MBL Regulates The Th17/Treg Axis In Mice Infected With Candida Albicans Through TGF-?/Smad Signaling Pathway

Background Mannan-binding lectin is an anti-infective immune molecule in the innate immune system.It can recognize the sugar structure on the surface of various pathogens and activate the complement system through its C-terminal sugar recognition domain.TGF-?is a key regulator in the development,proliferation,and differentiation of T cells.TGF-?-mediated signaling pathways are involved in Th17 and Treg cell subtype differentiation.Our previous research found that MBL can regulate the immune balance of Th17/Treg cells during Candida albicans infection,but the mechanism is unclear.In this study,we explored whether MBL has a regulatory effect on TGF-?-mediated signaling pathways and whether it has a correlation with changes of Th17/Treg cell immune balance,thereby revealing the regulatory mechanism of MBL on Th17/Treg cell immune balance.This study is of great significance to clarify the role and mechanism of MBL in regulating the immune response induced by C.albicans.Objective To study whether MBL affects Th17/Treg cell immune imbalance and its subsequent inflammatory effects by regulating TGF-?-mediated signaling pathways during C.albicans infection.Methods 1.The role of TGF-?/Smad signaling pathway in the regulation of Th17 and Treg cell proportion by MBL in vitro:CD4⁺T cells were isolated from the spleen of WT mice by immunomagnetic beads,and induced to differentiate into Th17 or Treg cells.MBL protein and pathway inhibitors were added to intervene the differentiation.The proportion of T cell subsets in different groups were detected by Flow cytometry.The expression of ROR?t,Foxp3 and key proteins in TGF-?/Smad pathway were detected by Western blot.2.Establishment of Candida albicans infection model using MBL gene knockout mice:WT mice and MBL^-/-mice were intraperitoneally injected with 5×10⁷CFUs of C.albicans to establish a systemic model of Candida albicans infection.Control groups of WT mice and MBL^-/-mice were injected with the same amount of saline.3.Detection of protein expression in four groups of mice by Western blot:CD4⁺T cells sorted by magnetic beads from each group of mice were used to extract total protein,and the expression level of key proteins of ROR?t?Foxp3 and TGF-?/Smad signaling pathways was detected.4.Detection of changes of gene expression levels by qRT-PCR:Total RNA were extracted from CD4⁺T cells of each group,and the expression of ROR?t and Foxp3 mRNA in different groups were detected.5.Detection of cytokines by ELISA:Collect intravenous anticoagulation of mice in each group,take the supernatant after centrifugation,and detect the changes of IL-17A,IL-10 and TGF?in the plasma of the four groups of mice.6.Flow cytometry detection:Flow cytometry was used to analyze the proportion of Th17?Treg cells in the blood of mice in each group was performed after 5 days of infection.7.Pathological examination:Mice tissues were collected 5 days after Candida albicans infection,and H&E?PAS staining were used to evaluate the differences in fungal infection among the groups;The changes of fungal load in mice tissues of each group were detected by plate dilution method.8.Survival analysis:WT mice and MBL^-/-mice were intraperitoneally injected with1×10⁸CFUs of Candida albicans,and the survival of the two groups of mice was observed for a period of 14 days.The survival time of the mice was recorded,and the survival curve was drew using GraphPadPrism software.Results 1.The results of vitro experiments showed that when CD4⁺T cells were induced to differentiate into Th17 cells,MBL protein can reduce the proportion of Th17 cells,and the expression levels of ROR?t protein and p-Smad2?S465/467?were weakened.The inhibitory effect of MBL was decreased after the treatment of signaling pathway inhibitor,which was statistically significant.When CD4⁺T cells were induced to differentiate into Treg cells,MBL can up-regulate the proportion of Treg cells,and the expression levels of Foxp3 protein and p-Smad3?S423/425?are enhanced.The enhancement effect of MBL was weakened after the treatment of signaling pathway inhibitor,which was statistically significant.2.The mouse model of C.albicans infection was successfully established by MBL knockout mice.3.Western blot and qRT-PCR results showed that compared with WT infected mice,the expressions of Smad7,p-Smad2?S465/467?and its downstream transcription factor ROR?t in the MBL^-/-infected group were increased at the protein and mRNA levels;the expression of p-Smad3?S423/425?and its downstream transcription factor Foxp3 were decreased.4.ELISA results showed that compared with WT infected mice,the levels of IL-17A and IL-21 in the plasma of MBL^-/-infected mice were significantly increased,and the levels of IL-10 and TGF-?were significantly reduced.5.The results of flow cytometry showed that compared with the WT infection group,the proportion of Th17 cells in peripheral blood of mice in the MBL^-/-infection group was significantly increased,and the proportion of Treg cells was significantly decreased.6.The results of pathological analysis showed that compared with WT infected mice,the tissue CFU value of MBL^-/-infected mice increased significantly,and histopathological fungal infection was more serious.7.Compared with WT infected mice,MBL^-/-infected mice had higher mortality.Conclusion MBL inhibits the differentiation of CD4⁺T cells into Th17 cell subtypes and promote the differentiation of CD4⁺T cells into Treg cell subtypes by regulating the expression levels of key signal molecules Smad2,Smad3 and Smad7 in the TGF-?/Smad signaling pathway in C.albicans infected mice.MBL further regulate the immune balance of Th17/Treg cells,inhibiting the inflammatory response induced by C.albicans,and reducing the lethality of infection.