HDAC6 Dictates NK Cell Homeostasis And Maturation Via Modulating HSP90-p-STAT5 Axis

Nature Killer Cell(NK)is an important component of innate immunity.NK cells develop in the bone marrow(BM)from hematopoietic stem cells(HSCs)that sequentially differentiate into common lymphoid progenitors(CLPs)and NK lineage-restricted progenitors(NKPs).NK cells that undergo maturation can be divided into three stages based on CD 11b and CD27 or KLRG1 expression:CD11b-CD27+,CD11b+CD27+,CD11b+CD27-or CD11b-KLRG1-,CD11b+KLRG1-,CD11b+KLRG1+.This development program parallels with a progressive acquisition of effector function.NK cells kill target cells with no antigen-specificity,no need for antigen pre-stimulation and no MHC restriction,and play an important role in anti-viral infection and anti-tumor.NK cells can not only directly induce apoptosis of target cells by releasing perforin,granzyme,and expressing apoptosis ligands such as FasL and TRAIL,but also regulate immune response by secreting cytokines.However,in tumor microenvironment,the number of NK cells decreaseds and their functions become impaired,which contribute to the tumor progression.Therefore,NK cell-immunotherapy has been widely concerned and has great potential in clinical anti-tumor treatment.In-depth study of regulatory mechanisms about NK cell development,maturation and biological functions would further improve the efficacy of NK cell-based immunotherapy.Protein acetylation is involved in many biological processes,such as immune response,by regulating protein localization,stability and functional activity.Some studies have shown that histone deacetylases(HDACs)inhibitors down-regulated the survival ability and function of NK cells,but the effects and mechanisms of HDACs members in regulating NK cells remain unclear.Objectives:1.To screen potential HDACs members involved in NK cell homeostasis and functional regulation.2.To explore the molecular mechanism of candidate HDACs molecule regulating NK cell homeostasis and function.Methods and results:1.The expression of HDAC6 is up-regulated in activated NK cells1.1 HDACs inhibitors down-regulate NK cell proliferation and functionTo preliminarily verify the role of HDACs in regulating NK cell homeostasis and function,we first selected two HDACs inhibitors:NK-92 cells were treated with TSA(Trichostatin A)and SAHA(Suberoylanilide hydroxamic acid).The proliferation,cytokine secretion and degranulation of NK cells were measured by flow cytometry.The proliferation level of NK-92 cells was decreased,and the secretion of cytokines and degranulation ability were decreased.These results suggest that inhibition of HDACs can down-regulate the proliferation level and effector function of NK cells.1.2 HDAC6 is significantly upregulated in NK cell activationIn order to preliminarily screen the HDACs molecules involved in regulating NK cell homeostasis and function,NK92 cells were stimulated by IL-12 and IL-15,and the expressions of HDACs members were detected.Results showed that the expression of HDAC6 was significantly upregulated during NK cell activation.In addition,HDAC6 expression also increased in NK92 cells stimulated with poly I:C)and anti-NKG2D antibodies.These results suggest that HDAC6 may be involved in regulating the activation and function of NK cells.2.HDAC6 is involved in maintaining NK cell homeostasis and functional maturation2.1 Deletion of Hdac6 gene down-regulates the proportion and number of NK cellsHdac6 knockout(Hdac6 KO)mice were bred,and the proportion and number of NK cells in the liver,spleen and blood of the mice were detected by flow cytometry.Compared with those of wild-type(WT)mice,the proportion and number of NK cells in Hdac6 KO mice were down-regulated.However,there was no significant difference in the ratio of bone marrow NK cell precursors,common lymphoid progenitor cell(CLP)precursors(pre-NKP)and redefined NK cell precursors(rNKP)between WT and Hdac6 KO mice.Mixed bone marrow(BM)chimera assay showed that the proportion of Hdac6 KO NK cells was lower than that of WT NK cells in in the spleen of recipient mice.These results indicated that HDAC6 endogenously regulates NK cell homeostasis.2.2 HDAC6 promotes the proliferative capacity of NK cellsTo clarify the mechanism of HDAC6 regulation of NK cell homeostasis,the proliferation and apoptosis levels of Hdac6 KO NK cells and WT NK cells were compared.The results showed that Hdac6 gene knockout inhibited the proliferation of NK cells,but did not affect the apoptosis of NK cells.Overexpression and knockdown of HDAC6 in NK92 cells further confirmed the above results.2.3 Deletion of Hdac6 gene inhibits NK cell maturationThe immune effect of NK cells depends on both their number and maturation.Thus,we further detected the effect of HDAC6 on NK cell maturation.The expression of CD27,CD11b or KLRG1 in liver,spleen and blood NK cells was detected by flow cytometry.The results showed that knockout of Hdac6 gene inhibited NK cell maturation.In addition,NK cells of Hdac6 KO mice had downregulated expression of maturation transcription factors,T-bet and Eomes,compared with WT NK cells.Moreover,mixed bone marrow(BM)chimera assay showed that Hdac6 KO NK cells in the spleen of recipient mice had lower KLRG1 expression than that of WT NK cells.These results indicated that HDAC6 endogenously promotes the maturation of NK cells.2.4 HDAC6 expression enhances the function of NK cellsNK cells undergo maturation and eventually achieve functional optimization.The study further examined the regulatory effect of HDAC6 on NK cell function.Purified splenic NK cells were stimulated with IL-12 and IL-15,or mononuclear cells from bone marrow,spleen and blood were stimulated with target cell Yac-1,respectively.The killing ability of NK cells and cytokine production ability were detected by flow cytometry.The results showed that compared with WT NK cells,Hdac6 KO NK cells had a decreased ability to secrete cytokines,and significantly reduced the killing ability against Yac-1 cells.Moreover,mixed bone marrow(BM)chimera assay showed that the cytokine expression of Hdac6 KO NK cells in the spleen of recipient mice was significantly lower than that of WT NK cells.Overexpression or knockdown of HDAC6 in NK92 further confirmed the above results.These results indicate that HDAC6 endogenously enhanced NK cell function.3.HDAC6 mediates HSP90 deacetylation to enhance STAT5 signaling pathway and effector functions of NK cells3.1 Deletion of Hdac6 gene impairs IL-15/STAT5 signaling pathway in NK cellsIL-15 signaling pathway plays an important role in NK cell proliferation,maturation and functional regulation.Therefore,we further explored the effect of HDAC6 on IL-15 signaling pathway in NK cells.First,NK cells were stimulated with 20ng/ml and 50ng/ml IL-15,respectively.The results showed that,compared with WT NK cells,Hdac6 KO NK cells had lower expression of CD 107a and IFN-y,but there was no significant differences in the expression of IL-15 receptors CD 122 and CD132.Meanwhile,the phosphorylation of STAT5 decreased in Hdac6 KO NK cells.STAT5 inhibitors abrogated the inhibition effect of Hdac6 gene deletion on NK cell function.These results suggest that HDAC6 may affect NK cell function by regulating IL-15/STAT5 signaling pathway.3.2 HDAC6 mediates HSP90 deacetylation to facilitate STAT5 phosphorylation and functional effects of NK cellsIt has been reported that phosphorylated STAT5(p-STAT5)is a client molecule of HSP90,which is the deacetylated substrate protein of HDAC6.The absence of HDAC6 leads to the hyperacetylation and the loss of chaperone activity of HSP90.Co-immunoprecipitation assay showed that IL-15 stimulation led to deacetylation of HSP90 in NK-92 cells and increased binding of HSP90 with phosphorylated STAT5 compared with control cells.Moreover,HSP90 inhibitor 17-AAG significantly downregulated the STAT5 phosphorylation level and cytokine secretion in NK92 cells stimulated by IL-15.More importantly,HDAC6 knockdown led to upregulation of HSP90 acetylation in NK-92 cells and decreased interaction between HSP90 and p-STAT5,while overexpression of HDAC6 exhibited the opposite effects.At the same time,17-AAG eliminated the effect of HDAC6 overexpression on NK cell function.These results prove that HDAC6 promotes the STAT5 signaling pathway and effector functions by regulating the acetylation of HSP90.Conclusion:1.The expression of HDAC6 is up-regulated during NK cell activation.2.Hdac6 knockout retards NK cells maturation,proliferation and function.3.HDAC6 mediates HSP90 deacetylation to promote the activation of STAT5 signaling pathway,proliferation and function and maturation of NK cells.Innovations and significances:1.It is first discovered that HDAC6 regulates NK cell homeostasis,maturation and effector functions.2.It is preliminarily demonstrated that HDAC6 mediates HSP90 deacetylation to regulate IL-15/STAT5 signaling and NK cell functional maturation.