Effects Of Different Invasiveness Of Candida Albicans On Vaginal Local Immunity In RVVC

Objective:In this study,the differences in invasiveness of vulvovaginal candidiasis(VVC)pathogenic strains and recurrent vulvovaginal candidiasis(RVVC)pathogenic strains were compared;the effects of C.albicans with different Sap activity on the infection rate of RVVC and vaginal local immunity in mice were investigated from animal experiments and in vitro cell experiments.To clarify the role of IL-17 in the occurrence and development of RVVC,and to explore how IL-17 and neutrophils interact with each other to participate in the local immunity of RVVC.The study provides a theoretical basis for elucidating the pathogenesis of RVVC,and provides a new direction for the diagnosis and treatment of RVVC.Methods:1.Collect 100 strains of VVC pathogenic strains and 92 strains of RVVC pathogenic strains,and isolate and purify them in Sabouraud medium.Antu chromogenic medium and VITEK 2 system selected C.albicans among the above strains for subsequent experiments.2.Sap,Plb enzyme activity analysis was performed on all C.albicans strains using milk medium and egg yolk medium.3.Select C.albicans with the strongest Sap enzyme activity(Sap=0.242)and the weakest Sap enzyme activity(Sap=0.731)to construct a mouse RVVC model,and calculate the infection rate at different stages of disease;Observation of vaginal epithelium in different stages of disease by HE staining and transmission electron microscope.4.Multi-factor analysis was performed on the vaginal lavage fluid of RVVC mice after the second infection using inflammatory factor antibody chip,and qRT-PCR was used to verify the differential factors.5.ELISA was used to determine the content of differential factors IL-4,IL-17 and IL-8 in vaginal lavage fluid in different stages of RVVC.6.Human and mouse primary vaginal epithelial cells were cultured in vitro by enzymatic digestion,and co-cultured with C.albicans with different Sap activity.The contents of IL-4,IL-8 and IL-17 in the cell supernatant after co-culture were detected by ELISA.7.The VVC mouse models with different Th17 contents were constructed by using halofuginone,which were divided into control group,0μg halofuginone group,5μg halofuginone group and 10μg halofuginone group.Flow cytometry was used to detect the content of Th17 in the mice in the four groups.Wright staining was used to observe the number of neutrophils in the vagina,the fungal load in the vagina was detected on the Sabouraud agar plate,and the damage of the vaginal epithelium was detected by HE staining.Chemical staining was used to detect the expression of IL-17 and Cleaved-Caspase3 in vaginal epithelium;Tunel staining was used to detect the number of apoptotic cells in vaginal epithelium.8.Flow cytometry was used to detect the expression of IL-17 in vaginal neutrophils of mice in the control group and halofuginone 0μg group;Tunel staining was used to detect the apoptosis of neutrophils;The expression of IL-17 and neutrophil apoptosis-related proteins in neutrophils were detected by Western Blot.9.IL-17,IL-17 antibody,control mouse serum,infected mouse serum and mouse neutrophils were co-cultured,and the migration of neutrophils was detected by Transwell;Flow cytometry,Tunel staining and Western Blot were used to detect the apoptosis rate and apoptosis-related protein expression of neutrophils after co-culture for 12 h.Results:1.The proportions of C.albicans in VVC pathogenic strains and RVVC pathogenic strains were 95%and 93.5%respectively,and there was no statistical difference between the two groups.2.The Sap activity of VVC pathogenic strains was significantly stronger than that of RVVC pathogenic strains;there was no significant difference in Plb activity between VVC strains and RVVC strains.3.C.albicans with weak Sap activity and strong Sap activity can construct mouse RVVC model.The third infection rate of the two groups of mice was the lowest,and the infection rate of the Sap strong group in the third time was significantly lower than that of the Sap weak group.HE staining and transmission electron microscopy showed that the vaginal epithelial injury did not aggravate with repeated infection,and there was no significant difference between the Sap strong group and the Sap weak group.4.Multi-factor analysis and qRT-PCR showed that IL-4 and IL-17 were highly expressed in vaginal epithelium after secondary infection with VVC,and the expression of these two factors was different between Sap strong group and Sap weak group.5.The contents of IL-4,IL-8 and IL-17 in vagina increased in different stages of RVVC.C.albicans with strong Sap activity can stimulate the vaginal epithelium to secrete more immune factors,especially IL-17;the high expression of IL-17 after the second infection in the strong Sap group is related to the low rate of the third infection.6.In vitro experiments confirmed that human and mouse vaginal epithelial cells have the ability to secrete IL-4,IL-8 and IL-17,and C.albicans with high Sap activity can stimulate human and mouse vaginal epithelial cells to produce higher immune factors,the increase of IL-17 was more significant.7.The number of C.albicans colonies/hyphal number and vaginal epithelial damage in the mouse vagina were inversely proportional to the Th17 content;the number of neutrophils in the mouse vagina decreased with the decrease of Th17.8.The content of IL-17 in neutrophils in VVC mice increased compared to control mice,and the apoptosis of neutrophils was enhanced too.9.IL-17 can chemotactic neutrophils and promote neutrophil apoptosis;VVC mouse serum also has chemotactic effects on neutrophils and enhances neutrophil apoptosis.Conclusion:1.C.albicans is the main pathogen causing VVC and RVVC.The Sap enzyme activity of VVC-causing C.albicans is stronger than that of RVVC-causing C.albicans.2.C.albicans with weak Sap enzyme activity is more likely to cause RVVC,but the morphological changes of vaginal epithelium in different stages of RVVC have no obvious relationship with Sap enzyme activity.3.During the pathogenesis of RVVC,the immune response of the vaginal epithelium was the strongest after the second infection.4.C.albicans can activate vaginal epithelial cells to produce a large number of cytokines in a short time.C.albicans with strong Sap enzyme activity induces more cytokines,especially IL-17,which reduces the infection rate of RVVC.5.The changes of cytokines produced by human and mouse vaginal epithelial cells are basically the same when infected by C.albicans.5.Th17 and IL-17 play a protective role in the pathogenesis of RVVC,and IL-17 can protect local vaginal tissue by promoting neutrophil apoptosis.6.During the pathogenesis of RVVC,neutrophils in the vagina can produce IL-17 in an autocrine manner.