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S1669 Gl1001 Attenuation of Acute Colitis in a Mouse DSS Model Is Associated with Inhibition of Its Target, ACE2
induced release of “illness behaviour” implicated cytokines such as IL-1β are responsible for the decreased clinical severity of disease.
S1668 P110δ Phosphoinositide 3-Kinase Uncouples Colitis from Colitis-Associated Cancer Klaartje Kok, Fiebo J. ten Kate, Johan Offerhaus, Maikel P. Peppelenbosch, Daniel Hommes
AGA Abstracts
S1665 SPARC-Associated Attenuation of Intestinal Inflammation in the Mouse Yoke Leng Ng, Frances Lloyd, Borut Klopcic, Ian C. Lawrance
Introduction: Mice that express an enzymatic inactive form of the class IA phosphoinositide 3-kinase isoform p110δ (p110δD910A/D910A) develop colitis. Given the now well established causal role between inflammation and cancer, this raises the question whether these mice predispose to colorectal cancer. This is especially an important issue since p110δ PI3K attracts widespread attention as a therapeutic target in inflammation and allergy and p110δspecific inhibitors are being developed. Methods: p110δD910A/D910A mice were compared to wildtype mice for their propensity to colitis in a specific-pathogen free environment versus conventional animal housing as well as in a dextran sulphate sodium colitis (DSS) model. p110δD910A/D910A mice were also compared to wildtype mice for their susceptibility to cancer, both spontaneously in aged mice and in a colitis-associated cancer model using the procarcinogen azoxymethane and DSS. Results: p110δD910A/D910A mice develop colitis but only in the presence of commensal flora, especially Helicobacter hepaticus. When subjected to a DSS colitis model these mice develop significantly more severe colitis. Aged p110δD910A/ D910A mice did not develop colorectal cancer or any other type of cancer. When subjected to the colitis-associated cancer model, p110δD910A/D910A mice did not develop more cancer compared to wildtype, despite developing more severe colitis. Furthermore it was shown that inactivation of the p110δ subunit of PI3K results in a specific inability of regulatory T cells to migrate. As a consequence thymic egress is compromised. Conclusion: We conclude that lack of active p110δ phosphoinositide 3-kinase predisposes to colitis, but only in the presence of commensal flora. Furthermore, lack of active p110δ does not increase the risk of cancer. Finally, lack of p110δ might protect against colitis-associated colon cancer through impairment of normal regulatory T cell activity.
Background: Colorectal cancer (CRC) is common and its incidence increases over time with the chronic intestinal inflammation observed in patients with Inflammatory Bowel Disease. Secreted Protein, Acidic and Rich in Cysteine (SPARC) has been show to affect tumour growth, cancer invasion and patient prognosis in numerous human cancers including bowel cancer. SPARC may also affect the colonic inflammatory response. Aim: To investigate SPARC's role on the development of colonic inflammation in the mouse. Methods: SPARC wild-type (WT) and knockout (KO) mice were treated with 3% dextran sodium sulphate (DSS) for 7 days. Intestinal inflammation was assessed on day 6, using the modified murine endoscopic score of colitis severity (MEICS), by high-resolution miniature video endoscopy. Mice were sacrificed and the colons assess histologically. In separate experiments, colons were harvested and total lymphocytes were isolated from the spleen, intra-epithelium (IE) and rest of colon (C) and were analysed for levels of CD4 and FoxP3 +ve cells by flow cytometry. Results: SPARC KO mice demonstrated less endoscopic colonic inflammation than WT mice (MEICS 4 vs 7; p=0.013) and less weight loss (KO 0.48% weight gain vs WT 6.2% weight loss p=0.074). Overall lymphocyte numbers isolated from spleen, IE and C from KO was markedly less than that isolated from WT mice (0.77x108/ml vs 1.28x108/ml; 0.82x106/ml vs 16.8x106/ml and 4.4x106/ml vs 8.8x106/ml respectively). The percentage of CD4 +ve cells from spleen, IE and C were between 10-15% from both KO and WT mice. The percentage of FoxP3 +ve cells from spleen, IE and C from both KO and WT mice were 0.05% vs 1.85%, 1.2% vs 2.3%, 1.22% vs 1.75% respectively. Conclusion: SPARC affects the endoscopic colonic inflammatory response. Total lymphocyte counts are lower in inflamed SPARC KO colons and spleen consistent with a lower level of chronic inflammation. The percentage of FoxP3 +ve cells from the spleen and IE were lower in KO mice suggesting an impact of regulation of the immune response. The mechanistic impact of SPARC requires further elucidation.
S1669 Gl1001 Attenuation of Acute Colitis in a Mouse DSS Model Is Associated with Inhibition of Its Target, ACE2 Luz-Maria Guzman, Carri A. Boiselle, Shobu Odate, Stefan B. Gross, Dominic Picarella, John Byrnes, Courtney Ellard, Leanne K. Bourque, Stephen Donahue
S1666
Background and Aims: Angiotensin-converting enzyme 2 (ACE2) is a metallopeptidase abundantly expressed in the brush border of human small intestine enterocytes and in epithelial and endothelial cells of the gastrointestinal (GI) tract. Previous studies suggested a beneficial role for ACE2 inhibition in inflammatory GI diseases while studies with GL1001, a potent and selective inhibitor of ACE2, revealed that subcutaneous administration was efficacious in a dextran sodium sulfate (DSS) model of acute colitis in mice. In the present study we propose to test the oral efficacy of GL1001 in DSS models of inflammatory bowel disease (IBD) and analyze ensuing pharmacodynamic effects. Methods: In Study 1, a model with mild DSS-induced disease, adult mice (n=15) were treated for 5 days with 5% DSS in the drinking water followed by 9 days of twice-daily oral dosing with vehicle or GL1001 (100, 300 or 600 mg/kg). Throughout the study, disease activity indices (DAI) were monitored: body weight, rectal prolapse, stool consistency, and fecal occult blood. At study termination, colon length and MAdCAM-1 were measured, as was ACE2 activity, GL1001 concentrations, and ACE2 protein levels in major organs and plasma. In Study 2, a model of severe DSS-induced disease was used involving 7 days of 5% DSS treatment, with survival as an endpoint. At study termination colon length, histopathology and colonic TNF-alpha levels were assessed. Results: Oral treatment with GL1001 produced significant improvements in several DAIs in both DSS models. In Study 1, GL1001 was found to accumulate in colon and cecum tissues at a time several hours after the last oral dose when it was absent in other surveyed organs and plasma. ACE2 activity in the cecum was inhibited in a dosedependent manner, and complete inhibition of enzymatic activity was observed in the high dose group (>99% inhibition vs. vehicle). Determinations of plasma MAdCAM-1 revealed significantly decreased levels in the high GL1001 dose group. In Study 2, GL1001 treatment increased survival (30%, p<0.016 high dose vs. DSS only) and decreased expression levels of colonic TNF-alpha. Conclusions: Oral administration of GL1001 in DSS models attenuates acute colonic symptoms with concomitant full inhibition of ACE2 activity and significantly decreased levels of IBD-related pro-inflammatory cytokines. The data support the proposed beneficial therapeutic role for GL1001 and ACE2 inhibition in inflammatory gastrointestinal diseases through target-mediated, anti-inflammatory effects.
The Role of TH17/IL-23 Pathway On Intestinal Inflammation in SKG Mice Chang Soo Eun, Dong Soo Han, Joo Hyun Choi, Sang Bong Ahn, Hyun Seok Cho, Tae Jun Byun, Jeehee Youn Background/Aims: Th17/IL-23 pathway with suppressive CD4+ Treg subsets have recently been identified to play an important role in the pathogenesis of inflammatory bowel disease. SKG mice have a point mutation of the zeta-associated protein of 70 kD (ZAP-70), a key signal transduction molecule in T cells, and spontaneously develop CD4+ T cell-mediated chronic autoimmune arthritis, mainly involving IL-17. The aims of this study were to evaluate dextran sulfate sodium (DSS) induced intestinal inflammation and to study the role of Th17/ IL-23 pathway on intestinal inflammation using SKG mice. Methods: Acute colitis was induced in SKG and BALB/c mice (control) by administering 2.7% DSS orally in drinking water for nine days under specific pathogen free condition. Colonic inflammation was assessed based on body weight change, total colon length and weight, and histological scores. IL-6, IFN-gamma, IL-17, IL-23, and IL12p40 levels were measured by ELISA in the supernatants of colonic tissue explants. Results: After DSS treatment, significant weight loss was developed both in SKG and BALB/c mice compared to mice not treated with DSS. Clinical and histological activities of colitis, and IL-6 levels in colonic tissues were attenuated in DSS-treated SKG mice compared to DSS-treated BALB/c mice. The level of IL-12p40 in colonic tissues was increased in DSS-treated SKG mice compared to DSS-treated BALB/c mice. IL-23 level in colonic tissues, by contrast, was decreased in DSS-treated SKG mice compared to DSS-treated BALB/c mice. Conclusion: Th17 cells and related cytokines may play an important role in mucosal homeostasis of intestinal inflammation. S1667 Resident “M2-Like” Intestinal Macrophages Express Eotaxin-1 in Colonic Injury Amanda B. Waddell, Richard Ahrens, Simon P. Hogan Rationale: Clinical and experimental studies have demonstrated a link between eotaxin-1/ CCL11, eosinophils and the inflammatory bowel diseases (IBD), Crohn's disease (CD) and ulcerative colitis (UC). However, the cellular source of CCL11 remains unclear. Methods: We examined colonic macrophage (MΦ) phenotype in BALB/c WT mice at baseline and following administration of 5.0% DSS treatment flow cytometry analysis. Colonic MΦ were purified by adherence and examined for alterative M2 activation (retlna, YM1 and arginase) and eotaxin-1 by RT-PCR. Bone marrow derived (BM) MΦ's were stimulated In Vitro by IL-4, IFNγ and LPS and CCL11, and alternative M2 activation was determined by RT-PCR and immunofluoresence analysis. Results: Phenotypic identification of resident colonic MΦ at baseline identified a heterogeneous non-inflammatory (F4/80+CD11b+Ly6C-) MΦ population consisting of CD206- and CD206+ M2-like MΦ (CD206- %: 79.8±2.6 vs 20.2±2.6; n=3 per group; mean ± SEM). DSS administration promoted an increase in inflammatory (F4/80+CD11b+Ly6C+) MΦ and not resident colonic F4/80+CD11b+Ly6C- MΦ. However, immunofluoresence analysis of adherence purified MΦ from the colon of DSS-treated mice revealed colocalization of M2-like markers, Retlna and arginase and CCL11. In Vitro analysis of BM MΦ's stimulated with IL-4 or IFN-γ + LPS led to development of classically activated “M1” nitric oxide-positive and alternatively activated “M2” Retlna+ Arginase+ macrophages. PCR analysis revealed CCL11 expression in M2 MΦ. Conclusions: Collectively, these In Vitro and In Vivo analyses demonstrate that CCL11 is predominantly expressed by resident intestinal M2-like MΦ.
AGA Abstracts
S1670 A Potential Role for Intestinal Alkaline Phosphatase in Colonic Inflammation Sundaram Ramasamy, Madhu S. Malo, Golam Mostafa, Jose L. Millan, Atul K. Bhan, Richard A. Hodin Background & Aims: An inadequate protective mechanism against gut-derived bacterial toxin may damage the colonic mucosal barrier resulting in intestinal inflammation. We have previously shown that intestinal alkaline phosphatase (IAP), a small intestinal brush border enzyme, is a gut mucosal defense factor that provides resistance to bacterial invasion by detoxifying lipopolysaccharides (LPS). The aim of this study was to elucidate a potential role for IAP in the context of dextran sodium sulphate (DSS)-induced chronic colitis. Methods: Chronic colitis was induced by oral administration of DSS in distilled water. IAPknockout (KO) and wild-type (WT) mice (n = 5) were treated with DSS (3%) for 7 days, followed by regular distilled water for 7 days; this cycle was repeated three times. Disease severity was determined by weight loss, loose stools/diarrhea, rectal bleeding and histopathology. Results: Following the first cycle of DSS treatment, both WT and IAP KO mice developed colitis symptoms (diarrhea/bloody stools), however, there were no differences between the two groups. Following the second cycle of DSS treatment, KO mice exhibited a significant mortality (2/5) compared with WT mice (0/5). Interestingly, WT mice, but not KO mice, showed significant weight loss compared to the control group. Following the third cycle of DSS treatment, KO mice again exhibited a high mortality (2/3) compared to the WT mice
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S1668 P110δ Phosphoinositide 3-Kinase Uncouples Colitis from Colitis-Associated Cancer Klaartje Kok, Fiebo J. ten Kate, Johan Offerhaus, Maikel P. Peppelenbosch, Daniel Hommes
AGA Abstracts
S1665 SPARC-Associated Attenuation of Intestinal Inflammation in the Mouse Yoke Leng Ng, Frances Lloyd, Borut Klopcic, Ian C. Lawrance
Introduction: Mice that express an enzymatic inactive form of the class IA phosphoinositide 3-kinase isoform p110δ (p110δD910A/D910A) develop colitis. Given the now well established causal role between inflammation and cancer, this raises the question whether these mice predispose to colorectal cancer. This is especially an important issue since p110δ PI3K attracts widespread attention as a therapeutic target in inflammation and allergy and p110δspecific inhibitors are being developed. Methods: p110δD910A/D910A mice were compared to wildtype mice for their propensity to colitis in a specific-pathogen free environment versus conventional animal housing as well as in a dextran sulphate sodium colitis (DSS) model. p110δD910A/D910A mice were also compared to wildtype mice for their susceptibility to cancer, both spontaneously in aged mice and in a colitis-associated cancer model using the procarcinogen azoxymethane and DSS. Results: p110δD910A/D910A mice develop colitis but only in the presence of commensal flora, especially Helicobacter hepaticus. When subjected to a DSS colitis model these mice develop significantly more severe colitis. Aged p110δD910A/ D910A mice did not develop colorectal cancer or any other type of cancer. When subjected to the colitis-associated cancer model, p110δD910A/D910A mice did not develop more cancer compared to wildtype, despite developing more severe colitis. Furthermore it was shown that inactivation of the p110δ subunit of PI3K results in a specific inability of regulatory T cells to migrate. As a consequence thymic egress is compromised. Conclusion: We conclude that lack of active p110δ phosphoinositide 3-kinase predisposes to colitis, but only in the presence of commensal flora. Furthermore, lack of active p110δ does not increase the risk of cancer. Finally, lack of p110δ might protect against colitis-associated colon cancer through impairment of normal regulatory T cell activity.
Background: Colorectal cancer (CRC) is common and its incidence increases over time with the chronic intestinal inflammation observed in patients with Inflammatory Bowel Disease. Secreted Protein, Acidic and Rich in Cysteine (SPARC) has been show to affect tumour growth, cancer invasion and patient prognosis in numerous human cancers including bowel cancer. SPARC may also affect the colonic inflammatory response. Aim: To investigate SPARC's role on the development of colonic inflammation in the mouse. Methods: SPARC wild-type (WT) and knockout (KO) mice were treated with 3% dextran sodium sulphate (DSS) for 7 days. Intestinal inflammation was assessed on day 6, using the modified murine endoscopic score of colitis severity (MEICS), by high-resolution miniature video endoscopy. Mice were sacrificed and the colons assess histologically. In separate experiments, colons were harvested and total lymphocytes were isolated from the spleen, intra-epithelium (IE) and rest of colon (C) and were analysed for levels of CD4 and FoxP3 +ve cells by flow cytometry. Results: SPARC KO mice demonstrated less endoscopic colonic inflammation than WT mice (MEICS 4 vs 7; p=0.013) and less weight loss (KO 0.48% weight gain vs WT 6.2% weight loss p=0.074). Overall lymphocyte numbers isolated from spleen, IE and C from KO was markedly less than that isolated from WT mice (0.77x108/ml vs 1.28x108/ml; 0.82x106/ml vs 16.8x106/ml and 4.4x106/ml vs 8.8x106/ml respectively). The percentage of CD4 +ve cells from spleen, IE and C were between 10-15% from both KO and WT mice. The percentage of FoxP3 +ve cells from spleen, IE and C from both KO and WT mice were 0.05% vs 1.85%, 1.2% vs 2.3%, 1.22% vs 1.75% respectively. Conclusion: SPARC affects the endoscopic colonic inflammatory response. Total lymphocyte counts are lower in inflamed SPARC KO colons and spleen consistent with a lower level of chronic inflammation. The percentage of FoxP3 +ve cells from the spleen and IE were lower in KO mice suggesting an impact of regulation of the immune response. The mechanistic impact of SPARC requires further elucidation.
S1669 Gl1001 Attenuation of Acute Colitis in a Mouse DSS Model Is Associated with Inhibition of Its Target, ACE2 Luz-Maria Guzman, Carri A. Boiselle, Shobu Odate, Stefan B. Gross, Dominic Picarella, John Byrnes, Courtney Ellard, Leanne K. Bourque, Stephen Donahue
S1666
Background and Aims: Angiotensin-converting enzyme 2 (ACE2) is a metallopeptidase abundantly expressed in the brush border of human small intestine enterocytes and in epithelial and endothelial cells of the gastrointestinal (GI) tract. Previous studies suggested a beneficial role for ACE2 inhibition in inflammatory GI diseases while studies with GL1001, a potent and selective inhibitor of ACE2, revealed that subcutaneous administration was efficacious in a dextran sodium sulfate (DSS) model of acute colitis in mice. In the present study we propose to test the oral efficacy of GL1001 in DSS models of inflammatory bowel disease (IBD) and analyze ensuing pharmacodynamic effects. Methods: In Study 1, a model with mild DSS-induced disease, adult mice (n=15) were treated for 5 days with 5% DSS in the drinking water followed by 9 days of twice-daily oral dosing with vehicle or GL1001 (100, 300 or 600 mg/kg). Throughout the study, disease activity indices (DAI) were monitored: body weight, rectal prolapse, stool consistency, and fecal occult blood. At study termination, colon length and MAdCAM-1 were measured, as was ACE2 activity, GL1001 concentrations, and ACE2 protein levels in major organs and plasma. In Study 2, a model of severe DSS-induced disease was used involving 7 days of 5% DSS treatment, with survival as an endpoint. At study termination colon length, histopathology and colonic TNF-alpha levels were assessed. Results: Oral treatment with GL1001 produced significant improvements in several DAIs in both DSS models. In Study 1, GL1001 was found to accumulate in colon and cecum tissues at a time several hours after the last oral dose when it was absent in other surveyed organs and plasma. ACE2 activity in the cecum was inhibited in a dosedependent manner, and complete inhibition of enzymatic activity was observed in the high dose group (>99% inhibition vs. vehicle). Determinations of plasma MAdCAM-1 revealed significantly decreased levels in the high GL1001 dose group. In Study 2, GL1001 treatment increased survival (30%, p<0.016 high dose vs. DSS only) and decreased expression levels of colonic TNF-alpha. Conclusions: Oral administration of GL1001 in DSS models attenuates acute colonic symptoms with concomitant full inhibition of ACE2 activity and significantly decreased levels of IBD-related pro-inflammatory cytokines. The data support the proposed beneficial therapeutic role for GL1001 and ACE2 inhibition in inflammatory gastrointestinal diseases through target-mediated, anti-inflammatory effects.
The Role of TH17/IL-23 Pathway On Intestinal Inflammation in SKG Mice Chang Soo Eun, Dong Soo Han, Joo Hyun Choi, Sang Bong Ahn, Hyun Seok Cho, Tae Jun Byun, Jeehee Youn Background/Aims: Th17/IL-23 pathway with suppressive CD4+ Treg subsets have recently been identified to play an important role in the pathogenesis of inflammatory bowel disease. SKG mice have a point mutation of the zeta-associated protein of 70 kD (ZAP-70), a key signal transduction molecule in T cells, and spontaneously develop CD4+ T cell-mediated chronic autoimmune arthritis, mainly involving IL-17. The aims of this study were to evaluate dextran sulfate sodium (DSS) induced intestinal inflammation and to study the role of Th17/ IL-23 pathway on intestinal inflammation using SKG mice. Methods: Acute colitis was induced in SKG and BALB/c mice (control) by administering 2.7% DSS orally in drinking water for nine days under specific pathogen free condition. Colonic inflammation was assessed based on body weight change, total colon length and weight, and histological scores. IL-6, IFN-gamma, IL-17, IL-23, and IL12p40 levels were measured by ELISA in the supernatants of colonic tissue explants. Results: After DSS treatment, significant weight loss was developed both in SKG and BALB/c mice compared to mice not treated with DSS. Clinical and histological activities of colitis, and IL-6 levels in colonic tissues were attenuated in DSS-treated SKG mice compared to DSS-treated BALB/c mice. The level of IL-12p40 in colonic tissues was increased in DSS-treated SKG mice compared to DSS-treated BALB/c mice. IL-23 level in colonic tissues, by contrast, was decreased in DSS-treated SKG mice compared to DSS-treated BALB/c mice. Conclusion: Th17 cells and related cytokines may play an important role in mucosal homeostasis of intestinal inflammation. S1667 Resident “M2-Like” Intestinal Macrophages Express Eotaxin-1 in Colonic Injury Amanda B. Waddell, Richard Ahrens, Simon P. Hogan Rationale: Clinical and experimental studies have demonstrated a link between eotaxin-1/ CCL11, eosinophils and the inflammatory bowel diseases (IBD), Crohn's disease (CD) and ulcerative colitis (UC). However, the cellular source of CCL11 remains unclear. Methods: We examined colonic macrophage (MΦ) phenotype in BALB/c WT mice at baseline and following administration of 5.0% DSS treatment flow cytometry analysis. Colonic MΦ were purified by adherence and examined for alterative M2 activation (retlna, YM1 and arginase) and eotaxin-1 by RT-PCR. Bone marrow derived (BM) MΦ's were stimulated In Vitro by IL-4, IFNγ and LPS and CCL11, and alternative M2 activation was determined by RT-PCR and immunofluoresence analysis. Results: Phenotypic identification of resident colonic MΦ at baseline identified a heterogeneous non-inflammatory (F4/80+CD11b+Ly6C-) MΦ population consisting of CD206- and CD206+ M2-like MΦ (CD206- %: 79.8±2.6 vs 20.2±2.6; n=3 per group; mean ± SEM). DSS administration promoted an increase in inflammatory (F4/80+CD11b+Ly6C+) MΦ and not resident colonic F4/80+CD11b+Ly6C- MΦ. However, immunofluoresence analysis of adherence purified MΦ from the colon of DSS-treated mice revealed colocalization of M2-like markers, Retlna and arginase and CCL11. In Vitro analysis of BM MΦ's stimulated with IL-4 or IFN-γ + LPS led to development of classically activated “M1” nitric oxide-positive and alternatively activated “M2” Retlna+ Arginase+ macrophages. PCR analysis revealed CCL11 expression in M2 MΦ. Conclusions: Collectively, these In Vitro and In Vivo analyses demonstrate that CCL11 is predominantly expressed by resident intestinal M2-like MΦ.
AGA Abstracts
S1670 A Potential Role for Intestinal Alkaline Phosphatase in Colonic Inflammation Sundaram Ramasamy, Madhu S. Malo, Golam Mostafa, Jose L. Millan, Atul K. Bhan, Richard A. Hodin Background & Aims: An inadequate protective mechanism against gut-derived bacterial toxin may damage the colonic mucosal barrier resulting in intestinal inflammation. We have previously shown that intestinal alkaline phosphatase (IAP), a small intestinal brush border enzyme, is a gut mucosal defense factor that provides resistance to bacterial invasion by detoxifying lipopolysaccharides (LPS). The aim of this study was to elucidate a potential role for IAP in the context of dextran sodium sulphate (DSS)-induced chronic colitis. Methods: Chronic colitis was induced by oral administration of DSS in distilled water. IAPknockout (KO) and wild-type (WT) mice (n = 5) were treated with DSS (3%) for 7 days, followed by regular distilled water for 7 days; this cycle was repeated three times. Disease severity was determined by weight loss, loose stools/diarrhea, rectal bleeding and histopathology. Results: Following the first cycle of DSS treatment, both WT and IAP KO mice developed colitis symptoms (diarrhea/bloody stools), however, there were no differences between the two groups. Following the second cycle of DSS treatment, KO mice exhibited a significant mortality (2/5) compared with WT mice (0/5). Interestingly, WT mice, but not KO mice, showed significant weight loss compared to the control group. Following the third cycle of DSS treatment, KO mice again exhibited a high mortality (2/3) compared to the WT mice
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