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Tryptophan Metabolome Signature Differences in Inflammatory Bowel Diseases
Tu1813
whereas TRPM8 is reported to be activated at temperatures below 25°C. Since cold temperature is commonly used as local anti-inflammatory treatment, we hypothesized that icilin would diminish colitis in mouse models of colonic inflammation. Colonic inflammation in C57BL6 mice was induced by treatment with either 2.5% dextran sodium sulphate (DSS) solution as drinking water or tri-nitro-benzene sulphonic acid (TNBS, 2mg in 100ul of 40% ethanol, intracolonically). Colitis was allowed to develop for 7 days and icilin (100μg in 3% DMSO/saline, intraperitoneally) was administered daily. Seven days after induction of colitis, macroscopic damage score, bowel thickness and myeloperoxidase (MPO) activity were measured. DSS or TNBS challenge induced potent colonic inflammation in mice leading to significant increases in bowel thickness, macroscopic damage scores and MPO activity. However, DSS- or TNBS-induced increase in bowel thickness and macroscopic damage scores were significantly reduced in mice that received daily icilin treatment. Lastly, mice challenged with DSS showed significantly reduced MPO activity when treated with icilin as compared to mice treated with vehicle. In conclusion, icilin confers protection against the development of colitis in mice. Thus, agents that can activate TRPA1 and/or TRPM8 may represent novel therapeutics for inflammatory bowel disease in the future.
AGA Abstracts
TH1/TH17 Profiles in Crohn's Disease: A Cross Sectional Single Centre Study in Postoperative Crohn's Disease Aravinth U. Murugananthan, David Bernardo Ordiz, Phil Tozer, Cheng T. Tee, Elizabeth R. Mann, Ailsa Hart, Naila Arebi, Stella C. Knight, Hafid O. Al-Hassi Introduction: Th1 and Th17 pathways are implicated in Crohn's disease (CD). In operative resection samples healthy ileum shows high TGFb levels in patients who develop recurrence, with TGFb being a known activator of the Th17 response. Other studies in CD show a dominant Th1 cytokine profile, with high levels of IFNg, which reduce Th17 response and augment Th1 response. The relationship of Th1/Th17 cytokine profiles in postoperative CD has not been examined. We aimed to study tissue Th1/Th17 cytokine secretion after invitro biopsy culture in postoperative CD. Methods: Colonoscopy was undertaken in postoperative CD patients. Recurrence graded as no/minimal inflammation (Rutgeert Score [RS] ≤1) or progressive inflammation (RS ≥2). Ileal biopsies were cultured overnight and cell free supernatants obtained. Supernatant cytokines (IL-2, IL-4, L10, IL17 TNF a, INFg and IL6) were assessed by flow cytometry using cytometric bead array TM(Becton Dickinson). Statistical analysis was via unpaired t-tests. Results: Consecutive patients attending endoscopy (n= 24, 9m/15f) were identified. Mean age 45.0 yrs and time from I-C resection was 5.8 years; 5 patients were smokers. Drugs were thiopurines 13, Infliximab 1 and nil 10. Endoscopic severity was i0 n=5, i1 n=6, i2 n=5, i3 n=3, i4 n=5. Mean cytokine concentrations from supernatants are shown in the table. Comparison between RS ≤1 and ≥2 showed that proinflammatory cytokines IL17a (p<0.02) and IFNg (p<0.03) were significantly higher in RS i2-i4 neo terminal ileum as compared with those with RS i0-i1. The regulatory cytokine IL10 was significantly higher in patients with RS ≤1 (p<0.038). Conclusion: Cytokine profiles in those with RS >2, show higher levels of IL 17a and IFNg and reduced IL10 compared to RS <1. This profile supports a Th17 and Th1 mediated response as one of the early instigators of endoscopic progression in postoperative CD. Our observation is consistent with recent findings of a T cell subset able to produce cytokines involved in both Th1 and Th17 responses. Previous therapies directed at Th1 pathway e.g. anti-IL-12p40 antibody ustekinumab and anti-IFNG Fontolizumab failed to show significant clinical benefit in CD. Given our findings targeting the Th17 response e.g. with anti-IL-23 antibodies and antiIL17 may deliver improved therapeutic outcome. Cytokine concentrations per Rutgeert Score
Tu1816 Evaluation of p38 MAPK Pathway as a Molecular Signature in Ulcerative Colitis Xinmei Zhao, Fachao Zhi, Bin Kang, Bo Jiang, Siqi Liu, Youyong Lu Early diagnosis and treatment of Ulcerative colitis (UC) is clinically challenging. In order to overcome this problem, we explored the interrelated multiplex signaling pathway to identify molecular signatures in UC by use of integrated strategy in proteomics. Twentyfour cases intestinal mucosa of UC and normal control underwent comparative proteomic analysis. A total of 30 unique differential proteins were identified, including 16 up-regulated and 14 down-regulated in UC group. A differential protein cluster, consisting of 11 proteins involved in p38 mitogen-actived protein kinase (MAPK) pathway, was deduced and validated by Western blot. Furthermore, the expression of three proteins elicited from the protein cluster, phosphorylated p38, MAWBP and galectin-3, as a molecular signature, were detected by immunohistochemistry on 120 cases UC and normal samples. The molecular signature was significantly different between UC and normal samples (P<0.001), correlates with disease progression of UC (P<0.01), and classified the UC risk with high sensitivity (95.00%±2.81%) and specificity (98.33%±1.65%). P38 MAPK pathway modulates the expression of the protein clusters in macrophages RAW264.7 cells as evidenced by the alteration of the expression pattern of the protein cluster with specific inhibitor SB203580. Therefore, molecular signature of P38 MAPK pathway may serve as a promising biomarker in evaluating UC risk. Tu1817 Tryptophan Metabolome Signature Differences in Inflammatory Bowel Diseases Yunki Yau, Sean S. Shin, Sonia Bustamante, Russell Pickford, Rupert W. Leong, Valerie C. Wasinger BACKGROUND: Metabolism of the essential amino acid tryptophan via the kynurenine pathway is associated with inflammatory neurotransmission and gut motility. Quinolinic acid (QA) and Picolinic acid (PA) are the terminal end products of this pathway. QA is known to preferentially activate a glutamate receptor subtype, while PA induces macrophage inflammatory proteins preceding acute neutrophilic inflammation. Metabolomics is a novel technique in IBD research and we present here the first metabolome assay of human IBD plasma. METHODS: We quantified and compared QA and PA expression levels and performed a whole plasma metabolome assay between populations of blinded CD, UC, and healthy controls using Gas Chromatography/Mass Spectrometry (GC/MS) and Liquid Chromatography (LC)/MS, respectively. GC/MS was performed on a HP 6890 gas chromatograph interfaced to a mass selective detector, and a Ultra High Pressure Liquid Chromatograph (UHPLC) with C18 reverse-phase LC and Orbitrap MS instrument was utilized in scanning for chemical identities between 50 - 1000 m/z for a whole metabolome assay. RESULTS: Nineteen UC, 16 CD and 9 control samples were analyzed. In CD, QA levels was significantly increased when compared to controls by a mean of 0.1348 pmol/ml (P=0.021), and by 0.1401pmol/ml in active disease from non-active (p=0.027). Total kynurenine metabolites in CD was also significantly increased against controls (mean increase of 0.1678 pmol/ml (P=0.015)), and once again in active disease when compared to non-active (0.1879 pmol/ ml mean increase (p=0.009)). Kynurenine metabolites did not differ significantly between UC and controls, or with UC disease activity. SIEVE software quantitated, and Chemspider search engine identified a large number of differentially expressed metabolites from whole metabolome assay. Of note, peroxisome dysfunction marker tetracosa hexaenoic acid (THA) was significantly more abundant in UC compared to CD (p<0.008). CONCLUSION: Significant overexpression of QA in CD suggests an overactivation of receptors involved in the gutbrain axis and possible irregularities in interleukin 2 (IL-2) induced T cells. Furthermore, relative overabundance of THA in UC may have implications in disease-specific mucin depletion.
Tu1814 Protease-Activated Receptor 1, 2 but Not 4 Sensitizes Transient Receptor Potential Vanilloid 4 in Human Intestinal Epithelial Cells Emilie D'Aldebert, Nicolas Cenac, Charlyne Casteras, Nathalie Vergnolle In a previous study, our laboratory has demonstrated that Transient Receptor Potential Vanilloid 4 (TRPV4) was expressed and functional in human intestinal epithelial cells. TRPV4 activation in those cells caused increases in intracellular calcium concentrations, chemokine release and its activation in mouse gastrointestinal tract caused colitis. TRPV4 appears to be an important actor in intestinal inflammation. In an inflammatory context, many mediators are released, such as serine proteases, which will activate protease-activated receptors (PAR). We hypothesized that these proteases released during inflammation, through activation of their receptors the PARs, may sensitize TRPV4. Methods: TRPV4 response was evaluated by measurement of calcium flow with the probe Fluo-8 in Normal derived Colon Mucosa (NCM460) cells. These cells were stimulated by PAR1-activating peptide (TFLLRN-NH2), PAR2-activating peptide (SLIGRL-NH2), PAR4-activating peptide (AYPGFK-NH2) or their vehicle, 3 minutes before a second stimulation with the specific agonist of TRPV4, 4αPDD (4α-phorbol-12,13-didenoate) or its vehicle. Results: TRPV4 specific activation involved a dose-dependent calcium flow in NCM460 cells. The response to 10 μM of 4αPDD was significantly increased when cells were pre-incubated with PAR1-activating peptide (100 μM) or PAR2-activating peptide (10 μM). Intestinal epithelial cells did not respond to low concentration (1 μM) of 4αPDD. However, if cells were pre-treated with the PAR1 agonist (100 μM) or the PAR2 agonist (10 μM), they became responsive to low concentration (1 μM) of the TRPV4 agonist 4αPDD. PAR4 agonist (200 μM) never modified TRPV4 response. Conclusions: Our study shows that activation of PAR1, PAR2 but not PAR4 potentiated the cellular response of TRPV4 but also sensitized the receptor. Overall, these results suggest that TRPV4 signals and potentially its pro-inflammatory effects in intestinal epithelial cells can be enhanced by proteases acting on PAR1 and PAR2.
Tu1818 Short and Long-Term Regulation of Intestinal Na+/H+ Exchange by TLR2 José Miguel Cabral, Fernando Magro, Patricio Soares-da-Silva
Tu1815
Type 2 toll-like receptors (TLR2) are expressed in the cell membrane and recognize a wide range of pathogen-associated molecular patterns derived from bacteria as lipoteichoic acid (LTA), a Gram-positive bacteria component. Aims: To evaluate the effect of TLR2 activation by LTA on activity of type 1 and 2 Na+/H+ exchanger (NHE) in T84 intestinal epithelial cells. Matherial and methods: Intracellular pH levels and NHE activity were measured using fluorimetric assays and protein expression was determined by western blot analysis. All experiments were performed in presence of S3226 (100 nM), a selective inhibitor of NHE3, to ensure an effective evaluation NHE1/NHE2 activity. Results: Short-term (0.5 h) TLR2
Icilin, an Agonist Cold Activated Transient Receptor Potential (TRP) Channels Protects Mice Against Colitis Eric Hyun, Rithwik Ramachandran, Morley D. Hollenberg Icilin is a known agonist for members of the transient receptor potential family of ion channels (TRPA1 and TRPM8), which are reported to play a role in various physiological events including temperature sensation. TRPA1 is activated by temperatures below 18°C,
AGA Abstracts
S-840
whereas TRPM8 is reported to be activated at temperatures below 25°C. Since cold temperature is commonly used as local anti-inflammatory treatment, we hypothesized that icilin would diminish colitis in mouse models of colonic inflammation. Colonic inflammation in C57BL6 mice was induced by treatment with either 2.5% dextran sodium sulphate (DSS) solution as drinking water or tri-nitro-benzene sulphonic acid (TNBS, 2mg in 100ul of 40% ethanol, intracolonically). Colitis was allowed to develop for 7 days and icilin (100μg in 3% DMSO/saline, intraperitoneally) was administered daily. Seven days after induction of colitis, macroscopic damage score, bowel thickness and myeloperoxidase (MPO) activity were measured. DSS or TNBS challenge induced potent colonic inflammation in mice leading to significant increases in bowel thickness, macroscopic damage scores and MPO activity. However, DSS- or TNBS-induced increase in bowel thickness and macroscopic damage scores were significantly reduced in mice that received daily icilin treatment. Lastly, mice challenged with DSS showed significantly reduced MPO activity when treated with icilin as compared to mice treated with vehicle. In conclusion, icilin confers protection against the development of colitis in mice. Thus, agents that can activate TRPA1 and/or TRPM8 may represent novel therapeutics for inflammatory bowel disease in the future.
AGA Abstracts
TH1/TH17 Profiles in Crohn's Disease: A Cross Sectional Single Centre Study in Postoperative Crohn's Disease Aravinth U. Murugananthan, David Bernardo Ordiz, Phil Tozer, Cheng T. Tee, Elizabeth R. Mann, Ailsa Hart, Naila Arebi, Stella C. Knight, Hafid O. Al-Hassi Introduction: Th1 and Th17 pathways are implicated in Crohn's disease (CD). In operative resection samples healthy ileum shows high TGFb levels in patients who develop recurrence, with TGFb being a known activator of the Th17 response. Other studies in CD show a dominant Th1 cytokine profile, with high levels of IFNg, which reduce Th17 response and augment Th1 response. The relationship of Th1/Th17 cytokine profiles in postoperative CD has not been examined. We aimed to study tissue Th1/Th17 cytokine secretion after invitro biopsy culture in postoperative CD. Methods: Colonoscopy was undertaken in postoperative CD patients. Recurrence graded as no/minimal inflammation (Rutgeert Score [RS] ≤1) or progressive inflammation (RS ≥2). Ileal biopsies were cultured overnight and cell free supernatants obtained. Supernatant cytokines (IL-2, IL-4, L10, IL17 TNF a, INFg and IL6) were assessed by flow cytometry using cytometric bead array TM(Becton Dickinson). Statistical analysis was via unpaired t-tests. Results: Consecutive patients attending endoscopy (n= 24, 9m/15f) were identified. Mean age 45.0 yrs and time from I-C resection was 5.8 years; 5 patients were smokers. Drugs were thiopurines 13, Infliximab 1 and nil 10. Endoscopic severity was i0 n=5, i1 n=6, i2 n=5, i3 n=3, i4 n=5. Mean cytokine concentrations from supernatants are shown in the table. Comparison between RS ≤1 and ≥2 showed that proinflammatory cytokines IL17a (p<0.02) and IFNg (p<0.03) were significantly higher in RS i2-i4 neo terminal ileum as compared with those with RS i0-i1. The regulatory cytokine IL10 was significantly higher in patients with RS ≤1 (p<0.038). Conclusion: Cytokine profiles in those with RS >2, show higher levels of IL 17a and IFNg and reduced IL10 compared to RS <1. This profile supports a Th17 and Th1 mediated response as one of the early instigators of endoscopic progression in postoperative CD. Our observation is consistent with recent findings of a T cell subset able to produce cytokines involved in both Th1 and Th17 responses. Previous therapies directed at Th1 pathway e.g. anti-IL-12p40 antibody ustekinumab and anti-IFNG Fontolizumab failed to show significant clinical benefit in CD. Given our findings targeting the Th17 response e.g. with anti-IL-23 antibodies and antiIL17 may deliver improved therapeutic outcome. Cytokine concentrations per Rutgeert Score
Tu1816 Evaluation of p38 MAPK Pathway as a Molecular Signature in Ulcerative Colitis Xinmei Zhao, Fachao Zhi, Bin Kang, Bo Jiang, Siqi Liu, Youyong Lu Early diagnosis and treatment of Ulcerative colitis (UC) is clinically challenging. In order to overcome this problem, we explored the interrelated multiplex signaling pathway to identify molecular signatures in UC by use of integrated strategy in proteomics. Twentyfour cases intestinal mucosa of UC and normal control underwent comparative proteomic analysis. A total of 30 unique differential proteins were identified, including 16 up-regulated and 14 down-regulated in UC group. A differential protein cluster, consisting of 11 proteins involved in p38 mitogen-actived protein kinase (MAPK) pathway, was deduced and validated by Western blot. Furthermore, the expression of three proteins elicited from the protein cluster, phosphorylated p38, MAWBP and galectin-3, as a molecular signature, were detected by immunohistochemistry on 120 cases UC and normal samples. The molecular signature was significantly different between UC and normal samples (P<0.001), correlates with disease progression of UC (P<0.01), and classified the UC risk with high sensitivity (95.00%±2.81%) and specificity (98.33%±1.65%). P38 MAPK pathway modulates the expression of the protein clusters in macrophages RAW264.7 cells as evidenced by the alteration of the expression pattern of the protein cluster with specific inhibitor SB203580. Therefore, molecular signature of P38 MAPK pathway may serve as a promising biomarker in evaluating UC risk. Tu1817 Tryptophan Metabolome Signature Differences in Inflammatory Bowel Diseases Yunki Yau, Sean S. Shin, Sonia Bustamante, Russell Pickford, Rupert W. Leong, Valerie C. Wasinger BACKGROUND: Metabolism of the essential amino acid tryptophan via the kynurenine pathway is associated with inflammatory neurotransmission and gut motility. Quinolinic acid (QA) and Picolinic acid (PA) are the terminal end products of this pathway. QA is known to preferentially activate a glutamate receptor subtype, while PA induces macrophage inflammatory proteins preceding acute neutrophilic inflammation. Metabolomics is a novel technique in IBD research and we present here the first metabolome assay of human IBD plasma. METHODS: We quantified and compared QA and PA expression levels and performed a whole plasma metabolome assay between populations of blinded CD, UC, and healthy controls using Gas Chromatography/Mass Spectrometry (GC/MS) and Liquid Chromatography (LC)/MS, respectively. GC/MS was performed on a HP 6890 gas chromatograph interfaced to a mass selective detector, and a Ultra High Pressure Liquid Chromatograph (UHPLC) with C18 reverse-phase LC and Orbitrap MS instrument was utilized in scanning for chemical identities between 50 - 1000 m/z for a whole metabolome assay. RESULTS: Nineteen UC, 16 CD and 9 control samples were analyzed. In CD, QA levels was significantly increased when compared to controls by a mean of 0.1348 pmol/ml (P=0.021), and by 0.1401pmol/ml in active disease from non-active (p=0.027). Total kynurenine metabolites in CD was also significantly increased against controls (mean increase of 0.1678 pmol/ml (P=0.015)), and once again in active disease when compared to non-active (0.1879 pmol/ ml mean increase (p=0.009)). Kynurenine metabolites did not differ significantly between UC and controls, or with UC disease activity. SIEVE software quantitated, and Chemspider search engine identified a large number of differentially expressed metabolites from whole metabolome assay. Of note, peroxisome dysfunction marker tetracosa hexaenoic acid (THA) was significantly more abundant in UC compared to CD (p<0.008). CONCLUSION: Significant overexpression of QA in CD suggests an overactivation of receptors involved in the gutbrain axis and possible irregularities in interleukin 2 (IL-2) induced T cells. Furthermore, relative overabundance of THA in UC may have implications in disease-specific mucin depletion.
Tu1814 Protease-Activated Receptor 1, 2 but Not 4 Sensitizes Transient Receptor Potential Vanilloid 4 in Human Intestinal Epithelial Cells Emilie D'Aldebert, Nicolas Cenac, Charlyne Casteras, Nathalie Vergnolle In a previous study, our laboratory has demonstrated that Transient Receptor Potential Vanilloid 4 (TRPV4) was expressed and functional in human intestinal epithelial cells. TRPV4 activation in those cells caused increases in intracellular calcium concentrations, chemokine release and its activation in mouse gastrointestinal tract caused colitis. TRPV4 appears to be an important actor in intestinal inflammation. In an inflammatory context, many mediators are released, such as serine proteases, which will activate protease-activated receptors (PAR). We hypothesized that these proteases released during inflammation, through activation of their receptors the PARs, may sensitize TRPV4. Methods: TRPV4 response was evaluated by measurement of calcium flow with the probe Fluo-8 in Normal derived Colon Mucosa (NCM460) cells. These cells were stimulated by PAR1-activating peptide (TFLLRN-NH2), PAR2-activating peptide (SLIGRL-NH2), PAR4-activating peptide (AYPGFK-NH2) or their vehicle, 3 minutes before a second stimulation with the specific agonist of TRPV4, 4αPDD (4α-phorbol-12,13-didenoate) or its vehicle. Results: TRPV4 specific activation involved a dose-dependent calcium flow in NCM460 cells. The response to 10 μM of 4αPDD was significantly increased when cells were pre-incubated with PAR1-activating peptide (100 μM) or PAR2-activating peptide (10 μM). Intestinal epithelial cells did not respond to low concentration (1 μM) of 4αPDD. However, if cells were pre-treated with the PAR1 agonist (100 μM) or the PAR2 agonist (10 μM), they became responsive to low concentration (1 μM) of the TRPV4 agonist 4αPDD. PAR4 agonist (200 μM) never modified TRPV4 response. Conclusions: Our study shows that activation of PAR1, PAR2 but not PAR4 potentiated the cellular response of TRPV4 but also sensitized the receptor. Overall, these results suggest that TRPV4 signals and potentially its pro-inflammatory effects in intestinal epithelial cells can be enhanced by proteases acting on PAR1 and PAR2.
Tu1818 Short and Long-Term Regulation of Intestinal Na+/H+ Exchange by TLR2 José Miguel Cabral, Fernando Magro, Patricio Soares-da-Silva
Tu1815
Type 2 toll-like receptors (TLR2) are expressed in the cell membrane and recognize a wide range of pathogen-associated molecular patterns derived from bacteria as lipoteichoic acid (LTA), a Gram-positive bacteria component. Aims: To evaluate the effect of TLR2 activation by LTA on activity of type 1 and 2 Na+/H+ exchanger (NHE) in T84 intestinal epithelial cells. Matherial and methods: Intracellular pH levels and NHE activity were measured using fluorimetric assays and protein expression was determined by western blot analysis. All experiments were performed in presence of S3226 (100 nM), a selective inhibitor of NHE3, to ensure an effective evaluation NHE1/NHE2 activity. Results: Short-term (0.5 h) TLR2
Icilin, an Agonist Cold Activated Transient Receptor Potential (TRP) Channels Protects Mice Against Colitis Eric Hyun, Rithwik Ramachandran, Morley D. Hollenberg Icilin is a known agonist for members of the transient receptor potential family of ion channels (TRPA1 and TRPM8), which are reported to play a role in various physiological events including temperature sensation. TRPA1 is activated by temperatures below 18°C,
AGA Abstracts
S-840