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M1669 Human Umbilical Cord Mesenchymal Stem Cell Transplantation Ameliorates Inflammation in a Mouse Model of Colitis
AGA Abstracts
PBS alone was performed three times in a week. After 2 weeks, theses animals were sacrificed, and histological evaluation and the gene expression of inflammatory cytokines (IL-12p40, TNFα, and IFNγ) in colonic mucosa were evaluated by RT-PCR. Results: (1) The production of TNFα and IL-6 were significantly decreased in supernatant from RAW264.7 cells treated with FK506 compared to non-treated cells. (2) Pretreatment of FK506 suppressed LPSinduced activation of both NFκB and p38MAPK in RAW264.7 cells. (3) The production of IL-12p40, TNFα and IL-6 were significantly decreased in supernatant from peritoneal macrophages treated with FK506 compared to non-treated cells. (4) Histological examination revealed that histological score was significantly decreased in mice treated with FK506 than in those with PBS. (5) The gene expression of IL-12p40, TNFα and IFNγ in colonic mucosa were significantly decreased in mice treated with FK506 compared to those with PBS. Conclusion: We firstly demonstrated the immunosuppressive effects of FK506 on macrophage, by which immune-mediated colonic inflammation was attenuated. Blockade of activation of NFκB and p38MAPK by FK506 could be associated with this immunosuppressive mechanism.
M1667 Evolution of Markers of Colitis in Mdr1a-/- Mice Housed in SPF and Conventional Conditions Miquel Moretó, Mònica Maijó, Lluïsa Miró, Javier Polo, Eric Weaver, Joe D. Crenshaw, Louis Russell, Joy Campbell, Anna Pérez-Bosque Background and aims: The mdr1a-/- mouse model develops a spontaneous colitis. The degree of severity of the pathology and the time of appearance of colitis signs strongly depend on housing conditions. In the present study we have assayed a protocol combining the stay of mice in specific pathogen frer (SPF) conditions followed by conventional housing, in order to define conditions were food intake and growth were not markedly affected. Methods: Knock out (KO) mice were obtained from the pathogen free colony of mdr1a-/at Taconic (Germantown, NY). Mice (n= 6-7) were grown and maintained in SPF conditions until week 4 when they where transferred to conventional housing for 4-5 additional weeks. Wild type (WT; n= 6-7) congenic FVB (+/+) mice were raised in conventional housing for the same time period. A disease activity index (DAI) was defined, accounting for changes in motor activity, body weight, consistency of faeces, and presence of blood in stools. After sacrifice, the weight of spleen and mesenteric lymph nodes, and the length and weight of the colon were recorded. The permeability of colon mucosa was analysed ex vivo by confocal microscopy using FITC-Dextran 10 kDa as marker. Tissue interferon-γ (IFN-γ) was measured by bioplex cytokine assay. Results: First signs of disease were observed when mice were 5wk old when DAI progressively increased reaching a value of 1.94 ± 0.23 at day 58 (DAI in WT was 0.31 ± 0.18; P<0.05). In KO mice, the weight of colon, spleen and mesenteric lymph nodes was 2-3 fold higher than in WT animals (P<0.05), and these changes were well correlated with DAI. No differences in body weight between groups were observed at sacrifice. The permeability of crypts to dextran in KO was 2 fold higher than WT animals (P<0.05). Production of IFN-γ in the colon was markedly stimulated in KO mice (1.7 ± 0.2 pg/mg prot) compared to WT group (0.3 ± 0.04 pg/mg prot). However, in the ileum IFNγ expression was the same in both groups. Conclusion: The protocol used in this study allowed development of mild colitis without affecting food intake and growth though several markers such as IFN- γ expression and crypt permeability were enhanced in the mdr1a-/mice. This indicates that this protocol is adequate to carry out studies on the effect of diets in prevention of inflammatory bowel disease.
M1665 Development and In Vivo Analysis of a New Generation of Optimized ThioGTP Analogues for Therapy of IBD Imke Atreya, Alexandra Diall, Christian Ostrau, Gerhard Fritz, Radovan Dvorsky, Mathias Gruen, Christoph Becker, Markus F. Neurath Introduction: Therapy with azathioprine (aza) and 6-mercaptopurine (6-MP) represents the gold standard for maintenance of remission in inflammatory bowel disease (IBD), although the use of these classic immunosuppressive agents in acute flares of Crohn's disease or ulcerative colitis is limited by the delayed onset of action of thiopurines. Based on 6-thioGTP, which could recently be identified as a key active metabolite of aza and is responsible for the pro-apoptotic capacity of thiopurines via inhibition of the small GTPase Rac in T cells, we now aim on the development of a new class of immunosuppressive drugs with an improved efficacy in acute IBD. Method: A rational development of new 6-thio-GTP analogues was performed by steric modelling in order to generate 6-thio-GTP analogues with an increased capacity to block Rac activity and to mediate an earlier onset of pro-apoptotic effects. 5 groups of chemically modified novel 6-thio-GTP analogues (Group A-F), a total of 48 new substances, were synthesized and tested on isolated human T cells for their proapoptotic capacity. The In Vivo immunosuppressive capacity of lead compounds was analysed in a murine model of TNBS-induced acute colitis and analysed by mini-endoscopy. The potential clinical applicability of the best candidates was evaluated by In Vitro toxicity tests. Results: Based on caspase-3 activity assays, 6-thio-GTP analogues of group B, D and E could be identified as strong inducers of T cell apoptosis, while analogues of group A, C and F failed to induce apoptosis. Finally, 4 lead compounds (B0, B0L, B0N, E1) were identified and characterized by a superior ability to induce increased levels of T cell apoptosis at earlier time points than classic thiopurines. With regard to In Vitro hepato- and mylotoxicity, especially B0 and B0N showed a reduced toxic potential compared to the classic drug 6MP. Results from a comet-assay indicated that In Vitro treatment of human T cells with B0N induced a significantly lower number of DNA strand breaks than 6-MP. Due to this promising In Vitro profile of B0N (high immunosuppressive capacity and reduced toxicity), the In Vivo immunosuppressive efficacy of B0N was evaluated in the model of TNBS-induced acute colitis. In Vivo application of B0N in TNBS-treated balb/c mice resulted in a significant reduction of mucosal inflammation (colitis-score=7,5) compared to untreated control group (colitis-score=10,3). Conclusion: Due to promising low In Vitro toxicity as well as high In Vitro and In Vivo immunosuppressive efficacy, B0N represents an excellent candidate for GLP safety studies and subsequent clinical development.
M1668 Defective CD8+ CD28- Regulatory T Cells in the Intestine of Patients with Crohn's Disease Keren M. Rabinowitz, Paul Smereka, Lloyd Mayer INTRODUCTION: In the normal state, the interaction between IEC and LP lymphocytes gives rise to a population of CD8+ CD28- T cells with regulatory function that can be identified phenotypically and functionally in normal LPL. These cells suppress by cell contact. AIM: Characterize the factors required for expansion of these regulatory LP CD8+ T cells and define how they mediate suppression METHODS: Freshly isolated LPLs from normal controls, UC and CD patients were stimulated with visilizumab (Nuvion), a humanized antiCD3 mAb (100ng/ml), for 5 days followed by addition of IL7 (10ng/ml) and IL15 (20ng/ ml) and irradiated PBMCs. After 14 days CD8+ T cells were purified by magnetic bead sorting, and cultured in the presence of IL7 and IL15. Cell lines were analyzed for suppressor activity (inhibition of CD4+ T cell proliferation in response to anti-CD3/CD28 beads), surface molecule expression (by flow cytometry) and cytokine secretion profile (CBA, ELISA). Suppressive cell lines were used as immunogens to generate murine mAbs against surface molecules involved in suppressor function. RESULTS: The cell lines did not express CD28, CD25, ICOS, PDL1, CD16, perforin, granzyme B, CTLA4 and FoxP3 but were positive for CD3, CD8β, CD2 and CD101. Following stimulation with anti-CD3/CD28 beads cell lines secreted substantial amounts of IFN-γ and TNF-α, IL13 and GM-CSF but only modest amounts of IL10, IL4 and IL2. With regard to suppressor function, lines generated from normal (15 lines) and UC patients (3 lines) were able to suppress CD4+ T cell proliferation in a contact dependent fashion. In contrast, lines generated from CD patients (9 lines) had a markedly decreased capacity to mediate suppression. Lastly, sera from 3 mice immunized with normal CD8+ LPL lines but not unimmunized control sera demonstrated the ability to block suppression. CONCLUSION: We developed a method for generating regulatory CD8+ T cell lines from the LP. CD8+ T cell lines from control and UC but not CD patients exhibit contact dependent regulatory function. Antisera from cell line immunized mice block this suppression and appear to recognize the molecule(s) mediating this suppression. These cells may play a role in control of local inflammation in the gut.
M1666 Anti-Inflammatory Effects of HMPL-004 Are Mediated By the Inhibition of Multiple Targets Tao Wang, Yuan Zhou, Yu Cai, Weihan Zhang, Weihua Gu, Yang Sai, Genhong Cheng, Xiaoqiang Yan HMPL-004 is a botanic drug in global phase II development for the treatment of inflammatory bowel disease (IBD). The botanic drug is derived from Andrographis Paniculata, an herb that has long been used in Asia for the treatment of acute infections. In a proof of concept (POC) study, we showed that HMPL-004, compared to Mesalamine, was effective in treating patients with mild to moderate Ulcerative Colitis (UC). In the current studies, we report pre-clinical results that may mechanistically explain the anti-inflammatory activities of HMPL-004. HMPL-004 was able to inhibit multiple cellular targets involved in the NF-κB and MAPK pathways. The inhibition of NF-κB was achieved by suppressing the phosphorylation of IκBα as observed in bacterial lipopolysaccharide (LPS) induced THP-1 cells and by interfering with the binding of NF-κB to DNA as seen in HEK293 cells over-expressing p65 or p50. The inhibition of MAPK pathway was demonstrated by the inhibiting the phorphorylation of Erk1/2 and MAKPAPK2. In a DNBS (2, 4 nitrobenzenesulfonic acid)-induced rat colitis model, HMPL-004 significantly suppressed the inflammation and the levels of TNFα and IL-1β in the colons. We further performed rat pharmacokinetics studies and found that the plasma exposures of active compounds, andrographolides and flavonoids, identified In Vitro, were at ng/mL levels that were too low to achieve effective target inhibition. Therefore, an ex vivo study was performed using isolated rat colons after a DNBS challenge. Significant inhibition of IL-1β production by HMPL-004 was observed in the colons ex vivo. In summary, these findings demonstrated that HMPL-004 is able to suppress multiple cellular targets including IκBα, p65, p50, and MAPKs. The ex vivo studies further suggests a local action of HMPL-004 that may have contributed to the biological effects observed in the DNBSinduced rat colitis model and also to the clinical benefits seen in UC patients.
AGA Abstracts
M1669 Human Umbilical Cord Mesenchymal Stem Cell Transplantation Ameliorates Inflammation in a Mouse Model of Colitis Xiao cang Cao, Bangmao Wang, Yingzi Cong, Fang Yan Background: Although significant advances have been made towards understanding the immunopathogenesis of inflammatory bowel disease (IBD), a major challenge of IBD research is to successfully develop new strategies for the treatment of IBD. The present therapeutic applications for IBD are limited by their side effects and unstable effectiveness. Accumulating data indicate stem cell transplantation (SCT) could be potentially used as therapy of the patients with IBD. Considering IBD susceptibility loci, allo-SCT should the appropriate candidate as it could “tinker up these bug”, and human umbilical cord mesenchymal stem cells (hUCMSCs) could be an ideal resource and more convenient in future application because of undemanding HLA match in transplantation. More recently it had been shown not only to be able to differentiate into multiple tissues, but also to exert a potent antiproliferative effect that result in the inhibition of immune responses. Therefore the purpose of this study is to determine the effects of transplantation of hUCMSCs on inflammation in SCID mice. Merthods: Colitis in SCID mice was induced by injection (i.p.) of CD4+CD25-T cells isolated from spleen of wild-type Balb/c mice using magnetic separation method. hUCMSCs obtained from volunteer, labeled by CM-DiI, were administrated (i.v.) to SCID mice for
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5*106 each mouse subsequently. Saline was used as a control. Colon sections were prepared for H&E staining to determine intestinal inflammation by DAI score. CM-DiI-labeled hUCMSCs were detected by CONFOCAL and Optical In Vivo Imaging system (XENOGEN IVIS200). The effects of hUCMSCs on T cells proliferation were studied In Vitro by BrdUlabeled method and concentrations of IL-17, IL-23 and IFN-g were measured by ELISA. Results: The SCID mice developed intestinal inflammation 3-5 weeks after first transplantation, which was similar to that in human IBD patients. The lesions of treated mice were improved in mice with CM-DiI hUCMSCs treatment after second transplantation, which DAI score decreased from 3.1 to 1.3. CM-DiI+ hUCMSCs, which were detected by In Vivo Imaging system, were also recovered in intestinal mucosa and submucosa of the colon by CONFOCAL. Furthermore, hUCMSCs inhibited mice T cell response to antigen stimulation In Vitro in a dose-dependent manner and production of proinflammatory cytokines was lower, indicating the immunosuppressive activity of hUCMSCs. CONCLUSION: Systemically administrated hUCMSCs responded to an adequate tissue lesion and resulted in accelerated tissue repair in mouse IBD.hUCMSC transplantation may serve as a potential approach for IBD treatment.
M1672
Background: Glycogen synthase kinase 3-β (GSK3-β) recently was identified as a critical regulator of Toll-like receptor (TLR) signaling mediating excessive inflammatory responses. Its activity enhances TLR-induced proinflammatory cytokine secretion from blood monocytes while IL-10 production is suppressed. In previous studies we provided evidence that In Vivo blockade of GSK3 β by LiCl attenuates excessive TLR-mediated immune responses to bacterial constituents during chronic intestinal inflammation. Aim of this study was to investigate whether LiCl directly impairs the function of freshly isolated human and mouse intestinal lymphocytes. Methods: For induction of chronic colitis, female Balb/c mice received 4 cycles of dextran sodium sulphate (DSS) treatment. After the onset of chronic colitis mesenteric lymph node cells (MLC) and lamina propria mononuclear cells (LPMC) were isolated and stimulated In Vitro with CpG-ODN or control-ODN (2μg/ml) in the presence or absence of 10 mM LiCl. Human LPMC were isolated from surgical specimens of control patients and patients with inflammatory bowel disease (IBD) and stimulated In Vitro with CpG-ODN, control-ODN (10 μg/ml), LPS (10-50 ng/ml) or anti-CD3/anti-CD28 beads in the presence or absence of 10 mM LiCl. Cytokine levels were determined by Luminex and ELISA. Results: CpG-ODN stimulation of murine MLC and LPMC resulted in production of large amounts of IL-6, TNF, and IFN-γ. Presence of LiCl significantly reduced CpG-ODN-induced IL-6 (60%; p<0.0001) and TNF (30%; p<0.0005) secretion from MLC and LPMC, while IFN-γ production of LPMCs was reduced by 90% (p<0.0001). Basal IL-10 production of LPMC was enhanced by 14% after GSK3-β blockade. In human LPMC isolated from control patients basal IL-6 production was not impaired by LiCl. However, IL-6 secretion induced by LPS (1.4-fold) or T cell stimulation (5.4-fold) were reduced by up to 30% in the presence of LiCl. In contrast, basal IL-6 production of LPMC isolated from inflamed IBD tissue was significantly (52%; p<0.001) diminished by LiCl and LPS-induced IL-6 levels were reduced by 40% (p<0.0001). A LiCl-dependent decrease of basal (17%; p=0.017) and CpG-ODNinduced (23%; p=0.0007) was observed in LPMC from non-inflamed IBD tissue. Conclusion: Blockade of GSK3-β by LiCl attenuates excessive proinflammatory TLR-mediated immune responses of intestinal lymphocytes from chronic inflamed tissue. GSK3-β inhibition therefore constitutes a promising therapeutic option for selectively reducing exaggerated intestinal immune reactions towards the luminal flora in IBD.
M1670 Decreased Production of IL-10 and TGF-β in TLR-Activated Intestinal B Cells in SAMP1/Yit Mice Yoshiyuki Mishima, Shunji Ishihara, Md. M. Aziz, Naoki Oshima, Akihiko Oka, Ryusaku Kusunoki, Aya Ohtani, Ichiro Moriyama, Yong-Yu Li, Yuji Amano, Yoshikazu Kinoshita (Background & Aim) A unique subset of B cells expressing interleukin (IL)-10 and transforming growth factor (TGF)-β plays an essential role in maintaining immune responses. These B cells produce IL-10 and TGF-β after sensing pathogen-associated molecules via toll-like receptors (TLRs), and modulate autoimmunity and inflammation in a variety of organs. However, little is known regarding their role in immune-related intestinal disorders. SAMP1/Yit mice spontaneously develop ileitis and are widely recognized as a murine model of Crohn's disease. In the present study, we investigated the presence of this cell subset in mice intestines and its role in the pathogenesis of ileitis in SAMP1/Yit mice. (Materials and Methods) Five-, ten- and thirty-week-old male SAMP1/Yit mice and age-matched male control AKR/J mice were used in this study. Development of ileitis in the SAMP1/Yit mice was confirmed by histological examinations. Mononuclear cells were isolated from mesenteric lymph nodes (MLNs) and spleens of the mice, and the expressions of B220, CD5, TLR4, TLR9, RP105, Mac-1, IgM, IgD, and CD1d in isolated cells were analyzed by flow cytometry. For In Vitro experiments, B cells were separated from isolated mononuclear cells magnetically by positive selection using B220 micro-beads. Purified B cells were cultured in 96-well plates and stimulated with LPS or CpG DNA for 3 days, then IL-10 and TGF-β contents in the culture media were measured with enzyme immunoassays. In addition, the intracellular contents of IL-10 and TGF-β in TLR ligand-stimulated B cells were analyzed by flow cytometry after staining with CD5, TLR4, TLR9, RP105, Mac-1, IgM, IgD, and CD1d. (Results) Ileitis was clearly observed in the 10- and 30-week-old SAMP1/Yit mice. The expression patterns of several cellular markers in B cells isolated from the MLNs and spleen of SAMP1/Yit mice were similar to those of AKR/J mice, as shown by flow cytometry. On the other hand, the production levels of IL-10 and TGF-β stimulated by LPS and CpG DNA were significantly lower in B cells separated from MLNs of SAMP1/Yit mice as compared to those from AKR/J mice. Furthermore, decreased production of those cytokines was also observed in B cells separated from the MLNs of 5-week-old SAMP1/Yit mice without typical ileitis. Also, a significant expression of CD1d was observed in B cells producing IL-10 and TGF-β in both SAMP1/Yit and AKR/J mice. (Conclusion) We observed decreased production of IL-10 and TGF-β by TLR-activated intestinal B cells in SAMP1/Yit mice. Our results suggest that these immuno-regulatory B cells are associated with the pathogenesis of Crohn's disease.
M1673 Wiskott-Aldrich Syndrome Protein (WASP) Is Critical for the Inducible Generation of FOXP3+ Regulatory T Cells Elisa K. Boden, Vineet Ahuja, Christopher J. Moran, Deanna D. Nguyen, Scott B. Snapper The Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency that results from mutations in WASP. WASP transduces cell surface receptor signals to the cytoskeleton via ARP2/3, a protein complex that has recently been linked to ulcerative colitis in a genomewide association study. WASP-deficient mice (WKO) develop a spontaneous colitis that is associated with both quantitative and qualitative defects in CD4+CD25+Foxp3+ regulatory T cells (Tregs) that control autoimmunity. As inducible Tregs (iTregs) are believed to control organ-specific inflammation, we asked whether reduced numbers of peripheral Tregs were a reflection of impaired iTreg generation in WKO mice. iTregs are generated by dendritic cells (DCs) in a process that is TGFβ-dependent and FoxP3 can be induced In Vitro when naive T cells are stimulated via the T cell receptor (TCR) in the presence of TGFβ. Therefore, CD4+CD25- T cells from wild type (WT) or WKO mice were cultured in the presence of TGFβ and plate-bound anti-CD3. After 72 hours, cells were analyzed by flow cytometry for the expression of FoxP3. The induction of FoxP3 was significantly decreased in WKO T cells (3.3 +/- 1.3%) compared to WT T cells (29.5 +/- 9.3%, p<.04). Defective TGFβ signaling has been described in mucosal T cells from patients with Crohn's disease, characterized by reduced SMAD3 phosphorylation and elevated expression of SMAD7, an inhibitor of TGFβ signaling. WKO CD4+ T cells, however, had no obvious alterations in TGFβ signaling. Addition of the costimulatory antibody anti-CD28 to anti-CD3 increased the number of iTregs generated from WT T cells (80.8 +/- 7.9%), but did not rescue iTreg generation in WKO T cells (1.8+/- 0.77%, p<.003). However, addition of the T cell mitogen, concanavalin A (ConA), which bypasses the early steps of TCR engagement partially rescued FoxP3 expression in WKO T cells (20.9+/- 3.2%). These data demonstrate that WASP-dependent TCR signals are critical for the generation of iTregs. Impaired peripheral generation of Tregs likely contributes to colitis in WKO mice, and may be operative in human inflammatory bowel disease (IBD). The elucidation of mechanisms that control Treg homeostasis may allow for targeted therapeutics in IBD.
M1671 All-Trans Retinoic Acid Downregulates Inflammatory Responses By Shifting the Treg/TH17 Profile in Human Ulcerative and Murine Colitis Aiping Bai, Nonghua Lu, Yuan Guo, Zhikang Peng Background and aims: Inflammatory bowel disease (IBD) is characterized by uncontrolled immune responses in inflamed mucosa, with dominance of IL-17 producing cells and deficiency of Treg cells. This study was conducted to explore the effect and mechanisms of all-trans retinoic acid, the ligand of retinoic acid receptor α (RARα), on immune responses in both human and murine colitis. Methods: Colonic biopsies from patients with ulcerative colitis were cultured and treated with all-trans retinoic acid as the agonist of RARα, or LE350 as the antagonist of RARα. Expressions of IL-17 and FOXP3 were detected by immunohistochemistry. Murine colitis was induced by intrarectal administration of 2,4,6,trinitrobenzene sulphonic acid (TNBS) at day 1. Mice were then intraperitoneally treated with all-trans retinoic acid or LE350 daily for 7 days. Cytokine levels in the cultures of mouse lamina propria mononuclear cells (LPMCs) were measured. Expressions of FOXP3 and IL-17 in colon tissues or mesenteric lymph nodes (MLN) were detected by immunohistological analysis. Body weight and colon inflammation were evaluated. Results: All-trans retinoic acid treatment upregulated FOXP3 expression and down-regulated IL-17 expression in colon biopsies of patients, and in colon tissues and MLN of mice with colitis, compared with controls. LPMCs from retinoic acid-treated mice produced lower levels of proinflammatory cytokines (TNFα, IL-1β, IL-17), but more regulatory cytokines (IL-10, TGFβ), compared with that of untreated mice. LE135-treated mice showed the opposite effect of all-trans retinoic acid. Furthermore, all-trans retinoic acid ameliorated TNBS-induced colitis in a dose-dependent manner as seen by improved body weight and colon inflammation. Conclusions: All-trans retinoic acid down-regulates colon inflammatory responses in patients with IBD In Vitro and in murine colitis In Vivo, representing a potential therapeutic approach in IBD treatment.
M1674 Gender-Specific Differences in CD4+CD25+FOXP3+ Natural T Regulatory Cells Modulate Chronic Intestinal Inflammation in Experimental Crohn's Disease Rekha R. Garg, Muhammadreza A. Sachedina, Brian K. Reuter, Theresa T. Pizarro Background: Previous studies report that the overall clinical status and severity of several autoimmune diseases, including CD, is worse in female patients compared to their male counterparts. Mechanistically, gender differences can be influenced by X/Y dimorphism as well as sex hormones. In addition, peripheral immune tolerance is maintained by the immunosuppressive role of natural T regulatory cells (nTregs), characterized by CD4+CD25+FoxP3+ cell surface markers, and nTreg depletion has been shown to lead to autoimmunity. Aim: To determine gender-specific phenotypic and functional differences among Treg populations in the SAMP1/YitFc (SAMP) model of CD-like ileitis. Methods: Ileal inflammation was histologically evaluated in F/M-SAMP and control AKR mice. Flow cytometry and In Vitro CFSE-based proliferation and sex hormone stimulation assays were performed on mesenteric lymph node (MLN) cells. Results: A significant gender difference in ileal inflammation was observed as early as 6 wks of age, with increased severity in F-
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AGA Abstracts
AGA Abstracts
In Vitro Blockade of GSK3-β Selectively Reduces the Proinflammatory Phenotype of Lymphocytes from Chronic Inflamed Intestinal Tissue Claudia Hofmann, Nadja Dunger, Jurgen Scholmerich, Werner Falk, Florian Obermeier
PBS alone was performed three times in a week. After 2 weeks, theses animals were sacrificed, and histological evaluation and the gene expression of inflammatory cytokines (IL-12p40, TNFα, and IFNγ) in colonic mucosa were evaluated by RT-PCR. Results: (1) The production of TNFα and IL-6 were significantly decreased in supernatant from RAW264.7 cells treated with FK506 compared to non-treated cells. (2) Pretreatment of FK506 suppressed LPSinduced activation of both NFκB and p38MAPK in RAW264.7 cells. (3) The production of IL-12p40, TNFα and IL-6 were significantly decreased in supernatant from peritoneal macrophages treated with FK506 compared to non-treated cells. (4) Histological examination revealed that histological score was significantly decreased in mice treated with FK506 than in those with PBS. (5) The gene expression of IL-12p40, TNFα and IFNγ in colonic mucosa were significantly decreased in mice treated with FK506 compared to those with PBS. Conclusion: We firstly demonstrated the immunosuppressive effects of FK506 on macrophage, by which immune-mediated colonic inflammation was attenuated. Blockade of activation of NFκB and p38MAPK by FK506 could be associated with this immunosuppressive mechanism.
M1667 Evolution of Markers of Colitis in Mdr1a-/- Mice Housed in SPF and Conventional Conditions Miquel Moretó, Mònica Maijó, Lluïsa Miró, Javier Polo, Eric Weaver, Joe D. Crenshaw, Louis Russell, Joy Campbell, Anna Pérez-Bosque Background and aims: The mdr1a-/- mouse model develops a spontaneous colitis. The degree of severity of the pathology and the time of appearance of colitis signs strongly depend on housing conditions. In the present study we have assayed a protocol combining the stay of mice in specific pathogen frer (SPF) conditions followed by conventional housing, in order to define conditions were food intake and growth were not markedly affected. Methods: Knock out (KO) mice were obtained from the pathogen free colony of mdr1a-/at Taconic (Germantown, NY). Mice (n= 6-7) were grown and maintained in SPF conditions until week 4 when they where transferred to conventional housing for 4-5 additional weeks. Wild type (WT; n= 6-7) congenic FVB (+/+) mice were raised in conventional housing for the same time period. A disease activity index (DAI) was defined, accounting for changes in motor activity, body weight, consistency of faeces, and presence of blood in stools. After sacrifice, the weight of spleen and mesenteric lymph nodes, and the length and weight of the colon were recorded. The permeability of colon mucosa was analysed ex vivo by confocal microscopy using FITC-Dextran 10 kDa as marker. Tissue interferon-γ (IFN-γ) was measured by bioplex cytokine assay. Results: First signs of disease were observed when mice were 5wk old when DAI progressively increased reaching a value of 1.94 ± 0.23 at day 58 (DAI in WT was 0.31 ± 0.18; P<0.05). In KO mice, the weight of colon, spleen and mesenteric lymph nodes was 2-3 fold higher than in WT animals (P<0.05), and these changes were well correlated with DAI. No differences in body weight between groups were observed at sacrifice. The permeability of crypts to dextran in KO was 2 fold higher than WT animals (P<0.05). Production of IFN-γ in the colon was markedly stimulated in KO mice (1.7 ± 0.2 pg/mg prot) compared to WT group (0.3 ± 0.04 pg/mg prot). However, in the ileum IFNγ expression was the same in both groups. Conclusion: The protocol used in this study allowed development of mild colitis without affecting food intake and growth though several markers such as IFN- γ expression and crypt permeability were enhanced in the mdr1a-/mice. This indicates that this protocol is adequate to carry out studies on the effect of diets in prevention of inflammatory bowel disease.
M1665 Development and In Vivo Analysis of a New Generation of Optimized ThioGTP Analogues for Therapy of IBD Imke Atreya, Alexandra Diall, Christian Ostrau, Gerhard Fritz, Radovan Dvorsky, Mathias Gruen, Christoph Becker, Markus F. Neurath Introduction: Therapy with azathioprine (aza) and 6-mercaptopurine (6-MP) represents the gold standard for maintenance of remission in inflammatory bowel disease (IBD), although the use of these classic immunosuppressive agents in acute flares of Crohn's disease or ulcerative colitis is limited by the delayed onset of action of thiopurines. Based on 6-thioGTP, which could recently be identified as a key active metabolite of aza and is responsible for the pro-apoptotic capacity of thiopurines via inhibition of the small GTPase Rac in T cells, we now aim on the development of a new class of immunosuppressive drugs with an improved efficacy in acute IBD. Method: A rational development of new 6-thio-GTP analogues was performed by steric modelling in order to generate 6-thio-GTP analogues with an increased capacity to block Rac activity and to mediate an earlier onset of pro-apoptotic effects. 5 groups of chemically modified novel 6-thio-GTP analogues (Group A-F), a total of 48 new substances, were synthesized and tested on isolated human T cells for their proapoptotic capacity. The In Vivo immunosuppressive capacity of lead compounds was analysed in a murine model of TNBS-induced acute colitis and analysed by mini-endoscopy. The potential clinical applicability of the best candidates was evaluated by In Vitro toxicity tests. Results: Based on caspase-3 activity assays, 6-thio-GTP analogues of group B, D and E could be identified as strong inducers of T cell apoptosis, while analogues of group A, C and F failed to induce apoptosis. Finally, 4 lead compounds (B0, B0L, B0N, E1) were identified and characterized by a superior ability to induce increased levels of T cell apoptosis at earlier time points than classic thiopurines. With regard to In Vitro hepato- and mylotoxicity, especially B0 and B0N showed a reduced toxic potential compared to the classic drug 6MP. Results from a comet-assay indicated that In Vitro treatment of human T cells with B0N induced a significantly lower number of DNA strand breaks than 6-MP. Due to this promising In Vitro profile of B0N (high immunosuppressive capacity and reduced toxicity), the In Vivo immunosuppressive efficacy of B0N was evaluated in the model of TNBS-induced acute colitis. In Vivo application of B0N in TNBS-treated balb/c mice resulted in a significant reduction of mucosal inflammation (colitis-score=7,5) compared to untreated control group (colitis-score=10,3). Conclusion: Due to promising low In Vitro toxicity as well as high In Vitro and In Vivo immunosuppressive efficacy, B0N represents an excellent candidate for GLP safety studies and subsequent clinical development.
M1668 Defective CD8+ CD28- Regulatory T Cells in the Intestine of Patients with Crohn's Disease Keren M. Rabinowitz, Paul Smereka, Lloyd Mayer INTRODUCTION: In the normal state, the interaction between IEC and LP lymphocytes gives rise to a population of CD8+ CD28- T cells with regulatory function that can be identified phenotypically and functionally in normal LPL. These cells suppress by cell contact. AIM: Characterize the factors required for expansion of these regulatory LP CD8+ T cells and define how they mediate suppression METHODS: Freshly isolated LPLs from normal controls, UC and CD patients were stimulated with visilizumab (Nuvion), a humanized antiCD3 mAb (100ng/ml), for 5 days followed by addition of IL7 (10ng/ml) and IL15 (20ng/ ml) and irradiated PBMCs. After 14 days CD8+ T cells were purified by magnetic bead sorting, and cultured in the presence of IL7 and IL15. Cell lines were analyzed for suppressor activity (inhibition of CD4+ T cell proliferation in response to anti-CD3/CD28 beads), surface molecule expression (by flow cytometry) and cytokine secretion profile (CBA, ELISA). Suppressive cell lines were used as immunogens to generate murine mAbs against surface molecules involved in suppressor function. RESULTS: The cell lines did not express CD28, CD25, ICOS, PDL1, CD16, perforin, granzyme B, CTLA4 and FoxP3 but were positive for CD3, CD8β, CD2 and CD101. Following stimulation with anti-CD3/CD28 beads cell lines secreted substantial amounts of IFN-γ and TNF-α, IL13 and GM-CSF but only modest amounts of IL10, IL4 and IL2. With regard to suppressor function, lines generated from normal (15 lines) and UC patients (3 lines) were able to suppress CD4+ T cell proliferation in a contact dependent fashion. In contrast, lines generated from CD patients (9 lines) had a markedly decreased capacity to mediate suppression. Lastly, sera from 3 mice immunized with normal CD8+ LPL lines but not unimmunized control sera demonstrated the ability to block suppression. CONCLUSION: We developed a method for generating regulatory CD8+ T cell lines from the LP. CD8+ T cell lines from control and UC but not CD patients exhibit contact dependent regulatory function. Antisera from cell line immunized mice block this suppression and appear to recognize the molecule(s) mediating this suppression. These cells may play a role in control of local inflammation in the gut.
M1666 Anti-Inflammatory Effects of HMPL-004 Are Mediated By the Inhibition of Multiple Targets Tao Wang, Yuan Zhou, Yu Cai, Weihan Zhang, Weihua Gu, Yang Sai, Genhong Cheng, Xiaoqiang Yan HMPL-004 is a botanic drug in global phase II development for the treatment of inflammatory bowel disease (IBD). The botanic drug is derived from Andrographis Paniculata, an herb that has long been used in Asia for the treatment of acute infections. In a proof of concept (POC) study, we showed that HMPL-004, compared to Mesalamine, was effective in treating patients with mild to moderate Ulcerative Colitis (UC). In the current studies, we report pre-clinical results that may mechanistically explain the anti-inflammatory activities of HMPL-004. HMPL-004 was able to inhibit multiple cellular targets involved in the NF-κB and MAPK pathways. The inhibition of NF-κB was achieved by suppressing the phosphorylation of IκBα as observed in bacterial lipopolysaccharide (LPS) induced THP-1 cells and by interfering with the binding of NF-κB to DNA as seen in HEK293 cells over-expressing p65 or p50. The inhibition of MAPK pathway was demonstrated by the inhibiting the phorphorylation of Erk1/2 and MAKPAPK2. In a DNBS (2, 4 nitrobenzenesulfonic acid)-induced rat colitis model, HMPL-004 significantly suppressed the inflammation and the levels of TNFα and IL-1β in the colons. We further performed rat pharmacokinetics studies and found that the plasma exposures of active compounds, andrographolides and flavonoids, identified In Vitro, were at ng/mL levels that were too low to achieve effective target inhibition. Therefore, an ex vivo study was performed using isolated rat colons after a DNBS challenge. Significant inhibition of IL-1β production by HMPL-004 was observed in the colons ex vivo. In summary, these findings demonstrated that HMPL-004 is able to suppress multiple cellular targets including IκBα, p65, p50, and MAPKs. The ex vivo studies further suggests a local action of HMPL-004 that may have contributed to the biological effects observed in the DNBSinduced rat colitis model and also to the clinical benefits seen in UC patients.
AGA Abstracts
M1669 Human Umbilical Cord Mesenchymal Stem Cell Transplantation Ameliorates Inflammation in a Mouse Model of Colitis Xiao cang Cao, Bangmao Wang, Yingzi Cong, Fang Yan Background: Although significant advances have been made towards understanding the immunopathogenesis of inflammatory bowel disease (IBD), a major challenge of IBD research is to successfully develop new strategies for the treatment of IBD. The present therapeutic applications for IBD are limited by their side effects and unstable effectiveness. Accumulating data indicate stem cell transplantation (SCT) could be potentially used as therapy of the patients with IBD. Considering IBD susceptibility loci, allo-SCT should the appropriate candidate as it could “tinker up these bug”, and human umbilical cord mesenchymal stem cells (hUCMSCs) could be an ideal resource and more convenient in future application because of undemanding HLA match in transplantation. More recently it had been shown not only to be able to differentiate into multiple tissues, but also to exert a potent antiproliferative effect that result in the inhibition of immune responses. Therefore the purpose of this study is to determine the effects of transplantation of hUCMSCs on inflammation in SCID mice. Merthods: Colitis in SCID mice was induced by injection (i.p.) of CD4+CD25-T cells isolated from spleen of wild-type Balb/c mice using magnetic separation method. hUCMSCs obtained from volunteer, labeled by CM-DiI, were administrated (i.v.) to SCID mice for
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5*106 each mouse subsequently. Saline was used as a control. Colon sections were prepared for H&E staining to determine intestinal inflammation by DAI score. CM-DiI-labeled hUCMSCs were detected by CONFOCAL and Optical In Vivo Imaging system (XENOGEN IVIS200). The effects of hUCMSCs on T cells proliferation were studied In Vitro by BrdUlabeled method and concentrations of IL-17, IL-23 and IFN-g were measured by ELISA. Results: The SCID mice developed intestinal inflammation 3-5 weeks after first transplantation, which was similar to that in human IBD patients. The lesions of treated mice were improved in mice with CM-DiI hUCMSCs treatment after second transplantation, which DAI score decreased from 3.1 to 1.3. CM-DiI+ hUCMSCs, which were detected by In Vivo Imaging system, were also recovered in intestinal mucosa and submucosa of the colon by CONFOCAL. Furthermore, hUCMSCs inhibited mice T cell response to antigen stimulation In Vitro in a dose-dependent manner and production of proinflammatory cytokines was lower, indicating the immunosuppressive activity of hUCMSCs. CONCLUSION: Systemically administrated hUCMSCs responded to an adequate tissue lesion and resulted in accelerated tissue repair in mouse IBD.hUCMSC transplantation may serve as a potential approach for IBD treatment.
M1672
Background: Glycogen synthase kinase 3-β (GSK3-β) recently was identified as a critical regulator of Toll-like receptor (TLR) signaling mediating excessive inflammatory responses. Its activity enhances TLR-induced proinflammatory cytokine secretion from blood monocytes while IL-10 production is suppressed. In previous studies we provided evidence that In Vivo blockade of GSK3 β by LiCl attenuates excessive TLR-mediated immune responses to bacterial constituents during chronic intestinal inflammation. Aim of this study was to investigate whether LiCl directly impairs the function of freshly isolated human and mouse intestinal lymphocytes. Methods: For induction of chronic colitis, female Balb/c mice received 4 cycles of dextran sodium sulphate (DSS) treatment. After the onset of chronic colitis mesenteric lymph node cells (MLC) and lamina propria mononuclear cells (LPMC) were isolated and stimulated In Vitro with CpG-ODN or control-ODN (2μg/ml) in the presence or absence of 10 mM LiCl. Human LPMC were isolated from surgical specimens of control patients and patients with inflammatory bowel disease (IBD) and stimulated In Vitro with CpG-ODN, control-ODN (10 μg/ml), LPS (10-50 ng/ml) or anti-CD3/anti-CD28 beads in the presence or absence of 10 mM LiCl. Cytokine levels were determined by Luminex and ELISA. Results: CpG-ODN stimulation of murine MLC and LPMC resulted in production of large amounts of IL-6, TNF, and IFN-γ. Presence of LiCl significantly reduced CpG-ODN-induced IL-6 (60%; p<0.0001) and TNF (30%; p<0.0005) secretion from MLC and LPMC, while IFN-γ production of LPMCs was reduced by 90% (p<0.0001). Basal IL-10 production of LPMC was enhanced by 14% after GSK3-β blockade. In human LPMC isolated from control patients basal IL-6 production was not impaired by LiCl. However, IL-6 secretion induced by LPS (1.4-fold) or T cell stimulation (5.4-fold) were reduced by up to 30% in the presence of LiCl. In contrast, basal IL-6 production of LPMC isolated from inflamed IBD tissue was significantly (52%; p<0.001) diminished by LiCl and LPS-induced IL-6 levels were reduced by 40% (p<0.0001). A LiCl-dependent decrease of basal (17%; p=0.017) and CpG-ODNinduced (23%; p=0.0007) was observed in LPMC from non-inflamed IBD tissue. Conclusion: Blockade of GSK3-β by LiCl attenuates excessive proinflammatory TLR-mediated immune responses of intestinal lymphocytes from chronic inflamed tissue. GSK3-β inhibition therefore constitutes a promising therapeutic option for selectively reducing exaggerated intestinal immune reactions towards the luminal flora in IBD.
M1670 Decreased Production of IL-10 and TGF-β in TLR-Activated Intestinal B Cells in SAMP1/Yit Mice Yoshiyuki Mishima, Shunji Ishihara, Md. M. Aziz, Naoki Oshima, Akihiko Oka, Ryusaku Kusunoki, Aya Ohtani, Ichiro Moriyama, Yong-Yu Li, Yuji Amano, Yoshikazu Kinoshita (Background & Aim) A unique subset of B cells expressing interleukin (IL)-10 and transforming growth factor (TGF)-β plays an essential role in maintaining immune responses. These B cells produce IL-10 and TGF-β after sensing pathogen-associated molecules via toll-like receptors (TLRs), and modulate autoimmunity and inflammation in a variety of organs. However, little is known regarding their role in immune-related intestinal disorders. SAMP1/Yit mice spontaneously develop ileitis and are widely recognized as a murine model of Crohn's disease. In the present study, we investigated the presence of this cell subset in mice intestines and its role in the pathogenesis of ileitis in SAMP1/Yit mice. (Materials and Methods) Five-, ten- and thirty-week-old male SAMP1/Yit mice and age-matched male control AKR/J mice were used in this study. Development of ileitis in the SAMP1/Yit mice was confirmed by histological examinations. Mononuclear cells were isolated from mesenteric lymph nodes (MLNs) and spleens of the mice, and the expressions of B220, CD5, TLR4, TLR9, RP105, Mac-1, IgM, IgD, and CD1d in isolated cells were analyzed by flow cytometry. For In Vitro experiments, B cells were separated from isolated mononuclear cells magnetically by positive selection using B220 micro-beads. Purified B cells were cultured in 96-well plates and stimulated with LPS or CpG DNA for 3 days, then IL-10 and TGF-β contents in the culture media were measured with enzyme immunoassays. In addition, the intracellular contents of IL-10 and TGF-β in TLR ligand-stimulated B cells were analyzed by flow cytometry after staining with CD5, TLR4, TLR9, RP105, Mac-1, IgM, IgD, and CD1d. (Results) Ileitis was clearly observed in the 10- and 30-week-old SAMP1/Yit mice. The expression patterns of several cellular markers in B cells isolated from the MLNs and spleen of SAMP1/Yit mice were similar to those of AKR/J mice, as shown by flow cytometry. On the other hand, the production levels of IL-10 and TGF-β stimulated by LPS and CpG DNA were significantly lower in B cells separated from MLNs of SAMP1/Yit mice as compared to those from AKR/J mice. Furthermore, decreased production of those cytokines was also observed in B cells separated from the MLNs of 5-week-old SAMP1/Yit mice without typical ileitis. Also, a significant expression of CD1d was observed in B cells producing IL-10 and TGF-β in both SAMP1/Yit and AKR/J mice. (Conclusion) We observed decreased production of IL-10 and TGF-β by TLR-activated intestinal B cells in SAMP1/Yit mice. Our results suggest that these immuno-regulatory B cells are associated with the pathogenesis of Crohn's disease.
M1673 Wiskott-Aldrich Syndrome Protein (WASP) Is Critical for the Inducible Generation of FOXP3+ Regulatory T Cells Elisa K. Boden, Vineet Ahuja, Christopher J. Moran, Deanna D. Nguyen, Scott B. Snapper The Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency that results from mutations in WASP. WASP transduces cell surface receptor signals to the cytoskeleton via ARP2/3, a protein complex that has recently been linked to ulcerative colitis in a genomewide association study. WASP-deficient mice (WKO) develop a spontaneous colitis that is associated with both quantitative and qualitative defects in CD4+CD25+Foxp3+ regulatory T cells (Tregs) that control autoimmunity. As inducible Tregs (iTregs) are believed to control organ-specific inflammation, we asked whether reduced numbers of peripheral Tregs were a reflection of impaired iTreg generation in WKO mice. iTregs are generated by dendritic cells (DCs) in a process that is TGFβ-dependent and FoxP3 can be induced In Vitro when naive T cells are stimulated via the T cell receptor (TCR) in the presence of TGFβ. Therefore, CD4+CD25- T cells from wild type (WT) or WKO mice were cultured in the presence of TGFβ and plate-bound anti-CD3. After 72 hours, cells were analyzed by flow cytometry for the expression of FoxP3. The induction of FoxP3 was significantly decreased in WKO T cells (3.3 +/- 1.3%) compared to WT T cells (29.5 +/- 9.3%, p<.04). Defective TGFβ signaling has been described in mucosal T cells from patients with Crohn's disease, characterized by reduced SMAD3 phosphorylation and elevated expression of SMAD7, an inhibitor of TGFβ signaling. WKO CD4+ T cells, however, had no obvious alterations in TGFβ signaling. Addition of the costimulatory antibody anti-CD28 to anti-CD3 increased the number of iTregs generated from WT T cells (80.8 +/- 7.9%), but did not rescue iTreg generation in WKO T cells (1.8+/- 0.77%, p<.003). However, addition of the T cell mitogen, concanavalin A (ConA), which bypasses the early steps of TCR engagement partially rescued FoxP3 expression in WKO T cells (20.9+/- 3.2%). These data demonstrate that WASP-dependent TCR signals are critical for the generation of iTregs. Impaired peripheral generation of Tregs likely contributes to colitis in WKO mice, and may be operative in human inflammatory bowel disease (IBD). The elucidation of mechanisms that control Treg homeostasis may allow for targeted therapeutics in IBD.
M1671 All-Trans Retinoic Acid Downregulates Inflammatory Responses By Shifting the Treg/TH17 Profile in Human Ulcerative and Murine Colitis Aiping Bai, Nonghua Lu, Yuan Guo, Zhikang Peng Background and aims: Inflammatory bowel disease (IBD) is characterized by uncontrolled immune responses in inflamed mucosa, with dominance of IL-17 producing cells and deficiency of Treg cells. This study was conducted to explore the effect and mechanisms of all-trans retinoic acid, the ligand of retinoic acid receptor α (RARα), on immune responses in both human and murine colitis. Methods: Colonic biopsies from patients with ulcerative colitis were cultured and treated with all-trans retinoic acid as the agonist of RARα, or LE350 as the antagonist of RARα. Expressions of IL-17 and FOXP3 were detected by immunohistochemistry. Murine colitis was induced by intrarectal administration of 2,4,6,trinitrobenzene sulphonic acid (TNBS) at day 1. Mice were then intraperitoneally treated with all-trans retinoic acid or LE350 daily for 7 days. Cytokine levels in the cultures of mouse lamina propria mononuclear cells (LPMCs) were measured. Expressions of FOXP3 and IL-17 in colon tissues or mesenteric lymph nodes (MLN) were detected by immunohistological analysis. Body weight and colon inflammation were evaluated. Results: All-trans retinoic acid treatment upregulated FOXP3 expression and down-regulated IL-17 expression in colon biopsies of patients, and in colon tissues and MLN of mice with colitis, compared with controls. LPMCs from retinoic acid-treated mice produced lower levels of proinflammatory cytokines (TNFα, IL-1β, IL-17), but more regulatory cytokines (IL-10, TGFβ), compared with that of untreated mice. LE135-treated mice showed the opposite effect of all-trans retinoic acid. Furthermore, all-trans retinoic acid ameliorated TNBS-induced colitis in a dose-dependent manner as seen by improved body weight and colon inflammation. Conclusions: All-trans retinoic acid down-regulates colon inflammatory responses in patients with IBD In Vitro and in murine colitis In Vivo, representing a potential therapeutic approach in IBD treatment.
M1674 Gender-Specific Differences in CD4+CD25+FOXP3+ Natural T Regulatory Cells Modulate Chronic Intestinal Inflammation in Experimental Crohn's Disease Rekha R. Garg, Muhammadreza A. Sachedina, Brian K. Reuter, Theresa T. Pizarro Background: Previous studies report that the overall clinical status and severity of several autoimmune diseases, including CD, is worse in female patients compared to their male counterparts. Mechanistically, gender differences can be influenced by X/Y dimorphism as well as sex hormones. In addition, peripheral immune tolerance is maintained by the immunosuppressive role of natural T regulatory cells (nTregs), characterized by CD4+CD25+FoxP3+ cell surface markers, and nTreg depletion has been shown to lead to autoimmunity. Aim: To determine gender-specific phenotypic and functional differences among Treg populations in the SAMP1/YitFc (SAMP) model of CD-like ileitis. Methods: Ileal inflammation was histologically evaluated in F/M-SAMP and control AKR mice. Flow cytometry and In Vitro CFSE-based proliferation and sex hormone stimulation assays were performed on mesenteric lymph node (MLN) cells. Results: A significant gender difference in ileal inflammation was observed as early as 6 wks of age, with increased severity in F-
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AGA Abstracts
AGA Abstracts
In Vitro Blockade of GSK3-β Selectively Reduces the Proinflammatory Phenotype of Lymphocytes from Chronic Inflamed Intestinal Tissue Claudia Hofmann, Nadja Dunger, Jurgen Scholmerich, Werner Falk, Florian Obermeier