The Role And The Underlying Mechanisms Of LncRNA-GM In Polarizing Th17 Differentiation To Mediate Autoimmune Inflammation

CD4~+T cells play crucial roles in immune defense against infections by participating in a variety of immune responses to clear pathogens.In response to stimulation from antigens or various cytokines,na(?)ve CD4~+T cells can differentiate into different types of helper T(Th)cell subsets,such as Th1,Th2,Th17,Tfh,Th9 and T regulatory cells(Treg).Among these Th cell subsets,Th17 cells are mainly responsible for induction of inflammation by secreting inflammatory cytokine IL-17.In contrast,Treg cells have immunomodulatory function to inhibit the inflammatory response or promote the resolution of inflammation.The balance of Th17 and Treg is critical for regulation of immune response and maintaining homeostasis.Dysregulation of Th17/Treg balance is related to development and pathogenesis of autoimmune diseases.Until now,the regulatory mechanisms of Th17/Treg balance remain to be fully understood.As an important kind of non-coding RNAs,long non-coding RNAs(lncRNAs)play essential roles in the regulation of immune response and inflammation.However,their functions in T cell subsets differentiation and T cells-mediated autoimmune diseases remain unclear.Our previous study revealed a new lncRNA(named lncRNA-GM),that could promote antiviral response of macrophages by enhancing TBK1 activity.lncRNA-GM is also highly expressed in CD4~+T cells,except for in macrophages and monocytes.Therefore,we speculated that lncRNA-GM might be involved in the differentiation and function of CD4~+T cells.Firstly,we found the expression of lncRNA-GM was remarkably high in Th17 cells among the CD4~+T cells subsets induced in vitro.Consistently,the ortholog of lncRNA-GM in human(lncRNA AK026392.1)was also highly expressed in Th17 cells derived from peripheral blood mononuclear cells(PBMCs).On the contrary,expression of lncRNA-GM was much lower in the induced Tregs(i Tregs)derived from mouse spleen or human blood,Herein,we show that lncRNA-GM is increased in Th17 cells for polarizing Th17 differentiation but decreased in Treg cells for inhibiting Treg differentiation.Next,we utilized lncRNA-GM knockout mice(lncRNA-GM-/-)to investigate its role in regulation of T cell differentiation in vivo.lncRNA-GM deletion did not influence the development of T and B cells in spleen and m LN,or the proliferation of CD4~+T and CD8~+T cells.However,lncRNA-GM deficiency significantly reduced the effector/memory T cells in spleen,which indicated lncRNA-GM is required for T cell function.Among induced Th subsets,deficiency of lncRNA-GM leads to significantly decrease of Th17 cells differentiation but sharply increase of i Treg cell differentiation.While overexpression of lncRNA-GM significantly promoted Th17 cell differentiation in CD4~+T cells from both WT and lncRNA-GM-/-mice.RNA-seq analysis showed that Th17 cell-specific genes such as Rorc,Il17 a,Il17f,Rora,Il23 r,Tbx21 were downregulated in lncRNA-GM-/-Th17 cells compared to that in WT Th17 cells.To explore the mechanism of lncRNA-GM in regulation of Th17/Treg cells differentiation,we examined the key signaling pathways via RNA-seq analysis and found that the lncRNA-GM promoted the expression of genes in mTOR signaling.Moreover,we found that transcription factor FoxO1 was highly expressed in lncRNA-GM-/-Th17 cells or Treg cells.It is reported that FoxO1 inhibits Th17 cell differentiation and promotes Treg cell differentiation.Therefore,the data suggest that lncRNA-GM promotes Th17 cell differentiation and inhibits Treg cell differentiation by targeting FoxO1.Besides,lncRNA-GM regulated the balance between Th17 and Treg cells independent of IL-6/ IL-2/ TGF-β signals.To investigate the physiological function of lncRNA-GM in regulating CD4 T cell subset differentiation in vivo,we established experimental autoimmune encephalomyelitis(EAE)mouse model with WT and lncRNA-GM-/-mice respectively.We found that deficiency of lncRNA-GM efficiently attenuated development of EAE,accompanied with the decreased Th17/Th1 cells infiltration and increased generation of Treg cells in brain,spleen and spinal cord.Furthermore,we established passive EAE mouse model by T cell adoptive transfer through isolating WT or lncRNA-GM-/-na(?)ve CD4~+T cells and adoptively transferring these T cells into Rag2-/-mice via tail vein injection.The recipient Rag2-/-mice transferred with lncRNA-GM-/-na(?)ve CD4~+T cells exhibited significantly decreased disease severity,accompanied with the less infiltrating Th17/Th1 cells and more Treg cells in brain,spleen and spinal cord.At last,we sorted CD4~+T cells in human PBMCs from healthy volunteers,and used electroporation assay to interfere with the human lncRNA-GM ortholog(AK026392.1).Consistently,knockdown of human lncRNA-GM significantly inhibited Th17 cell differentiation and promoted Treg cell differentiation.In summary,our study demonstrates that lncRNA-GM plays an important role in T cell differentiation and function.lncRNA-GM could promote Th17 cell differentiation as well as inhibit Treg cell differentiation by targeting FoxO1/mTOR axis.Knockout of lncRNA-GM significantly attenuates the pathological progression of EAE model.Our work reveals a new mechanism of lncRNA-GM in regulating the balance of Th17/Treg differentiation,and will shed new lights on the potential application for treatment of autoimmune diseases.