Immunomodulatory Role Of Nicotinamide Phosphoribosyltransferase(NAMPT) In Chronic Hepatitis B And Hepatocellular Carcinoma

BackgroundChronic hepatitis B(CHB)is a chronic progressive disease caused by infection with the hepatitis B virus(HBV).The natural course of CHB infection is divided into five phases based on specific biochemical,serological and virological characteristics:immune tolerant(IT),immune active(IA),immune control,HBeAg negative chronic hepatitis and functional cure.The infection status and disease progression of CHB depend on the interaction between viral replication and host immune regulation.Sustained stimulation with high viral antigen load leads to functional failure of lymphocyte subsets,which in turn causes defects in the body’s antiviral function and persistent viral infection.Hepatitis B is closely related to the development of hepatocellular carcinoma and is a high-risk factor for the development of hepatocellular carcinoma,with more than 80%of hepatocellular carcinoma patients having hepatitis B in combination.Hepatocellular carcinoma(HCC)is the most common type of liver cancer and ranks as the 2nd most common cancer mortality worldwide.Most patients with hepatocellular carcinoma are already in the middle to late stages at the time of initial diagnosis and are deprived of surgical opportunities.In recent years,immunotherapy represented by immune checkpoint blockade(ICB)has provided a new idea for the treatment of solid tumors.Immune cells undergo metabolic reprogramming during proliferation,differentiation and execution of effector functions.Nicotinamide adenine dinucleotide(NAD)metabolism is a key determinant of NK cell and T cell activation or effector functions.Enhanced NAD metabolism was found in activated NK and T cells,suggesting an important role for NAD metabolism in immune regulation.Nicotinamide phosphoribosyl transferase(NAMPT)is a rate-limiting enzyme in the NAD salvage pathway,which can regulate cellular metabolism by affecting NAD.Recent studies have reported that NAMPT can promote HBV replication and hepatocyte proliferation by regulating aerobic glycolysis and can convert the immunosuppressive phenotype of tumor-infiltrating T lymphocytes to an immune activating phenotype.However,the expression profile of NAMPT in CHB and hepatocellular carcinoma is unknown,and it is unclear whether it affects immune regulation.Part Ⅰ:NAMPT enhances antiviral immunity by modulating immune cell functionObjectiveThe aim of this study was to investigate the regulatory role of NAMPT in antiviral immunity in CHB patients by single cell sequencing,bioinformatics analysis and flow cytometry(FCM).Methods1.Single cell dataset download and downstream analysisThe CHB single cell dataset GSE182159 was downloaded from the GEO database and imported into R software(version 4.0.2)for analysis,filtered by the Seurat package quality control,and high-quality cells containing 600-10,000 genes and no more than 10%of mitochondrial genes were selected.We use Find VariableFeatures function to reduce the dimensionality of the data and extract the principal components.Clustering analysis was performed using the FindClusters function in the Seurat package.Cell subpopulations were annotated based on previous literature reports and manual verification,and the distribution of cells under different infection stages and infection sources was visualized using RunUAMP.2.Identification of signature genes in each group and enrichment analysis of GSVA pathwayThe characteristic genes of healthy control(HC),IT stage,IA stage and functional cure stage were extracted using FindAllMarkers function.AUCell was applied to the processed single cell RNA-sequencing(scRNA-seq)data and the pathway enrichment score was calculated for each cell using the AUCell_calcAUC function in the AUC package.3.Consensus clustering analysis and single-sample gene set enrichment analysis based on NAD metabolismThe GSE83148 dataset was downloaded and the data were normalized to extract the expression values of genes related to NAD metabolism.Using ConsensusClusterPlus package and k-means algorithm,the samples were clustered and sub-clustered according to the expression of NAD metabolism related genes.Meanwhile,the median NAMPT expression was used as the cut-off value to divide the samples into high and low expression groups,and the differences in the abundance of immune cell infiltration among the clustered subgroups and between the high and low NAMPT expression groups were compared using single sample gene set enrichment analysis(ssGSEA).4.Detection of NAMPT expression and analysis of correlation with clinical indexesPeripheral blood was collected from 65 samples of patients with a primary diagnosis of CHB.The expression levels of NAMPT in immune cells under different disease courses of HC and CHB were detected by FCM.Clinical information was collected from CHB patients and correlation between NAMPT expression in immune cells and clinical indices was analyzed.5.Correlation analysis of NAMPT expression and functional cytokine secretionThe NAMPT was divided into two groups,NAMPT-high MFI and NAMPT-low MFI,according to the mean fluorescence intensity(MFI)of NAMPT,and the expression of functional cytokines in CD8+T cells,CD4+T cells,CD56bright NK cells,CD56dim NK cells and NKT cells in both groups were compared.6.Validation of cellular experiments in vitroPeripheral blood mononuclear cells were extracted and cultured,stimulating with different concentrations of β-nicotinamide mononucleotide(NMN).The ability of CD8+T cells,CD4+T cells,NK cells and NKT cells to secrete functional cytokines was examined under different concentrations of NMN stimulation.Results1.Differences in the identification and proportion of each cell subpopulation in peripheral blood and liver133858 and 102214 cells were obtained from peripheral blood and intrahepatic,respectively.We found that the immune cell subsets in peripheral blood were mainly CD8+T cells,NK and NKT cells,and dendritic cells,while the intrahepatic immune cell subsets were mainly exhausted T cells,mucosa-associated invariant T cells and cytotoxic T cells distributed.Further analysis of the distribution of immune cells in different disease stages revealed that the proportion of CD8+T cells,NK and NKT cells in peripheral blood was highest in stage IA of CHB,while the proportion of exhausted T cell subsets and suppressor T cell subsets in the liver was highest in stage IA,and the proportion of cytotoxic T cell subsets was lower in stage IA than that in the remaining stages.2.Identification of characterized genes of immune cells and differences in functional pathwaysTen differentially expressed genes(DEGs)were obtained for each cell type population in different disease process origins.For the major differentially enriched pathways across disease stages,the mitochondrial biosynthetic pathway,glutamine and glutamate metabolism,oxidative stress response and NAD metabolism were found to be upregulated in CD8+T cells and NK and NKT cells in peripheral blood in stage IA compared to the remaining stages.In addition,NAD metabolism and oxidative stress response were upregulated in effector T cells in the liver in phase IA,and mitochondrial biosynthetic pathway,oxidative phosphorylation and NAD metabolism were less enriched in phase IA of exhausted T cell subtypes and memory T cell subtypes in the liver than in the remaining phases.3.Both NAD metabolic pathway and NAMPT expression affect immune cell infiltrationBased on the expression of NAD metabolic genes,CHB patients were clustered into two subpopulations,and the abundance of immune cell infiltration was significantly different between the two subpopulations.Also,when comparing between high and low NAMPT expression groups,the abundance of immune cell subsets(CD56dim NK cells,CD56bright NK cells,activated CD4+T cells and activated CD8+T cells,etc)was higher in the high NAMPT expression group than in the low NAMPT expression group.4.NAMPT expression was upregulated in peripheral blood immune cells,and NAMPT expression correlated with clinical indicesThe FCM results showed that the expression of NAMPT in CD8+T cells,CD4+T cells,CD56dim NK cells,CD56bright NK cells and NKT cells was significantly higher in CHB than that in HC.Further examination of the expression of NAMPT in different disease stages showed that its expression was significantly higher in stage IA than in other stages.The correlation between NAMPT expression in the above immune cells and virological,hepatic inflammatory and metabolic indices was analyzed,and it was found that NAMPT in CD8+T cells and NKT cells was negatively correlated with HBV DNA quantification and positively correlated with AST,ALB and TBIL,respectively.5.NAMPT enhances the effector functions of CD8+T cells,CD4+T cells,NK cells and NKT cells in CHB patientsThe FCM results showed that IFN-y,perforin,natural killer cell activation receptor 2D(NKG2D),programmed death 1(PD-1),T cell membrane protein-3(TIM-3)and killer cell lectin like receptor subfamily G memberl(KLRG1)in CD8+T cells,CD4+T cells,CD56bright NK cells,CD56dim NK cells and NKT cells of the NAMPT-high MFI group were significantly higher than those of the NAMPT-low MFI group.6.NMN supplementation effectively improves the ability of immune cells to secrete cytokinesCellular experiments confirmed that the ability of CD8+Tcells,CD4+T cells,NK cells and NKT cells to secrete TNF-α,IFN-γ and perforin gradually increased with the increase of NMN concentration.ConclusionsNAD metabolism was significantly enriched in immune cells of different disease stages.CD8+T cells and NK and NKT cell subsets in peripheral blood showed high enrichment in NAD metabolic pathway during the immune activation phase,while subtypes such as intrahepatic exhausted T cells and memory T cells showed lower enrichment levels of NAD metabolic pathway during the immune activation phase,which presumably NAD metabolism may be related to immune cell function.In addition,NAMPT,the key rate-limiting enzyme of NAD metabolism,exerts antiviral immune effects by enhancing the functions of cytotoxic T cells,NK cells and NKT cells,which provides valuable clues to achieve a functional cure for CHB.Part Ⅱ:Correlation analysis of NAMPT with pathological features and immune infiltration in hepatocellular carcinomaObjectiveTo analyze the expression of nicotinamide phosphate ribosyltransferase NAMPT in hepatocellular carcinoma(HCC)and to investigate its relationship with the pathological features and immune infiltration of HCC patients.Methods1.Data acquisitionThe TIMER database was used to obtain the expression data of NAMPT in HCC.The HCC dataset with clinical information was downloaded from the TCGA database to analyze the differential expression of NAMPT in different staging of HCC(different etiologies;different pathological grades).2.Estimation algorithm and single-sample gene set enrichment analysis based on NAMPT expressionThe median NAMPT expression was used as the cut-off value to classify HCC patients into high and low expression groups.The abundance of stromal cells,immune cells and tumor cell purity between the high and low NAMPT expression groups were assessed using an estimation algorithm.The ssGSEA algorithm was used to assess the infiltration abundance of 28 immune cells in both groups.Meanwhile,the expression of NAMPT in each immune cell subpopulation was analyzed in combination with hepatocellular carcinoma scRNA-seq data.3.Correlation analysis of NAMPT expression and immune-related molecules expression in hepatocellular carcinomaThe mRNA expression of the target gene NAMPT and immune-related molecules were extracted,and the differences in the expression of immune-related molecules between the high and low NAMPT expression groups were compared.Results1.Expression characteristics of NAMPT mRNA in HCCNAMPT expression was lower in HCC compared to paraneoplastic tissues.The expression of NAMPT was low in each pathological grade of tissues compared with that in normal liver tissues,while no differences were found when comparing between pathological grades of hepatocellular carcinoma.Analysis of NAMPT expression in HCC in relation to different etiologies(HBV and non-HBV;HCV and non-HCV)showed no statistical difference.2.NAMPT is closely associated with immune cell infiltrationThe proportion of stromal cells,immune cells in the NAMPT high expression group was higher than that in the low expression group.20 immune cells were more infiltrated in the NAMPT high expression group than in the low expression group.When combined with the scRNA-seq data of hepatocellular carcinoma,NAMPT was found to be highly expressed in T cells and NK cells.3.The expression of immune-related molecules was upregulated in the NAMPT high expression groupNKG2D,CD40,FAS,ELANE,PDL1 and PDL2 expression levels were increased in the NAMPT high expression group compared with the NAMPT low expression group,while TNF,KLRG1,perforin and CTLA-4 were not different between both groups.ConclusionsThe expression of NAMPT in hepatocellular carcinoma is low,and its expression correlates with clinicopathological parameters and reflects to some extent the status of the immune microenvironment in patients with hepatocellular carcinoma.This study provides new clues to identify the population benefiting from immunotherapy in hepatocellular carcinoma.