2015 IBD Abstracts
in vivo, but IL-21RKOxIL-6KO mice still showed GPR15 expression in vivo. Antibiotic
treatment in mice severely reduced GPR15 expression level in vivo, suggesting that
the presence of gut microbiota affects GPR15 expression. However, in vitro treatment
of short-chain fatty acids did not induce GPR15 expression in T cells. In human T cells,
iTregs and Th1 cells showed a significant expression of GPR15 in vitro.
Conclusions: GPR15 is preferentially expressed in Tregs. In mice, GPR15 is induced by
gut microbiota in vivo or by a combination of Stat3-dependent cytokines and
TGFbeta in vitro.
P-128
Ven120, A Plant Derived Human Protein, Attenuates Murine Ileitis Through
Activation of Treg Function
Chris MacManus*, Tom Nguyen†, Colm Collins‡, Randall Alfano*, Edwin de Zoeten§
*
Ventria Bioscience, Fort Collins, Colorado, †University of Colorado, Mucosal
Inflammation Program, Aurora, Colorado, ‡University of Colorado Anschutz Medical
Campus, Aurora, Colorado; and, §University of Colorado Denver, Aurora, Colorado.
Background: Current hypotheses suggest that Crohns disease (CD) may develop
through a dysregulated mucosal immune response toward the commensal enteric
flora in genetically susceptible individuals. Multiple animal studies indicate that
regulatory T cells (Treg) regulate the immune response in normal intestinal mucosa
and thereby prevent colitis development. A breakdown of this tolerance to luminal
antigens plays a pivotal role in IBD development. We hypothesize that a plant
produced human milk protein, Ven120, can modulate the adaptive immune system
in Crohn’s disease to ameliorate inflammation.
Methods: We aimed to validate this hypothesis using a TNF-driven model of CD the
TNFDARE mouse model, in which a 69bp deletion of the AU-rich element confers
increased TNF mRNA stability leading to spontaneous chronic murine ileitis. The
Ven120 was provided by oral gavage or by subcutaneous Alzet pump to 10-12 week
old TNFDARE mice with ileitis. After 2 weeks these mice were evaluated for intestinal
permeability by FITC dextran flux, inflammation by histology, cell isolation and flow
cytometry as well as Treg function. Finally, IL-10 and IL17 output was measured both
by ELISA and by intracellular cytokine staining.
Results: Isolated CD4+CD252 T cells had decreased proliferation in the presence of
Ven120. These cells produced increased quantities of IL-10. In the TNFDARE mouse
model of IBD after 2 weeks of treatment with Ven120 versus vehicle there was
a significant drop in influx of Naïve CD44lowCD62Lhi T cells into the lamina propria
of the intestine. In addition to this there was a significant increase in Treg numbers
and these cells produced increased quantities of IL-10. Histology of the intestines
from these mice was evaluated by a pathologist in blinded fashion and all indices of
inflammation were improved. Similar studies were carried out in the DSS model of
colitis and similar anti-inflammatory effects were noted.
Conclusions: Our studies have effectively demonstrated the therapeutic potential of
Ven120 in murine models of inflammatory bowel disease. Specifically, Ven120
administration significantly decreased naïve T cell infiltration and proliferation in the
intestinal lamina propria, mesenteric lymph nodes and spleen of 12 weeks old
TNFDARE mice. An increase in CD4+Foxp3+ regulatory T cells was noted in the
Ven120-treated mice. These Ven120 treated mice also exhibited improved histolog-
ical indices and had improved intestinal barrier function, indicating efficacy for
Ven120 to decrease inflammation in a preclinical model of CD.
P-129
Gimap5 Is Required for GSK3ß Inhibition Controlling the Transcriptional Program
Required for T Cell Proliferation/Differentiation While Maintaining Gut
Homeostasis
Andrew Patterson*, Mehari Endale*, Kristin Lampe*, Jim Woodgett†, Kasper Hoebe*
Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio; and, †Lunenfeld-
* colitis model, mice that received mPGES12/2 CD4+CD25+ cells showed an increased
Tanenbaum Research Institute Mount Sinai Hospital, Toronto, Canada.
Background: Polymorphisms in human GIMAP5 have been associated with auto-
immune/inflammatory diseases, while in mice loss of Gimap5 causes impaired T cell
survival/proliferation and severe early onset CD4+ T cell dependent colitis. The latter
was associated with an abnormal Treg/Th17 balance. Despite the important role of
Gimap5 in T cell survival and peripheral tolerance, the underlying mechanism(s)
have remained unclear. Using a forward genetics approach, we have identified
a point mutant in Gimap5, so-called “sphinx” mice. Gimap5sph/sph mice have a mis-
sense mutation in Gimap5 that results in essentially a null allele. We found Gimap5-
deficieny resulted in 2 major immunological effects. First, the sphinx mice progres-
sively lose Treg function and are unable to induce Treg cells in vivo. Second, the
mice progressively develop lymphopenia, a dramatic loss of Foxo expression, and
the remaining CD4+ T cells all have an activated Th17 phenotype, but yet fail to
undergo proliferation. These immunologic defects result in a spontaneous colitis
that is preventable with either (1) CD4+ T cell depletion; (2) Treg transplant; (3) or
antibiotic therapy. Despite these effective therapies, the cell-intrinsic defects in
Gimap5sph/sph CD4+ T cells are not restored. Here we test the hypothesis that
Copyright © 2016 Crohn’s & Colitis Foundation of America, Inc. Unauthorized reproduction of this article is prohibited.
S49
Gimap5 is required for inhibition of glycogen Synthase Kinase-3 (GSK3) following
TCR-signaling. A reduced inhibition of GSK3-activity causes an impaired transcription
factor (TF) program unable to support T cell proliferation and iTreg development
ultimately causing severe early onset gut inflammation.
Methods: Studies involve flow cytometric/Imagestream analysis of isolated WT or
Gimap5sph/sph mice; Cd4-cre/ERT2; GSK3bfl/fl mice and therapeutic targeting of
GSK3 by using LiCl or 6-bromoindirubin-3’-oxime both in vitro and in vivo.
Results: Genetic or therapeutic targeting of GSK3 in Gimap5-deficient mice recovered T
cell proliferation in vitro and resulted in normal survival of CD4+ T cells in vivo. Moreover,
while Gimap5-deficient T cells exhibit a reduced expression of c-Myc and Foxo, GSK3
inhibitors completely restored the expression of these TFs. Importantly, genetic/chem-
ical targeting of GSK3b in vivo completely prevented gut pathology and improved
lymphocyte survival in Gimap5-deficient mice. Further imagestream analyses suggest
Gimap5 to control inhibition of GSK3 through a unique mechanism involving seques-
tration of GSK3 in multivesicular bodies and lysosomes.
Conclusions: Our studies uncover a novel regulatory pathway required for T cell
activation and establish Gimap5 as an essential negative regulator of GSK3 activity
controlling the TF program required for CD4+ T cell proliferation/differentiation. The
uncovered pathway is critical for maintaining peripheral tolerance in the gut.
P-130
Production of Inflammatory Cytokines Is Regulated by mPGES-1-Dependent
PGE2 in T Cells
Damian Maseda*, Elizabeth Johnson†, Leslie Crofford*
Vanderbilt University Medical Center, Nashville, Tennessee; and, †Vanderbil
*
University, Nashville, Tennessee.
Background: The integration of inflammatory signals is paramount in controlling the
intensity and duration of immune responses. Eicosanoids, particularly prostaglandin E2
(PGE2), are critical molecules in the initiation and resolution of inflammation and in the
transition from innate to acquired immune responses. Additionally, PGE2 is associated
with modulating autoimmune features through altering the IL-23/IL-17 axis and
regulatory T cell development, whose balance is critical for keeping potential pathogenic
T cell responses under control. PGE2 has pleiotropic effects on many cell types, but it is
still unclear how it affects T cell differentiation and function. Microsomal prostaglandin E
synthase 1 (mPGES-1) is a membrane enzyme that controls local PGE2 levels and is highly
expressed at sites of inflammation. mPGES-1 is part of the PGE2 biosynthetic pathway
and, due to its inducible nature, it controls PGE2 concentrations during activation of the
inflammatory program. We hypothesize that mPGES1 plays a role in lymphoid and non-
lymphoid intestinal tissues in controlling inflammatory responses, especially in controlling
regulatory and pathogenic T cell responses.
Methods: We performed magnetic bead isolation of T cells and subsequent Treg and
Th17 polarization assays with freshly isolated naïve T cells (CD4+CD442CD62L+). We also
sorted different T cell populations from primary lymphoid organs and analyzed markers
for inflammation, T cell fate decision and PGE2-receptors by Real Time-PCR. Using LC-MS
and ELISA we analyzed prostaglandin production by different T cells upon activation. The
Rag2/2 transfer model of T-cell driven colitis was used to examine regulatory T cell
function and T cell tropism to the intestinal tissues by flow cytometry.
Results: Different T cell populations were analyzed for receptors involved in inflammatory
responses. We found that mPGES12/2 naïve T cells showed a significant decrease in
tgfbr1, ptger2 and ptger4 (PGE2 receptors EP2 and EP4) expression compared with WT
naive T cells. Since TGF-b is a key cytokine for the generation of both regulatory and Th17
cells we addressed the effect of mPGES-1 deficiency during in vitro T cell polarization. We
observed that mPGES12/2 naive CD4+ T cells generate more IL-17A+ cells under Th17
polarizing conditions, but exogenous addition of PGE2 shifted the production of IL-17A to
IFNg. In vitro activation of purified T cells showed that mPGES12/2 CD4+CD252 cells
were less capable of secreting PGE2 than their WT counterparts, and CD4+CD25+ T cells
produced less PGE2 than CD4+CD252 cells overall. Furthermore, in the Rag-/- transfer
percentage of CD4+FoxP3+ cells in the colonic lamina propria but not in the mesenteric
lymph nodes when compared to mice transferred with WT CD4+CD25+ cells, although
colitis incidence was not different between groups.
Conclusions: Endogenous mPGES-1-mediated production of PGE2 by T cells
modulates IL-17A and IFNg responses. T cell deficiency of mPGES-1 also reduces
regulatory T cell seeding of the colonic lamina propria, which might have
implications during colitis.
P-131
YI
Chitinase 3-Like 1 and RAGE Interaction Induces Survival and Proliferation of
Intestinal Epithelial Cells in Colitis-Associated Cancer
Renuka Subramaniam*, Daren Low*, Li Lin†, Tomoki Aomatsu‡, Atsushi Mizoguchi§,
Aylwin Ng*, Arianna Degrutttola*, Chun Geun Leek, Jack Eliask, Akira Andoh¶,
Mari Mino-Kenudson*, Emiko Mizoguchi*
S50 2015 IBD Abstracts
*
Massachusetts General Hospital and Harvard Medical School, Boston,
Massachusetts, †National Institutes of Aging, Baltimore, Maryland, ‡Shiga University
of Medical Sciences, Otsu, Japan, §Kurume University School of Medicine, Kurume-
shi, Japan, kWarren Alpert School of Medicine, Brown University, Providence, Rhode
Island; and, ¶Division of Mucosal Immunology, Shiga University of Medical Science,
Otsu, Japan.
Background: Patients with inflammatory bowel disease (IBD) for more than 10
years are at a higher risk of getting colitis-associated cancer (CAC) at an
increased rate of approximately 0.5% per year. Dysregulated host factors which
are inducibly expressed during the IBD development may have pathogenic
effects that can contribute to CAC. Our group demonstrated that inflammation-
associated molecule, chitinase 3-like 1 (CHI3L1), contributes to neoplastic
progression of intestinal epithelial cells (IECs) in IBD patients. However, little is
known about the mechanistic involvement of CHI3L1 in CAC.
Methods: CHI3L1 KO and wild type (WT) mice were treated with azoxymethane
(AOM) and 3 cycles of dextran sulphate sodium (DSS), and the development of
colitis and CAC were examined. To delineate the roles of CHI3L1 expression in
hematopoietic versus non-hematopoietic cells, we generated bone marrow (BM)-
transplanted chimeric mice. Pull-down assay and functional analyses were
performed to determine the specific interaction between CHI3L1 and RAGE
(Receptor for Advance Glycation End product), a putative binding partner of CHI3L1.
In addition, CHI3L1 levels in stool samples from healthy controls and IBD or CAC
patients were analyzed by ELISA.
Results: CHI3L1 KO mice treated with AOM/DSS developed severe colitis but
lesser incidence of CAC as compared to that of WT mice. CHI3L1 KO-derived
BM-transplanted WT (expressing CHI3L1 in non-hematopoietic cells only) and
WT mice developed higher incidence of CAC with milder colitis. In contrast,
WT-derived BM-transplanted CHI3L1 KO (expressing CHI3L1 in hematopoietic
cells only) and CHI3L1 KO mice showed significantly severe colitis but less
incidence of CAC as compared to that of other 2 groups. Since another
inflammation-associated molecule, S100A9, is also inducibly expressed in the
intestine during IBD, we examined the potential association among CHI3L1,
S100A9, and their common receptor RAGE. Concentration of CHI3L1 and
S100A9 in the colon was inversely correlated under inflammatory conditions:
High-CHI3L1/low-S100A9 and low-CHI3L1/high-S100A9 levels in colon were
observed during the chronic and the acute phase of colitis, respectively.
Subsequently, RAGE pull-down and immunoblotting assays showed that RAGE
binds with CHI3L1 in the presence of higher concentrations of CHI3L1 and result
in the proliferation of IECs in vitro. In contrast, presence of higher concen-
trations of S100A9 induces apoptotic cell death after ligating with RAGE. These
results indicate that both CHI3L1 and S100A9 can bind to RAGE competitively
depending upon the concentration gradient between the 2 proteins. In vitro
findings were confirmed in vivo by immunohistochemistry. CHI3L1-RAGE
interaction activates multiple signaling pathways including STAT3, b -catenin,
NF-kB and MAPK. Pearson correlation analysis showed positive correlation
between CHI3L1 level in stools and tumor multiplicity/size in AOM/DSS-treated
WT mice. These results were recapitulated in human fecal samples obtained
from healthy individuals, inactive-IBD, and CAC patients.
Conclusions: This study presents the essential role of CHI3L1/RAGE ligation in
promoting CAC by enhancing survival and proliferation of IECs and neoplastic cells
through down regulation of S100A9 and its effects during preceding inflammation.
We also demonstrate that CHI3L1 levels in non-invasively collected feces serves as
a convenient and reliable modality to predict the neoplastic changes during the
course of chronic colitis.
P-132
Malignant Potential of Colonic Tumor-Associated Fibroblasts in a Model of
Inflammation-Induced Carcinogenesis
Deep Patel, Valerie Kalter, Olivia Questore, Sagar Desai, Logan Rutch,
Linda Gutierrez
Wilkes University, Wilkes Barre, Pennsylvania.
Background: The crosstalk between epithelial cells and the stromal components of
the intestine is quite important to maintain its homeostasis. Stromal cells play
a critical role in the development of inflammatory bowel disease and colorectal
cancer as well. Thrombospondin 1 (TSP-1) is an anti-angiogenic protein that mainly
interacts with a variety of cells and growth factors in the stroma. In this study,
colonic inflammation-induced cancers were developed and tumor associated
fibroblasts (TAF) lacking TSP-1 were isolated. These fibroblasts were characterized
in vitro and its tumorigenic properties tested in vivo by using a syngeneic implan-
tation model.
Methods: Wild type (WT) and TSP-1 deficient (TSP-12/2) mice were injected with
a single dose of azoxymethane (AOM), while 2% dextran sodium sulfate was admin-
istered during multiple cycles. Tumors were dissected and TAF were isolated and
cultured. Proliferation assays were carried out, as well as the wound-healing assay,
which was used to test the migratory properties of these cells. WT (n ¼ 18) and
Copyright © 2016 Crohn’s & Colitis Foundation of America, Inc. Unauthorized reproduction of this article is prohibited.
TSP-12/2 (n ¼ 13) mice were subcutaneously injected with 1 · 106 WT and TSP-1
deficient fibroblasts into the flanks. Tumor growth was monitored for 5 weeks
before tumors were harvested, fixed, embedded in paraffin, cut and stained for
histological evaluation. Immunohistochemistry for leucine-rich repeat-containing
G-protein coupled receptor 5 (LgR5) was performed as well, since these fibroblasts
expressed LgR5 in the original colonic cancers.
Results: In vitro studies showed no major differences in proliferation and cell
migration between WT and TSP-1 deficient TAF. However, TSP-1 deficient colonic
fibroblasts were able to generate fibrosarcomas with moderate invasiveness in
100% of the mice injected reaching diameters up to 28 mm. Conversely, WT
fibroblasts were unable to develop as tumors after multiple attempts. Immuno-
histochemical evaluation of LgR5 in these fibrosarcomas showed focal positive
staining in the periphery but expression of LgR5 was negative in most of these
malignant fibroblasts.
Conclusions: Colonic TAF with a deficiency in TSP-1 has high tumorigenic
potential. This malignant phenotype is developed only upon contact with the
stromal environment in vivo, resulting in moderate invasive fibrosarcomas.
These results underline the importance of matricellular proteins such as TSP-
1, which may regulate the mesenchymal epithelial interactions in the colonic
environment.
P-133
YI
Real-Time Monitoring of Reactive Oxygen Species in Intestine During Ischemia-
Reperfusion Induced Injury and Infectious Colitis Using Electrochemical
Biosensors
Ekaterina Gubernatorova*, Xiaobo Liu†, Ali Othman†, Wayne Muraoka‡,
Silvana Andreescu†, Alexei Tumanov‡
*
Engelhardt Institute of Molecular Biology, Moscow, Russia, †Clarkson University,
Potsdam, New York; and, ‡Trudeau Institute, Saranac Lake, New York.
Background: Accumulating evidence suggests that formation of reactive oxygen
and reactive nitrogen species (ROS/RNS) is associated with inflammatory bowel
disease (IBD). However, real-time measurement of these markers is currently
challenging as most ROS/RNS are highly reactive and short lived, and their
concentrations change dynamically over time. There is thus a need for new
methods that can accurately measure these species in live tissue in order to better
understand their role in pathogenesis of IBD. To better understand the role of ROS/
RNS in pathogenesis of IBD, we developed electrochemical bioelectrodes as a novel
approach enabling real-time continuous tracking of ROS/RNS species in live
intestine.
Methods: To define the role of ROS in early intestinal inflammation, we analyzed the
expression of superoxide and nitric oxide levels in mouse model of intestinal
ischemia-reperfusion-induced injury, and infectious colitis induced by mucosal
pathogen Campylobacter jejuni. Ischemia-reperfusion injury of the small intestine
was achieved by inducing ischemia in the region of the distal ileum by temporally
occluding the peripheral and terminal collateral branches of the superior mesenteric
artery for 60 minutes using microvascular clips following 1 hour or 2 hours
reperfusion. Kinetics of superoxide and nitric oxide concentrations in intestinal
tissue during the ischemia-reperfusion were measured electrochemically using
novel microelectrode biosensors. To test the therapeutic potential of cerium oxide
and europium doped-cerium oxide nanoparticles as ROS scavenging agents,
nanoparticles were directly injected to the intestinal lumen during the beginning
of ischemia.
Results: For superoxide measurements in intestine, a miniaturized biosensor,
comprising gold electrode modified with cytochrome c, a redox protein that is
known to react with superoxide anion radical, silver reference electrode and
platinum counter electrode was developed. Electrochemical current generated
due to reduction of cytochrome c is proportional to the concentration of the
superoxide at the electrode surface. For measurement of nitric oxide, a modified
carbon fiber microelectrode was developed. An increased production of
superoxide radical was detected in intestine during the ischemia stage and
the beginning of reperfusion stage. In contrast, production of both pro-
inflammatory (TNF, IL-1b, CXCL2) and anti-inflammatory cytokines (IL-10, IL-
22) was upregulated in intestine at 2 hours after reperfusion but not during
ischemia. These results indicate that increased production of superoxide
precedes the upregulation of inflammatory cytokines in intestine. An increased
production of superoxide was also detected in intestine during the infectious
colitis induced by mucosal pathogen Campylobacter jejuni. Administration of
europium-doped cerium oxide nanoparticles during the beginning of ischemia
effectively blocked superoxide accumulation, promoted the expression of IL-10
and IL-22 and ameliorated the intestinal pathology examined by the analysis of
hematoxylin and eosin stained sections.
Conclusions: Our results suggest that early increased production of ROS during the
ischemia-reperfusion promotes the intestinal pathology and that mucosal delivery
of cerium oxide nanoparticles can be a potential therapeutic approach to block ROS