Mo1731 Pathogenic Role of PI3K P85α Subunit in a Murine Colitis Model Through the Regulation of...
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AGA Abstracts Mo1729 Differential Effect of Cigarette Smoke Extract Modifies CD103 and CXCR3 Expressions of Monocyte-Derived Dendritic Cells From Crohn's Disease and Ulcerative Colitis Patients Aito Ueno, Humberto Jijon, Suzanne Traves, Ronald Chan, Kim Ford, Herman Barkema, Miriam Fort Gasia, Rajveer Hundal, Travis B. Murdoch, Paul L. Beck, Gilaad Kaplan, Marietta Iacucci, Remo Panaccione, David Proud, Subrata Ghosh INTRODUCTION: Smoking has a paradoxical influence upon two types of Inflammatory Bowel Disease (IBD), it is protective in ulcerative colitis (UC) but adversely affects Crohn's disease (CD). Dendritic cells (DC) play a critical role in modulating and orchestrating T cell-mediated inflammation such as IBD. DC surface markers, which are responsible for gut- homing, enable the categorization of these cells into inflammatory and regulatory phenotypes. CXCR3 is an inflammatory marker that induces Th-1 polarization, whereas CD103 and CCR9 are considered regulatory markers. We hypothesized that smoking differentially modifies the immune response of DC in CD and UC patients. Our aim is to compare the in vitro effects of cigarette smoke extract (CSE) on cell surface marker expression by monocyte-derived DC (Mo-DC) from patients with CD and UC. METHODS: Blood monocytes (CD14+ leuko- cytes) from 6 CD and 7 UC patients with diagnosed IBD and no history of smoking were cultured with GM-CSF and IL-4 for 7 days to differentiate into DC. CSE was prepared fresh each time by bubbling cigarette smoke into the culture media and the concentration was adjusted to an absorbance of 0.15 at 320 nm (considered as 100%) using a spectrophotometer. Mo-DC were then exposed to 50% CSE for 24 hours. Cell surface expressions of CD103, CCR9, and CXCR3 were assessed by flow cytometry and secretion of CXCL10 (IP-10, a ligand for CXCR3) from Mo-DC were assessed by Luminex. Data from un-treated and CSE- exposed cells were analyzed using Mann-Whitney U test to compare between CD and UC samples. RESULTS: Baseline CD103 and CCR9 expression showed no significant differences between CD and UC patients. CSE exposure increased CD103 expression in UC but decreased it in CD, and this difference was statistically significant (p=0.008, Fig. A). CSE decreased CCR9 expression in both CD and UC samples but no significant difference was seen between CD and UC (Fig. B). UC samples showed significantly higher CXCR3 at baseline level than CD (p=0.041). CSE exposure increased CXCR3 expression in CD samples but decreased it in UC, causing a significant difference in CSE-driven CXCR3 expression between CD and UC (p=0.012, Fig.C). This was accompanied by CSE-driven decreases in CXCL10 secretion in UC samples compared to CD (p=0.008). CONCLUSION: CSE exposure differentially affected Mo-DC expression of CD103 and CXCR3 in CD and UC. CSE skewed Mo-DC into a more pro-inflammatory type in CD but induced a regulatory phenotype in UC patients. These differences may be relevant in part to the contrasting effects of cigarette smoking on the inflammation in CD and UC. CSE-driven modulation of DC gut-homing receptor expres- sion may be considered a therapeutic target in future. Figure (Left panel) CSE increased CD103 expression in UC samples but decreased in CD samples. There was a significant difference in CSE-derived CD103 modification between CD and UC. (Middle panel) CSE decreased CCR9 expression in both CD and UC samples. There was no difference in CSE-derived CCR9 decrease between CD and UC. (Right panel) Baseline of CXCR3 expression. There was a significant difference in CXCR3 expression between CD and UC. CSE increased CXCR3 expression in CD samples but decreased in UC samples. There was a significant difference in CSE-derived CXCR3 modification between CD and UC. Mo1730 Helminth Colonization Increases pSTAT3 and ILGRα Expression in Murine Terminal Ileum Epithelial Cells Jessica E. Stumphy, Ahmed Metwali, Sarah Winckler, M. Nedim Ince, Steven Polyak, David E. Elliott Background: Helminth exposure inhibits colitis and decreases the proinflammatory response in murine models. We hypothesize that helminth infection alters epithelial response to injury. The interleukin (IL)6/phosphorylated-signal transducers and activators of transcription 3 (pSTAT3) pathway contributes to epithelial cell repair and is also involved in inflammatory bowel disease. IL6 is a growth factor for intestinal epithelial cells (IEC) and signals by activating STAT3 through phosphorylation (pSTAT3). Therefore, we investigated the effect of helminth colonization on intestinal epithelial cell (IEC) STAT3 activation and IL6Rα (CD126) expression. Materials and methods: Wild type C57Bl/6 mice were infected with Heligmosomides polygyrus bakeri larvae via gastric lavage. Uninfected age-matched wild type mice were used as controls. Two weeks after helminth infection, the mice were sacrificed and their terminal ileums were harvested. The IEC were collected and analyzed by western blot and flow cytometry for pSTAT3 and IL6Rα expression. Results: There was statistically significant elevation of pSTAT3 expression in terminal ileum IEC from C57Bl/6 mice exposed to helminths compared to uninfected control mice. This increase occurred without histologic change. Helminth exposure increased IEC pSTAT3 expression by three to four fold. About 7.5 ± 0.2 % of IEC isolated from control mice expressed pSTAT3 compared to 27.7 ± 0.6 % from colonized mice (p < 0.05). Colonization also significantly increased IEC pSTAT3 expression as measured by western blot (see figure). In addition, expression of CD126 (IL6rα) was significantly increased in terminal ileum IEC of infected mice (from 0.3 ± 0.1% to 1.4 ± 0.2% (p<0.05)) as measured by flow cytometry. We repeated these experiment using Rag1-deficient mice that lack mature B and T cells. In these mice, helminth exposure had no effect on pSTAT3 or IL6Rα expression by terminal ileum IEC. Conclusion: IEC STAT3 activation and IL6Ra expression is increased in the terminal ileum of mice exposed to helminths. This suggests that helminth colonization may promote epithelial cell growth S-646 AGA Abstracts and repair through IL6-mediated activation of STAT3. The absence of increased pSTAT3 and CD126 expression in Rag1-deficient mice suggests that helminth-mediated activation of the IEC IL6/STAT3 pathway requires participation from the adaptive immune system. Ongoing research will elucidate if IEC pSTAT3 activation requires IL6 or IL10 signals provided by mucosal lymphocytes. Helminth infection increases IEC pSTAT3 expression. Representative results from 3 or more experiments Mo1731 Pathogenic Role of PI3K P85α Subunit in a Murine Colitis Model Through the Regulation of Intestinal Macrophage Functions Takayuki Hamada, Shusaku Hayashi, Makoto Kadowaki Background/Aim: Although the precise etiology of IBD has not yet been fully defined, recent studies have revealed that intestinal macrophages (MΦs) play a pivotal role in the pathogenesis of IBD. Phosphatidylinositol 3-kinase (PI3K) contributes to the regulation of various cellular functions and, in particular, p85α subunit of PI3K is crucial for MΦ functions such as migration, phagocytosis, and proliferation. We have recently found that PI3K p85α hetero deficient (p85α+/-) mice exhibit reduced susceptibility to dextran sulfate sodium (DSS)- induced colitis. In the present study, we examined the role of PI3K p85α subunit in the intestinal MΦ functions using DSS-induced colitis mice. Methods: p85α+/- mice (BALB/c background) and WT mice were used. Experimental colitis was induced by giving 3% DSS (36-50 kDa) in drinking water for 7 days ad libitum. F4/80+CD11b+ MΦs were isolated from colonic lamina propria (cLP) and examined for cytokine mRNA expression by real- time PCR. Results: The treatment with DSS for 7 days caused severe damage in the colon, with body weight loss, an increase in disease activity index and myeloperoxidase activity as well as shortening of colon length. These manifestations of DSS-induced colitis were significantly attenuated in p85α+/- colitis mice compared with WT colitis mice. The histologi- cal examination of the colonic mucosa showed deep erosion and infiltration of inflammatory cells in WT colitis mice and these histological abnormalities were diminished in p85α+/- colitis mice. Furthermore, TNF-α and IL-6 mRNA expression in the colon of WT colitis mice was greatly elevated, which was higher than that in the colon of p85α+/- colitis mice (WT colitis vs p85α+/- colitis, TNF-α: 1.00 ± 0.19 vs 0.41 ± 0.12; IL-6: 1.00 ± 0.38 vs 0.22 ± 0.08, P<0.05). Interestingly, the proportion of F4/80+CD11b+ MΦs in the cLP was increased equivalently in both WT and p85α+/- colitis mice. The LPS-induced expression of TNF-α and IL-6 mRNA in F4/80+CD11b+ MΦs isolated from inflamed colonic mucosa of WT colitis mice were higher than those in the normal colonic mucosa of WT mice. However, the LPS-induced upregulation of TNF-α and IL-6 mRNA in F4/80+CD11b+ MΦs of colonic mucosa were significantly suppressed in p85α+/- colitis mice compared with WT colitis mice (WT colitis vs p85α+/- colitis, TNF-α: 1.00 ± 0.12 vs 0.50 ± 0.13, P<0.05; IL- 6: 1.00 ± 0.19 vs 0.44 ± 0.18, P=0.099). Conclusion: These results demonstrated that p85α+/- mice were resistant to the development of DSS-induced colitis, and the colonic MΦs in p85α+/- colitis mice expressed lower levels of TNF-α and IL-6 mRNA than those in WT mice. These findings suggest that PI3K p85α subunit is involved in the development of DSS-induced colitis through the regulation of proinflammatory cytokine production in the intestinal MΦs of inflamed mucosa. Mo1732 The Role of EBI2 and Oxysterols in the Pathogenesis of Inflammatory Bowel Diseases (IBD) and the Generation of Lymphoid Structures in Mouse Intestine Annika Wyss, Tina Raselli, Kirstin Atrott, Isabelle Frey-Wagner, Andreas Sailer, Michael Fried, Gerhard Rogler, Benjamin Misselwitz Background: The oxysterol 7alpha,25-dihydroxycholesterol (7alpha,25-OHC) is a chem- oattractant for immune cells expressing Epstein-Barr virus-induced gene 2 (EBI2) and directs their migration. EBI2 is a G-protein coupled receptor which is primarily expressed in lymphoid tissue and leukocytes. Mice lacking EBI2 or enzymes responsible for production of 7alpha,25-OHC show severe defects in the generation of T cell dependent antibody responses. Recently EBI2 was identified as a risk gene for inflammatory bowel diseases (IBD). Furthermore, EBI2 was found amongst genes upregulated in the ileum of CD patients with mutations in NOD2 compared to CD patients with a NOD2 wildtype genotype. Methods: We used mice with and without a functional EBI2-7alpha,25-OHC system in models of acute and chronic colitis. Colitis was induced by administration of 2-3% DSS for 7 days (acute) or 4 cycles of 7 days interspersed with 10 days recovery periods (chronic) in EBI2 and CH25H knockout mice and wildtype controls. Disease severity was determined by colonoscopy, colon length, histopathological analyses, MPO activity and expression of typical inflammation markers. Results: In the acute colitis model expression of EBI2 and both enzymes responsible for 7alpha,25-OHC synthesis, CH25H and CYP7B1, were significantly upregulated in the colon after DSS treatment. In chronic DSS colitis histological scoring revealed significantly stronger mucosal inflammation in CH25H knockout mice as compared
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Differential Effect of Cigarette Smoke Extract Modifies CD103 and CXCR3Expressions of Monocyte-Derived Dendritic Cells From Crohn's Disease andUlcerative Colitis PatientsAito Ueno, Humberto Jijon, Suzanne Traves, Ronald Chan, Kim Ford, Herman Barkema,Miriam Fort Gasia, Rajveer Hundal, Travis B. Murdoch, Paul L. Beck, Gilaad Kaplan,Marietta Iacucci, Remo Panaccione, David Proud, Subrata Ghosh
INTRODUCTION: Smoking has a paradoxical influence upon two types of InflammatoryBowel Disease (IBD), it is protective in ulcerative colitis (UC) but adversely affects Crohn'sdisease (CD). Dendritic cells (DC) play a critical role in modulating and orchestrating Tcell-mediated inflammation such as IBD. DC surface markers, which are responsible for gut-homing, enable the categorization of these cells into inflammatory and regulatory phenotypes.CXCR3 is an inflammatory marker that induces Th-1 polarization, whereas CD103 and CCR9are considered regulatory markers. We hypothesized that smoking differentially modifies theimmune response of DC in CD and UC patients. Our aim is to compare the in vitro effectsof cigarette smoke extract (CSE) on cell surface marker expression by monocyte-derivedDC (Mo-DC) from patients with CD and UC. METHODS: Blood monocytes (CD14+ leuko-cytes) from 6 CD and 7 UC patients with diagnosed IBD and no history of smoking werecultured with GM-CSF and IL-4 for 7 days to differentiate into DC. CSE was prepared fresheach time by bubbling cigarette smoke into the culture media and the concentration wasadjusted to an absorbance of 0.15 at 320 nm (considered as 100%) using a spectrophotometer.Mo-DC were then exposed to 50% CSE for 24 hours. Cell surface expressions of CD103,CCR9, and CXCR3 were assessed by flow cytometry and secretion of CXCL10 (IP-10, aligand for CXCR3) from Mo-DC were assessed by Luminex. Data from un-treated and CSE-exposed cells were analyzed using Mann-Whitney U test to compare between CD and UCsamples. RESULTS: Baseline CD103 and CCR9 expression showed no significant differencesbetween CD and UC patients. CSE exposure increased CD103 expression in UC but decreasedit in CD, and this difference was statistically significant (p=0.008, Fig. A). CSE decreasedCCR9 expression in both CD and UC samples but no significant difference was seen betweenCD and UC (Fig. B). UC samples showed significantly higher CXCR3 at baseline level thanCD (p=0.041). CSE exposure increased CXCR3 expression in CD samples but decreased itin UC, causing a significant difference in CSE-driven CXCR3 expression between CD andUC (p=0.012, Fig.C). This was accompanied by CSE-driven decreases in CXCL10 secretionin UC samples compared to CD (p=0.008). CONCLUSION: CSE exposure differentiallyaffected Mo-DC expression of CD103 and CXCR3 in CD and UC. CSE skewed Mo-DC intoa more pro-inflammatory type in CD but induced a regulatory phenotype in UC patients.These differences may be relevant in part to the contrasting effects of cigarette smoking onthe inflammation in CD and UC. CSE-driven modulation of DC gut-homing receptor expres-sion may be considered a therapeutic target in future.
Figure (Left panel) CSE increased CD103 expression in UC samples but decreased in CDsamples. There was a significant difference in CSE-derived CD103 modification betweenCD and UC. (Middle panel) CSE decreased CCR9 expression in both CD and UC samples.There was no difference in CSE-derived CCR9 decrease between CD and UC. (Right panel)Baseline of CXCR3 expression. There was a significant difference in CXCR3 expressionbetween CD and UC. CSE increased CXCR3 expression in CD samples but decreased inUC samples. There was a significant difference in CSE-derived CXCR3 modification betweenCD and UC.
Mo1730
Helminth Colonization Increases pSTAT3 and ILGRα Expression in MurineTerminal Ileum Epithelial CellsJessica E. Stumphy, Ahmed Metwali, Sarah Winckler, M. Nedim Ince, Steven Polyak,David E. Elliott
Background: Helminth exposure inhibits colitis and decreases the proinflammatory responsein murine models. We hypothesize that helminth infection alters epithelial response to injury.The interleukin (IL)6/phosphorylated-signal transducers and activators of transcription 3(pSTAT3) pathway contributes to epithelial cell repair and is also involved in inflammatorybowel disease. IL6 is a growth factor for intestinal epithelial cells (IEC) and signals byactivating STAT3 through phosphorylation (pSTAT3). Therefore, we investigated the effectof helminth colonization on intestinal epithelial cell (IEC) STAT3 activation and IL6Rα(CD126) expression. Materials and methods: Wild type C57Bl/6 mice were infected withHeligmosomides polygyrus bakeri larvae via gastric lavage. Uninfected age-matched wild typemice were used as controls. Two weeks after helminth infection, the mice were sacrificedand their terminal ileums were harvested. The IEC were collected and analyzed by westernblot and flow cytometry for pSTAT3 and IL6Rα expression. Results: There was statisticallysignificant elevation of pSTAT3 expression in terminal ileum IEC from C57Bl/6 mice exposedto helminths compared to uninfected control mice. This increase occurred without histologicchange. Helminth exposure increased IEC pSTAT3 expression by three to four fold. About7.5 ± 0.2 % of IEC isolated from control mice expressed pSTAT3 compared to 27.7 ± 0.6% from colonized mice (p < 0.05). Colonization also significantly increased IEC pSTAT3expression as measured by western blot (see figure). In addition, expression of CD126(IL6rα) was significantly increased in terminal ileum IEC of infected mice (from 0.3 ± 0.1%to 1.4 ± 0.2% (p<0.05)) as measured by flow cytometry. We repeated these experimentusing Rag1-deficient mice that lack mature B and T cells. In these mice, helminth exposurehad no effect on pSTAT3 or IL6Rα expression by terminal ileum IEC. Conclusion: IECSTAT3 activation and IL6Ra expression is increased in the terminal ileum of mice exposedto helminths. This suggests that helminth colonization may promote epithelial cell growth
S-646AGA Abstracts
and repair through IL6-mediated activation of STAT3. The absence of increased pSTAT3and CD126 expression in Rag1-deficient mice suggests that helminth-mediated activationof the IEC IL6/STAT3 pathway requires participation from the adaptive immune system.Ongoing research will elucidate if IEC pSTAT3 activation requires IL6 or IL10 signalsprovided by mucosal lymphocytes.
Helminth infection increases IEC pSTAT3 expression. Representative results from 3 ormore experiments
Mo1731
Pathogenic Role of PI3K P85α Subunit in a Murine Colitis Model Through theRegulation of Intestinal Macrophage FunctionsTakayuki Hamada, Shusaku Hayashi, Makoto Kadowaki
Background/Aim: Although the precise etiology of IBD has not yet been fully defined, recentstudies have revealed that intestinal macrophages (MΦs) play a pivotal role in the pathogenesisof IBD. Phosphatidylinositol 3-kinase (PI3K) contributes to the regulation of various cellularfunctions and, in particular, p85α subunit of PI3K is crucial for MΦ functions such asmigration, phagocytosis, and proliferation. We have recently found that PI3K p85α heterodeficient (p85α+/-) mice exhibit reduced susceptibility to dextran sulfate sodium (DSS)-induced colitis. In the present study, we examined the role of PI3K p85α subunit in theintestinal MΦ functions using DSS-induced colitis mice. Methods: p85α+/- mice (BALB/cbackground) and WT mice were used. Experimental colitis was induced by giving 3% DSS(36-50 kDa) in drinking water for 7 days ad libitum. F4/80+CD11b+ MΦs were isolatedfrom colonic lamina propria (cLP) and examined for cytokine mRNA expression by real-time PCR. Results: The treatment with DSS for 7 days caused severe damage in the colon,with body weight loss, an increase in disease activity index and myeloperoxidase activityas well as shortening of colon length. These manifestations of DSS-induced colitis weresignificantly attenuated in p85α+/- colitis mice compared with WT colitis mice. The histologi-cal examination of the colonic mucosa showed deep erosion and infiltration of inflammatorycells in WT colitis mice and these histological abnormalities were diminished in p85α+/-colitis mice. Furthermore, TNF-α and IL-6 mRNA expression in the colon of WT colitismice was greatly elevated, which was higher than that in the colon of p85α+/- colitis mice(WT colitis vs p85α+/- colitis, TNF-α: 1.00 ± 0.19 vs 0.41 ± 0.12; IL-6: 1.00 ± 0.38 vs0.22 ± 0.08, P<0.05). Interestingly, the proportion of F4/80+CD11b+ MΦs in the cLP wasincreased equivalently in both WT and p85α+/- colitis mice. The LPS-induced expressionof TNF-α and IL-6 mRNA in F4/80+CD11b+ MΦs isolated from inflamed colonic mucosaof WT colitis mice were higher than those in the normal colonic mucosa of WT mice.However, the LPS-induced upregulation of TNF-α and IL-6 mRNA in F4/80+CD11b+ MΦsof colonic mucosa were significantly suppressed in p85α+/- colitis mice compared with WTcolitis mice (WT colitis vs p85α+/- colitis, TNF-α: 1.00 ± 0.12 vs 0.50 ± 0.13, P<0.05; IL-6: 1.00 ± 0.19 vs 0.44 ± 0.18, P=0.099). Conclusion: These results demonstrated thatp85α+/- mice were resistant to the development of DSS-induced colitis, and the colonicMΦs in p85α+/- colitis mice expressed lower levels of TNF-α and IL-6 mRNA than thosein WT mice. These findings suggest that PI3K p85α subunit is involved in the developmentof DSS-induced colitis through the regulation of proinflammatory cytokine production inthe intestinal MΦs of inflamed mucosa.
Mo1732
The Role of EBI2 and Oxysterols in the Pathogenesis of Inflammatory BowelDiseases (IBD) and the Generation of Lymphoid Structures in Mouse IntestineAnnika Wyss, Tina Raselli, Kirstin Atrott, Isabelle Frey-Wagner, Andreas Sailer, MichaelFried, Gerhard Rogler, Benjamin Misselwitz
Background: The oxysterol 7alpha,25-dihydroxycholesterol (7alpha,25-OHC) is a chem-oattractant for immune cells expressing Epstein-Barr virus-induced gene 2 (EBI2) and directstheir migration. EBI2 is a G-protein coupled receptor which is primarily expressed inlymphoid tissue and leukocytes. Mice lacking EBI2 or enzymes responsible for productionof 7alpha,25-OHC show severe defects in the generation of T cell dependent antibodyresponses. Recently EBI2 was identified as a risk gene for inflammatory bowel diseases (IBD).Furthermore, EBI2 was found amongst genes upregulated in the ileum of CD patients withmutations in NOD2 compared to CD patients with a NOD2 wildtype genotype. Methods:We used mice with and without a functional EBI2-7alpha,25-OHC system in models ofacute and chronic colitis. Colitis was induced by administration of 2-3% DSS for 7 days(acute) or 4 cycles of 7 days interspersed with 10 days recovery periods (chronic) in EBI2and CH25H knockout mice and wildtype controls. Disease severity was determined bycolonoscopy, colon length, histopathological analyses, MPO activity and expression of typicalinflammation markers. Results: In the acute colitis model expression of EBI2 and bothenzymes responsible for 7alpha,25-OHC synthesis, CH25H and CYP7B1, were significantlyupregulated in the colon after DSS treatment. In chronic DSS colitis histological scoringrevealed significantly stronger mucosal inflammation in CH25H knockout mice as compared
