Mannan-Binding Lectin Regulates The Treg/Th17 Axis In Mice Infected With Candida Albicans

BackgroundMannose-binding lectin（MBL）is a key molecule in the complement activation pathway of the innate immune system;Recently the studies have found that MBL also plays an indispensable role in the adaptive immune response.In the case of infection,CD4⁺T cell plays an important role in acquired immune regulation inside the body,especially the dynamic changes of the Treg/Th17 cell immune axis,which affects the occurrence,development and outcome of inflammatory reactions.C.albicans is an opportunistic pathogenic fungus that usually colonizes the gastrointestinal tract,skin,and mucosa of humans.Under certain conditions,it can cause fatal invasive fungal infections with high pathogenicity and mortality.Therefore,in-depth study of the body’s regulation of Treg/Th17 cell immune balance in C.albicans infection,is important to elucidate the role of MBL in acquired immunity.ObjectiveTo investigate the regulation of Th17 and Treg differentiation of CD4⁺T cells by Mannose-binding lectin（MBL）in mice infected with C.albicans and related mechanisms.Methods1.C.albicans culture and CFU colony count:C.albicans was cultured in Sacraco fungi medium and YPD solid medium,and colony counts were performed by CFU colony counting method.2.Infected mice by intraperitoneal injection of C.albicans:MBL^-/-C57BL/6 mice and WT C57BL/6 mice were divided into four groups.C.albicans（2×10⁷CFU）were injected intraperitoneally into the infected mice.3.Paraffin section:Pathological sections were taken from the mice liver and kidneys after 3 days.H&E staining and PAS staining confirmed the establishment of mouse systemic infection models.4.Flow cytometry（FACS）detect:After 7 days,the mouse eyeball was removed and the venous blood was taken.Flow cytometry was used to analyze Treg and Th17 cells in mice.5.Quantitative real-time PCR（q RT-PCR）analysis:The mice were sacrificed by cervical dislocation and spleen cell was taken.CD4⁺T cell was obtained by MACS magnetic separation.Total RNA was extracted and the expression of Foxp3,RORγt m RNA was detected.6.Enzyme-linked immunosorbent assay（ELISA）detect:Intravenous anticoagulation was taken from different groups of mice,and the levels of cytokines IL-10 and IL-17A in mice were measured by ELISA.、7.Western blotting:CD4⁺T cell was extracted from mouse spleen,total protein was extracted,and the expression levels of Foxp3 and RORγt were detected by Western blotting.The phosphorylation of JAK/STAT pathway-related protein was detected.Results1.Successfully established a mouse model of C.albicans infection2.Compared with the WT-infected group,flow cytometry showed that the proportion of Th17 cell subtypes increased,and the proportion of Treg cell subtypes decreased in the MBL^-/-infected mice.3.Compared with the WT-infected group,the MBL^-/-mice expressed significantly lower Foxp3 m RNA levels,while showing higher RORγt m RNA levels by q RT-PCR.4.Compared with the WT-infected group,The results of ELISA showed that the IL-17A level in MBL^-/-mice was significantly higher and the IL-10 level was lower.5.Compared with the WT-infected group,Western blotting results show that the MBL^-/-infection group RORγt protein expression quantity increased obviously,Th17negative regulatory factors SOCS3 protein levels reduced,and reduced the volume of Foxp3 protein expression,the MBL^-/-infected mice STAT3^Y705 protein expression levels and STAT3 upstream P-JAK2^Y1007+Y1008 expression increased,the downstream RORγt is found the same change,SOCS3 expression is abate.P-STAT5^Y694 and P-JAK3^Tyr980/981protein expression levels decreased,and the expression of Foxp3 decreased.ConclusionMannan-binding lectin inhibited the differentiation of Th17 in mice infected with C.albicans and promoted the differentiation of Treg.The regulatory effect of mannan-binding lectin can be achieved possibly via JAK/STAT signaling pathway...