The Role Of Intestinal Mucosal Lymphocytes In Dextran Sulphate Sodium Induced Colitis In Mice

Inflammatory bowel diseases (IBD) include ulcerative colitis (UC) and Crohn's disease (CD), which are the major chronic inflammatory diseases of the gastrointestinal tract in humans. Although the causes of these disorders remain unknown, various features strongly suggest the involvement of immune responses, particularly autoimmune reactions, in the pathogenesis of IBD. Tolerance to normal flora seems to be disrupted in IBD patients, suggesting an active immune response against luminal antigens. IBD patients have elevated serum antibodies against dietary antigens. Therefore, the study of oral tolerance may have a significant clinical impact on IBD management. Several low doses of antigens induce tolerance through the negative immunoregulatory cytokines secreted by regulatory T cells. It was reported that oral colitis-extracted proteins (CEP) or normal colon-extracted proteins (NCEP) with five low doses can prevent experimental colitis induced by 2,4, 6-trinitrobenesulfonic acid (TNBS) in rodent animals. Tolerance induction was mediated by immunosuppressive cytokines such as TGF-β1, IL-4, IL-10 and so on.γδT cells are preferentially localized in the epithelial tissues. Multiple functions have been ascribed toγδT cells. They induce cytolysis of infected or transformed intestinal epithelial cells, support mucosal IgA production and maintain homeostasis of the intestinal epithelium through an intranet withαβT and epithelial cells.γδT cells may also play an important regulatory role in oral tolerance induction. However, both the beneficial and detrimental roles ofγδT cells in the inflammatory process are evident. The role ofγδT cells in IBD patients and animal models is still controversial. The relative contributions of intestinal mucosal y8 T cells in the protective effect of oral tolerance on IBD also remain unclear. The CD8+T cells are abundant in the intestinal mucosa, especially in the intraepithelial compartment. It was reported that CD8+ regulatory T cells could play suppressive effects on IBD. Splenic CD8+Treg are induced upon oral antigen feeding. Despite of much recent interest in the intestinal mucosal CD8+T cells, the immunological function of these cells in oral tolerance is less understood.Therefore, we evaluated the changes of CD8+T cells and TCRγδ+T cells in gut-associated lymphoid tissues (GALT) and spleens in dextran sulfate sodium (DSS)-induced colitis mice and CEP-fed DSS-induced colitis mice by flow cytometry (FCM). Then we evaluated the roles of SI-IEL or SI-γδIEL sorted by magnetic activated cell sorting (MACS) technology in DSS-induced colitis by adoptive transfer of SI-IEL or SI-y8 IEL from untreated, colitis and CEP-fed colitis mice.Part I Improvement of colitis is associated with increases of CD8α+and TCRγδ+T cells in large intestinal mucosaAcute colitis was induced in BALB/c mice by treatment with 4% DSS in drinking water for 7 days. The daily disease activity index (DAI) was evaluated and the large intestine from the anus to the splenic flexure area was used to estimate histological scores. DAI and histological scores in DSS-treated mice were significantly higher than those in untreated mice.The colons were removed from DSS-induced colitis mice and used for preparation of oral antigen. CEP was orally administered into mice using a feeding atraumatic needle,250μg/mouse in 200μl, every other day for five doses. Bovine serum albumin (BSA) was used in parallel with a protocol identical to that used with CEP. After three days of rest, mice were given 4% DSS for 7days. The daily monitored disease activity index was reduced in CEP-fed colitis mice (n=12) compared with that of BSA-fed colitis mice (n=12) from day 4 to 7. There is significant difference in histological scores between two groups (33.6±2.8 vs 25.4±5.0, p<0.01).The relationship between histological scores and DAI of the last day correlated well (r=0.78,p<0.01).Serum TGF-β1 and IFN-y levels were measured in CEP-fed colitis mice, colitis mice, and untreated mice by enzyme-linked immunosorbent assay (ELISA). Compared to untreated mice, colitis mice demonstrated significantly lower levels of serum TGF-β1. The feeding of CEP induced a two-to three-fold increase in serum TGF-β1 levels, compared to the BSA-fed colitis mice. However, no significant change was found in serum IFN-y levels.Then percentages of CD3+, TCRγδ+, CD8α+and CD8α+TCRγδ+T cells in intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL) of small intestine (SI) and large intestine (LI), Peyer's patches (PP), the spleens and mesenteric lymph nodes (MLN) from model mice (n=8 each group) were determined by flow cytometry.The proportion of TCRγδ+T cells and CD8a+TCRγδ+T cells in SI-IEL and SI-LPL from colitis mice was significantly higher compared to untreated mice. Accordingly, TCRγδ- T cells and CD8a+TCRγδ- T cells were significantly reduced in SI-IEL and SI-LPL from colitis mice. The proportion of TCRγδ+T cells, CD8α+TCRyγδ+T cells, CD8α+T cells and CD8a+TCRγδ- T cells in LI-IEL and LI-LPL from CEP-fed colitis mice was significantly higher compared to BSA-fed colitis mice. Accordingly, TCRγδ-T cells and CD8αTCRγδ-T cells were significantly reduced. The relationship between DAI of the last day and percentages of TCRγδ+T cells/CD8α+T cells in large intestinal mucosa was also negatively correlated well (p<0.05).From the results of CEP-fed DSS mice, it seemed that improvement of colitis was accompanied by increases of CD8α+T cells and TCRγδ+T cells in large intestinal mucosal lymphocytes. Hence, the percentages of these cells in repair-period mice five days after termination of DSS treatment were also investigated. The proportion of TCRγδ+T cells, CD8α+TCRγδ+T cells, CD8α+T cells and CD8α+TCRγδ- T cells in LI-IEL and LI-LPL from repair-period mice was significantly higher compared to untreated mice. Accordingly, TCRγδ- T cells and CD8α-TCRγδ- T cells were significantly reduced.The proportion of CD8α+T cells in spleens from CEP-fed colitis mice was significantly higher compared to BSA-fed colitis mice. The proportion of CD3+T cells in PP from colitis mice was significantly higher than that from untreated mice. The proportion of CD8α+T cells was also increased. The proportion of CD3+T cells and CD8α+T cells from CEP-fed colitis mice was significantly lower compared to BSA-fed colitis mice. The proportion of CD3+T cells in MLN from colitis mice was significantly lower than that from untreated mice. The proportion of CD8α+T cells was also reduced. The proportion of CD3+T cells and CD8α+T cells from CEP-fed colitis mice was significantly higher compared to BSA-fed colitis mice.In conclusion, oral administration of colitis-extracted proteins could alleviate experimental colitis. Improvement of colitis resulted from oral administration of antigen was accompanied by increases of CD8α+T cells and TCRγδ+T cells in large intestinal mucosal lymphocytes. Mucosal repair in repair-period mice was also accompanied by increases of CD8α+T cells and TCRγδ+T cells in large intestinal mucosal lymphocytes. These results support protective regulatory role of intestinal mucosal CD8α+T cells and TCRγδ+T cells in DSS-induced colitis. Oral tolerance induced an increase of CD8α+T cells in spleens. PP and MLN may play differential roles not only in DSS-induced colitis but also in oral immune regulation.PartⅡIsolation of murine small intestinal intraepithelialγδT cellsγδT cells represent a small leukocyte subpopulation in the peripheral blood and most lymphoid organs. However, they are enriched at epithelial barriers such as the gastrointestinal mucosa. The experiments presented here describe a purification procedure for small intestinal intraepithelialγδT lymphocytes in BALB/c mice.Each small intestine was removed from BALB/c mice, everted and shaken to individualize epithelial content. In some experiments, the discrete epithelium was directly centrifuged using discontinuous 40%-70% Percoll gradients. In other experiments, the discrete epithelium was first centrifuged in 10 ml of 30% Percoll to remove the most of epithelial cells and then centrifuged in a discontinuous 40%-70% Percoll gradient. The time required for the SI-IEL isolation using two-step Percoll centrifugation was-2 h, while less half an hour for one-step Percoll centrifugation. From a large series of SI-IEL isolates (n=8 each group), there was a significant increase in the purity of SI-IEL after two-step purification (93.2%) compared to the purity following one-step purification (88.8%), thus representing an overall increase in purity of 4.4%. The contaminating cells were mainly epithelial cells. There was no significant difference in the yields between two groups. The recovery of SI-IEL remained both high, with 0.92-1.34×107 cells (mean value of 1.123×107) after two-step Percoll and 0.81-1.48×107 cells (mean value of 1.141×107) after one-step Percoll centrifugation procedure. Percentages of CD3+, TCRγδ+, CD8α+ and CD8a+TCRγδ+T cells in SI-IEL from BALB/c mice were determined by three color fluorescence flow cytometry. SI-IEL contained mostly T lymphocytes and majority of these cells were CD8a+. TCRγδ+T cells on CD3+gating were abundant, comprising up to 30-50% of the total cell population. Approximately 90%of TCRγδ+T cells were CD8a+. The phenotypic composition of the cell populations was also compared between the two SI-IEL purification protocols. Results showed no significant difference in the proportional distribution of SI-IEL subsets from the two purification protocolsSI-IEL isolated following two-step Percoll purification procedure was used for separation of TCRγδ+T cells. During the process, the amount of antibody and microbeads needed to be optimized to get sufficient purity and recovery. Optimal purity and recovery were achieved when 1.5×10 cells were stained with 3μg of PE-conjugated anti-TCRδ-chain antibody and sorted with 45μl of anti-PE microbeads. SI-IEL could produce much more IFN-y and TGF-β1 than sortedγδT cells upon stimulation with plate-bound anti-CD3εmAb. y8 T cells secrete moderate amounts of TGF-β1 and minimal amounts of IFN-γ.In summary, the SI-IEL isolation protocol described here is a reliable and simple method with high purity and high yields. One-step Percoll purification by discontinuous 40%-70% Percoll gradients is less laborious yet it is sufficient to get exact profiles for use in phenotypic analysis of cell subsets. SI-IEL isolated after two-step Percoll centrifugation by 10 ml of 30% Percoll and then by discontinuous 40%-70% Percoll gradients are fit to purify y8 T cells by MACS. Purification of y8 T cells using an appropriate method is helpful in investigating the phenotype and function of intestinal mucosalγδT cells.PartⅢProtective role of small intestinal intraepithelial lymphocytes in murine colitis induced by dextran sulfate sodiumTo research the roles of SI-IEL and y8 T cells in DSS-induced colitis, SI-IEL (3×106) or SI-y8 IEL (9×105) from untreated mice, colitis mice, CEP-fed colitis mice and BSA-fed colitis mice were injected into naive mice (n= 8 each group) via tail vein in 150μl of PBS. Then mice were given 4% DSS for 7 days. Results showed that transfer of SI-IELs or sorted y8 T cells from untreated and colitis mice alleviated experimental colitis. Transfer of SI-IELs or sortedγδT cells from CEP-fed and BSA-fed colitis mice also alleviated experimental colitis. The protective effects of SI-IEL from CEP-fed colitis mice were stronger than SI-IEL from BSA-fed colitis mice. The DAI and histological scores of colitis mice upon transfer of SI-IEL were less than those of colitis mice upon transfer of SI-γδIEL on day 7, but differences were not statistically significant in this experiment. SI-IEL and sorted y8 T cells from CEP-fed colitis mice produced more TGF-β1 than SI-IEL and sortedγδT cells from BSA-fed colitis mice upon stimulation with plate-bound anti-CD3εmAb. No difference in IFN-y production was found in supernatants of SI-IELs or sortedγδT cells between CEP-fed and BSA-fed colitis mice, or untreated and colitis mice. Serum TGF-β1 and IFN-y levels were measured in cell-transferred colitis mice by ELISA. Transfer of cells, including SI-IEL and sortedγδT cells from untreated, colitis, CEP-fed colitis and BSA-fed colitis mice, induced a two-to three-fold increase in serum TGF-β1 levels of the recipient mice. The production of serum TGF-β1 was negatively related to weight loss and histological scores of colitis (r=-0.762 and r=-0.770, p= 0.002 and p= 0.002). There were higher levels in colitis mice following transfer of SI-IEL from CEP-fed colitis mice than from BSA-fed colitis mice (p= 0.04). No significant difference was detected in serum IFN-y levels among cell-transferred mice. In vitro culture showed SI-IEL responded poorly to anti-CD3s antibody and ConA.In summary, SI-IEL and sortedγδT cells play protective roles in DSS-induced colitis in B ALB/c mice. Oral tolerance strengthens the suppressive effects of regulatory subsets in SI-IEL, includingγδT cells. TGF-β1 seems to be the best candidate for mediating these suppressive effects. Future studies should explore the therapeutic potential of oral immune regulation, regulatory cells and immunosuppressive cytokines in human IBD.