TNF-α induced Neuropeptide Y (NPY) expression in the enteric nervous system influences intestinal inflammation, epithelial barrier functions and colonic motility

Neuropeptides released from the enteric nervous system can modulate inflammation (neurogenic inflammation), and thus play a significant role in the pathogenesis of Inflammatory Bowel Disease (IBD). We investigated the changes in Neuropeptide Y (NPY) expression in the enteric nervous system in animal models of experimental colitis, and in an enteric neuronal cell line. We also investigated the downstream impact of NPY expression on epithelial barrier functions. Finally the impact of NPY expression and inflammation on colonic motility was assessed. Colitis was induced by dextran sodium sulfate (DSS) or tri nitro benzene sulfonic acid (TNBS) in the mouse strain 129 S3/SvImJ. A group of TNBS treated mice were injected with an NPY Y1 receptor antagonist (BIBP-3222, 10 mg/ kg body weight). Colonic NPY expression was assessed by immuno staining and real time PCR. In addition, ganglia isolated by laser capture micro dissection from colonic cryo sections were also assessed for NPY expression. Mice were evaluated for disease severity through clinical and histological scores and myeloperoxidase assay. Enteric neuronal cell lines were treated with TNF, in presence/ absence of inhibitors of various signaling pathways and assessed for NPY expression. NPY promoter deletion constructs were transfected into the neuronal cells and luciferase activity was used to determine TNF-induced promoter response. Further, the TNF-induced transcription factors that modulate NPY promoter activity was determined via chromatin immunoprecipitation (ChIP). Site directed mutagenesis was done to determine the specificity of the factor binding. WT and NPY knockout mice (NPY KO) were gavaged with FITC-Dextran (FD-4) and colonic permeability was assessed. FD-4 permeability across the isolated colonic muscle strips from these mice were tested in Ussing chamber. Caco2-BBE cells were treated with NPY and transepithelial resistance (TER), FD-4 flux and claudin-2 protein were measured. Neuronal cell lines treated with TNF (+/- NPY SiRNA) were assessed for apoptosis via cleaved caspase-3 staining. Neuronal nitric oxide synthase (nNOS) expression was determined in TNF-treated neurons. Colonic motility in control and DSS- colitic mice were assessed by isometric muscle recording and number of nitrergic and cholinergic neurons were assessed in the colon. Colonic NPY was up regulated during colitis and in TNF-a-treated enteric neurons in vitro via phosphatidyl-inositol-3-kinase (PI-3-K) and mitogen activated protein kinase pathways. Inflammation was attenuated in mice treated with Y1 receptor antagonist compared to untreated TNBS colitis. NPY promoter activity was significantly induced by TNF-α via increased c-Jun phosphorylation. NPY increased epithelial permeability in vitro by increasing Claudin-2 expression and protein via P-I-3-K. WT mice exhibited increased permeability than NPY KO as observed from macromolecular flux and TER. Murine chronic colitis involved enteric neuronal apoptosis that resulted in impaired colonic contraction and relaxation. We demonstrate that TNF-α-induced apoptosis in enteric neurons requires NPY and involves neuronal nitric oxide synthase (nNOS) upregulation. To our knowledge, this study is the first to demonstrate that NPY expression, inducible by pro-inflammatory signals like TNF-α, can orchestrate epithelial barrier function and colonic motility. Hence NPY may be a useful therapeutic target for treating inflammatory disorders like IBD.

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