The Effect And Mechanism Of FKBP51 Or Its Mutation On Th17 Cell Differentiation

Objectives:T helper 17(Th17)is a subgroup of CD4+T cells that express RORyt and secrete the effector cytokine interleukin 17(IL-17).Thl7 cells are important immune cells in the body's immune defense.They play an important role in the body's elimination of exogenous bacterial and fungal infections,and are also involved in the occurrence and development of inflammatory diseases and a variety of autoimmune diseases.The mechanism of Th17 cell differentiation and function regulation is complex.Relevant studies have shown that cytokines,transcription factors and metabolic factors,such as glycolytic pathway and epigenetics,are involved in the differentiation regulation of Th17 cells.A large number of studies have shown that PI3K/Akt/mTOR is a key signaling pathway to promote Th17 cell differentiation,which regulates Th17 cell differentiation by upregulation of phosphorylation and expression of ribosomal protein S6 kinase 1/2(S6K1/2)to promote nuclear translocation of RORyt,a key transcription factor for Th17 cell differentiation.FK506 binding protein 51(FKBP51)is a member of the immunophilin family and has a variety of biological functions.As a scaffold protein,FKBP51 promotes the combination of phosphatase PHLPP and AKT,mediates the dephosphorylation of AKT,and inhibits the activity of AKT signaling pathway.Our previous study found that a new FKBP5 gene mutation(c.163G>C,p.Va155Leu)is related to Paget's disease of bone(PDB).Studies have shown that FKBP51V55L mutation promotes the activation of AKT signaling pathway,thereby promoting the proliferation and activation of osteoclasts.However,the regulatory effect of FKBP51 and its mutation on Th17 differentiation is unclear.In this paper,FKBP51V55L mutation and FKBP51 knockout(FKBP51ko)CD4+T cells were used as models to study the regulatory effect of FKBP51 on Thl7 cell differentiation,and to elucidate its regulatory mechanism on Th17 cell differentiation,so as to provide experimental basis for the discovery of new targets for the diagnosis and treatment of Th17 cell-related autoimmune diseases.Methods:1.Identification of genetically modified mice:FKBP51V55L gene mutant mice and FKBP51 gene knockout mice were bred in the laboratory.The mouse tail was clipped to 2-3mm,and the DNA was extracted by lysis with the mouse tail lysate.FKBP51vssL mutant primers and FKBP51KO primers(primer sequences were shown in the experimental materials)were amplified by PCR,and the mouse genotype was identified by agarose-gel electrophoresis.2.Isolation and purification of CD4+T cells:Take mouse spleens,prepare a single cell suspension,and use magnetic bead sorting to separate and purify CD4+T cells for subsequent induction and differentiation experiments in vitro.3.Th17 cell induced differentiation:CD4+T cells were suspended in Th17 cell induced differentiation medium with a cell concentration of 2×106 cells/2ml/well.The cells were inoculated in a 6-well cell culture plate with anti-mouse CD3 antibody at 37? in a cell culture box with a concentration of 5%C02 for 5 days to induce differentiation.4.Flow cytometry:Fluorescent antibody anti-CD4(PerCp-Cy5.5),anti-IL17A(PE),anti-Foxp3(AFP647)labeled cells were subjected to flow cytometry to determine the proportion of Th17 cells and Treg cells.The effects of FKBP51V55L mutation and analyze FKBP51 knockout on the differentiation of CD4+T cells into Th17 cells.5.RT-PCR:Real-time fluorescence quantitative polymerase chain reaction was used to detect the mRNA levels of Th17 cell-specific transcription factors RORyt,RORa,Stat3 and function-related cytokines IL17a and IL17f.At the mRNA level,the effect of FKBP51V55L and FKBP51-/-on the differentiation of CD4+T cells into Th17 cells was further verified.6.Western blot:The expression levels of Th17 cell differentiation-specific transcription factor RORyt,Cytokine IL17,Th17 cell differentiation-related AKT signaling pathway-related protein phosphorylation and T cell activation-related signaling pathway NF-?B,NFAT-related proteins were detected by Western blot.To explore the mechanism of FKBP51V55L and FKBP51-/-on the differentiation of Th17 cells.7.Luminex multi-factor detection:detect the content of cytokines IFN-?,IL2,IL4?IL5,IL10,TNF-?,IL17A secreted by Th1,Th2,Th17 cells in the cell culture supernatant by Luminex multi-factor detection kit.The effects of FKBP51V55L on Th17 cell function were analyzed.Results:1.Isolation and purification of CD4+T cells in mouse spleen cells:Flow cytometry analysis of magnetic bead sorting cells showed that the proportion of CD4+cells was greater than 97%,which can be used for subsequent induction of Th17 differentiation experiments.2.FKBP51V55L mutation promotes the differentiation of CD4+T cells into Th17 cells:After in vitro CD4+T cells were cultured under Th17 induced differentiation conditions for 5 days,flow cytometry analysis found that the proportion of mutant and wild mouse Th17 cells was 17.96 ± 1.94%And 8.36 ±0.73%,the difference was significant(P<0.001);the proportion of mutant mice and wild mice Treg cells were 1.97 ± 0.35%and 2.87 ± 0.33%,respectively,no significant difference(p=0.087).The results suggested that FKBP51V55L mutation promoted Th17 cell differentiation.3.FKBP5ko promoted the differentiation of CD4+ T cells into Th17 cells:Afte CD4+T cells were cultured under Th17 induced differentiation conditions for 5 days in vitro,flow cytometry analysis found that the ratio of knockout mice and wild mice Th17 cells were 7.33 ± 0.72%and 4.70 ± 0.35%,the difference was statistically significant,(P<0.05);the proportion of Treg cells in gene knockout mice and wild mice were 5.93 ± 1.22%and 4.67 ± 2.27%,respectively,no significant difference,(p=0.649).The results suggested that FKBP51 inhibited TH17 cell differentiation.4.FKBP51V55L mutation up-regulated the expression of Th17 cell differentiation-specific transcription factors and function-related cytokines:V55L mutation and wild mouse CD4+T cells were induced differentiation in vitro for 5 days.Western blot showed that mutant mouse Th17 cell-specific transcription factor RORyt protein expression level was significantly higher than wild mice(P<0.01);mutant mice induced cells with higher levels of IL17a and IL17f mRNA than wild group(P<0.05),mutant mice induced cells with higher concentration of IL17a in cell culture supernatant than wild group(P<0.01).The results suggested that FKBP51V55L mutation promoted Th17 cell differentiation through upregulation of RORyt expression.5.FKBP5ko upregulate the protein levels of Th17 cell differentiation specific transcription factor RORyt and intracellular IL17:CD4+T cells of FKBP5 gene knockout and wild mice were induced differentiation in vitro for 5 days,western blot showed that the protein expression level of RORyt in FKBP5ko group was significantly higher than that in wild mice(P<0.001);The intracellular IL17a protein level of FKBP5ko group was higher than that of the wild group(P<0.01),suggesting that FKBP51 inhibited Th 17 cell differentiation by down-regulating RORyt expression.6.AKT-mTOR signaling pathway is involved in FKBP51-mediated differentiation of Th17 cells:After CD4+T cells were cultured under Thl7 induced differentiation in vitro for 5 days,the phosphorylation level of AKT Thr308 in FKBP51V55L mutant group was higher than that in wild group(P<0.05);The mTOR protein level was higher than that in the wild group(P<0.01);It suggested that FKBP51V55L mutation activated AKT/mTOR signaling pathway by upregulation of AKT T308 phosphorylation level and upregulation of ROR yt expression to promote Th17 differentiation.The phosphorylation level of AKT Thr308 in FKBP51 gene knockout group was higher than that in wild group(P<0.01),and the level of mTOR protein was higher than that in wild group(P<0.01),It suggested that FKBP51 inhibited AKT/mTOR signaling pathway by down-regulating the phosphorylation level of AKT T308 and down-regulated RORyt expression to inhibit Th17 differentiation.Conclusion:FKBP51V55L mutation can up-regulate the expression of Th17 cell-specific nuclear transcription factor RORyt by activating AKT/mTOR signaling pathway,and promote Th17 differentiation.By inhibiting AKT/mTOR signaling pathway,FKBP51 reduces the expression of Th17 cell-specific nuclear transcription factor ROR?t and inhibits Th17 differentiation.