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Mo1781 Smoking is Associated With Disordered Homeostasis of Small Intestinal and Proximal Colonic Mucosal Mast Cells
mechanisms that may explain the enrichment of these cell populations in the cecum are currently under active investigation.
AGA Abstracts
P62 accumulation was reported in CD patients also indicating that the autophagic flux is active in CD Paneth cells. TEM high magnification observation revealed that autophagy activation was associated with a significant decrease in the number of secretory granules in CD patients. The granules appeared to be wrapped around autophagolysosomal vacuoles and abnormally degraded. Conclusion: This work describes of a novel selective autophagy pathway, called crinophagy that targets specifically secretory granules of CD Paneth cells. This process consists of the self-digestion of secretory granules by the autophago-lysosomal pathway. Crinophagy may explain the decrease of CD secretory granule observed in our study as well as the disorganization of the secretory granules and the exocytosis defect previously reported in CD. Mo1779 Interleukin-33 Induces Distinct Gastric Epithelial Alterations and Plays an Important Role in the Pathogenesis of Murine Gastritis Luca Pastorelli, Carlo De Salvo, Rekha R. Garg, Benedetta Mattioli, Jon Meddings, Maurizio Vecchi, Theresa T. Pizarro Background & Aim: Gastric inflammatory conditions are associated with the development of gastric adenocarcinoma, with published work indicating that cytokines and pro-inflammatory mediators, including interleukin (IL)-1 family members, play a key role in gastric carcinogenesis. IL-33 is a novel IL-1 family member inducing Th2 immune responses and epithelial hyperplasia (EH) in the gut. We recently reported IL-33 overexpression in SAMP1/YitFc (SAMP) mice, a spontaneous model of intestinal inflammation, also displaying Crohn's-like gastritis. The aim of this study was to evaluate the potential role of IL-33 in the development of gastritis and gastric epithelial changes in SAMP mice. Materials & Methods: Recombinant IL-33 (33 μg/kg, i.p.) was administered daily to 4- and 12-wk-old AKR (parental control) and SAMP mice for one wk (N=6/grp). IL-33 neutralization was performed using an antiST2-Fc fusion protein administered to 14 wk-old SAMP for 4 wks (N=4/exp grp). In Vivo gastric permeability was measured by fractional excretion (FE) of sucrose using an establish assay, prior to, and after, IL-33 treatment. BrdU (120 mg/kg, i.p.) was administered to mice 2h before sacrifice in order to assess cell proliferation. Stomachs were collected and evaluated histologically. IHC and qRT-PCR were performed for BrdU and ST2 (IL-33 receptor) localization, as well as tight junction protein expression, respectively. Results: IL-33 treatment induced gastritis in 4- and 12-wk-old AKR mice vs. vehicle controls (VC) (3.66±0.61 vs 0.33±0.33, P<0.001 and 6.40±1.20 vs 1.83±0.30, P<0.005, respectively), as well as in 4wk-old SAMP vs VC (5.66±0.66 vs 1.16±0.40, P<0.0005). IL-33 treated mice displayed striking macroscopic changes in the gastric mucosa, consisting of hypertrophic longitudinal folds; histologic evaluation showed increased epithelial hyperplasia in 4- (2.33±0.33 vs 0.16±0.16, P<0.0005) and 12-wk-old AKR (3.60±0.40 vs 1.167±0.30; P<0.001) and in 4(2.5±0.56 vs 0.33±0.21, P<0.005) and 12-wk-old SAMP (2.33±0.49 vs. 0.83±0.47; P=0.054) compared to VC. Actively proliferating cells, identified by BrdU staining, were dramatically increased in IL-33 treated mice and co-localized with ST2. Increased gastric epithelial permeability was detected in IL-33 treated mice (FE=0.0042±0.0011 vs 0.0010±0.0002; P<0.05), which correlated to an increased trend in claudin-2 and a reduction in occludin, ZO-1, claudin-3 and -4 mRNA expression. IL-33 blockade did not affect gastric inflammation in SAMP, but specifically reduced epithelial hyperplasia in treated SAMP vs VC (1.25±0.47 vs 3.50±0.50, P<0.05). Conclusion: IL-33 specifically induces gastritis and marked epithelial alterations leading to epithelial barrier dysfunction, which may represent early changes in the pathogenesis of inflammation-associated gastric cancers.
A. Simultaneous multi-color flow cytometry gating strategy from ileocecal valve pinch biopsies of one subject. From left to right, singlet live CD3+ CD4+ lymphocytes were gated in stepwise fashion. B. CD4+ lymphocytes were analyzed for production of IL-17, IL-22, IL4, TNF-α, IFN-γ, and FoxP3.
Mo1780 TH17 and FOXP3+ Cells are Enriched in the Healthy Human Cecum Martin J. Wolff, Jacqueline M. Leung, Michael Davenport, Ilseung Cho, Michael A. Poles, P'ng Loke
A. A sample analysis of IL-17 producing CD4+ cells from the ileocecal valve of one subject. B. This analysis was repeated for IL-17 producing CD4+ cells from the terminal ileum (TI), the ileocecal valve (ICV), the appendiceal orifice (AO), and 20 centimeters (Sig) of all enrolled subjects as shown on the right. Median values represented. Relationships not shown were non-significant. * P < 0.05. ** P < 0.005. C and D. Similar analysis of CD4+FoxP3+ cells within the human intestine of endoscopically normal subjects. Median values represented. * P < 0.05. ** P < 0.005.
BACKGROUND: Regional variations of cytokine-producing effector T helper cell populations in the healthy human colon and distal small intestine have not been systematically wellcharacterized. This baseline information is essential for making comparisons to intestinal immune responses under disease conditions. We utilized flow cytometric analysis of pinch biopsy samples obtained during screening colonoscopies of an average-risk population to assess regional differences in CD4+ T cell homeostasis in the human intestinal tract. METHODS: Institutional review board approval at the New York Harbor VA Hospital was obtained before enrolling patients in the study. Six to eight pinch biopsies were obtained at colonoscopy in 20 consecutive patients from the terminal ileum (TI), the ileocecal valve (ICV), the appendiceal orifice (AO), and 20 centimeters (Sig) from the anal verge. Not every location was biopsied in all subjects. Biopsies were digested with collagenase and leukocytes were isolated using Percoll separation. Following stimulation with phorbol 12-myristate 13acetate (PMA) and ionomycin for 4 hours, cells were stained with extracellular markers for CD3, CD4, CD8, CD56, and CD25. Cells were then permeabilized and stained with an intracellular cytokine panel for interleukin (IL)-4, IL-17, IL-22, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and with a nuclear antigen panel that included forkhead box P3 (FoxP3). Simultaneous multi-color flow cytometry was performed (BD LSR II). Results were analyzed in Flowjo (Treestar, Inc.) and statistical analysis was performed using Prism (GraphPad Software, Inc.) RESULTS: There were significant differences in IL-17 producing CD4+ cells (TI, N = 18, Median 8.2%; ICV, N = 16, 12.2%; AO, N = 15, 10.4%; Sig, N = 19, 6.7%, Kruskall-Wallis P = 0.0079), as well as in IL-22 producing CD4+ cells (TI 6.0%; ICV 11.5%; AO 12.2%; Sig 5.2%, P = 0.0012) among the sites biopsied. CD4+FoxP3+ populations were also significantly higher in the ileocecal valve and appendiceal orifice in comparison to the terminal ileum and sigmoid colon (TI 9.6%, ICV 15.2%, AO 13.1%, Sig 9.7% P = 0.0034). The relative proportions of IFN-γ, TNF-α, and IL-4 producing CD4+ cells were not significantly different based on biopsy location (P = 0.0834, P = 0.1044, P = 0.9100, respectively). CONCLUSIONS: The cecum and luminal appendiceal orifice are not often considered immunologically active structures in the human gastrointestinal tract. We found that the relative populations of CD4+IL-17+ and CD4+Foxp3+ cells were significantly higher at the ileocecal valve and appendiceal orifice than at the terminal ileum or distal large intestine in an average risk population without clinically apparent mucosal inflammation. The
Mo1781 Smoking is Associated With Disordered Homeostasis of Small Intestinal and Proximal Colonic Mucosal Mast Cells Nick Powell, Marjorie M. Walker, Nicholas J. Talley, Jukka Ronkainen, Pertti Aro, Tom Storskrubb, Anna N. Andreasson, Lars Agreus INTRODUCTION: Inflammatory bowel disease (IBD) is characterized by dysregulation of mucosal immunity. Genetic and environmental factors are implicated in IBD. Genetic variation at about 100 distinct loci are linked with IBD, and polymorphisms at loci encoding innate immune genes strongly linked to Crohn's disease (CD). Smoking has a clear association with IBD[1], exacerbates CD, and paradoxically is protective in ulcerative colitis. Smoking is also associated with irritable bowel syndrome[2]. How smoking influences gastrointestinal immune function In Vivo is poorly understood. We hypothesized that smoking would result in altered profiles of gastrointestinal mucosal innate immune cells. METHODS: This was a retrospective analysis in two random samples from the general population in Sweden, Kalixanda[3] and PopCol[4], gastrointestinal epidemiology by endoscopy and biopsy. The prevalence of smoking was ascertained and innate immunity (mast cell and eosinophil counts) established in the first (D1) and second (D2) parts of the duodenum (Kalixanda, 175 subjects) and terminal ileum (TI) and colon (PopCol, 194 subjects). Immunostaining was performed with CK117 to identify mast cells. Mast cells and eosinophils (H&E) were quantified in 5 high power fields at x 40 magnification in D1 and D2, TI, cecum, transverse colon, sigmoid colon and rectum. We report preliminary data: the Kalixanda cohort, H&E staining in 120 non-smokers, 26 smokers and 20 snuff users; CK117 immunostaining in 62 non-smokers, 13 smokers and 10 snuff users, and in the Popcol cohort, H&E staining in 81 non-smokers, 63 ex-smokers and 31 current smokers; and immunostaining in 72 non-smokers, 53 smokers and 24 current smokers. RESULTS: In current smokers mucosal
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AGA Abstracts
CD20, CD56) CD14+ cells and Lin- CD33int CD11b+ cells. Peripheral blood obtained from healthy volunteers was used for purified Naïve T cells. Naïve T cells were co-cultured with or without DC like macrophages and/or MDSCs. Culture supernatant was collected and analyzed using cytometric beads array (CBA) to assess the cytokine production. Results; we found that Lin- CD33int CXCR1+ cells are markedly upregulated in inflamed mucosa of UC patients. These cells were also CD11b+, CD15+. Morphologically, these new innate cells subset resembles granulocytic MDSCs. Although CD14+ macrophages proliferate naïve T cells, this reaction was inhibited when co-cultured with MDSCs. Furthermore, MDSCmediated T-cell suppression was observed as MDSC dose dependent manner. Culture supernatant, in naive T cells with CD14+ macrophages and MDSCs, contains less inflammatory cytokines, such as IFN-γ, than that in naive T cells with CD14+ macrophages. Conclusions; Dysregulated APCs found in intestinal LP of IBD patients induce colitogenic T cells. This vicious way was inhibited only by regulatory T cells, but our findings suggest that new suppressive MDSCs contribute to negative feedback of chronic inflammation in IBD. All the data suggest that novel subset of innate cells are recruited at the inflamed site in UC patients and prevent excessive inflammation.
mast cells were significantly reduced in the TI compared to ex-smokers (median counts: 231 vs 324, p=<0.004) and non-smokers (median: 231 vs 342, p=<0.03); in the cecum of smokers, compared to ex-smokers (median: 116 vs 158, p=<0.007) or non-smokers (median: 116 vs 170, p=<0.0002). Mast cells were significantly reduced in D1 of smokers, compared to non-smokers (median: 167 vs 238, p=<0.01). There was a tendency for eosinophilia in D1 of smokers (p=0.055). Eosinophils were not significantly altered in the TI/colon of smokers. CONCLUSIONS: Smoking is associated with disordered homeostasis of gastrointestinal mucosal mast cells, seen in small intestinal and ileocolic regions, sites most commonly affected in CD. Our data suggest that chronic exposure to tobacco may subtly alter the balance of mucosal innate immunity, particularly mast cells. 1. Cosnes J. Best Pract Res Clin Gastroenterol 2004; 18:481-96 2. Nam SY et al. J Neurogastroenterol Motil. 2010; 16:4751 3. Aro P et al. Scand J Gastroenterol. 2004; 39:1280-8 4. Walter SA et al Scand J Gastroenterol. 2010; 45:556-66
Mo1784 The In Vivo Transcriptional Response of Intestinal Epithelium to C. Parvum Infection is Dominated by Interferon-Alpha Signaling Pathways Derek Foster, Dahlia Nielsen, Stephen Stauffer, Jody Gookin Cryptosporidium parvum, a cause of profound malabsorptive diarrhea, induces both innate and adaptive immune responses to limit the infection and clear the parasite. As C. parvum infection is confined to the epithelium, the host response is dependent on signaling by the intestinal epithelial cells (IEC). The current understanding of the IEC response to C. parvum is predominantly derived from reductionist cell culture studies or immunodeficient mouse models, neither of which recapitulate the clinical infection or the influence of the lamina propria. We hypothesized that the transcriptional response of C. parvum-infected IEC In Vivo using an unparalleled animal model of human cryptosporidiosis will identify novel mechanisms of innate immunity activation and host defense against C. parvum infection. Neonatal piglets were infected (n=8) or not (n=5) with 108 C. parvum oocysts, and at peak infection, IEC were harvested by exfoliation from the ileum using a calcium chelation solution containing ethylene diamine tetraacetic acid. RNA was extracted, processed, and hybridized to Affymetrix porcine genomic microarrays. The arrays were normalized using a Loess normalization technique, and differentially expressed genes were indentified using an ANOVA with a false discovery rate set at 0.05. Differentially expressed genes with a two-fold change in expression were probed using Ingenuity Pathway Analysis and the expression of select genes was confirmed with qRT-PCR. Compared to control IEC, C. parvum infection differentially altered the expression of 82 genes (61 upregulated, 21 downregulated). Upregulated genes were represented by 24 targets of interferon-alpha (IFNa) including the ubiquitinlike protein modifier ISG15 (21.23-fold) and guanylate binding proteins-1 (7.8-fold) and 2 (11.9-fold). Transcription of IFNa was not increased by C. parvum suggesting a subepithelial origin that directs IEC innate immune response via paracrine effects. These findings demonstrate a previously undiscovered mechanism of innate IEC defense in C. parvum infection dominated by IFNa-dependent signaling. Investigations of this select group of highly differentially expressed genes may disclose novel pharmacological targets for control of C. parvum infection.
Mast cells are decreased in the ileocolic region in smokers compared to non smokers Mo1782 Disruption of the TLR4 Pathway Leads to Dysfunction of the Intestinal Barrier in an Escherichia coli Nissle Monoassociation Model - Implication for Experimental Inflammatory Bowel Disease Susceptibility Marijana Basic, Lydia M. Keubler, Anna Smoczek, Andrea Liese, Hans J. Hedrich, Andre Bleich Background and Aim: Escherichia coli Nissle 1917 (EcN), a well described probiotic bacterium, has been demonstrated to induce severe and lethal inflammation in C3H/HeJZtm (Tlr4defective) monoassociated mice. A major component of the toll-like receptor 4 (TLR4) signaling pathway, CD14, has been demonstrated to provide a protective mechanism in experimental inflammatory bowel disease (IBD) as Cd14-deficient mice display increased colitis susceptibility. Therefore, the aim of this study was to elucidate the role of CD14 in the EcN monoassociation model. Material and methods: Germfree Tlr4-defective, Tlr4-intact (C3H/HeOuJ) and Cd14-deficient (C3H/HeN.129S1-Cd14tm1Smg) mice received 109 cfu of EcN or PBS by oral gavage. 72 hours post inoculation cfu were determined in spleen, liver, cecum and colon and expression levels of tight junction proteins were assessed by qRTPCR and Western Blot analysis. cDNA was generated from small intestines to be used in microarray analysis. Results: cfu were not detected in spleens and livers of Tlr4-intact mice, whereas up to 103 cfu were found in spleens and livers of Cd14-deficient mice. Even more pronounced numbers of cfu were found in organs of Tlr4-defective mice. Expression levels of the tight junction proteins occludin, zona occludens 1 and claudin-8 were reduced in small intestines of Cd14-deficient and Tlr4-defective mice. Microarray analysis demonstrated an upregulation of genes associated with immune activation in Tlr4-intact mice. In Tlr4defective mice an upregulation of genes associated with inflammatory response was observed. Conclusions: EcN was demonstrated to invade Cd14-deficient mice analogous to Tlr4defective mice and disseminate beyond the intestinal barrier. The observed reduction of tight junction proteins in Cd14-deficient and Tlr4-defective mice indicates that the TLR4 pathway is critical for the function of the epithelial barrier in this model. These findings might explain increased IBD susceptibility of Cd14-deficient mice and demonstrate the important role of CD14 in intestinal homeostasis.
Mo1785 A Key Regulatory Role of Vav1 in Controlling Septic Shock and Experimental Peritonitis via IL-6 Production in Macrophages Stefanie Zenker, Stefan J. Wirtz, Tadamitsu Kishimoto, Markus F. Neurath, Imke Atreya Introduction: Sepsis is a complex disease, which is often associated with severe peritonitis, causes a very high rate of morbidity and mortality in patients. Severe peritonitis is, in particular, accelerate by massive infiltration of innate immune cells like macrophages (Mθ). Hyperinflammatory responses mediated by activated Mθ play key role in peritonitis and thus in septic shock. The Vav family of RhoGEFs consists of three family members, Vav1, Vav2 and Vav3. Vav1, which is mainly expressed in hematopoietic system, is an important intracellular adapter molecule and modulates cell activation in T cells. Furthermore, evidence suggested a function of Vav1 in Mθ chemotaxis, adhesion, migration and cytoskeleton reorganization. However, the capacity of Vav1 to influence early Mθ activity in sepsis has not been investigated extensively yet. Method: In order to analyze the function of Vav1 in Mθ, we analyzed Vav1-/- mice in the experimental model of LPS-induced septic shock. ELISA, qRT-PCR, Western blotting, CHIP and FACS analysis, amongst others, were used to specify In Vivo and In Vitro behavior of thioglycollate elicited peritoneal CD11b+ Vav1-/Mθ. Results: Unexpectedly, treatment with LPS resulted in significantly increased susceptibility of Vav1-/- mice for LPS -induced septic shock compared to wild-type (WT) BALB/c mice. Analysis of LPS-stimulated Vav1-/- macrophages revealed a significantly enhanced secretion of the pro-inflammatory cytokine IL-6 compared to WT-Mθ at early time points. At the same time, TNFα secretion by Mθ was not markedly affected by the loss of Vav1 in our experimental setting. LPS/Actinomycin D treatment of WT- and Vav1-/- Mθ emphasized that IL-6 is closely regulated on transcriptional level. Regarding the underlying intracellular signalling pathway, our data excluded classical IL-6 regulation factors, such as NFκB, as source of enhanced IL-6 secretion in Vav1-/- Mθ. Interestingly, the translocation of Vav1 in the nucleus and furthermore the interaction with HSF1 and the HSE2 region of the IL6 promoter seemed to be of central importance for the observed inhibitory effects of Vav1 on the expression of IL-6 in Mθ. Conclusion: Vav1 emerges as a crucial regulator protein of the early state of peritonitis and sepsis via secretion of the pro-inflammatory cytokine IL6 in Mθ. Our results indicate that Vav1 diminishes the initiate activation of Mθ response via preventing the maximal expression of IL-6 on transcriptional level. Overwhelming IL6 release in the absence of Vav1 resulted in an excessive innate immune reaction. Induction of increased Vav1 signalling might therefore represent an effective tool to regulate overwhelming IL-6 secretion in the context of septic peritonitis. In a nutshell, Vav1 exhibits a protective key role as guardian of excessive innate immune reaction.
Mo1783 New Immunosuppressive System by Myeloid-Derived Suppressor Cells in the Lamina Propria of Ulcerative Colitis Patients Yohei Mikami, Takanori Kanai, Shinta Mizuno, Tomohisa Sujino, Tango Handa, Atsuhiro Matsumoto, Toshiro Sato, Nobuhiko Kamada, Katsuyoshi Matsuoka, Tadakazu Hisamatsu, Akira Sonoda, Masakazu Takazoe, Rikisaburo Sahara, Kazutaka Koganei, Akira Sugita, Toshifumi Hibi Background ; Intestinal macrophages play a central role in regulation of intestinal immune responses. Interestingly, intestinal macrophages lack the expression of innate immune receptor CD14 and do not produce proinflammatory cytokines against commensal bacteria. However, we previously reported that CD14+CD33+CD205+ macrophages are markedly increased in inflamed mucosa of IBD patients, and these cells produce larger amounts of proinflammatory cytokines, and induce proinflammatory colitogenic T cells. Conversely, myeloid-derived suppressor cells (MDSC) are supposed to be one of responsible cells for immune suppression in patients with advanced cancer and these cells suppress T cell expansion. To connect stimulatory and supprresive control of intestinal immune responses, we here clarify the new suppressive cells contribute to the immunosuppression of chronic intestinal inflammation in patients with IBD. Methods; Mucosal samples were obtained from surgically resected specimens (controls, n=12; UC, n=8; CD, n=12). DC like macrophages and MDSCs from lamina propria (LP) were sorted by flowcytometry as Lin- (CD3e, CD19,
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P62 accumulation was reported in CD patients also indicating that the autophagic flux is active in CD Paneth cells. TEM high magnification observation revealed that autophagy activation was associated with a significant decrease in the number of secretory granules in CD patients. The granules appeared to be wrapped around autophagolysosomal vacuoles and abnormally degraded. Conclusion: This work describes of a novel selective autophagy pathway, called crinophagy that targets specifically secretory granules of CD Paneth cells. This process consists of the self-digestion of secretory granules by the autophago-lysosomal pathway. Crinophagy may explain the decrease of CD secretory granule observed in our study as well as the disorganization of the secretory granules and the exocytosis defect previously reported in CD. Mo1779 Interleukin-33 Induces Distinct Gastric Epithelial Alterations and Plays an Important Role in the Pathogenesis of Murine Gastritis Luca Pastorelli, Carlo De Salvo, Rekha R. Garg, Benedetta Mattioli, Jon Meddings, Maurizio Vecchi, Theresa T. Pizarro Background & Aim: Gastric inflammatory conditions are associated with the development of gastric adenocarcinoma, with published work indicating that cytokines and pro-inflammatory mediators, including interleukin (IL)-1 family members, play a key role in gastric carcinogenesis. IL-33 is a novel IL-1 family member inducing Th2 immune responses and epithelial hyperplasia (EH) in the gut. We recently reported IL-33 overexpression in SAMP1/YitFc (SAMP) mice, a spontaneous model of intestinal inflammation, also displaying Crohn's-like gastritis. The aim of this study was to evaluate the potential role of IL-33 in the development of gastritis and gastric epithelial changes in SAMP mice. Materials & Methods: Recombinant IL-33 (33 μg/kg, i.p.) was administered daily to 4- and 12-wk-old AKR (parental control) and SAMP mice for one wk (N=6/grp). IL-33 neutralization was performed using an antiST2-Fc fusion protein administered to 14 wk-old SAMP for 4 wks (N=4/exp grp). In Vivo gastric permeability was measured by fractional excretion (FE) of sucrose using an establish assay, prior to, and after, IL-33 treatment. BrdU (120 mg/kg, i.p.) was administered to mice 2h before sacrifice in order to assess cell proliferation. Stomachs were collected and evaluated histologically. IHC and qRT-PCR were performed for BrdU and ST2 (IL-33 receptor) localization, as well as tight junction protein expression, respectively. Results: IL-33 treatment induced gastritis in 4- and 12-wk-old AKR mice vs. vehicle controls (VC) (3.66±0.61 vs 0.33±0.33, P<0.001 and 6.40±1.20 vs 1.83±0.30, P<0.005, respectively), as well as in 4wk-old SAMP vs VC (5.66±0.66 vs 1.16±0.40, P<0.0005). IL-33 treated mice displayed striking macroscopic changes in the gastric mucosa, consisting of hypertrophic longitudinal folds; histologic evaluation showed increased epithelial hyperplasia in 4- (2.33±0.33 vs 0.16±0.16, P<0.0005) and 12-wk-old AKR (3.60±0.40 vs 1.167±0.30; P<0.001) and in 4(2.5±0.56 vs 0.33±0.21, P<0.005) and 12-wk-old SAMP (2.33±0.49 vs. 0.83±0.47; P=0.054) compared to VC. Actively proliferating cells, identified by BrdU staining, were dramatically increased in IL-33 treated mice and co-localized with ST2. Increased gastric epithelial permeability was detected in IL-33 treated mice (FE=0.0042±0.0011 vs 0.0010±0.0002; P<0.05), which correlated to an increased trend in claudin-2 and a reduction in occludin, ZO-1, claudin-3 and -4 mRNA expression. IL-33 blockade did not affect gastric inflammation in SAMP, but specifically reduced epithelial hyperplasia in treated SAMP vs VC (1.25±0.47 vs 3.50±0.50, P<0.05). Conclusion: IL-33 specifically induces gastritis and marked epithelial alterations leading to epithelial barrier dysfunction, which may represent early changes in the pathogenesis of inflammation-associated gastric cancers.
A. Simultaneous multi-color flow cytometry gating strategy from ileocecal valve pinch biopsies of one subject. From left to right, singlet live CD3+ CD4+ lymphocytes were gated in stepwise fashion. B. CD4+ lymphocytes were analyzed for production of IL-17, IL-22, IL4, TNF-α, IFN-γ, and FoxP3.
Mo1780 TH17 and FOXP3+ Cells are Enriched in the Healthy Human Cecum Martin J. Wolff, Jacqueline M. Leung, Michael Davenport, Ilseung Cho, Michael A. Poles, P'ng Loke
A. A sample analysis of IL-17 producing CD4+ cells from the ileocecal valve of one subject. B. This analysis was repeated for IL-17 producing CD4+ cells from the terminal ileum (TI), the ileocecal valve (ICV), the appendiceal orifice (AO), and 20 centimeters (Sig) of all enrolled subjects as shown on the right. Median values represented. Relationships not shown were non-significant. * P < 0.05. ** P < 0.005. C and D. Similar analysis of CD4+FoxP3+ cells within the human intestine of endoscopically normal subjects. Median values represented. * P < 0.05. ** P < 0.005.
BACKGROUND: Regional variations of cytokine-producing effector T helper cell populations in the healthy human colon and distal small intestine have not been systematically wellcharacterized. This baseline information is essential for making comparisons to intestinal immune responses under disease conditions. We utilized flow cytometric analysis of pinch biopsy samples obtained during screening colonoscopies of an average-risk population to assess regional differences in CD4+ T cell homeostasis in the human intestinal tract. METHODS: Institutional review board approval at the New York Harbor VA Hospital was obtained before enrolling patients in the study. Six to eight pinch biopsies were obtained at colonoscopy in 20 consecutive patients from the terminal ileum (TI), the ileocecal valve (ICV), the appendiceal orifice (AO), and 20 centimeters (Sig) from the anal verge. Not every location was biopsied in all subjects. Biopsies were digested with collagenase and leukocytes were isolated using Percoll separation. Following stimulation with phorbol 12-myristate 13acetate (PMA) and ionomycin for 4 hours, cells were stained with extracellular markers for CD3, CD4, CD8, CD56, and CD25. Cells were then permeabilized and stained with an intracellular cytokine panel for interleukin (IL)-4, IL-17, IL-22, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and with a nuclear antigen panel that included forkhead box P3 (FoxP3). Simultaneous multi-color flow cytometry was performed (BD LSR II). Results were analyzed in Flowjo (Treestar, Inc.) and statistical analysis was performed using Prism (GraphPad Software, Inc.) RESULTS: There were significant differences in IL-17 producing CD4+ cells (TI, N = 18, Median 8.2%; ICV, N = 16, 12.2%; AO, N = 15, 10.4%; Sig, N = 19, 6.7%, Kruskall-Wallis P = 0.0079), as well as in IL-22 producing CD4+ cells (TI 6.0%; ICV 11.5%; AO 12.2%; Sig 5.2%, P = 0.0012) among the sites biopsied. CD4+FoxP3+ populations were also significantly higher in the ileocecal valve and appendiceal orifice in comparison to the terminal ileum and sigmoid colon (TI 9.6%, ICV 15.2%, AO 13.1%, Sig 9.7% P = 0.0034). The relative proportions of IFN-γ, TNF-α, and IL-4 producing CD4+ cells were not significantly different based on biopsy location (P = 0.0834, P = 0.1044, P = 0.9100, respectively). CONCLUSIONS: The cecum and luminal appendiceal orifice are not often considered immunologically active structures in the human gastrointestinal tract. We found that the relative populations of CD4+IL-17+ and CD4+Foxp3+ cells were significantly higher at the ileocecal valve and appendiceal orifice than at the terminal ileum or distal large intestine in an average risk population without clinically apparent mucosal inflammation. The
Mo1781 Smoking is Associated With Disordered Homeostasis of Small Intestinal and Proximal Colonic Mucosal Mast Cells Nick Powell, Marjorie M. Walker, Nicholas J. Talley, Jukka Ronkainen, Pertti Aro, Tom Storskrubb, Anna N. Andreasson, Lars Agreus INTRODUCTION: Inflammatory bowel disease (IBD) is characterized by dysregulation of mucosal immunity. Genetic and environmental factors are implicated in IBD. Genetic variation at about 100 distinct loci are linked with IBD, and polymorphisms at loci encoding innate immune genes strongly linked to Crohn's disease (CD). Smoking has a clear association with IBD[1], exacerbates CD, and paradoxically is protective in ulcerative colitis. Smoking is also associated with irritable bowel syndrome[2]. How smoking influences gastrointestinal immune function In Vivo is poorly understood. We hypothesized that smoking would result in altered profiles of gastrointestinal mucosal innate immune cells. METHODS: This was a retrospective analysis in two random samples from the general population in Sweden, Kalixanda[3] and PopCol[4], gastrointestinal epidemiology by endoscopy and biopsy. The prevalence of smoking was ascertained and innate immunity (mast cell and eosinophil counts) established in the first (D1) and second (D2) parts of the duodenum (Kalixanda, 175 subjects) and terminal ileum (TI) and colon (PopCol, 194 subjects). Immunostaining was performed with CK117 to identify mast cells. Mast cells and eosinophils (H&E) were quantified in 5 high power fields at x 40 magnification in D1 and D2, TI, cecum, transverse colon, sigmoid colon and rectum. We report preliminary data: the Kalixanda cohort, H&E staining in 120 non-smokers, 26 smokers and 20 snuff users; CK117 immunostaining in 62 non-smokers, 13 smokers and 10 snuff users, and in the Popcol cohort, H&E staining in 81 non-smokers, 63 ex-smokers and 31 current smokers; and immunostaining in 72 non-smokers, 53 smokers and 24 current smokers. RESULTS: In current smokers mucosal
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AGA Abstracts
CD20, CD56) CD14+ cells and Lin- CD33int CD11b+ cells. Peripheral blood obtained from healthy volunteers was used for purified Naïve T cells. Naïve T cells were co-cultured with or without DC like macrophages and/or MDSCs. Culture supernatant was collected and analyzed using cytometric beads array (CBA) to assess the cytokine production. Results; we found that Lin- CD33int CXCR1+ cells are markedly upregulated in inflamed mucosa of UC patients. These cells were also CD11b+, CD15+. Morphologically, these new innate cells subset resembles granulocytic MDSCs. Although CD14+ macrophages proliferate naïve T cells, this reaction was inhibited when co-cultured with MDSCs. Furthermore, MDSCmediated T-cell suppression was observed as MDSC dose dependent manner. Culture supernatant, in naive T cells with CD14+ macrophages and MDSCs, contains less inflammatory cytokines, such as IFN-γ, than that in naive T cells with CD14+ macrophages. Conclusions; Dysregulated APCs found in intestinal LP of IBD patients induce colitogenic T cells. This vicious way was inhibited only by regulatory T cells, but our findings suggest that new suppressive MDSCs contribute to negative feedback of chronic inflammation in IBD. All the data suggest that novel subset of innate cells are recruited at the inflamed site in UC patients and prevent excessive inflammation.
mast cells were significantly reduced in the TI compared to ex-smokers (median counts: 231 vs 324, p=<0.004) and non-smokers (median: 231 vs 342, p=<0.03); in the cecum of smokers, compared to ex-smokers (median: 116 vs 158, p=<0.007) or non-smokers (median: 116 vs 170, p=<0.0002). Mast cells were significantly reduced in D1 of smokers, compared to non-smokers (median: 167 vs 238, p=<0.01). There was a tendency for eosinophilia in D1 of smokers (p=0.055). Eosinophils were not significantly altered in the TI/colon of smokers. CONCLUSIONS: Smoking is associated with disordered homeostasis of gastrointestinal mucosal mast cells, seen in small intestinal and ileocolic regions, sites most commonly affected in CD. Our data suggest that chronic exposure to tobacco may subtly alter the balance of mucosal innate immunity, particularly mast cells. 1. Cosnes J. Best Pract Res Clin Gastroenterol 2004; 18:481-96 2. Nam SY et al. J Neurogastroenterol Motil. 2010; 16:4751 3. Aro P et al. Scand J Gastroenterol. 2004; 39:1280-8 4. Walter SA et al Scand J Gastroenterol. 2010; 45:556-66
Mo1784 The In Vivo Transcriptional Response of Intestinal Epithelium to C. Parvum Infection is Dominated by Interferon-Alpha Signaling Pathways Derek Foster, Dahlia Nielsen, Stephen Stauffer, Jody Gookin Cryptosporidium parvum, a cause of profound malabsorptive diarrhea, induces both innate and adaptive immune responses to limit the infection and clear the parasite. As C. parvum infection is confined to the epithelium, the host response is dependent on signaling by the intestinal epithelial cells (IEC). The current understanding of the IEC response to C. parvum is predominantly derived from reductionist cell culture studies or immunodeficient mouse models, neither of which recapitulate the clinical infection or the influence of the lamina propria. We hypothesized that the transcriptional response of C. parvum-infected IEC In Vivo using an unparalleled animal model of human cryptosporidiosis will identify novel mechanisms of innate immunity activation and host defense against C. parvum infection. Neonatal piglets were infected (n=8) or not (n=5) with 108 C. parvum oocysts, and at peak infection, IEC were harvested by exfoliation from the ileum using a calcium chelation solution containing ethylene diamine tetraacetic acid. RNA was extracted, processed, and hybridized to Affymetrix porcine genomic microarrays. The arrays were normalized using a Loess normalization technique, and differentially expressed genes were indentified using an ANOVA with a false discovery rate set at 0.05. Differentially expressed genes with a two-fold change in expression were probed using Ingenuity Pathway Analysis and the expression of select genes was confirmed with qRT-PCR. Compared to control IEC, C. parvum infection differentially altered the expression of 82 genes (61 upregulated, 21 downregulated). Upregulated genes were represented by 24 targets of interferon-alpha (IFNa) including the ubiquitinlike protein modifier ISG15 (21.23-fold) and guanylate binding proteins-1 (7.8-fold) and 2 (11.9-fold). Transcription of IFNa was not increased by C. parvum suggesting a subepithelial origin that directs IEC innate immune response via paracrine effects. These findings demonstrate a previously undiscovered mechanism of innate IEC defense in C. parvum infection dominated by IFNa-dependent signaling. Investigations of this select group of highly differentially expressed genes may disclose novel pharmacological targets for control of C. parvum infection.
Mast cells are decreased in the ileocolic region in smokers compared to non smokers Mo1782 Disruption of the TLR4 Pathway Leads to Dysfunction of the Intestinal Barrier in an Escherichia coli Nissle Monoassociation Model - Implication for Experimental Inflammatory Bowel Disease Susceptibility Marijana Basic, Lydia M. Keubler, Anna Smoczek, Andrea Liese, Hans J. Hedrich, Andre Bleich Background and Aim: Escherichia coli Nissle 1917 (EcN), a well described probiotic bacterium, has been demonstrated to induce severe and lethal inflammation in C3H/HeJZtm (Tlr4defective) monoassociated mice. A major component of the toll-like receptor 4 (TLR4) signaling pathway, CD14, has been demonstrated to provide a protective mechanism in experimental inflammatory bowel disease (IBD) as Cd14-deficient mice display increased colitis susceptibility. Therefore, the aim of this study was to elucidate the role of CD14 in the EcN monoassociation model. Material and methods: Germfree Tlr4-defective, Tlr4-intact (C3H/HeOuJ) and Cd14-deficient (C3H/HeN.129S1-Cd14tm1Smg) mice received 109 cfu of EcN or PBS by oral gavage. 72 hours post inoculation cfu were determined in spleen, liver, cecum and colon and expression levels of tight junction proteins were assessed by qRTPCR and Western Blot analysis. cDNA was generated from small intestines to be used in microarray analysis. Results: cfu were not detected in spleens and livers of Tlr4-intact mice, whereas up to 103 cfu were found in spleens and livers of Cd14-deficient mice. Even more pronounced numbers of cfu were found in organs of Tlr4-defective mice. Expression levels of the tight junction proteins occludin, zona occludens 1 and claudin-8 were reduced in small intestines of Cd14-deficient and Tlr4-defective mice. Microarray analysis demonstrated an upregulation of genes associated with immune activation in Tlr4-intact mice. In Tlr4defective mice an upregulation of genes associated with inflammatory response was observed. Conclusions: EcN was demonstrated to invade Cd14-deficient mice analogous to Tlr4defective mice and disseminate beyond the intestinal barrier. The observed reduction of tight junction proteins in Cd14-deficient and Tlr4-defective mice indicates that the TLR4 pathway is critical for the function of the epithelial barrier in this model. These findings might explain increased IBD susceptibility of Cd14-deficient mice and demonstrate the important role of CD14 in intestinal homeostasis.
Mo1785 A Key Regulatory Role of Vav1 in Controlling Septic Shock and Experimental Peritonitis via IL-6 Production in Macrophages Stefanie Zenker, Stefan J. Wirtz, Tadamitsu Kishimoto, Markus F. Neurath, Imke Atreya Introduction: Sepsis is a complex disease, which is often associated with severe peritonitis, causes a very high rate of morbidity and mortality in patients. Severe peritonitis is, in particular, accelerate by massive infiltration of innate immune cells like macrophages (Mθ). Hyperinflammatory responses mediated by activated Mθ play key role in peritonitis and thus in septic shock. The Vav family of RhoGEFs consists of three family members, Vav1, Vav2 and Vav3. Vav1, which is mainly expressed in hematopoietic system, is an important intracellular adapter molecule and modulates cell activation in T cells. Furthermore, evidence suggested a function of Vav1 in Mθ chemotaxis, adhesion, migration and cytoskeleton reorganization. However, the capacity of Vav1 to influence early Mθ activity in sepsis has not been investigated extensively yet. Method: In order to analyze the function of Vav1 in Mθ, we analyzed Vav1-/- mice in the experimental model of LPS-induced septic shock. ELISA, qRT-PCR, Western blotting, CHIP and FACS analysis, amongst others, were used to specify In Vivo and In Vitro behavior of thioglycollate elicited peritoneal CD11b+ Vav1-/Mθ. Results: Unexpectedly, treatment with LPS resulted in significantly increased susceptibility of Vav1-/- mice for LPS -induced septic shock compared to wild-type (WT) BALB/c mice. Analysis of LPS-stimulated Vav1-/- macrophages revealed a significantly enhanced secretion of the pro-inflammatory cytokine IL-6 compared to WT-Mθ at early time points. At the same time, TNFα secretion by Mθ was not markedly affected by the loss of Vav1 in our experimental setting. LPS/Actinomycin D treatment of WT- and Vav1-/- Mθ emphasized that IL-6 is closely regulated on transcriptional level. Regarding the underlying intracellular signalling pathway, our data excluded classical IL-6 regulation factors, such as NFκB, as source of enhanced IL-6 secretion in Vav1-/- Mθ. Interestingly, the translocation of Vav1 in the nucleus and furthermore the interaction with HSF1 and the HSE2 region of the IL6 promoter seemed to be of central importance for the observed inhibitory effects of Vav1 on the expression of IL-6 in Mθ. Conclusion: Vav1 emerges as a crucial regulator protein of the early state of peritonitis and sepsis via secretion of the pro-inflammatory cytokine IL6 in Mθ. Our results indicate that Vav1 diminishes the initiate activation of Mθ response via preventing the maximal expression of IL-6 on transcriptional level. Overwhelming IL6 release in the absence of Vav1 resulted in an excessive innate immune reaction. Induction of increased Vav1 signalling might therefore represent an effective tool to regulate overwhelming IL-6 secretion in the context of septic peritonitis. In a nutshell, Vav1 exhibits a protective key role as guardian of excessive innate immune reaction.
Mo1783 New Immunosuppressive System by Myeloid-Derived Suppressor Cells in the Lamina Propria of Ulcerative Colitis Patients Yohei Mikami, Takanori Kanai, Shinta Mizuno, Tomohisa Sujino, Tango Handa, Atsuhiro Matsumoto, Toshiro Sato, Nobuhiko Kamada, Katsuyoshi Matsuoka, Tadakazu Hisamatsu, Akira Sonoda, Masakazu Takazoe, Rikisaburo Sahara, Kazutaka Koganei, Akira Sugita, Toshifumi Hibi Background ; Intestinal macrophages play a central role in regulation of intestinal immune responses. Interestingly, intestinal macrophages lack the expression of innate immune receptor CD14 and do not produce proinflammatory cytokines against commensal bacteria. However, we previously reported that CD14+CD33+CD205+ macrophages are markedly increased in inflamed mucosa of IBD patients, and these cells produce larger amounts of proinflammatory cytokines, and induce proinflammatory colitogenic T cells. Conversely, myeloid-derived suppressor cells (MDSC) are supposed to be one of responsible cells for immune suppression in patients with advanced cancer and these cells suppress T cell expansion. To connect stimulatory and supprresive control of intestinal immune responses, we here clarify the new suppressive cells contribute to the immunosuppression of chronic intestinal inflammation in patients with IBD. Methods; Mucosal samples were obtained from surgically resected specimens (controls, n=12; UC, n=8; CD, n=12). DC like macrophages and MDSCs from lamina propria (LP) were sorted by flowcytometry as Lin- (CD3e, CD19,
AGA Abstracts
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