Kai Fang

Application of Comparative Transcriptional Genomics to Identify Molecular Targets for Pediatric IBD

Experimental models of colitis in mice have been used extensively for analyzing the molecular events that occur during inflammatory bowel disease (IBD) development. However, it is uncertain to what extent the experimental models reproduce features of human IBD. This is largely due to the lack of precise methods for direct and comprehensive comparison of mouse and human inflamed colon tissue at the molecular level. Here, we use global gene expression patterns of two sets of pediatric IBD and two mouse models of colitis to obtain a direct comparison of the genome signatures of mouse and human IBD. By comparing the two sets of pediatric IBD microarray data, we found 83 genes were differentially expressed in a similar manner between pediatric Crohn’s disease and ulcerative colitis. Up-regulation of the chemokine (C–C motif) ligand 2 (CCL2) gene that maps to 17q12, a confirmed IBD susceptibility loci, indicates that our comparison study can reveal known genetic associations with IBD. In comparing pediatric IBD and experimental colitis microarray data, we found common signatures amongst them including: (1) up-regulation of CXCL9 and S100A8; (2) cytokine–cytokine receptor pathway dysregulation; and (3) over-represented IRF1 and IRF2 transcription binding sites in the promoter region of up-regulated genes, and HNF1A and Lhx3 binding sites were over-represented in the promoter region of the down-regulated genes. In summary, this study provides a comprehensive view of transcriptome changes between different pediatric IBD populations in comparison with different colitis models. These findings reveal several new molecular targets for further study in the regulation of colitis.

MicroRNA-31-3p Is Involved in Substance P (SP)-Associated Inflammation in Human Colonic Epithelial Cells and Experimental Colitis

Substance P (SP) mediates colitis. SP signaling regulates the expression of several miRNAs, including miR-31-3p, in human colonocytes. However, the role of miR-31-3p in colitis and the underlying mechanisms has not been elucidated. We performed real-time PCR analysis of miR-31-3p expression in human colonic epithelial cells overexpressing neurokinin-1 receptor (NCM460 NK-1R) in response to SP stimulation and in NCM460 cells after IL-6, IL8, tumor necrosis factor (TNF)-α, and interferon-γ exposure. Functions of miR-31-3p were tested in NCM460-NK-1R cells and the trinitrobenzene sulfonic acid (TNBS) and dextran sodium sulfate (DSS) models of colitis. Targets of miRNA-31-3p were confirmed by Western blot analysis and luciferase reporter assay. Jun N-terminal kinase inhibition decreased SP-induced miR-31-3p expression. miR-31-3p expression was increased in both TNBS- and DSS-induced colitis and human colonic biopsies from ulcerative colitis, compared with controls. Intracolonic administration of a miR-31-3p chemical inhibitor exacerbated TNBS- and DSS-induced colitis and increased colonic TNF-α, CXCL10, and chemokine (C-C motif) ligand 2 (CCL2) mRNA expression. Conversely, overexpression of miR-31-3p ameliorated the severity of DSS-induced colitis. Bioinformatic, luciferase reporter assay, and Western blot analyses identified RhoA as a target of miR-31-3p in NCM460 cells. Constitutive activation of RhoA led to increased expression of CCL2, IL6, TNF-α, and CXCL10 in NCM460-NK-1R cells on SP stimulation. Our results reveal a novel SP-miR-31-3p-RhoA pathway that protects from colitis. The use of miR-31-3p mimics may be a promising approach for colitis treatment.

Application of Comparative Functional Genomics to Identify New Pathways of Pediatric IBD Pathogenesis: P-185 YI

human immune cells in vivo in a xenobiotic setting. Using this approach, we have developed two novel mouse models of intestinal inflammation that are dependent on human T cells. METHODS: For anti-CD3 enteritis, 8-10 week old NSG mice were injected with 2x10human CD4þ (hCD4þ) T cells, and12 days later, mice received either a single i.p. injection of OKT3 or control IgG. Serum was collected at 0,1,6, and 24 hours and analyzed for human cytokines by luminex. Intestinal inflammation was assessed after 24 hours based on clinical and histological scores. T cell activation was quantified by flow cytometry for spleen and small intestine lamina propria. qPCR was performed on RNA isolated from splenic T cells and small intestine RNA. For TNBS-induced colitis, two days following transfer of 2 x10hCD4þ T cells, mice were sensitized with 1% TNBS solution applied to 1 cm2 patch of skin at the base of the neck. One week post-sensitization, mice were administered a single intrarectal dose of 0.125 mg TNBS in 50% ethanol. Weight loss was monitored daily and mice were sacrificed three days following the TNBS enema. Colonic inflammation was assessed by H&E stained colon sections. RESULTS: OKT administration resulted in diarrhea and severe small intestine villous atrophy within 24 hours, only in hCD4þ reconstituted NSG mice. In addition, OKT3 promoted T cell recruitment into the SI lamina propria, reduced cell surface CD3 expression and concomitant increase in CD25, a marker of activation. NSG mice receiving OKT3 in the absence of hCD4þ T cells were free of clinical or histological signs of inflammation, indicating that hCD4þ T cells are necessary for OKT3-induced enteritis. hCD4þ reconstituted NSG mice, following sensitization and exposure to TNBS, exhibited clinical and histological features of colitis in contrast to control NSG mice lacking hCD4þ T cells. CONCLUSION(S): Human CD4þ T cells adoptively transferred into NSG mice can induce small bowel and colonic inflammation in mice following exposure to OKT3 and TNBS respectively. These models will facilitate preclinical assessment of therapeutic applications targeting T cells and aid in mechanistic studies evaluating pathways controlling mucosal homeostasis.

Leukocyte Recruitment Alters Pathological Angiogenesis Gene Expression During DSS Colitis: P-184 YI

human immune cells in vivo in a xenobiotic setting. Using this approach, we have developed two novel mouse models of intestinal inflammation that are dependent on human T cells. METHODS: For anti-CD3 enteritis, 8-10 week old NSG mice were injected with 2x10human CD4þ (hCD4þ) T cells, and12 days later, mice received either a single i.p. injection of OKT3 or control IgG. Serum was collected at 0,1,6, and 24 hours and analyzed for human cytokines by luminex. Intestinal inflammation was assessed after 24 hours based on clinical and histological scores. T cell activation was quantified by flow cytometry for spleen and small intestine lamina propria. qPCR was performed on RNA isolated from splenic T cells and small intestine RNA. For TNBS-induced colitis, two days following transfer of 2 x10hCD4þ T cells, mice were sensitized with 1% TNBS solution applied to 1 cm2 patch of skin at the base of the neck. One week post-sensitization, mice were administered a single intrarectal dose of 0.125 mg TNBS in 50% ethanol. Weight loss was monitored daily and mice were sacrificed three days following the TNBS enema. Colonic inflammation was assessed by H&E stained colon sections. RESULTS: OKT administration resulted in diarrhea and severe small intestine villous atrophy within 24 hours, only in hCD4þ reconstituted NSG mice. In addition, OKT3 promoted T cell recruitment into the SI lamina propria, reduced cell surface CD3 expression and concomitant increase in CD25, a marker of activation. NSG mice receiving OKT3 in the absence of hCD4þ T cells were free of clinical or histological signs of inflammation, indicating that hCD4þ T cells are necessary for OKT3-induced enteritis. hCD4þ reconstituted NSG mice, following sensitization and exposure to TNBS, exhibited clinical and histological features of colitis in contrast to control NSG mice lacking hCD4þ T cells. CONCLUSION(S): Human CD4þ T cells adoptively transferred into NSG mice can induce small bowel and colonic inflammation in mice following exposure to OKT3 and TNBS respectively. These models will facilitate preclinical assessment of therapeutic applications targeting T cells and aid in mechanistic studies evaluating pathways controlling mucosal homeostasis.