Role And Underlying Mechanism Of Liver X Receptor β In The Survival Of Single-positive Thymocytes

T lymphocytes,including CD8~+and CD4~+T cells,which are essential mediators for cellular and humoral immune response,are produced following sequential developmental stages in the thymus.T-cell precursors migrate from the bone marrow(BM)to the thymus where they differentiate into CD4~–CD8~–double-negative(DN)thymocytes.DN thymocytes futher develop into CD4~+CD8~+double-positive(DP)thymocytes,which undergo positive-and negative-selection,and then differentiate into CD4~+or CD8~+single-positive(SP)thymocytes.SP thymocytes migrate to the peripheral lymphoid organs,where they become na?ve CD4~+or CD8~+T cells.Interleukin-7(IL-7)signaling is essential for the development and survival of thymocytes.IL-7 receptorα(IL-7Rα,encoded by Il7r gene)and commonγchain form IL-7 receptor(IL-7R),which is distributed in cell membrane and mediates IL-7signal transduction.SP thymocytes express IL-7Rαand survive depending on IL-7.Although a mount of transcription factors which control the transcription of IL-7Rαin other cells have been reported,the transcription factors regulating IL-7Rαexpression in SP thymocytes remain to be elucidated.Liver X receptors(LXRs)are part of the nuclear receptor family and are known to regulate cholesterol and lipid homeostasis.LXRs consist of two subtypes,LXRα(encoded by Nr1h3)and LXRβ(encoded by Nr1h2).LXRαis mainly expressed in tissues with active metabolism,such as liver,intestine,kidney,spleen,and adipose tissue,while LXRβis ubiquitously expressed.LXRs can be activated by endogenous ligands including oxysterols,desmosterol,as well as synthetic agonists,such as GW3965 and T0901317.Apart from their roles in lipid metabolism,LXRs also exert several important functions in the regulation of immune responses.For example,LXR agonists display potently anti-inflammatory actions by transrepressing the expressions of several inflammatory genes in several different mouse models of inflammatory disease.Moreover,upon bacterial infection,LXRαpromotes macrophage survival via up-regulating the expression of SPα,an anti-apoptosis factor.The expression of Mer induced by LXR agonists is crucial for macrophage to phagocytize apoptotic cells.In addition,LXR agonists exhibit an anti-proliferative action by promoting cholesterol efflux via the induction of ABCG1 expression in T lymphocytes.LXR activation inhibits the T_H17 differentiation through SREBP1c,which physically interacts with aryl hydrocarbon receptor(Ahr)and inhibits Ahr-controlled IL-17 expression.Despite all these progresses,little is known regarding the roles of LXRs in T-cells development.This project focuses on the role and underlying mechanism of LXRs in T-cells development.The main conclusions are described as three parts:1.LXRβis essential for the maintenance of SP thymocytes.Compared with wild-type(WT)mice,the frequencies and numbers of CD4~+and CD8~+SP thymocytes(CD4SP and CD8SP,respectively)in LXRβknock-out(βKO)mice were significantly reduced,while the frequencies and numbers of DP and DN thymocytes were not altered.Meanwhile,the frequencies and numbers of na?ve CD4~+and CD8~+T cells in secondary lymphoid organs fromβKO mice,including peripheral blood lymphocytes(PBLs),spleen,and lymph nodes(LNs)were obviously lower than those from WT mice,and the frequency and number of RTE(recent thymic emigrant)cells in spleen fromβKO mice were also dramatically less than those from WT mice.Moreover,the competitive bone marrow(BM)chimeric experiment showed that,compared with those from WT origin,both CD4SP and CD8SP thymocytes fromβKO origin were significantly decreased and the CD4~+and CD8~+splenocytes fromβKO origin were dramaticly diminished,although the survival and homeostasis proliferation of T splenocytes looked similar.In addition,the LXR agonist T0901317 treatment increased the frequency of SP thymocytes in WT mice,but failed inβKO mice.2.LXRβis required for the survival of SP thymocytes.To find out the cause of reduction in SP thymocytes due to LXRβdeficiency,we firstly examined the expression of CD69,TCRβ,CD24 and CD5 markers in DP thymocytes and DP thymocyte death by a gradient of TCR stimulation or neglect,which revealed that WT and LXRβ-null DP thymocytes exhibited similar expressions of aforementioned markers and sensitivities to death,suggesting that the reduction of SP thymocytes caused by LXRβdeficiency is probably not the result of the developmental dysfunction from DP to SP thymocytes.We then tested the survival and proliferation of SP thymocytes and found that the survival of SP thymocytes was significantly imparied inβKO mice,whereas the proliferation was similar.Since IL-7 signaling is essential for the survival of SP thymocytes,we next investigated the IL-7Rαexpression,Stat5 phosphorylation,and Bcl2 expression in SP thymocytes.As expected,all of them inβKO SP thymocytes were obviously lower than those in WT SP thymocytes.Collectively,these findings suggest that LXRβis required for the survival of SP thymocytes by regulating IL-7Rα-Bcl2 signaling.3.LXRβpositively regulates the transcription of IL-7Rα.The Il7r mRNA transcript inβKO SP thymocytes was lower than that in WT SP thymocytes,and the Il7r mRNA transcript increased after LXR agonist treatment,which indicated that LXRβmay regulate the transcription of IL-7Rα.On the one hand,the expressions of transcription factor Foxo1,Foxp1 and TCF-1(encoded by Tcf7),which are known as key transcriptional regulators of Il7r transcription,were not obviously altered inβKO SP thymocytes.On the other hand,a published Ch IP-seq data showed that LXRβcould bind to the 5’regulatory region of Il7r locus in murine macrophages.Thus,we performed a Ch IP-q PCR assay and found that LXRβalso bound to this region of Il7r gene in SP thymocytes.Moreover,ex vivo reporter gene assay exhibited that LXRβpromoted Il7r transcription in SP thymocytes.In addition,overexpression either LXRβor IL-7Rα-Bcl2 axis rescues the defects of SP thymocytes caused by LXRβdeficiency,confirming that LXRβis required for the survival of SP thymocytes by regulating IL-7Rαexpression.In conclusion,we investigated the role and underlying mechanism of LXRs in T-cells development,and have demonstrated that LXRβ,as an important transcription factor upstream of IL-7Rα,is required for the survival of SP thymocytes by controlling IL-7Rα-Bcl2axis.This project has identified IL-7Rαas a novel LXRβtarget gene in SP thymocytes,has improved our knownledge of LXRs function in T-cell development,and has revealed new insights into the cell type-dependent and metabolism-independent functions of LXRβin the regulation of IL-7Rαexpression.