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Interleukin-10 Receptor Gene Polymorphisms Associate with Early-Onset Inflammatory Bowel Disease
AGA Abstracts
with 12 non-IBD family controls in EBV-transformed B cells. The mRNA expression levels of PTPN2 were increased in diseased tissues compared with adjacent normal tissues in 32 CD patients. The overall mean PTPN2/GAPDH expression values were: 4.36 ± 6.5 (diseased), vs. 2.45 ± 3.2 (normal) (p < 0.05). However, mRNA expression levels of PTPN2 were not significantly altered in the 30 UC diseased tissues vs adjacent normal tissues. Our previous cDNA microarray results showed that PTPN2 could be down-regulated by Nkx2-3 knockdown in B cells from CD and UC patients, and human intestinal microvascular endothelial cells (HIMECs). We confirm these results by RT-PCR in Nkx2-3 knockdown B cells from CD and UC, and Nkx2-3 knockdown HIMECs. The mRNA expression levels of Nkx2-3 were increased in 14 CD compared with 14 non-IBD family controls in EBV-transformed B cells (p < 0.05), and were increased in diseased tissues compared with adjacent normal tissues in 32 CD patients (p < 0.05). However, mRNA expression levels of Nkx2-3 were not significantly altered in EBV-transformed B cells and intestinal tissues from UC patients. mRNA expression levels of PTPN2 were positively correlated with those of Nkx2-3 in 14 CD (r = 0.6024, p < 0.01) and 12 UC (r = 0.5928, p < 0.01) from EBV-transformed B cells. Conclusions PTPN2 SNP rs2542152 was associated with CD. mRNA expression of PTPN2 was up-regulated in CD patients from EBV-transformed B cells and intestinal tissues. Expression of PTPN2 was regulated by Nkx2-3 in CD patients.
anti-inflammatory effects of LL-37. Conclusion: Cathelicidins are important modulators of colonic inflammation by mechanisms involving inhibition of NF-kappaB activity in colonocytes. Our results also indicate that exogenous cathelicidin administration may represent a novel approach for treatment of colonic inflammation, including inflammatory bowel disease. Supported by a Pilot and Feasibility Study grant from UCLA-CURE Center, Career Development Award from Crohn's and Colitis Foundation of America and K01DK084256 from National Institute of Health to HWK. Su1742 Targeting Human CD2 by the CB.219 Monoclonal Antibody Protects From Intestinal Inflammation in a Humanized Colitis Model Anja A. Kuehl, Nina N. Pawlowski, Katja Grollich, Ulrike Erben, Martin Zeitz, Joerg C. Hoffmann, Christoph Loddenkemper Purpose: In mouse models, a deficiency in the costimulatory molecule CD2 or simultaneous treatment with anti-mouse CD2 monoclonal antibodies (mAb) reduced intestinal inflammation. Anti-human CD2 mAb inhibited the proliferation of human lymphocytes and their interferon (IFN) γ synthesis In Vitro. Linking both observations, we here asked whether the anti-human CD2 mAb CB.219 counteracts intestinal inflammation in a humanized model of transfer colitis. Methods: Colitis was induced in immmunodeficient RAG 1 ko mice by transfer of CD4CD45RBhi T cells derived from human CD2 transgenic mice. Treatment with CB.219 started either simultaneously with colitis induction (preventive treatment) or after onset of colitis (therapeutic treatment). Mice received 200 μg CB.219 or an isotype control mAb every third day. Development of colitis was observed by body weight and hemoccult assay over time. After 87 days, histological scores were assessed in a blinded fashion. Cytokine profiles were measured by cytometric bead array from supernatants of short-term colon cultures. Results: Mice receiving CB.219 mAb ahead of T cell transfer continuously gained weight, survived throughout the experiment and were completely protected from colitis development. Underlining the protective effect of the mAb, CB.219-treated animals showed significantly reduced histological scores (0.3 ± 0.2 vs. 2.8 ± 0.5, p < 0.01) and increased transforming growth factor (TGF) β secretion in colon cultures compared to controls (19.6 ± 13.5 vs. 5.0 ± 0.4 pg/μg colon, p < 0.05). The CB.219 mAb also worked in a therapeutic setting. Again, the histological colon damage was less severe upon CB.219 treatment (histological score: 0.9 ± 0.2 vs. 2.9 ± 0.4, p < 0.01). Colon organ cultures from CB.219-treated mice revealed a trend to increased TGF β (p = 0.064) and significantly decreased tumor necrosis factor (TNF) α production compared to controls (23.6 ± 5.1 vs. 154.5 ± 54.8 pg/ mg colon, p < 0.01). Attributing this capacity to intestinal immune cells, diminished TNF α secretion was found in lymphocytes isolated from the lamina propria of CB.219-treated mice after short-term In Vitro stimulation via CD3/CD28. These supernatants also contained less interleukin (IL) 2, IL 17A, and IFN γ than the respective controls. Discussion: In our humanized mouse model of colitis the CB.219 mAb specific for human CD2 protected from colitis by altering the local proinflammatory milieu even if applied in established colitis. Thus, we here show that targeting CD2+ T cells provides an interesting new approach for the treatment of colitis in man and further experiments will address gut specificity, underlying mechanisms and general applicability of CB.219 treatment.
Su1740a Interleukin-10 Receptor Gene Polymorphisms Associate with Early-Onset Inflammatory Bowel Disease Christopher J. Moran, Subra Kugathasan, Christoph Klein, Mark S. Silverberg, Aleixo M. Muise, Scott B. Snapper Introduction: Recently, several infants with inflammatory bowel disease (IBD) were found to have homozygous mutations in interleukin-10 receptor (IL-10R) genes (IL10RA or IL10RB). These patients presented with severe enterocolitis refractory to traditional medical and surgical therapies. Their symptoms resolved following allogeneic bone marrow transplantation. We hypothesized that IL-10R genetic mutations play a role in very early onset IBD (VEO-IBD) but less so in the broader IBD population. While single nucleotide polymorphisms (SNPs) in IL-10R have not been reported to associate with risk for IBD in genome-wide association studies (GWAS), current GWAS strategies may not detect at-risk SNPs that are poorly tagged in the GWAS, possess a minor allele frequency <5%, or rare private polymorphisms. We report the largest sequencing effort of the IL-10R genes in early onset IBD to date. Methods: IL-10R genes (IL-10RA and IL-10RB) were deep sequenced by Sanger method in 122 previously collected samples from early onset IBD patients (5 years old and younger) and a group of 185 healthy controls. The odds ratio (OR) for IBD (as well as Crohn’s and ulcerative colitis risk) was calculated under a dominant genetic model using Fisher’s exact test. Results: In IBD patients, 48 SNPs were detected in IL-10RA and 24 SNPs were detected in IL-10RB. rs2228055 (Ile224Val) was associated with IBD (OR 2.38, p= 0.032) and ulcerative colitis (OR 2.52, p=0.029). An additional 4 IL-10RA SNPs were associated with UC. Two SNPs in the 3’ untranslated region of IL-10RB were associated with Crohn’s disease, rs8178561 (OR 0.10, p<0.001) and rs1058867 (OR 2.12, p=0.004). Conclusions: This is the largest sequencing effort to date of the IL-10R genes in early-onset IBD. These results suggest that polymorphisms in IL-10R genes are associated with IBD. Replication of these SNPs in an independent cohort as well as functional studies are required to determine the importance of these identified polymorphisms.
Su1743 Mechanism of Immune Regulation by CD11b+ Myeloid Cells in the Intestine Masako Murai, Petra Krause, Jr-Wen Shui, Rajat Madan, Christopher L. Karp, Mitchell Kronenberg Background and aims: We found that a subset of CD11b+ myeloid cells in the intestinal lamina propria constitutively produce IL-10, and adoptive transfer of these IL-10 producing cells to recipients helped to maintain Foxp3 expression and the function of Treg. Although much emphasis has been placed on the regulatory role of T cell derived IL-10, our data show that IL-10 synthesis by intestinal myeloid cells has a nonredundant and dramatic effect on maintaining Treg function and preventing T cell-mediated colitis in mice. Here, we further investigate the role of intestinal myeloid cell derived IL-10 in the regulation of gut inflammation. Methods: We defined the phenotype of the IL-10-producing myeloid cells in the intestine using IL-10 reporter mice, with reporter+ cells analyzed by quantitative PCR and flow cytometry. The requirement of IL-10 expression by CD11b+ myeloid cells was assessed using mice with a cell type specific-IL-10 gene deletion, under both steady conditions and following Citrobacter rodentium infection. Results: IL-10 producing intestinal myeloid cells expressed higher CD11b, CD11c, F4/80, Gr-1 and CX3CR1 by both messenger RNA and protein determination, compared to their non-IL-10 producing intestinal myeloid counterparts. However, TGFβ1, TGFβ3 and PD-L1 expression was not different between the groups. Furthermore, there were no differences in the ability of reporter+ and negative cells for induction of regulatory T cells (iTreg) In Vitro. Myeloid cell specific IL-10 deficient mice had fewer Treg in the large intestine, but not in the spleen. Futhermore, C. rodentium caused a decrease in the number of Treg in the large intestine, but this defect was more pronounced in mice with a myeloid cell specific (LysM-Cre driven) IL-10 deficiency. Conclusions: IL10 producing myeloid cells have a pivotal role in maintaining Treg population in the intestine especially under inflammatory conditions as exemplified by C. rodentium infection. Supported by grants from CCFA and PO1 grant AI 89624
The numbers associated with each SNP reflect the number of patients possessing at least one variant allele at a given locus. Su1741 Roles of Endogenous and Exogenous Cathelicidin During Colonic Inflammation Hon Wai Koon, David Q. Shih, Tressia C. Hing, Dezheng Zhao, Hua Xu, Mary Pat Moyer, Richard Gallo, Charalabos Pothoulakis Background and Aims: Cathelicidin (LL-37 in human and CRAMP in mice) is a family of endogenous anti-microbial peptides that protect the host from infection as part of the innate immune response. Recent evidences suggest that cathelicidins may modulate inflammation. Here we determine whether cathelicidin can modulate colonic inflammation in animal model of colitis as well as human colonic epithelial cells. Methods: Wild-type and mCRAMP deficient mice were administered with trinitrobenzene sulphonic acid (TNBS) to induce acute (7 days) colitis. Some mice were treated intracolonically with cathelicidin and colonic histological changes and cytokine levels during colonic inflammation were evaluated. Results: TNBS induced acute colitis in mice that was reduced by exogenous intracolonic cathelicidin (mCRAMP) administration (5 mg/kg every 2 days). This inhibitory mCRAMP effect was associated with lower colonic histological damage and reduced levels of the proinflammatory mediators KC, PGE2 and TNFalpha compared to controls. Compared to wild-type mice, mCRAMP deficient mice developed more severe TNBS-induced colonic inflammation associated with higher levels of colonic proinflammatory mediators. Exposure of human colonic epithelial NCM460 cells to LL-37 (1 microM) significantly reduced basal levels of inflammatory mediators (IL-8, PGE2 and TNFalpha), cyclooxygenase-2 protein expression, as well as NF-kappaB phosphorylation and activity. Induction of NF-kappaB activity by phorbol ester (PMA) abolished the anti-inflammatory effects and NF-kappaB activity in response to LL37 in colonic epithelial cells, suggesting that reduced NF-kappaB activity is involved in the
AGA Abstracts
S-486
with 12 non-IBD family controls in EBV-transformed B cells. The mRNA expression levels of PTPN2 were increased in diseased tissues compared with adjacent normal tissues in 32 CD patients. The overall mean PTPN2/GAPDH expression values were: 4.36 ± 6.5 (diseased), vs. 2.45 ± 3.2 (normal) (p < 0.05). However, mRNA expression levels of PTPN2 were not significantly altered in the 30 UC diseased tissues vs adjacent normal tissues. Our previous cDNA microarray results showed that PTPN2 could be down-regulated by Nkx2-3 knockdown in B cells from CD and UC patients, and human intestinal microvascular endothelial cells (HIMECs). We confirm these results by RT-PCR in Nkx2-3 knockdown B cells from CD and UC, and Nkx2-3 knockdown HIMECs. The mRNA expression levels of Nkx2-3 were increased in 14 CD compared with 14 non-IBD family controls in EBV-transformed B cells (p < 0.05), and were increased in diseased tissues compared with adjacent normal tissues in 32 CD patients (p < 0.05). However, mRNA expression levels of Nkx2-3 were not significantly altered in EBV-transformed B cells and intestinal tissues from UC patients. mRNA expression levels of PTPN2 were positively correlated with those of Nkx2-3 in 14 CD (r = 0.6024, p < 0.01) and 12 UC (r = 0.5928, p < 0.01) from EBV-transformed B cells. Conclusions PTPN2 SNP rs2542152 was associated with CD. mRNA expression of PTPN2 was up-regulated in CD patients from EBV-transformed B cells and intestinal tissues. Expression of PTPN2 was regulated by Nkx2-3 in CD patients.
anti-inflammatory effects of LL-37. Conclusion: Cathelicidins are important modulators of colonic inflammation by mechanisms involving inhibition of NF-kappaB activity in colonocytes. Our results also indicate that exogenous cathelicidin administration may represent a novel approach for treatment of colonic inflammation, including inflammatory bowel disease. Supported by a Pilot and Feasibility Study grant from UCLA-CURE Center, Career Development Award from Crohn's and Colitis Foundation of America and K01DK084256 from National Institute of Health to HWK. Su1742 Targeting Human CD2 by the CB.219 Monoclonal Antibody Protects From Intestinal Inflammation in a Humanized Colitis Model Anja A. Kuehl, Nina N. Pawlowski, Katja Grollich, Ulrike Erben, Martin Zeitz, Joerg C. Hoffmann, Christoph Loddenkemper Purpose: In mouse models, a deficiency in the costimulatory molecule CD2 or simultaneous treatment with anti-mouse CD2 monoclonal antibodies (mAb) reduced intestinal inflammation. Anti-human CD2 mAb inhibited the proliferation of human lymphocytes and their interferon (IFN) γ synthesis In Vitro. Linking both observations, we here asked whether the anti-human CD2 mAb CB.219 counteracts intestinal inflammation in a humanized model of transfer colitis. Methods: Colitis was induced in immmunodeficient RAG 1 ko mice by transfer of CD4CD45RBhi T cells derived from human CD2 transgenic mice. Treatment with CB.219 started either simultaneously with colitis induction (preventive treatment) or after onset of colitis (therapeutic treatment). Mice received 200 μg CB.219 or an isotype control mAb every third day. Development of colitis was observed by body weight and hemoccult assay over time. After 87 days, histological scores were assessed in a blinded fashion. Cytokine profiles were measured by cytometric bead array from supernatants of short-term colon cultures. Results: Mice receiving CB.219 mAb ahead of T cell transfer continuously gained weight, survived throughout the experiment and were completely protected from colitis development. Underlining the protective effect of the mAb, CB.219-treated animals showed significantly reduced histological scores (0.3 ± 0.2 vs. 2.8 ± 0.5, p < 0.01) and increased transforming growth factor (TGF) β secretion in colon cultures compared to controls (19.6 ± 13.5 vs. 5.0 ± 0.4 pg/μg colon, p < 0.05). The CB.219 mAb also worked in a therapeutic setting. Again, the histological colon damage was less severe upon CB.219 treatment (histological score: 0.9 ± 0.2 vs. 2.9 ± 0.4, p < 0.01). Colon organ cultures from CB.219-treated mice revealed a trend to increased TGF β (p = 0.064) and significantly decreased tumor necrosis factor (TNF) α production compared to controls (23.6 ± 5.1 vs. 154.5 ± 54.8 pg/ mg colon, p < 0.01). Attributing this capacity to intestinal immune cells, diminished TNF α secretion was found in lymphocytes isolated from the lamina propria of CB.219-treated mice after short-term In Vitro stimulation via CD3/CD28. These supernatants also contained less interleukin (IL) 2, IL 17A, and IFN γ than the respective controls. Discussion: In our humanized mouse model of colitis the CB.219 mAb specific for human CD2 protected from colitis by altering the local proinflammatory milieu even if applied in established colitis. Thus, we here show that targeting CD2+ T cells provides an interesting new approach for the treatment of colitis in man and further experiments will address gut specificity, underlying mechanisms and general applicability of CB.219 treatment.
Su1740a Interleukin-10 Receptor Gene Polymorphisms Associate with Early-Onset Inflammatory Bowel Disease Christopher J. Moran, Subra Kugathasan, Christoph Klein, Mark S. Silverberg, Aleixo M. Muise, Scott B. Snapper Introduction: Recently, several infants with inflammatory bowel disease (IBD) were found to have homozygous mutations in interleukin-10 receptor (IL-10R) genes (IL10RA or IL10RB). These patients presented with severe enterocolitis refractory to traditional medical and surgical therapies. Their symptoms resolved following allogeneic bone marrow transplantation. We hypothesized that IL-10R genetic mutations play a role in very early onset IBD (VEO-IBD) but less so in the broader IBD population. While single nucleotide polymorphisms (SNPs) in IL-10R have not been reported to associate with risk for IBD in genome-wide association studies (GWAS), current GWAS strategies may not detect at-risk SNPs that are poorly tagged in the GWAS, possess a minor allele frequency <5%, or rare private polymorphisms. We report the largest sequencing effort of the IL-10R genes in early onset IBD to date. Methods: IL-10R genes (IL-10RA and IL-10RB) were deep sequenced by Sanger method in 122 previously collected samples from early onset IBD patients (5 years old and younger) and a group of 185 healthy controls. The odds ratio (OR) for IBD (as well as Crohn’s and ulcerative colitis risk) was calculated under a dominant genetic model using Fisher’s exact test. Results: In IBD patients, 48 SNPs were detected in IL-10RA and 24 SNPs were detected in IL-10RB. rs2228055 (Ile224Val) was associated with IBD (OR 2.38, p= 0.032) and ulcerative colitis (OR 2.52, p=0.029). An additional 4 IL-10RA SNPs were associated with UC. Two SNPs in the 3’ untranslated region of IL-10RB were associated with Crohn’s disease, rs8178561 (OR 0.10, p<0.001) and rs1058867 (OR 2.12, p=0.004). Conclusions: This is the largest sequencing effort to date of the IL-10R genes in early-onset IBD. These results suggest that polymorphisms in IL-10R genes are associated with IBD. Replication of these SNPs in an independent cohort as well as functional studies are required to determine the importance of these identified polymorphisms.
Su1743 Mechanism of Immune Regulation by CD11b+ Myeloid Cells in the Intestine Masako Murai, Petra Krause, Jr-Wen Shui, Rajat Madan, Christopher L. Karp, Mitchell Kronenberg Background and aims: We found that a subset of CD11b+ myeloid cells in the intestinal lamina propria constitutively produce IL-10, and adoptive transfer of these IL-10 producing cells to recipients helped to maintain Foxp3 expression and the function of Treg. Although much emphasis has been placed on the regulatory role of T cell derived IL-10, our data show that IL-10 synthesis by intestinal myeloid cells has a nonredundant and dramatic effect on maintaining Treg function and preventing T cell-mediated colitis in mice. Here, we further investigate the role of intestinal myeloid cell derived IL-10 in the regulation of gut inflammation. Methods: We defined the phenotype of the IL-10-producing myeloid cells in the intestine using IL-10 reporter mice, with reporter+ cells analyzed by quantitative PCR and flow cytometry. The requirement of IL-10 expression by CD11b+ myeloid cells was assessed using mice with a cell type specific-IL-10 gene deletion, under both steady conditions and following Citrobacter rodentium infection. Results: IL-10 producing intestinal myeloid cells expressed higher CD11b, CD11c, F4/80, Gr-1 and CX3CR1 by both messenger RNA and protein determination, compared to their non-IL-10 producing intestinal myeloid counterparts. However, TGFβ1, TGFβ3 and PD-L1 expression was not different between the groups. Furthermore, there were no differences in the ability of reporter+ and negative cells for induction of regulatory T cells (iTreg) In Vitro. Myeloid cell specific IL-10 deficient mice had fewer Treg in the large intestine, but not in the spleen. Futhermore, C. rodentium caused a decrease in the number of Treg in the large intestine, but this defect was more pronounced in mice with a myeloid cell specific (LysM-Cre driven) IL-10 deficiency. Conclusions: IL10 producing myeloid cells have a pivotal role in maintaining Treg population in the intestine especially under inflammatory conditions as exemplified by C. rodentium infection. Supported by grants from CCFA and PO1 grant AI 89624
The numbers associated with each SNP reflect the number of patients possessing at least one variant allele at a given locus. Su1741 Roles of Endogenous and Exogenous Cathelicidin During Colonic Inflammation Hon Wai Koon, David Q. Shih, Tressia C. Hing, Dezheng Zhao, Hua Xu, Mary Pat Moyer, Richard Gallo, Charalabos Pothoulakis Background and Aims: Cathelicidin (LL-37 in human and CRAMP in mice) is a family of endogenous anti-microbial peptides that protect the host from infection as part of the innate immune response. Recent evidences suggest that cathelicidins may modulate inflammation. Here we determine whether cathelicidin can modulate colonic inflammation in animal model of colitis as well as human colonic epithelial cells. Methods: Wild-type and mCRAMP deficient mice were administered with trinitrobenzene sulphonic acid (TNBS) to induce acute (7 days) colitis. Some mice were treated intracolonically with cathelicidin and colonic histological changes and cytokine levels during colonic inflammation were evaluated. Results: TNBS induced acute colitis in mice that was reduced by exogenous intracolonic cathelicidin (mCRAMP) administration (5 mg/kg every 2 days). This inhibitory mCRAMP effect was associated with lower colonic histological damage and reduced levels of the proinflammatory mediators KC, PGE2 and TNFalpha compared to controls. Compared to wild-type mice, mCRAMP deficient mice developed more severe TNBS-induced colonic inflammation associated with higher levels of colonic proinflammatory mediators. Exposure of human colonic epithelial NCM460 cells to LL-37 (1 microM) significantly reduced basal levels of inflammatory mediators (IL-8, PGE2 and TNFalpha), cyclooxygenase-2 protein expression, as well as NF-kappaB phosphorylation and activity. Induction of NF-kappaB activity by phorbol ester (PMA) abolished the anti-inflammatory effects and NF-kappaB activity in response to LL37 in colonic epithelial cells, suggesting that reduced NF-kappaB activity is involved in the
AGA Abstracts
S-486