Study Of The Immunologic Mechanism Of Plasmid PGP3 Mediating Chlamydia Descending Spread From Small Intestine To Large Intestine

BackgroundChlamydia trachomatis(CT)is one of the most common pathogens of sexually transmitted diseases in the world.Its infection is stealthily,and patients often delay treatment due to lack of conscious symptoms.Chlamydia is also frequently detected in the gastrointestinal(GI)tract,and the chlamydia colonized in the small intestine is usually cleared by the intestinal mucosal immune response of the host within 28 days after infection.Once chlamydia crosses the small intestine and reaches the large intestine,it can be colonized in the large intestine for a long time and indirectly aggravate the inflammatory lesions of the genital tract through trans-mucosal immunity.Therefore,it is very important to explore the immune mechanism that prevents chlamydia from descending spread and colonizing in the large intestine for a long time.As genital tract virulence factors,chlamydia-plasmid p GP3 can aggravate tubal lesions of upper genital tract.When the p GP3 protein encoded by chlamydia plasmid is lost,chlamydia can only be colonized in the small intestine,but cannot be detected in the large intestine.It is not clear whether chlamydia without plasmid p GP3 loses the ability to establish stable colonization in the large intestine or the ability to descending spread from the small intestine to the large intestine.Therefore,the purpose of this research was to analyze the reasons why chlamydia without plasmid p GP3 could not be detected in the large intestine,to reveal the immune mechanism of interaction between plasmid pgp3 deficient chlamydia and intestinal tissue,and to provide new ideas for the prevention and treatment of persistent lesions in the genital tract caused by long-term latent infection of chlamydia.Objective:1.To determine the main reason why chlamydia without plasmid p GP3 could not be detected in the large intestine.2.To reveal the immune mechanism of interaction between plasmid pgp3 deficient chlamydia and intestinal tissue,and to provide a new idea for the prevention and treatment of long-term latent chlamydia infection caused by persistent lesions in the genital tract.Methods:1.To analyze the reason why CMpgp3 s could not be detected in large intestine tissue,different doses of wild-type chlamydia(CMwt)or plasmidfree chlamydia(CMpf)or pgp3 deficient chlamydia(CMpgp3s)were inoculated into C57BL/6J mice by oral /intrajejunal /intracolumn inoculation,establishing chlamydia infection mouse model.The quantity of chlamydia in rectal swabs or GI tract of mice including stomach,duodenum,jejunum,ileum,cecum,colon and rectum was quantitatively monitored by immunofluorescence method.chlamydia inclusion body number(IFUs)was used to represent the colonization of chlamydia in the GI tract.2.To determine the type of immunity that blocks detection of CMpgp3 s in large intestine tissue,TLR2/4/13 KO,My D88 KO,STING KO,Rag1 KO,CD4 KO,CD8 KO and μ chain KO mice were be selected to establish chlamydia CMpgp3 s infection mouse model,wild-type C57BL/6J mice were be used as control.IFUs of chlamydia in rectal swabs or GI tract of mice were quantitatively monitored by immunofluorescence method.3.To identify immune cells that block detection of CMpgp3 s in large intestine tissue,splenic lymphocytes were extracted from wild-type C57BL/6J mice by magnetic beads negative selection method,then donor cells were transplanted into corresponding immune cell gene knockout mice by inner canthus injection.Groups of non-transplanted donor cells were used as control,to establish chlamydia CMpgp3 s infection mouse model.IFUs in rectal swabs or GI tract of mice were quantitatively monitored by immunofluorescence method.Results:1.C57BL/6J mice oral inoculated with 107 IFUs CMwt could be detected from the rectal swabs of mice on day 3 of inoculation and lasted until day 28.CMpf could be detected from rectal swabs on the day 7 of inoculation,and the detected amount was lower than CMwt until the day28.CMpgp3 s could not be detected from the rectal swabs of mice regardless of the dose and detection time.Dissection of gastrointestinal tissues of mice showed that CMwt could be detected in stomach,small intestine and large intestine at 7,14 and 21 days after inoculation,while CMpf could be detected in small intestine and large intestine at 7 and 14 days after inoculation,and only in large intestine at 21 days after inoculation.CMpgp3 s were detected in the small intestine of mice at 7,14 and 21 days after inoculation,but still could not be detected in the large intestine.2.C57BL/6J mice intrajejunal inoculated with 10~5 IFUs CMwt could be detected from the rectal swabs of mice on the day 3 of inoculation,and CMpf were detected from the rectal swabs of mice on the day 7 of inoculation and lasted until the day 28,while CMpgp3 s were still not detected in the rectal swab at each detection time point.Dissection of the gastrointestinal tissues of mice showed that CMwt and CMpf were detected in the small intestine and large intestine of mice on the day 3 and day 7 of inoculation,while CMpgp3 s were detected in the small intestine of mice on the day 3 and day 7 of inoculation,but could not be detected in the large intestine.3.C57BL/6J mice intracolumn inoculated with different chlamydia(10~5 IFUs),and the results of CMpgp3 s were similar with CMwt and CMpf,which could be detected in the rectal swabs of mice on day 3 of inoculation and lasted until day 28.CMpgp3 s could still be detected continuously in rectal swabs of mice after lowering the dose of CMpgp3 s to 103 IFUs.4.After a total of 106 IFUs CMpf and 10~5 IFUs CMpgp3 s were intrajejunal inoculated of C57BL/6J mice,Neither CMpf nor CMpgp3 s could be detected from rectal swabs and large intestine of mice.These results suggest that CMpgp3 s can activate the intestinal mucosal barrier and prevent descending spread in the intestine of CMpgp3 s and CMpf.5.CMpgp3 s of 10~5 IFUs were intrajejunal inoculated with different gene knockout mice,CMpgp3 s could be detected from large intestine in Rag1 KO mice but not in TLR2/4/13 KO,My D88 KO and STING KO mice.Since T lymphocytes of Rag1 KO mice lack TCR and B lymphocytes lack BCR,it is suggested that the intestinal mucosal barrier mainly relies on lymphocyte recognition antigen for immune effect,rather than the expression of innate immune signaling molecules such as TLR or My D88 or STING.6.CMpgp3 s of 10~5 IFUs were intrajejunal inoculated with different gene knockout mice,CMpgp3 s could be detected from large intestine in CD4 KO,CD8 KO and μ chain KO mice.The detection amount of CMpgp3 s in large intestine of CD4 KO mice was significantly higher than that of CD8 KO and μ chain KO mice.After lowering the dose to 104 IFUs,it could only be detected from large intestine in CD4 KO mice but not CD8 KO and μ chain KO mice.7.CD4 KO mice were intrajejunal inoculated with 10~5 IFUs CMpgp3 s after they were supplemented with spleen derived naive CD4~+T lymphocytes,CMpgp3 s could no longer be detected from large intestine,while CMpgp3 s could still be detected in the large intestine of mice that were not supplemented with naive CD4~+T lymphocytes.These results suggest that CD4~+T lymphocytes are sufficient to maintain the intestinal mucosal barrier and prevent chlamydia descending spread in the intestine.Conclusions:Chlamydia-plasmid p GP3 is a key protein for chlamydia spreading from small intestine to large intestine and establish stable colonization.Anti-chlamydia immune response mediated by CD4~+T lymphocytes can prevent the spread of pgp3 deficient chlamydia from small intestine to large intestine.