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Suppression of Experimental Ulcerative Colitis by Topical Delivery of Specific Dnazyme Blocking Thetranscription Factor GATA3
Mo1711
AGA Abstracts
A MECHANISM OF ACTION FOR CYCLOSPORINE A IN ULCERATIVE COLITIS: THE ROLE OF THE TEC KINASE ITK IN DISEASE PATHOGENESIS Benno Weigmann, Stefanie Mott, Raja Atreya, Markus Neurath The immunosuppressant Cyclosporine A (CsA) mediates rapid anti-inflammatory effects in the treatment of acute steroid-refractory ulcerative colitis (UC), inducing impetuous remission. In the treatment of Crohn's disease (CD) however, CsA does possess little if any any therapeutic potential. The mechanism of action by which CsA exerts its therapeutic effect in UC has not yet been identified. The aim of our study was therefore to elucidate the molecular mechanism of CsA in UC. Lamina propria mononuclear cells (LPMCs) were isolated from biopsies taken from inflammatory bowel disease (IBD) and control patients. The rate of apoptosis induction upon treatment with CsA was assessed via flow cytometric analysis of annexin V/propidium iodide staining. Cytokine concentration (IFN-g, IL-13, IL2) in the supernatant of cell cultures were analyzed using ELISA. Immunofluorescence staining for itk of cytospins of LPMCs isolated from gut specimen of UC patients and controls were performed. Oxazolone colitis was induced in itk deficient mice and controls. Disease activity was evaluated by means of body weight, histological and endoscopic score of inflammatory activity. Upon administration of CsA there is significant induction of apoptosis in LPMCs from patients with UC but not in CD or corresponding controls. Analysis of cytokine concentrations in UC revealed a significant inhibition of IFN-g and IL-13 production, while IL-2 was not suppressed after CsA treatment. Histological analysis of gut cytospins showed a significant increase of itk expression in UC compared to controls. Importantly in the oxazolone induced colitis model, itk deficient mice were significantly protected against the development of intestinal inflammation compared to control mice. Our results indicate, that the rapid therapeutic effect of CsA in UC is due to its capability to induce apoptosis in LPMCs. The augmented intestinal itk expression in UC and the protection of itk deficient mice in an experimental model of colitis, demonstrate a pivotal role of the T cell associated tec kinase itk in disease pathogenesis. These data show that in UC, an itk mediated pathway leads to the augmented resistance of LPMCs to apoptosis and that the interaction of CsA with itk leads to the induction of apoptosis explaining its clinical efficacy
Mo1709 A ROLE OF IRHOM2 IN CHRONIC INTESTINAL INFLAMMATION IN IL-10DEFICIENT MICE Jee Hyun Kim, Jihye Kim, Jooyoung Lee, Jaeyoung Chun, Changhyun Lee, Jong Pil Im, Joo Sung Kim Background and aims: TNF-α converting enzyme (TACE) regulates the release of TNF-α, and inactive rhomboid protein 2 (iRhom2) was identified as an essential molecule for TACE maturation in immune cells. We have previously shown that iRhom2 could play a critical role for TACE maturation and TNF-α secretion in immune cells of colitis. We investigated a role of iRhom2 in chronic intestinal inflammation in IL-10-deficient mice. Methods: To study the function of iRhom2 in chronic intestinal inflammation in IL-10-deficient mice, IL-10-/- mice [IL-10 knockout (KO)] were crossed with iRhom2-/- [iRhom2 KO] mice to generate IL-10-/- × iRhom2-/- double knockout [DKO] mice. Colitis was induced with piroxicam, and colitis was quantified by disease activity index, colon length and histopathologic evaluation. Results: IL-10 KO and IL-10-/- × iRhom2-/- DKO mice spontaneously developed colitis with a comparable time course, however, DKO mice showed more severe colonic inflammation histologically. IRhom2 KO mice did not exhibit any sign of active colitis. In colitis induced with piroxicam, DKO mice also showed higher disease activity index as compared with IL-10 KO. The colon length in the DKO group was significantly shorter than that in the IL-10 KO. Histologic score in DKO mice was significantly higher than that in IL-10 KO. Conclusions: This result suggests that dysregulation of iRhom2-mediated control of intestinal homeostasis exacerbates intestinal inflammation in the absence of immunoregulatory IL-10. The IL-10-/- × iRhom2-/- DKO mouse model may further provide a pivotal insight into the pathogenesis of inflammatory bowel disease.
Mo1712 BALANCING JAK/STAT-SIGNALING WITH TOFACITINIB IN MONOCYTES OF HEALTHY CONTROLS AND IBD PATIENTS Anna F. Cordes, Eva Lenker, Toni Weinhage, Georg Varga, Dirk Foell Introduction. Monocytes are bridging natural and acquired immunity. Information about JAK signaling in monocytes is scarce especially in an inflammatory milieu. JAK-inhibition is a promising new anti-inflammatory treatment option. However, JAK/STAT activation may be involved both in pro- and anti-inflammatory monocyte programs. We have shown that GM-CSF-activated regulatory monocytes (GMaM) induce Treg-differentiation in co-cultures with naive T-cells in vitro. Inflammatory T-cells produce high amounts of GM-CSF in vivo, not leading to anti-inflammatory monocytes, likely because of pro-inflammatory cytokines in the environment. We used JAK-inhibitor Tofacitinib to explore mechanisms that block proinflammatory pathways and still allow anti-inflammatory functions in monocytes. Methods. Primary monocytes from healthy human donors were isolated and phenotyped by FACS after treatment with GM-CSF and JAK-inhibitor Tofacitinib. Monocytes were co-cultured with autologous naïve T-cells and differentiation of Foxp3+ regulatory T-cells was evaluated by FACS. Furthermore, primary monocytes from IBD patients with active disease were used to investigate JAK/STAT signaling and inhibition. JAK1 activation (represented by IFNγinduced phospho-STAT-1), JAK2 activation (represented by GM-CSF-induced phosphoSTAT5), and JAK3 activation (represented by IL-4-induced phospho-STAT6) was analyzed by FACS. Non-toxic dosages of 1-1000 nM Tofacitnib were used. Results. We aimed to define the dose of JAK inhibition that keeps JAK2 activity (GM-CSF-induced pSTAT5) intact. At 10-100 nM Tofacitinib we found GM-CSF-induced phospho-STAT5 while phosphoSTAT1 and phospho-STAT6 were still blocked. Concentrations above 100 nM Tofacitinib led to inhibition of GM-CSF-induced CD39-, CD206-, CD209-expression. 10-100 nM allowed CD39-, CD206-, CD209 expression and IL-10 release while TNFα was still blocked. Co-culture of GMaM and T-cells resulted in increased differentiation of Foxp3+ Treg that was even enhanced when 10nM Tofacitinib was used. Investigation of JAK/STAT activation in monocytes from IBD patients revealed a higher base line phosphorylation of STATs with lower increase after stimulation compared to healthy controls. Furthermore, TNFα expression was not inhibited in monocytes of IBD patients using a similar Tofacitinib dosage which significantly inhibited TNFα expression in healthy controls (10-100 nM). Conclusion. In summary, Tofacitinib (10-100 nM) facilitates GM-CSF-induced reprogramming of monocytes to anti-inflammatory cells. This could not be confirmed in monocytes from active IBD patients as blockage with similar doses did not inhibit pro-inflammatory cytokine expression. Thus, pro-inflammatory activation in IBD does depend on more complex interplays of multiple factors and solely blocking JAK/STAT activation by Tofacitinib cannot fully restore the GMaM phenotype.
Mo1710 SUPPRESSION OF EXPERIMENTAL ULCERATIVE COLITIS BY TOPICAL DELIVERY OF SPECIFIC DNAZYME BLOCKING THETRANSCRIPTION FACTOR GATA3 Benno Weigmann, Vanessa Popp, Katharina Gerlach, Raja Atreya, Agnieszka M. Turowska, Holger Garn, Harald Renz, Markus Neurath GATA3 has been identified as a major transcription factor of Th2-cell derived cytokines. Here, we investigated the role of GATA3 in inflammatory bowel diseases and experimental colitis in-vivo. First, we measured the expression of GATA3 in human specimens by qPCR and immunostainings. In experimental oxazolone colitis, we used mice with T-cell specific deletion of GATA3 and treated wild-type colitic mice with the GATA3 specific DNAzyme hgd40 or a control DNAzyme. The distribution of labelled DNAzyme in the colon was monitored by in-vivo imaging. GATA3 was highly expressed in colonic specimens of patients with ulcerative colitis but not Crohn's disease and GATA3-levels correlated with proinflammatory cytokine production. In oxazolone-induced colitis, increased GATA3- expression in lamina propria T-cells was observed. GATA3 deficiency in T-cells suppressed intestinal inflammation and proinflammatory cytokine production (IL-6, IL-9, and IL-13). Furthermore, topical administration of a specific DNAzyme resulted in homogeneous distribution throughout the inflamed colon and downregulated GATA3- expression. GATA3-DNAzyme treatment protected mice from oxazolone colitis associated with suppression of IL-6, IL-9, and IL-13 production by lamina propria cells. Finally, GATA3 specific DNAzyme significantly suppressed TNFindependent oxazolone colitis in TNF-receptor double knockout mice. The transcription factor GATA3 plays a crucial regulatory role in experimental colitis and is overexpressed in human ulcerative colitis. Targeting of GATA3 blocks Th2 and Th9 cytokine production and is effective for therapy of both TNF-dependent and -independent colitis. GATA3- specific DNAzyme emerges as a novel treatment approach for ulcerative colitis in humans.
AGA Abstracts
Mo1713 THE ROLE OF THE RS8005161 POLYMORPHISM ON PH-SENSING G PROTEIN-COUPLED RECEPTOR GPR65 (TDAG8) SIGNALLING IN INTESTINAL INFLAMMATION Irina Tcymbarevich, Nicole M. Obialo, Jesus Cosin-Roger, Klaus Seuwen, Gerhard Rogler, Benjamin Misselwitz, Cheryl de Valliere Background. Inflammatory bowel diseases (IBD), Crohn's disease (CD) and ulcerative colitis (UC), are typically associated with a decrease in local pH. Genome-wide association studies (GWAS) identified over 200 non-overlapping single-nucleotide polymorphism (SNP) genetic risk loci for IBD. The proton-sensing G-protein-coupled receptor T-cell death associated gene 8 (TDAG8 or GPR65) has been found to be a genetic risk factor for IBD in recent GWASs. Thereby, the T genotype of the SNP rs8005161 within the GPR65 gene confers
S-758
AGA Abstracts
A MECHANISM OF ACTION FOR CYCLOSPORINE A IN ULCERATIVE COLITIS: THE ROLE OF THE TEC KINASE ITK IN DISEASE PATHOGENESIS Benno Weigmann, Stefanie Mott, Raja Atreya, Markus Neurath The immunosuppressant Cyclosporine A (CsA) mediates rapid anti-inflammatory effects in the treatment of acute steroid-refractory ulcerative colitis (UC), inducing impetuous remission. In the treatment of Crohn's disease (CD) however, CsA does possess little if any any therapeutic potential. The mechanism of action by which CsA exerts its therapeutic effect in UC has not yet been identified. The aim of our study was therefore to elucidate the molecular mechanism of CsA in UC. Lamina propria mononuclear cells (LPMCs) were isolated from biopsies taken from inflammatory bowel disease (IBD) and control patients. The rate of apoptosis induction upon treatment with CsA was assessed via flow cytometric analysis of annexin V/propidium iodide staining. Cytokine concentration (IFN-g, IL-13, IL2) in the supernatant of cell cultures were analyzed using ELISA. Immunofluorescence staining for itk of cytospins of LPMCs isolated from gut specimen of UC patients and controls were performed. Oxazolone colitis was induced in itk deficient mice and controls. Disease activity was evaluated by means of body weight, histological and endoscopic score of inflammatory activity. Upon administration of CsA there is significant induction of apoptosis in LPMCs from patients with UC but not in CD or corresponding controls. Analysis of cytokine concentrations in UC revealed a significant inhibition of IFN-g and IL-13 production, while IL-2 was not suppressed after CsA treatment. Histological analysis of gut cytospins showed a significant increase of itk expression in UC compared to controls. Importantly in the oxazolone induced colitis model, itk deficient mice were significantly protected against the development of intestinal inflammation compared to control mice. Our results indicate, that the rapid therapeutic effect of CsA in UC is due to its capability to induce apoptosis in LPMCs. The augmented intestinal itk expression in UC and the protection of itk deficient mice in an experimental model of colitis, demonstrate a pivotal role of the T cell associated tec kinase itk in disease pathogenesis. These data show that in UC, an itk mediated pathway leads to the augmented resistance of LPMCs to apoptosis and that the interaction of CsA with itk leads to the induction of apoptosis explaining its clinical efficacy
Mo1709 A ROLE OF IRHOM2 IN CHRONIC INTESTINAL INFLAMMATION IN IL-10DEFICIENT MICE Jee Hyun Kim, Jihye Kim, Jooyoung Lee, Jaeyoung Chun, Changhyun Lee, Jong Pil Im, Joo Sung Kim Background and aims: TNF-α converting enzyme (TACE) regulates the release of TNF-α, and inactive rhomboid protein 2 (iRhom2) was identified as an essential molecule for TACE maturation in immune cells. We have previously shown that iRhom2 could play a critical role for TACE maturation and TNF-α secretion in immune cells of colitis. We investigated a role of iRhom2 in chronic intestinal inflammation in IL-10-deficient mice. Methods: To study the function of iRhom2 in chronic intestinal inflammation in IL-10-deficient mice, IL-10-/- mice [IL-10 knockout (KO)] were crossed with iRhom2-/- [iRhom2 KO] mice to generate IL-10-/- × iRhom2-/- double knockout [DKO] mice. Colitis was induced with piroxicam, and colitis was quantified by disease activity index, colon length and histopathologic evaluation. Results: IL-10 KO and IL-10-/- × iRhom2-/- DKO mice spontaneously developed colitis with a comparable time course, however, DKO mice showed more severe colonic inflammation histologically. IRhom2 KO mice did not exhibit any sign of active colitis. In colitis induced with piroxicam, DKO mice also showed higher disease activity index as compared with IL-10 KO. The colon length in the DKO group was significantly shorter than that in the IL-10 KO. Histologic score in DKO mice was significantly higher than that in IL-10 KO. Conclusions: This result suggests that dysregulation of iRhom2-mediated control of intestinal homeostasis exacerbates intestinal inflammation in the absence of immunoregulatory IL-10. The IL-10-/- × iRhom2-/- DKO mouse model may further provide a pivotal insight into the pathogenesis of inflammatory bowel disease.
Mo1712 BALANCING JAK/STAT-SIGNALING WITH TOFACITINIB IN MONOCYTES OF HEALTHY CONTROLS AND IBD PATIENTS Anna F. Cordes, Eva Lenker, Toni Weinhage, Georg Varga, Dirk Foell Introduction. Monocytes are bridging natural and acquired immunity. Information about JAK signaling in monocytes is scarce especially in an inflammatory milieu. JAK-inhibition is a promising new anti-inflammatory treatment option. However, JAK/STAT activation may be involved both in pro- and anti-inflammatory monocyte programs. We have shown that GM-CSF-activated regulatory monocytes (GMaM) induce Treg-differentiation in co-cultures with naive T-cells in vitro. Inflammatory T-cells produce high amounts of GM-CSF in vivo, not leading to anti-inflammatory monocytes, likely because of pro-inflammatory cytokines in the environment. We used JAK-inhibitor Tofacitinib to explore mechanisms that block proinflammatory pathways and still allow anti-inflammatory functions in monocytes. Methods. Primary monocytes from healthy human donors were isolated and phenotyped by FACS after treatment with GM-CSF and JAK-inhibitor Tofacitinib. Monocytes were co-cultured with autologous naïve T-cells and differentiation of Foxp3+ regulatory T-cells was evaluated by FACS. Furthermore, primary monocytes from IBD patients with active disease were used to investigate JAK/STAT signaling and inhibition. JAK1 activation (represented by IFNγinduced phospho-STAT-1), JAK2 activation (represented by GM-CSF-induced phosphoSTAT5), and JAK3 activation (represented by IL-4-induced phospho-STAT6) was analyzed by FACS. Non-toxic dosages of 1-1000 nM Tofacitnib were used. Results. We aimed to define the dose of JAK inhibition that keeps JAK2 activity (GM-CSF-induced pSTAT5) intact. At 10-100 nM Tofacitinib we found GM-CSF-induced phospho-STAT5 while phosphoSTAT1 and phospho-STAT6 were still blocked. Concentrations above 100 nM Tofacitinib led to inhibition of GM-CSF-induced CD39-, CD206-, CD209-expression. 10-100 nM allowed CD39-, CD206-, CD209 expression and IL-10 release while TNFα was still blocked. Co-culture of GMaM and T-cells resulted in increased differentiation of Foxp3+ Treg that was even enhanced when 10nM Tofacitinib was used. Investigation of JAK/STAT activation in monocytes from IBD patients revealed a higher base line phosphorylation of STATs with lower increase after stimulation compared to healthy controls. Furthermore, TNFα expression was not inhibited in monocytes of IBD patients using a similar Tofacitinib dosage which significantly inhibited TNFα expression in healthy controls (10-100 nM). Conclusion. In summary, Tofacitinib (10-100 nM) facilitates GM-CSF-induced reprogramming of monocytes to anti-inflammatory cells. This could not be confirmed in monocytes from active IBD patients as blockage with similar doses did not inhibit pro-inflammatory cytokine expression. Thus, pro-inflammatory activation in IBD does depend on more complex interplays of multiple factors and solely blocking JAK/STAT activation by Tofacitinib cannot fully restore the GMaM phenotype.
Mo1710 SUPPRESSION OF EXPERIMENTAL ULCERATIVE COLITIS BY TOPICAL DELIVERY OF SPECIFIC DNAZYME BLOCKING THETRANSCRIPTION FACTOR GATA3 Benno Weigmann, Vanessa Popp, Katharina Gerlach, Raja Atreya, Agnieszka M. Turowska, Holger Garn, Harald Renz, Markus Neurath GATA3 has been identified as a major transcription factor of Th2-cell derived cytokines. Here, we investigated the role of GATA3 in inflammatory bowel diseases and experimental colitis in-vivo. First, we measured the expression of GATA3 in human specimens by qPCR and immunostainings. In experimental oxazolone colitis, we used mice with T-cell specific deletion of GATA3 and treated wild-type colitic mice with the GATA3 specific DNAzyme hgd40 or a control DNAzyme. The distribution of labelled DNAzyme in the colon was monitored by in-vivo imaging. GATA3 was highly expressed in colonic specimens of patients with ulcerative colitis but not Crohn's disease and GATA3-levels correlated with proinflammatory cytokine production. In oxazolone-induced colitis, increased GATA3- expression in lamina propria T-cells was observed. GATA3 deficiency in T-cells suppressed intestinal inflammation and proinflammatory cytokine production (IL-6, IL-9, and IL-13). Furthermore, topical administration of a specific DNAzyme resulted in homogeneous distribution throughout the inflamed colon and downregulated GATA3- expression. GATA3-DNAzyme treatment protected mice from oxazolone colitis associated with suppression of IL-6, IL-9, and IL-13 production by lamina propria cells. Finally, GATA3 specific DNAzyme significantly suppressed TNFindependent oxazolone colitis in TNF-receptor double knockout mice. The transcription factor GATA3 plays a crucial regulatory role in experimental colitis and is overexpressed in human ulcerative colitis. Targeting of GATA3 blocks Th2 and Th9 cytokine production and is effective for therapy of both TNF-dependent and -independent colitis. GATA3- specific DNAzyme emerges as a novel treatment approach for ulcerative colitis in humans.
AGA Abstracts
Mo1713 THE ROLE OF THE RS8005161 POLYMORPHISM ON PH-SENSING G PROTEIN-COUPLED RECEPTOR GPR65 (TDAG8) SIGNALLING IN INTESTINAL INFLAMMATION Irina Tcymbarevich, Nicole M. Obialo, Jesus Cosin-Roger, Klaus Seuwen, Gerhard Rogler, Benjamin Misselwitz, Cheryl de Valliere Background. Inflammatory bowel diseases (IBD), Crohn's disease (CD) and ulcerative colitis (UC), are typically associated with a decrease in local pH. Genome-wide association studies (GWAS) identified over 200 non-overlapping single-nucleotide polymorphism (SNP) genetic risk loci for IBD. The proton-sensing G-protein-coupled receptor T-cell death associated gene 8 (TDAG8 or GPR65) has been found to be a genetic risk factor for IBD in recent GWASs. Thereby, the T genotype of the SNP rs8005161 within the GPR65 gene confers
S-758