Role Of Intestinal Glucocorticoid In The Regulation Of Anti-viral CD8~+ T Cell Immune Responses

Intestine is not only the main organ for digestion and absorption,but also an important place for immune response.CD8⁺T cells are recognized as cytotoxic T lymphocyte（CTL）once they become activated by viruses or intracellular bacteria.The uncontrolled activation of intestinal CTL leads to disrupt the intestine epithelial barrier integrity,which may have either a direct cell death-promoting effect on intestinal epithelial cells,or may disrupt tight junctions.These observations clearly point out the need of a stringent control of immune responses in the intestinal mucosa,in order to protect the host from invasion by pathogens.Glucocorticoids（GC）are steroid hormones predominantly produced in the adrenal glands to regulation of strong immune responses and inflammation in body.Recently,studies has revealed that intestine is also capable of producing GC.The role of local GC synthesis in the regulation of anti-viral immune responses in the gut is presently less well studied.The role of local GC synthesis in the regulation of normal protective immune responses in the gut is presently less well studied.LRH1（liver receptor homolog 1）,which is highly expressed in the intestinal epithelium and regulates the expression of steroidogenic enzymes.SHP（small heterodimer partner）is a target gene of LRH1,but at the same time it interacts with LRH1 and inhibits its transcriptional activity.1.CD8⁺T cell activation promoted intestinal GC synthesis.CD3εmonoclonal antibody was injected intraperitoneally into C57BL/6 mice.Flow cytometry was employed to detect the activation of CD8⁺T cells.The results showed that CD69 and CD25 were expressed on CD8⁺T cells in mesenteric lymph node lymphocyte（MLNL）,Peyer’s patches lymphocyte（PPL）and intraepithelial lymphocyte（IEL）.There were 80.9%,86.9%,72.97%of CD8αβ⁺T cells expressing CD69 in MLNL,PPL,IEL respectively and 48.1%,45.47%,41.8%of CD8αβ⁺T cells expressing CD25 in MLNL,PPL,IEL respectively.Radioimmunoassay（RIA）showed that the concentration of small and large intestinal GC in mice injected with anti-CD3εwas 29 and 26 ng corticosterone/g intestine,respectively,while the control group was only 0.6 and 0.2 ng corticosterone/g intestine,respectively.LRH1^+/+mice and LRH1^-/-mice were injected with anti-CD3ε.When the LRH1 gene was knockout in the intestinal epithelial cells,the synthesis of intestinal GC was inhibited.While in the small intestine was about 60 ng corticosterone/g intestine in LRH1^+/+mice and only about 15 ng corticosterone/g intestine in LRH1^-/-mice（p<0.05）,GC synthesis in the large intestine was about 33 ng corticosterone/g intestine in LRH1^+/+mice and only about 4.5 ng corticosterone/g intestine in LRH1^-/-mice（p<0.01）.Additionally,the intestinal GC could inhibit the expression of CD25 in the CD8⁺T cells but promote the expression of CD69 in CD8⁺T cells.This meaned the intestinal GC could regulate local immune responses in two-ways.2.The intestinal GC synthesis was time-dependent after LCMV infection.C57BL/6 mice were injected with Lymphocytic choriomeningitis virus（LCMV）via tail vein.The virus could stimulate the activation of intestinal CD8αβ⁺T cells and the expansion of intestinal CD8αβ⁺TCRαβ⁺T cells.The peak numbers of CD69⁺CD8αβ⁺T cells and CD25⁺CD8αβ⁺T cells were detected at 4 dpi and 6 dpi respectively by flow cytometry.The number of CD8αβ⁺TCRαβ⁺T cells in MLNL,PPL and IEL at 0,4,6,8,and 10 dpi were 19%,2.7%,7.4%;15.3%,5.4%,6.1%;25.5%,11%,20.2%;26.2%,22.9%,23.8%;20.6%,11.2%,11.1%,respectively,and of which the highest numbers were observed at 8dpi.The steroidogenic enzymes of intestinal GC were dectected by qRT-PCR.The results showed that the highest value of CYP11A1 was dectected at 4 dpi in small intestine and large intestine,and the peak value of CYP11B1 was measured at 6dpi in the small intestine and at 4 dpi in the large intestine.Accordingly,intestinal GC synthesized post LCMV infection,and the highest value of GC was measured at 6 dpi in small intestine with around 12 ng corticosterone/g intestine and at 8 dpi in large intestine with around 13ng corticosterone/g intestine.The virus titers were detected by plaque assay and the results showed that the highest titers in the spleen,liver and intestine were detected at 6 dpi.It could be seen that CD8⁺T cell immune response and intestinal GC synthesis changed along with LCMV infection.3.Inhibition of intestinal GC synthesis promoted CD8⁺T cell immune responsesAfter LCMV infection in LRH1^+/+mice and LRH1^-/-mice,qRT-PCR assay was used to detect the expression of steroidogenic enzymes of CYP11A1 and CYP11B1.The results showed that the expression of steroidogenic enzymes of CYP11A1 and CYP11B1 in the LRH1^-/-mice was lower than those in the LRH1^+/+mice.Accordingly the synthesis of intestinal GC in the LRH1^-/-mice was inhibited after LCMV infection.The values of intestinal GC in small intestine of LRH1^+/+mice and LRH1^-/-mice were 10.4,2.7 ng corticosterone/g intestine at 6 dpi respectively and were 5.7,1.2 ng corticosterone/g intestine at 8 dpi respectively.There were 7.1,1.1 ng corticosterone/g intestine at 6dpi respectively and 16.5,1.7 ng corticosterone/g intestine at 8dpi respectively in large intestine of LRH1^+/+mice and LRH1^-/-mice.It could be seen that the amounts of intestinal GC synthesis in LRH1^-/-mice were decreased（p<0.05）.LCMV could activate intestinal CD8αβ⁺T cells not only in LRH1^+/+mice but also in LRH1^-/-mice.The number of intestinal CD69⁺CD8αβ⁺T cells or CD8αβ⁺TCRαβ⁺T cells in LRH1^-/-mice was more than that in LRH1^+/+mice.However,the number of intestinal CD25⁺CD8αβ⁺T cells in LRH1^-/-mice was less than that in LRH1^+/+mice.The expression of granzyme B,perforin and IFNγwere increased in LRH1^-/-mice.MLNL was stimulated with LCMV peptide GP33 in vitro,and around 7.5%and 14.3%CD8⁺T cells could express IFNγin LRH1^+/+mice and LRH1^-/-mice at 8dpi respectively（p<0.05）.4.Enhancement of intestinal GC synthesis inhibited CD8⁺T cell immune responsesAfter LCMV infection,the expressions of LRH1,CYP11A1 and CYP11B1 genes by qRT-PCR were enhanced in small and large intestines in SHP^-/-mice compared those in SHP^+/+mice.The results of RIA showed that the intestinal GC synthesis in SHP^-/-mice was stronger than that in SHP^+/+mice.The amounts of small intestinal GC synthesis in SHP^+/+mice and SHP^-/-mice were 2.8,5.8 ng corticosterone/g intestine at 6 dpi,respectively and were 5.5,9.4 ng corticosterone/g intestine at 8 dpi,respectively.The values of large intestinal GC synthesis in SHP^+/+mice and SHP^-/-mice were 3.4,7.4 ng corticosterone/g intestine at 6 dpi,respectively and were 11.9,21.4 ng corticosterone/g intestine at 8 dpi,respectively.The numbers of CD69⁺CD8αβ⁺T cells and CD8αβ⁺TCRαβ⁺T cells were lower in SHP^-/-mice than in SHP^+/+mice,but the amounts of CD25⁺CD8αβ⁺T cells were higher in SHP^-/-mice than in SHP^+/+mice.The expression of granzyme B,perforin and IFNγin small and large intestines of SHP^-/-mice were less than those in the intestines of SHP^+/+mice.The percentages of anti-LCMV IFNγ⁺CD8⁺T cells were 11.8%,7.7%respectively at 6 dpi and were 6.12%,3.2%respectively at 8 dpi in MLNL of SHP^-/-mice and SHP^+/+mice（p<0.05）.Collectively,intestinal GC synthesized after CD8⁺T cell activation.The local GC regulated the intestinal anti-viral CD8⁺T cell immune response.Moreover,intestinal GC played both inhibitory and stimulative effects in regulating intestinal CD8⁺T cell activation.It was noteworthy that intestinal GC inhibited the cytotoxic effector functions in intestine.LRH1 and SHP critically regulated intestinal GC synthesis in the intestinal epithelium could represent an interesting targets in the treatment of inflammatory disorders.