Lkb1 Maintains T_reg Cell Lineage Identity And Homeostasis

Objective:Regulatory T(Treg)cells are recognized a distinct T-cell lineage characterized by suppressing autoreactive immune responses and preserving immune homeostasis.The Treg cell lineage can be defined by two core characteristics,stable expression of transcriptional factor Foxp3(forkhead box P3)and potent suppressive capacity.There are important theoretical value and clinical practical significance in exploring the regulatory network to maintain Treg lineage identity.It was reported that Lkbl could control the development and activation of T cells to maintain immune equilibrium,while the role of Lkbl in Treg cells is still unknown.Our previous study found that the protein of Lkbl was upregulated in Treg cells under TCR stimulation,which indicates Lkbl might play a functional role in Treg cells.In this study,we seek to investigate the functional phenotype and molecular mechanisms of Lkbl in Treg cells in controlling immune homeostasis,which might provide scientific indication for clinical intervention against auto-immune diseases.Methods:We generated Foxp3Cre-YFP Lkb1f/f(cKO)mice to conditionally knock out lkb1 gene in Treg cells.We examined the general symptoms,lymphoid organ and lymphocyte cytology,survival analysis and histopathological analysis of WT and cKO mice.We detected the T cell and Treg cell cytology by flow cytometry,including Thl,2,17 cell differentiation subsets,T cell activation,population,cell cycle,apoptosis and phosphorylated transcriptional factor of Treg cells.We generated Foxp3CreAMPKa1f/fAMPKa2f/f mice to determine whether the function of Lkbl in Treg cells is dependent on,the canonical downstream protein of Lkb1,AMPK.We examined the expression of Foxp3 in CD4+YFP+cells from Foxp3CreRosa26YFP,and Foxp3CreLkb1f/fRosa26YFPmice,or that in WT and Lkb1-deficient Treg cells from Rag1-/-mice adoptively transferred the same amounts of WT and Lkbl-deficient Treg cells together with Tcon cells.To examine the impact of immediate induced deletion of Lkbl in Treg cells,we detected the expression of Foxp3 in 4-hydrotamoxifen-treated CD4+CD25+Treg cells from ERT2CreRosa26YFPand ERT2CreLkblf/fRosa26YFP mice in vitro.We also detected the expression of Foxp3 in CD4+YFP+cells from Foxp3Cr/+and Foxp3Cre/+Lkb1f/f female mice,in which there are no obvious immune-activation and about half WT Treg cells and half Lkbl-deficient Tree cells,to determine whether the phenotype of Lkbl-deficient Treg cells is dependent on endogenous mechanisms.We cultured WT or Lkbl-deficient Treg cells with dendritic cells under the stimulation of transcriptional factor STATs-related cytokines in vitro respectively and detected the expression of Foxp3.We detected the DNA methylation on Foxp3 CNS2 locus in Treg cells by bisulfite treatment and sequencing.We performed co-immunoprecipitation(co-IP)assay to examine the binding of DNMT1 or DNMT3 to STAT4 or STAT5,and chromatin immunoprecipitation(ChIP)assay to determine the binding of STAT4 or STAT5 to Foxp3 CNS2 DNA locus.Results:Loss of Lkbl in Treg cells caused mice early moribund at-35 days of age.Foxp3Cre-YFPLkb1f/f mice exhibited smaller size and barely increased in body Weight with age.cKO mice displayed splenomegaly and lymphadenopathy,and had increased cell numbers in secondary lymphoid organs.Histopathological analysis revealed massive infiltration of immune cells into multiple organs such as skin,lung,liver and stomach in cKO mice.cKO mice had stronger expansion,activation,and more Thl,Th2 differentiation of CD4+and CD8+T cells,but substantial decrease in Treg cell population.More than 80%CD4+YFP+cells did not express Foxp3 any more in Foxp3CreLkb1f/fRosa26YFP mice.We did not observe the similar phenotype in Foxp3CreAMPKa1f/fAMPKa2f/f mice.Lkb1-deficient Treg cells did not stably express Foxp3 compared with WT Treg cells in the presence of IL-2 and IL-12 in vitro.The DNA methylation of Foxp3 CNS2 locus and phosphorylation of STAT4 were upregulated in Lkbl-deficient Treg cells.STAT4 could bind to DNMT1 and recruit to Foxp3 CNS2 locus by co-IP and ChIP assay in Lkbl-deficient Treg cells.The inhibitor of DNA methylation could reverse the methylation in Foxp3 CNS2 DNA locus and the expression of Foxp3 in Lkbl-deficient Treg cells.Conclusion:1.Deletion of Lkbl in Treg cells leads to fatal autoimmunity in mice,2.Lkbl maintains Foxp3 expression and CNS2 demethylation in Treg cells.3.The function of Lkbl in Treg cells is independent of AMPK.4.Lkbl prevents STAT4 activation and binding to CNS2.5.Activated STAT4 could bind and recruit DNMT1 to promote DNA methylation on Foxp3 CNS2 locus.Objective:Immune homeostasis depends on the balance between immune activation and immunosuppression.In steady state,the frequencies of regulatory T cells(Treg)in multiple organs remain relatively stable,which not only maintains immune system equilibrium,but also ensures the immediate immune response.Treg population is determined by the biological processes of thymic generation,proliferation,apoptosis and peripheral induction.Under inflammation,Treg cell number increases correspondingly with T cell activation,promoting immune tolerance and preventing excessive inflammatory damage.However,the signaling pathway and molecular mechanisms controlling the size of Treg cell pool in steady-state and inflammation have not been clearly elucidated.We previously found that mice with conditionally deletion of Lkbl in DCs exhibited higher proportion of Treg cells in multiple organs in steady state,without alterring DC population and their classical surface markers.In this study,we seek to investigate the regulatory mechanisms of Lkbl in DCs on Treg cell population under the inflammatory environment.Methods:We established LPS-induced and E.coli-induced peritonitis mice models;qPCR and western blot were used to detect the mRNA and protein levels of Lkbl in the splenic DCs from control and peritonitis mice;We detected the proportion,absolute number and derivation of Treg cells in the spleen and lymph nodes from the peritonitis model by flow cytometry;OX40L expression on DCs from control and peritonitis mice were detected by flow cytometry;We co-cultured control or LPS-treated DCs with Treg cells to detect the ability to promote Treg proliferation of DCs in vitro,and whether it is mediated by upregulated OX40L;We introduced Lkb1f/f(WT),CDllcCreLkb1f/f(cKO)mice and crossed them with Treg cell specifically-depleted mice Foxp3DTR to generate Lkblf/fFoxp3DTR(WT-DTR)and CD11caCreLkb1f/f/Foxp3DTR(cKO-DTR),and LysMCre mice were also introduced.The tolerance of WT,cKO,WT-DTR and cKO-DTR mice to lethal LPS or E.coli challenge after DT treatment was observed and recorded;The tolerance of low-dose LPS or E.coli pretreated mice to lethal homogeneous challenge after DT treatment was observed and recorded.Microairay data sets of control,Lkbl-deficient and LPS-treated DCs were analyzed and verified by Real-time qPCR.Results:The proportion and number of Treg cells were significantly increased in the spleen and lymph nodes of LPS and E.coli-induced peritonitis mice;The expression level of Nrp1 and Helios in increased Treg cells from LPS and E.coli-treated mice were comparable with that in control mice;Our previous study proved that the DCs with Lkb1 specifically deletion would trigger stronger Treg proliferation by up-regulating OX40L expression;Lkbl mRNA in splenic DCs from LPS-treated mice was comparable to control counterpart,but Lkbl protein was significantly decreased after LPS or E.coli treatment;LPS or E.coli induced Lkbl protein degradation were only observed in DCs and macrophages,but not in B cells,T cells,and Treg cells;Treg cell increase was not observed in LysMCreLkb1f/fmice;The expression of OX40L was up-regulated on DCs treated with LPS;LPS-treated DC could driven stronger Tree cell augmentation in vitro,but blocked by OX40L neutralizing antibody;After intraperitoneal injection of lethal dose LPS or E.coli,cKO mice showed strongest tolerance than WT mice,but the difference was abolished after specific Treg cell depletion in cKO mice;The WT-DTR mice with specific Treg cell depletion but without pretreatment exhibited shortest lifetime after lethal chanllenge,LPS-pretreated mice had strongest protective tolerance but was abolished after removal of Treg cells;In E.coli survival experiment,despite pretreated-WT mice displayed the highest survival rate,this protective effect was not dependent of Treg cells increase since specific Treg cell depletion did not significantly impaire the survival rate;We detected and analyzed the microarray data from control,Lkbl-deficient and LPS-treated DCs,and found that the Lkbl deletion and LPS treatment could consistently significantly upregulate 0x401 expression compared with control DCs;GSEA analysis showed that pathways related to inflammatory responses,inflammatory effector processes,immune responses and acute immune responses were significantly upregulated in DCs from LPS-treated mice,while no significant enrichment in these gene sets appeared in Lkbl-deficient DCs.Conclusion:1.DCs from LPS and E.coli-imduced peritonitis mice could promote tTreg proliferation by degrading Lkb1 protein;2.Lkbl-deficient DCs could promote tTreg angmentation by upregulating OX40L expression;3.Specific deletion of Lkbl in DCs could protect mice from lethal LPS or E.coli chanllenge by augmenting Treg population;4.LPS pretreatment could protect mice from secondary lethal LPS chanllenge by augmenting Treg population;5.Lkbl discriminates regulatory programmes from inflammatory programmes in LPS-treated DCs.