Study on the effect of wild bitter melon on regulatory T cells and further modulation of intestinal immune system

Chronic inflammation and immune disorder are associated with insulin resistance, allergy, inflammatory bowel disease, etc. Regulatory T lymphocytes (Treg) suppress a variety of inflammatory and pathological immune responses. Wild bitter melon (WBM) has been demonstrated to improve the insulin resistance, but its effect on immune is still unclear. Therefore, the aim of this study is to investigate the effect of WBM on the intestinal immune system and Treg development. First, to investigate the improvement in insulin resistance of wild bitter melon is involved with its immune modulatory effects. High-fat diet-induced obese C57BL/6J mice were fed the diet containing 5% WBM powder for 12 weeks. The results showed that WBM treatment reduced fasting glucose and triglyceride in the serum, and improved insulin resistance. WBM reduced the M1/M2 ratio and CD4+ T cells infiltration in stromal vascular fraction (SVF). Moreover, WBM increased the percentage of CD4+Foxp3+ Treg in mesenteric lymph nodes (MLN). To confirm the effects of WBM on intestinal immune system and Foxp3+ Treg cells population, Foxp3-EGFP mice co-expressing EGFP and Foxp3 in Treg were fed WBM experimental diet. The results showed that WBM also increased the percentage of CD4+ Foxp3+ Treg in MLN and interleukin (IL)-10 and TGF (transforming growth factor)-β secretion by ConA-stimulated Peyer’s patches and MLN cells. These results suggested that WBM could enhance the Foxp3+ Treg development in the intestinal immune system. The bioactive compounds of WBM and mechanisms of promoting the development of Treg were investigated by using the primary spleen cells, MLN cells, CD4+ T cells and bone marrow derived dendritic cells (BMDC) culture in vitro. The results showed that M-Res and BuE extract of WBM increased IL-10 and TGF-β secretion by splenocytes and promoted the development of Foxp3+ Treg through the aryl hydrocarbon receptor (AhR) pathway. WE extract increased IL-10 secretion by BMDC and promoted CD4+ T cells differentiate into IL-10+ T cells and Foxp3+ T cells in CD4+ T cells cocultured with WE pretreated BMDC. Moreover, tryptophan is the bioactive compound from BuE extract was found that increase Foxp3+ Treg development in vitro. The anti-inflammatory and immunomodulatory effects of WBM and tryptophan on inflammatory bowel disease were investigated by using dextran sodium sulphate (DSS)- induced colitis murine model. A diet containing 5% WBM or 0.5% tryptophan was administered to mice for 2 or 6 weeks and then gave 7 days DSS treatment. WBM intake significantly improved weight loss, disease activity index and colon shortening. Colonic cytokine IL-6 and IL-1β were significantly decreased, and the anti-inflammatory cytokine IL-10 was increased in WBM-fed mice. The mRNA expression of TGF-β and Foxp3 in colon, and Foxp3+ Treg cell population in the MLN were also significantly higher in the WBM group. Tryptophan supplement ameliorated disease activity index and reduced the colonic IL-6 levels. The significantly negative correlation between Foxp3 mRNA expression and pro-inflammatory cytokines in colon is in accordance with the critical role of Treg cell for regulation of intestinal immune homeostasis. These results suggested that WBM reduces the inflammatory response and induces higher level of Treg cells in DSS-induced colitic mice. In summary, WBM enhanced Treg development in intestinal immune system in both in vitro and in vivo. The mechanism of enhancing Treg development by WBM are not only through AhR pathway, but also increased the CD103+ dendritic cells and IL-10 secretion. Furthermore, WBM inhibited the inflammatory response caused by colitis and induces higher level of Treg cells in the intestinal immune system. These results suggested that WBM could exert the anti-inflammatory and immunoregulatory effects in maintaining intestinal immune balance and alleviating the severity of the symptoms on inflammatory bowel disease.