Effect Of E-prostanoid 3 Receptor In NK Cells On Liver Fibrosis

Objective:Liver fibrosis is a pathological process in which extracellular matrix is excessively deposited in the liver.It is associated with chronic hepatic virus infection,alcohol,and nonalcoholic fatty liver disease,which eventually results in cirrhosis,portal hypertension,and liver failure.Hepatic stellate cells?HSC?overactivation plays the central role in the development of hepatic fibrosis.Inhibition of HSC activation and proliferation,or promotion of HSC apoptosis alleviates liver fibrosis.As one of important properties of innate immune against liver fibrosis,natural killer cells?NK?in immune system can kill directly activated HSCs.Increasing recruitment of NK cells in the liver significantly suppresses HSC activity and extracellular matrix deposition,thereby liver fibrosis is relieved.Prostaglandin?PG?is a class of important inflammatory mediator.It is currently found to be involved in the regulation of NK cell activity,but it is unclear whether it facilitates the clearance of HSC by NK cells and the progression of liver fibrosis.This study aims to elucidate the effects of PG and its receptors on NK cell activity and its role in liver fibers,and may provide a potential target for the treatment of liver fibrosis.Method:1.Expression of PG receptors was examined by RT-PCR in cytokine-stimulated NK cells,and then IFN-?expression was analysis in NK cells treated by different PG receptor inhibitors.,.2.The effect of PG receptor inhibitor on NK cell-medated HSC lysis was investigated by using co-culture system.3.Effect of EP3 deletion or inhibition on NK development and NK subpopulation distribution was examined by using flow cytometry.4.Liver fibrosis in mice was induced using carbon tetrachloride?CCL4?treament,and was evaluated by Western blot,HE staining,and Sirius red staining.5.NK cell activativatin induced by antibody coating was examined by analyzing IFN-?gene expression,.6.Finally,by using cell flow sorting and tail vein injection methods,NK cell subsets were transplanted into CCL4-treated mice,and liver fibrosis was analyzed by Western blot,HE staining,and Sirius red staining to evaluate the degree of liver fibrosis reversal.Result:1.Human NK cell line NK-92MI expressed high level of prostaglandin receptors EP2,EP3,EP4 and DP1.Upon IL-2 challenge,EP4 and DP1 inhibitors increased,while EP3 inhibitor significantly reduced NK cell activity.The effect of EP3 inhibitor was dose dependent.In mouse primary NK cells,TP,EP2,EP3,EP4,DP1 and DP2expression was detected.Similar to results in human NK cells,EP4 and DP1inhibitors increased,while EP3 inhibitors significantly reduced the activity of mouse NK cellsat dose-dependent manner..2.EP3 inhibitors significantly inhibited the killing effect of NK cells on HSCs.The killing effect of EP3^-/-motuse primary NK cells on HSC was significantly attenuated compared with that in WT control cells.3.The proportion of immature NK?iNK?in the spleen of EP3^-/-mice was increased,whereas mature NK?mNK?population was decreased in lymph nodes and spleens.CD27⁺CD11b⁺subgroup?Double posiive,DP?in the third stage was decreased significantly lymph nodes and spleens.Consistent with those in lymphatic organs,CD27⁺CD11b⁺subpopulations recruited liver from CCL4-treated mice were also decreased.4.In the model of carbon tetrachloride-induced liver fibrosis,total NK cells in the spleen of EP3^-/-mice was increased in lymphatic organs.The iNK proportion was increased while mNK proportion was decreased in spleens and lymph nodes.CD27⁺CD11b⁺subgroups in the third stage?DP?were significantly reduced in livers and lymphatic organs..5.In the model of liver fibrosis induced by carbon tetrachloride,the liver fibrosis of EP3^-/-mice was aggravated as evidenced by HE staining.The expression of hepatic collagen III in EP3^-/-was significantly increased and the index of HSC activation was greatly increased as compared to that in WT mice.6.The IFN-?secretion of hepatic CD27⁺CD11b⁺NK cells isolated from EP3^-/-mice was significantly reduced compared to that of WT upon NK1.1 antibody coating.Consistently,in vitro co-culture with mature HSC,the ability of NK cell CD27⁺CD11b⁺NK cells from EP3^-/-secreted less IFN-?compared to WT controls.7.Adoptive transfer of WT CD27⁺CD11b⁺NK cells significantly reduced collagen III expression,and the stellate cell activation,and improved liver fibrosis in EP3knockout mice.Conclusion:1.Deletion of EP3 receptor attenuates the activity of CD27⁺CD11b⁺NK cells.2.CD27⁺CD11b⁺NK cells is the major cell subpopulation of NKs that suppresses HSC-mediated liver fibrosis.3.Deletion of EP3 receptor promotes CCL4-induced liver fibrosis by suppressing the capacity of CD27⁺CD11b⁺NK cells subpopulations to eliminate hepatic HSCs.