Role Of Siglec-9 In NK Dysfunction In Chronic HBV Infection And The Identification Of Small Molecule Inhibitor Against Siglec-9

Background and ObjectivesPersistent Hepatitis B virus(HBV)infection is an important cause of chronic hepatitis B(CHB),cirrhosis and hepatocellular carcinoma(HCC).The currently available drugs such as nucleoside analogues(NAs)and interferon-α(IFN-α),are not effective in clearing HBV reservoir.It has been well documented that HBV infection leads to immune dysfunction which is crucial for the occurrence of related chronic liver diseases such as cirrhosis and HCC.Therefore,further explorations of mechanisms promoting HBV mediated immune dysfunction would provide effective strategies for the treatment of HBV-related liver disease.NK cells are abundant in the liver and play an important role in anti-HBV infection.However,NK cells from CHB patients are dysfunctional,expressing fewer activating receptors and more inhibitory receptors.Thus,revealing the molecular mechanism and restoring NK cell function are expected.Sialic acid-binding immunoglobulin-like lectins(Siglec),which are widely expressed on immune cells,belong to a new family of immune checkpoints that have been extensively studied in recent years.Siglec proteins have been reported to be involved in various pathophysiological processes,such as autoimmune diseases,inflammation,and tumors,by regulating immune cells.Among the Siglec family members,Siglec-7 and Siglec-9 are abundantly expressed in NK cells.As an inhibitory receptor,Siglec-9 is selectively expressed in CD56dim NK cells resulting in a mature phenotype and enhanced chemotactic potential.However,the role of Siglec-9 in chronic HBV infection remains unclear.Here,we explored the Siglec-9 mediated regulation of NK cells and its role in chronic hepatitis B.and defined small molecule inhibitor of Siglec-9 by virtual screening.Our data provide not only a new insight for understanding mechanism of NK dysfunction during HBV infection but also a strategy for treatment of CHB and HCC by enhancing NK cell function.Methods and ResultsFresh peripheral blood was collected from 79 CHB patients and 59 healthy controls.Siglec-9 expression in different subpopulation of NK cells was detected by flow cytometry while its correlation with viral replication and clinical indicators was analyzed.The results showed that Siglec-9 was mainly expressed in the CD3-CD56dim NK subpopulation.Compared with healthy controls,Siglec-9 expression in both CD3-CD56+NK cells and CD3-CD56dimNK subpopulation was significantly decreased in CHB patients,and Siglec-9 expression in NK cells was negatively correlated with serum HBeAg and HBV DNA levels.Phenotypic assays revealed that a more activated phenotype in Siglec-9+NK cells compared with Siglec-9-NK cells from CHB patients,exhibiting low expression of NKG2A but high expression of NKG2D,NKp46,and NKp30.Further functional assays showed that blockade of Siglec-9 significantly restored the function of both primary NK cells and human NK cell line NK92 in CHB patients.Immunofluorescence staining results showed that HBV infection and inflammatory factor stimulation significantly promoted the expression of Siglec-9 ligands.Consistent with this,Siglec-9Fc positive cells(expressing Siglec ligand)were significantly increased in liver tissues of CHB patients compared to normal liver tissues,suggesting that abnormal Siglec-9 ligand expression in CHB patients mediates Siglec-9 pathway activation and may be a potential target for the treatment of CHB.In order to obtain an effective method to block Siglec-9,enhance NK cell function,and block HBV chronic infection,we identified a small molecule inhibitor of human Siglec-9 from existing small molecule libraries through the following process:since the structure of Siglec-9 has not been resolved so far,the homologous protein model of human Siglec-9 was constructed by using the crystal structure of Siglec-7 as a template;then a docking-based virtual screening of the SPECS commercial library containing more than 300,000 small chemical molecules was performed using AutoDock Vina software,which provided 33 small molecule compounds as the candidates for further evaluation through a multi-level filtering strategy.A small molecule inhibitor SMSI-1 with strong Siglec-9 affinity was identified by protein-ligand affinity assay of biofilm layer interference technique.In vitro cell functional assays showed that SMSI-1 could effectively promote the production of killing molecules and cytokine secretion in NK92 cells and peripheral blood NK cells of CHB patients,and this effect could be disrupted by Siglec-9 Fc.All these results confirm that the small molecule inhibitor SMSI-1 can block Siglec-9 and restore the function of NK cells,providing a new strategy for the treatment of CHB.ConclusionsTaken together,this study demonstrated that the immunosuppressive receptor Siglec-9 pathway was abnormal in NK cells of CHB patients through clinical specimen detection combined with functional assays.Blocking the Siglec-9 pathway significantly restored NK cell function in CHB patients,suggesting that Siglec-9 plays an important role in the regulation of NK cell function in the chronic HBV infection.Furthermore,we identify a small molecule inhibitor of Siglec-9,which can significantly improve the function of NK cells in CHB patients.All the above results not only revealed a new mechanism of abnormal NK function in CHB patients,but also provided a potential novel strategy for the treatment of CHB and HCC.