a mutated gene that encodes for a chloride ion
channel, which results in the inability to make surfactant that moistens the lungs
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(Dekkers et al., 2013). Because this global mutation occurs throughout the genome,
intestinal organoids derived from cystic fibrosis patients can be cultured and monitored
over time, screened for compounds that restore proper activity of the intestinal organoid,
and advance chemotherapeutic options for patients with genetic diseases (Dekkers et
al., 2013).
An exciting application with ex vivo organoids is the concept of microinjection of
individual bacterial strains directly within the organoid lumen. This application is a novel
concept, and one of relevance to help simplify complex variables that occur between the
intestinal microbiota with the intestinal epithelial cells. A method has been described
that utilizes this very concept with gastric organoids, and the bacterium H. pylori
(Bartfeld et al., 2015), but can also be applied to colon derived organoids. Specific
species of bacteria have been associated with the induction of colorectal cancer
(Castellarin et al., 2012), (Abed et al., 2016), and introduction of these pro-carcinogenic
bacterial strains with intestinal organoids may have utility as a primary model to
determine the full contribution of these carcinogenic bacteria. Additionally, organoids
from genetically modified mice can be used as a model to study growth and proliferation
of intestinal stem cells ex vivo.
5.2.4 Future Directions with Organoids: a model system to study both genetic
and host-microbiome interactions
The alternative NF-kB pathway and principally NF-kB inducing kinase (NIK) is known for
its role in immune cell function and B-cell maturation (Eden et al., 2017), (Claudio et al.,
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2002), yet its role in intestinal epithelial cells is currently under investigation. The results
in the present dissertation suggest NIK to be critical for proper proliferation of intestinal
organoids and maintenance of epithelial cells. Since NIK has been shown to contribute
to cellular proliferation in the adaptive immune system (Claudio et al., 2002), (Li et al.,
2016), the results shown herein are consistent with these prior studies. Importantly,
however, our research findings are novel, as we have demonstrated NIK to be important
in intestinal stem cell (ISC) proliferation. Moreover, it was recently shown that Nik-/-
mice develop spontaneous eosinophilic esophagitis (Eden et al., 2017); this association
in combination with the results of the present dissertation further bridges the importance
of NIK to epithelial cell barrier function. It will be an interesting line of further
investigation to determine whether the alternative NF-kB pathway and the Wnt signaling
pathway collaborate in regulating the expression of LGR5. Indeed, future mechanistic
studies will be critical in uncovering the role of NIK with proper stem cell proliferation.
Intestinal organoids are a great model system to further study host-pathogen
interactions, or host-commensal interactions between GI epithelial cells with respective
microorganisms. One particular bacterium that our group has worked with, in
collaboration with Dr. Dan Slade (Virginia Tech, Biochemistry Department), is
Fusobacterium nucleatum. F. nucleatum has received attention in recent years because
of its association and presence in tumors of human patients with colon cancer. Thus,
this bacterium is implicated with contributing to the oncogenic transformation of
intestinal epithelial cells. Our group has been interested in determining the functional
role that F. nucleatum plays in colorectal cancer with the use of mouse colonic
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organoids. The interesting phenomenon of F. nucleatum is that it is a normal portion of
the microflora found in the human mouth, and is not normally found in the GI tract;
which provokes several intriguing questions: how is it that this bacterium is localized to
colonic tumors? Does it actually cause colon cancer or does it localize to a colon tumor
after a tumor has been initiated by alternative mechanisms? One hypothesis is that F.
nucleatum is able to migrate through the bloodstream, entering from the gums and can
localize to the colon through this route, ultimately resulting in its tumorigenic potential. A
likely reason is that F. nucleatum levels increase in the blood after the brushing of one’s
teeth, thus, it is possible that humans receive a substantial septic F. nucleatum dose
twice a day, depending on hygienic habits. The use of colonic organoids is a strategic
model to test this hypothesis, because it is relatively straightforward to treat colonic
organoids in vitro with this bacterium. One practical use of this technique is that colonic
epithelial cells that compose the organoid demonstrate cellular polarity when grown in
matrigel, similar to that of the actual organ under in vivo conditions (Sato et al., 2009).
Thus, the basolateral portion of the epithelial organoid would be the point of entry the
bacteria would exploit when traveling from the bloodstream to the tissue.
5.2.5 Gene Therapy Targeting Multi-potent Stem Cells
One can envision stem cell based therapeutics, in combination with gene therapy, to
advance treatments for human patients that suffer from genetic mutations, specifically
point mutations. One major hurdle with gene therapy is the ability to correct a mutation
that becomes retained throughout the genome of future daughter cells. This can be
overcome if the mutation is corrected in the multi-potent stem cells themselves,
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because it is these cells that are retained in adult tissue, and further give rise to fully
differentiated cell types that populate the organ. In this respect, the corrected mutation
will be retained in future daughter cells, and essentially cure that aberrant genetic
disease. This is one of the very promising aspects of CRISPR-Cas9 based gene
therapy; however, if this strategy is to be used in adult humans, not all multipotent stem
cells have been able to be isolated, so currently, only a limited number of diseases
could be addressed with this approach.
5.3 Conclusion
It will be exciting to view what the future holds regarding research advances in IBD,
mucosal immunology, and cancer fields. With the use of intestinal organoids, many
novel compounds may be discovered that improve therapeutic options for patients with
inflammatory related diseases. Furthermore, stem cell based approaches in
combination with gene therapy offer a promising methodology to treat or potentially cure
diseases caused by point mutations. Though the cost of this type of treatment may be
expensive, this is the type of personalized medical approach that could be utilized to
treat an individual’s particular type of malady.
There remain many unanswered question regarding cellular signaling and how
modulation of one single molecule can affect so many aspects related to the cell. In
conjunction with our current findings, continued research efforts focused on IRAK-M will
be essential and contribute to the global understanding of how this particular molecule
modulates aspects of the immune system. In the larger clinical context, the elucidation
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of the mechanistic role of IRAK-M offers significant promise towards the development of
novel therapeutic strategies for the treatment of GI pathologies, such as IBD and
colorectal cancer.
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Appendix
A. Works Completed
1. Rothschild, D. E., McDaniel, D. K., Ringel-Scaia, V. M., and Allen, I. C. (2018)
Modulating inflammation through the negative regulation of NF-kappaB signaling.
Journal of leukocyte biology
2. Rothschild, D. E., Zhang, Y., Diao, N., Lee, C. K., Chen, K., Caswell, C. C., Slade,
D. J., Helm, R. F., LeRoith, T., Li, L., and Allen, I. C. (2017) Enhanced Mucosal
Defense and Reduced Tumor Burden in Mice with the Compromised Negative
Regulator IRAK-M. EBioMedicine 15, 36-47
3. Eden, K., Rothschild, D. E., McDaniel, D. K., Heid, B., and Allen, I. C. (2017)
Noncanonical NF-kappaB signaling and the essential kinase NIK modulate crucial
features associated with eosinophilic esophagitis pathogenesis. Dis Model Mech 10,
1517-1527
4. Scott, G. K., Chu, D., Kaur, R., Malato, J., Rothschild, D. E., Frazier, K.,
Eppenberger-Castori, S., Hann, B., Park, B. H., and Benz, C. C. (2016) ERpS294 is a
biomarker of ligand or mutational ERalpha activation and a breast cancer target for
CDK2 inhibition. Oncotarget
5. Rothschild, D. E., Srinivasan, T., Aponte-Santiago, L. A., Shen, X., and Allen, I. C.
(2016) The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse
Intestinal Organoids. Journal of visualized experiments : JoVE
6. McDaniel, D. K., Jo, A., Ringel-Scaia, V. M., Coutermarsh-Ott, S., Rothschild, D. E.,
Powell, M., Zhang, R., Long, T. E., Oestreich, K., Riffle, J. S., Davis, R. M., and Allen,
I. C. (2016) TIPS pentacene loaded PEO-PDLLA core- shell nanoparticles have
similar cellular uptake dynamics in M1 and M2 macrophages and in corresponding in
vivo microenvironments. Nanomedicine
7. Brickler, T., Gresham, K., Meza, A., Coutermarsh-Ott, S., Williams, T. M.,
Rothschild, D. E., Allen, I. C., and Theus, M. H. (2016) Nonessential Role for the
NLRP1 Inflammasome Complex in a Murine Model of Traumatic Brain Injury.
182
Mediators Inflamm 2016, 6373506
8. Williams, T. M., Leeth, R. A., Rothschild, D. E., McDaniel, D. K., Coutermarsh-Ott,
S. L., Simmons, A. E., Kable, K. H., Heid, B., and Allen, I. C. (2015) Caspase-11
attenuates gastrointestinal inflammation and experimental colitis pathogenesis.
American journal of physiology. Gastrointestinal and liver physiology 308, G139-150
9. Williams, T. M., Leeth, R. A., Rothschild, D. E., Coutermarsh-Ott, S. L., McDaniel,
D. K., Simmons, A. E., Heid, B., Cecere, T. E., and Allen, I. C. (2015) The NLRP1
inflammasome attenuates colitis and colitis-associated tumorigenesis. Journal of
immunology 194, 3369-3380
183
B. Copyright Permissions
1. Rothschild, D.E., Zhang Y., Diao N., et al. “Enhanced Mucosal Defense and Reduced Tumor Burden in Mice with the Compromised Negative Regulator IRAK-M.” EBioMedicine. 2017 Feb;15:36-47.
Please note that, as the author of this Elsevier article, you retain the right to include it in a thesis or dissertation, provided it is not published commercially. Permission is not required, but please ensure that you reference the journal as the original source. For more information on this and on your other retained rights, please visit: https://www.elsevier.com/about/our-business/policies/copyright#Author-rights.
2. Rothschild D.E., Srinivasan T., Aponte-Santiago L.A., et al. “The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids.” J Vis Exp. 2016 May 18;(111), e54033.
In general, authors own the copyright to the written article, but grant JoVE the exclusive, royalty-free, perpetual use of the article, regardless of access type. Video copyrights are owned by JoVE unless the author selects open access, where it is then published under the creative commons license(CRC 3.0, by,nc,nd). The author may use the video and article for non-commercial purposes. Full copyright information is available in our Author License Agreement, or for UK-funded authors our Author License Agreement UK.
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3. Rothschild D.E., McDaniel D.K., Ringel-Scaia V.M., and Allen I.C. “Modulating inflammation through the negative regulation of NF-κB signaling.” J Leukocyte Bio. 2018 Jun;103(6):1131-1150.
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4. FIgure 1.1 (MOWAT, A. M. & AGACE, W. W. 2014. Regional specialization within the
intestinal immune system. Nat Rev Immunol, 14, 667-85.)
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v1.10 Last updated September 2015
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6. Figure 1.3 (KOO, B. K. & CLEVERS, H. 2014. Stem cells marked by the R-spondin
receptor LGR5. Gastroenterology, 147, 289-302.)
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4423400502072Sep 06, 2018ElsevierGastroenterologyStem Cells Marked by the
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Negative Regulation of Inflammation: Implications for Inflammatory Bowel
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