Effects And Mechanism Of Activin A On Regulation Of Activities Of NK Cells In Mouse

NK cells are large granular lymphocytes that originate from bone marrow precursor cells and are important innate immune cells in the body.When the body is infected by pathogens or tumors grow and invade,NK cells are rapidly recruited to damaged tissues and organs to remove infected cells or malignant cells,NK cells can also regulate other immune cell functions by producing cytokines.Activin A belongs to the TGF-β（transforming growth factor-β,TGF-β）superfamily and can be produced by a variety of immune cells,such as mononuclear macrophages,mast cells,neutrophils and Th2 cells.IL-9（Interleukin-9,IL-9）is an important proinflammatory cytokine,the main source of IL-9 are Th9 cells.However,whether activin A and IL-9can be produced by NK cells and what is the effect of activin A on mouse NK cell function?Is still not clear.Therefore,this study first analyzes whether activin A and IL-9 can be produced by NK cells,and determines whether mouse NK cells belong to activin A responsive cells,and then revealed that activin A regulated the activity of mouse NK cells through autocrine/paracrine manner and related mechanismsⅠ.METHODS1.Detection of cytokinesThe production of activin A,IL-2,IL-9,IL-10,and IFNγby Balb/c mice NK cells,were analyzed by intracellular cytokine staining.The release of mature activin A and IL-9 by NK cells was detected by ELISA.2.Detection of activin receptorsImmunofluorescence staining and flow cytometry were used to analyze the expressions of ActRIIA and ActRIIB on the surface of mouse NK cells.3.Smad3 knockdownThe mouse NK cells were transfected with pGCsi-U6-Neo-GFP and pGCsi-U6-Neo-GFP-Smad3 shRNA,respectively.And then the effect of Smad3knockdown on cytokines production in NK cells was examined.4.Identification of IIA^high and IIA^lowow NK cell subsetsFlow cytometry was used to detect ActRIIA^high(IIA^high)and ActRIIA^low(IIA^low)NK cells ratio in mouse peripheral blood,spleen,and liver NK cells.The morphological characterisation of IIA^high and IIA^low NK subsets were observed by Giemsa staining.The IIA^high and IIA^low NK cells proliferation were detected by BrdU assay.The NK cells subsets were cocultured with YAC-1 to analysis NK cells’cytotoxicity.And adhesion assays were used to test the adhesion ability of the NK cells subsets.Activin A was injected intratumorally into NS-1 cells transplanted tumor,and the effect of Activin A on the ratio of IIA ^highNK cells in the transplanted tumor was examined by flow cytometry.Ⅱ.RESULTS1.Secretion of activin A by mouse NK cellsRT-PCR results showed that not only activinβA mRNA but also ActRIIA,ActRIIB and Smad2,3,4 mRNA were expressed in NK cells of mouse.Intracellular cytokine staining revealed the presence of activinβA chain protein in mouse NK cells.The results of ELISA showed IL-2 promoted cultured NK cells to secrete mature activin A.2.The mouse NK cells express ActR proteinDouble immunofluorescent staining showed that Babl/c mice NK cells expressed both NK cell markers CD49b and ActRIIA.Flow cytometry results further showed that ActRIIA,ActRIIB were expressed on Babl/c mice CD3^-CD49b⁺NKp46⁺NK cells.3.Effect of activin A on NK cell functionThe study also showed that activin A can down-regulate the production of IFNγin mouse NK cells,but up-regulate IL-2 production,but has no significant effect on IL-10 synthesis.The BrdU proliferation kit showed that IL-2 can significantly promote the proliferation and cytotoxicity of NK cells,while activin A has an inhibitory effect on the proliferation of NK cells,but has no significant effect on cytotoxicity.As a positive control,CXCL12 can promote the adhesion of mouse NK cells,and activin A can also increase the adhesion of NK cells.4.Inhibitory effects of activin A on the release of IL-9 by NK cellsThe results of intracellular cytokine staining showed that the peripheral blood and spleen CD3⁺CD4⁺Th cells of mouse expressed IL-9,and the peripheral blood and spleen CD3^-CD49b⁺NKp46⁺NK cells also expressed IL-9.Activin A promoted the production of IL-9 in CD3⁺CD4⁺Th cells of mouse but inhibited the production of IL-9 in NK cells of the mouse.Western blot results showed that activin A can significantly up-regulate the levels of ActRIIA,Smad3 and p-Smad3 proteins in mouse NK cells.The inhibitory effects of activin A on IL-9 production by NK cells were attenuated in the presence of activin antagonist Follistatin（FST）or Smad3knockdown.5.IIA^high and IIA^low NK cell subpopulation characteristicsFlow cytometry was used to detect the expression of ActRIIA on peripheral blood、spleen、lymph nodes and liver NK cells in mice.Flow cytometry results showed that NK cells in different tissues and organs have different degrees of ActRIIA（IIA）expression,which can be divided into IIA^high subsets and IIA^low subsets.Studies have shown that the IIA^high NK cells have higher expression of CD25,Arg1,and iNOS than IIA^low NK cells but the expression level of CD11b on IIA^low NK cells is higher than IIA^high NK cells.Studies have also shown that IIA^high NK cells have a greater capacity to produce IFNγ,IL-2,IL-10,and IL-9 than IIA^low NK cells,and the cytotoxicity of IIA^highigh NK subpopulation was higher than IIA^low NK subsets;there was no difference in the proliferative capacity between the two groups in the natural state,while the IIA^lowow NK cells were more proliferative than the IIA^high NK cells treated by IL-2.In vivo experiments showed activin A was injected intratumorally into NS-1cells transplanted tumor,significantly increased the ratio of IIA^high NK cells with cytotoxicity.Ⅲ.CONCLUSIONIn summary,NK cells of the mouse were not only the source of activin A,but also the target cells in response to activin A.NK cells produced IL-9.Activin A could promote the IL-9 production by CD4⁺Th cells,but inhibited the IL-9 production of NK cells.It suggests that activin A exerts different biological effects on different cells.According to the expression of activin IIA receptor,mouse NK cells could be divided into IIA^high and IIA^lowNK subsets,and the biological characteristics and activities of the two cell subsets are different.The above information indicates that,activin A is an important regulator of NK cells,regulating NK cell function in an autocrine or paracrine manner.However,the physiopathological significance of the two subsets of NK cells remains to be further studied.