The Mechanisms Of STAT3 In Regulating NK Cell Functions Of HBeAg-negative CHB Patients

Object:Chronic hepatitis B virus(HBV)infection is a major health problem worldwide,which can trigger a wide spectrum of liver diseases such as liver fibrosis,cirrhosis and hepatocellular carcinoma(HCC),responsible for approximately one million deaths annually.The aims of anti-chronic HBV(CHB)therapy are persistent suppression of viral replication,and the prevention of fibrotic progression and oncogenesis.CHB is divided into the hepatitis B envelope antigen(HBeAg)-positive and HBeAg-negative phases.The HBeAg+ patients have demonstrated immune tolerance,and HBeAg seroconversion used to be the ideal endpoint of CHB therapy.However,recent studies indicate that the HBeAg" CHB patients,including the inactive hepatitis B surface antigen(HBsAg)carriers,have a high risk of progressing to HCC,and do not show any immune regulatory mechanism.Natural killer(NK)cells are crucial mediators of the innate immune system,and respond to viral infection rapidly without antigen presentation via the major histocompatibility complex(MHC).NK cell functions are regulated by several surface recognition receptors,with both activating and inhibitory functions,which need to be maintained in a balanced state for proper NK cell response.Blocking the activating receptors NKp46 and DNAM-1 impaired NK cell cytotoxicity against cytomegalovirus-infected macrophages.In CHB patients,the levels of activating receptors such as 2B4 and NKG2D were reduced by TGF-?,while that of the inhibitory receptor NKG2A was elevated,resulting in dysfunctional IFN-y production and NK cell cytotoxicity.HBsAg sero-clearance by pegylated-IFNa-2a and nucleos(t)ide analogues restored NK cell function in the HBeAg-negative CHB patients.However,the mechanisms underlying CHB-mediated NK cell dysfunction are not completely known.Signal transducers and activators of transcription 3(STAT3)is a multifunctional transcription factor that regulates the transcription of target genes associated with apoptosis,proliferation,and differentiation of immune cells,stem cells and cancer cells.Exogenous STAT3 expression,as well as its activation in tumor cells,is known to activate NK cells by increasing the secretion of IL-6,EGF and HGF in the tumor microenvironment.On the other hand,STAT3 also inhibited the migration and cytotoxicity of NK cells by downregulating the expression of chemokines and NKG2D ligands.However,the role of intrinsic STAT3 in the NK cells remains controversial.Zhu et al reported that intrinsic STAT3 can directly activate NK cells by promoting the transcription of NKG2D in healthy donors compared to HIES patients carrying a STAT3 mutation.However,STAT3 knockout enhanced the NK cell-dependent tumor surveillance to melanoma B16 cells in a mouse model.Therefore,differences in the disease microenvironment may confound the effect of intrinsic STAT3 in NK cells.In this study,we found that the function of NK cells isolated from the peripheral blood of HBeAg-negative CHB patients was positively correlated with the STAT3 levels.Furthermore,HBsAg downregulated STAT3 and inhibited NK cell function in CHB patients.Finally,intrinsic STAT3 activation in NK cells directly regulated the transcription of NKp46 and STING(Stimulator of Interferon genes)to enhance its cytotoxic functions and immune responses.Our study reveals a new mechanism of NK cell dysfunction induced by CHB infection.Methods:1.Samples of CHB patients were collected in Shandong University Qilu Hospital,without other infection or autoimmune diseases.2.PBMC were purified by Ficoll Isolation Kit,and the primary NK cells were isolated by the Human NK Cell Isolation Kit.3.The relationship between two molecules were extimates by correlation analysis.4.STAT3-knockdown and STAT3-overexpressed NK-92 cell lines were established respectively by transfecting the cells with LMP-STAT3-shRNA and pLEGFP-STAT3(#71450,Addgene)by electroporation,and selected with puromycin(2 ?g/ml,invivogen,San Diego,CA,USA)or G418(800 pg/mL,Sigma,USA)for three weeks.5.The levels of STAT3,p-STAT3,STING,receptors and functional molecules in NK cells were detected by FACS.6.The activation of STAT3 signal pathway was detected by Western blot.7.The binding rate of FITC-HBsAg to NK-92 cells was detected by FACS.8.The mRNA levels of STAT3,STING,Cyclin D1,receptors and functional molecules were detected by qPCR?9.The proliferation of NK-92 cells was detected by MTT.10.The binding of STAT3 to the promoter of NKp46 and STING was detected by ChIP.Results:1.Low level of STAT3 is associated with impaired NK cell function in CHB patients.a.The STAT3 levels of NK cells were significantly decreased in HBeAg-CHB patients.The expression and activation levels of STAT3 in the CD56 CD3-NK cells were both significantly decreased in NK cells of CHB patients compared to controls.Further analysis showed the levels of STAT3 decreased similarly in both CD56bright CD16-and CD56dimCD16+subsets of CHB patients.b.STAT3 was positively associated with NK cell function in CHB patients.STAT3 expression levels was positively correlated with NK cell cytolysis associated molecules,such as CD 107a,granzyme B,perforin and IFN-?.2.HBsAg inhibited the expression and activation of STAT3.a.HBsAg inhibited STAT3 signal in NK cells.The peripheral blood mononuclear cells(PBMCs)isolated from HDs were incubated with HBsAg,leading to significant decrease in STAT3 expression of NK cells.The expression and phosphorylation of STAT3 in the NK-92 cells were also significantly depressed by HBsAg incubationb.HBsAg bound to the surface of NK cells.HBsAg-FITC bound to more than 60%NK-92 cells.The proportion of FITC NK-92 cells was competitively decreased by pre-incubation with HBsAg but was not affected by BSA.3.STAT3 regulated the proliferation of NK cells.a.STAT3-knockdown and-overexpressed NK-92 cells were successfully establishedThe GFP NK-92 cells was established by transfection,and the STAT3 levels of NK-92-shSTAT3 and NK-92-shSTAT3 were respectively knockdown and overexpressed.b.STAT3 positively regulated the proliferation of NK-92 cellsThe proliferation ability of NK-92 cells was significantly inhibited upon STAT3-knockdown compared to the control cells,whereas STAT3-overexpression significantly increased the proliferation rate.Consistent with this,Cyclin D1 expression was also significantly downregulated by STAT3-knockdown but upregulated by STAT3-overexpression.4.STAT3 regulated the cytotoxicity of NK cellsKnockdown of STAT3 significantly decreased the surface expression of CD 107a,along with downregulating granzyme B,perforin and IFN-y mRNA and protein levels.In contrast,the above factors were upregulated in STAT3-overexpressing NK-92 cells.Consistent with this,the expression of CD 107a on primary NK cells of CHB patients was significantly restored by transfecting the cells with the STAT3-overexpression vector.Furthermore,knockdown of STAT3 inhibited rhIL-21-mediated activation of NK-92 cells.Taken together,STAT3 activation is correlated to cytolytic functions of NK cells.5.STAT3 modulated the balance between the activating and inhibitory NK cell receptors.The activating receptors NKp46 and NKp44 were significantly downregulated,and the inhibitory receptor NKG2A was upregulated in the STAT3 knockdown-NK-92 cells,while STAT3 overexpression only upregulated NKp46 and NKG2D.The proportion of circulating NKp46+NK cells in the CHB patients was positively correlated to that of the STAT3 NK cells.STAT3 knockdown inhibited the IL-21-induced increase in the proportion of NKp46+NK-92 cells.6.STAT3 directly bound to the promoter of NKp46 in NK cells.The NKp46 mRNA levels were decreased in both the primary NK cells of CHB patients and STAT3-knockdown NK-92 cells,but increased in STAT3-overexpressing NK-92 cells.We detected that p-STAT3 pull down material contained the predicted upstream DNA fragment(Binding Site 1 and Binding Site 2)of the NKp46 translational start site,which was verified by sequencing.In addition,incubation with HBsAg inhibited the binding capacity of STAT3.7.The STING levels of NK cells were significantly decreased in CHB patients.We first analyzed its expression levels of STING in the CD56 CD3-isolated from the peripheral blood of CHB patients and healthy donors(HD),and it significantly decreased in NK cells of patients compared to controls.8.STAT3 regulated the STING expression and response of NK cellsWe analyzed the expression levels of STING in the STAT3-knockdown and-overexpressing NK-92 cells.Knockdown of STAT3 significantly decreased the expression of STING.In contrast,STING levels were upregulated in STAT3-overexpressing NK-92 cells.In addition,knockdown of STAT3 inhibited cGAMP-mediated activation of NK-92 cells.9.STAT3 bound to the promoter of STING in NK cells.Specific primers were designed for 5 of those sites to identify the sequences in chromatin immuno-precipitated NK-92 cells with anti-p-STAT3 antibody.We detected that p-STAT3 pull down material contained the predicted upstream DNA fragment of the STING translational start site.Conclusion and Significance:1.In the study,we found that HBsAg could bind NK cell surface and inhibited STAT3 expression and activation of NK cells in HBeAg-CHB.2.STAT3 could positively regulate the proliferation,cytotoxicity and IFN-y production of NK cells.3.STAT3 was demonstrated to bind the promoter of both NKp46 and STING,directly regulating NK cell functions and immune responses.4.This study provided new mechanisms by which chronic HBV infection impaired NK cell functions,and new target for clinic therapy of CHB patients.