Stimulation of T-Cells in Irritable Bowel Syndrome (IBS) Mucosal Biopsy Tissue Releases Cytokines Which Selectively Activate Submucosal Neurons

Abstract

Background: Irritable Bowel Syndrome (IBS) is a common gastrointestinal disorder of unknown aetiology affecting 10-20% of adults. Symptoms include abdominal pain or discomfort and disordered defecation. There is evidence of a disturbed nerve function along the brain-gut axis with an altered immune profile in the blood, intestinal mucosa and submucosa of IBS patients. Subtle increases in plasma cytokines and immune cells including T-lymphocytes in the intestine of IBS patients have been reported. Aim: In this study we investigated the activation status of inflammatory signalling pathways and T-cells in colonic tissue from IBS patients. We also examined the effect of T-cell activation on submucosal neuronal function. Methods: Total RNA was isolated from colon biopsy tissue of healthy subjects, Inflammatory Bowel Disease (IBD) and IBS patients (n=10) and assayed for the expression of 34 genes from the Interferon inflammatory signalling pathway and for T-cell transcription factor genes including T-bet, GATA3, RORgammat and FOXP3. Colon biopsies from healthy (n=13) and IBS (n=30) were cultured ex vivo for 24 hours, left unstimulated or were stimulated with anti-CD3/CD28 to active T-cells within the tissue. Supernatants were assayed for cytokine and chemokine content using the MSDTMplatform and ELISA. A pool of supernatant (n=10) from the ex vivo stimulated healthy and IBS biopsies was added to rat submucosal neuronal cultures and Ca+ responses were measured. Results: There was significant upregulation of genes (STAT1, IRF-1, GBP1) from the Interferon pathway in IBD biopsy tissue relative to healthy controls but no such increase was detected in IBS tissue. In fact, decreased expression of Interferon pathway genes and T-cell transcription factor genes were detected in IBS tissue. Additionally, there was also a significant decrease in the secretion of IL-12p70 (p<0.03), TNFalpha (p<0.02) and IFNalpha2alpha (p<0.002) from IBS biopsies relative to healthy controls at basal levels. In contrast, ex vivo stimulated IBS biopsies displayed a hyperresponsiveness to anti-CD3/CD28 stimulation for the production of IL-17, CXCL9, CXCL10, MCP-1 and MIP-1beta compared to stimulated healthy controls. There was also a selective increase in calcium response from rat submucosal neurons exposed to conditioned media from IBS biopsies stimulated with anti-CD3/CD28. This effect was blocked by neutralizing antibodies to IL-17, IL-6 and CXCL10. Conclusion: Decreased expression of inflammatory and T-cell transcription factor genes in IBS patients compared to IBD patients and healthy controls suggests that IBS mucosa is not classically inflamed. However, a hyper-responsiveness to T-cell activation and T-cell induced neuronal responses suggests an altered immune response to stimuli in IBS. This capacity for altered responsiveness within the tissue may contribute to IBS pathogenesis and symptomology.