The Association And Transcriptome Analysis Between Circulating Mononuclear Myeloid Suppressor Cells And Acute Decompensation Of Cirrhosis

Acute decompensation of cirrhosis（AD）refers to the transition from compensated to decompensated cirrhosis,which is a stage of acute development of one or more clinical events,including ascites,gastrointestinal bleeding,hepatic encephalopathy,and bacterial infection.Decompensated cirrhosis is characterized by recurrent episodes of AD.Patients with AD have different clinical courses.Recurrent multiple acute decompensation clinical events or combined acute-on-chronic liver failure（ACLF）increase the risk of short-term death.Therefore,preventing the occurrence of acute decompensation clinical events,controlling the progression of decompensated cirrhosis and improving the prognosis have been the focus of attention.Immune dysfunction is the main pathogenesis of decompensated cirrhosis and affects the outcome of the disease.Myeloid-derived suppressor cells（MDSCs）are heterogeneous subsets of myeloid-derived,undifferentiated and immature immune cells with negative regulatory effects.In recent years,the immunosuppressive effect of MDSCs in chronic liver diseases has been paid more and more attention by experts at home and abroad.The profile of myeloid-derived suppressor cells（MDSCs）and their major subsets,namely polymorphonuclear-（PMN-MDSCs）and mononuclear-（M-MDSCs）subset,and their associations with different clinical courses in AD are still unclear.In the first part of this study,the distribution of MDSCs,PMN-MDSCs and M-MDSCs subsets in peripheral blood of patients with AD was clarified through case-control study.The correlation between MDSCs and their subsets and clinical indexes was further analyzed.Furthermore,the differences in the distribution of MDSCs,PMN-MDSCs and M-MDSCs subsets among different clinical course groups in patients with AD were further investigated,and the risk factors for the occurrence and prognosis of liver failure with AD were analyzed.The second part of this study conducted transcripome analysis of single cell of the circulating M-MDSCs clusters through single-cell RNA-sequencing（sc RNA-seq）.Single-cell characteristics and heterogeneity information of M-MDSCs,and differential gene expression were analyzed.The biological function characteristics,and the signaling pathways involved in metabolism were screened out,providing new ideas and new targets for precise immunotherapy.This study explained the etiology and pathogenesis of AD from the perspective of MDSCs and immunosuppression and provided a scientific basis for intervention immunotherapy,slowing the occurrence of AD and the progression of liver failure,reducing the mortality of patients with AD and prolonging the survival of patients.1.The association between myeloid immunosuppressive cells and acute decompensated cirrhosisThe study included 129 patients with AD,20 patients compensated cirrhosis（CC）and 36 age-and sex-matched healthy controls（HC）.Data on age,sex,cause of cirrhosis,history of decompensation,and acute clinical events were collected.Physical examination,laboratory examination,endoscopy,imaging examination and previous liver biopsy were recorded after admission.The following disease severity scores were calculated:Child-Pugh,model for end-stage liver disease（MELD）,chronic liver failure consortium-acute decompensated score（CLIF-C ADs）.Peripheral anticoagulant whole blood samples were collected,and peripheral blood mononuclear cell（PBMCs）determination was isolated from peripheral blood samples by density gradient separation.The proportion of circulating MDSCs,PMN-MDSCs and M-MDSCs subsets in PBMCs in peripheral blood was detected by flow cytometry.The differences in clinical features between the case group and the control group and the differences in the distribution of MDSCs,PMN-MDSCs and M-MDSCs subsets among each group were analyzed using Nonparametric tests.Pearson correlation analysis was used to analyze the correlation between MDSCs and clinical features in CC and AD populations.Logistic regression analysis was further used to analyze the association between MDSCs and the occurrence of AD.The results showed that the proportion of CD33⁺CD11b⁺myeloid cells in peripheral blood PBMCs of patients with AD was not significantly different from that of HC and CC groups.However,in CD33⁺CD11b⁺myeloid cells,the proportion of MDSCs in patients with AD was significantly higher than that in HC and CC groups.The proportion of PMN-MDSCs and M-MDSCs subgroups also increased significantly.The proportion of circulating MDSCs,PMN-MDSCs and M-MDSCs subsets in peripheral blood CD33⁺CD11b⁺myeloid cells in patients with CC was higher than that in HC group,but the difference was not statistically significant.In addition,in the CC and AD groups,MDSCs were significantly positively correlated with indicators of systemic inflammation and indicators of liver failure.Multivariate analysis included clinical parameters with significant differences in non-parametric tests between the CC and AD groups.After adjusting for confounding factors,logistic regression analysis showed that Child-Pugh grade,lactic acid level and circulating MDSCs were risk factors for AD.The OR value of correlation analysis between circulating MDSCs and AD occurrence was 1.301（95%CI:1.059,1.599,P=0.012）.According to the disease assessment at enrollment and the 90-day follow-up period,the patients with AD was divided into different clinical stages,namely stable decompensated cirrhosis（SDC）group（n=61）,unstable decompensated cirrhosis,UDC group（n=33）,pre-acute-on-chronic liver failure（Pre-ACLF）group（n=14）and AD-acute-on-chronic liver failure（AD-ACLF）group（n=21）.Non-parametric test was used to analyze the difference of clinical features in different clinical course groups.The results showed that the proportion of males in the Pre-ACLF and AD-ACLF groups was significantly increased.The mean age of the AD-ACLF group was lower than that of the other groups.Hepatitis B was the main etiology in all groups,and the proportion of other etiologies of liver diseases was relatively lower,such as autoimmune liver disease,hepatitis C,and schistosoma liver disease;however,the difference were not statistically significant.The proportion of the etiology of alcoholic liver disease in the Pre-ACLF and AD-ACLF groups was higher than that in other groups,and the difference was statistically significant.A higher proportion of precipitants of ascites and bacterial infection in patients with AD was observed.There was no significant difference in the incidence of ascites among the four groups.The incidence of bacterial infection events,the levels of systemic inflammation indicators and the score of disease severity in Pre-ACLF and AD-ACLF groups were significantly higher than those in UDC and SDC groups.The incidence of gastrointestinal bleeding was higher in UDC group,but there was no significant difference compared with other groups.The incidence of hepatic encephalopathy in AD-ACLF group was significantly higher than that in other groups.This study further analyzed the distribution differences of MDSCs,PMN-MDSCs and M-MDSCs subsets among the AD cohort.The results showed that there was no significant difference in the proportion of CD33⁺CD11b⁺myeloid cells in PBMCs of patients with different clinical course.A higher proportion of patients in the Pre-ACLF group and the AD-ACLF group were detected,but there was no significant difference among all groups.The proportion of PMN-MDSCs subset was the highest in UDC group,but there was no significant difference among all groups.The proportion of M-MDSCs in Pre-ACLF and AD-ACLF groups was significantly higher than that in UDC and SDC groups.The proportion of M-MDSCs in AD-ACLF group was the highest.This study further explored the relationship between MDSCs,PMN-MDSCs subset and M-MDSCs subset and the progression of liver failure in patients with AD.We analyzed the incidence of liver failure at baseline in peripheral blood of MDSCs,PMN-MDSCs and M-MDSCs subsets.The result showed that the percentage of MDSCs in CD33⁺CD11b⁺myeloid cells>13.8%（compared with≤13.8%,P<0.05）and the percentage of M-MDSCs in MDSCs>28.4%（compared with≤28.4%,P<0.001）had higher rate of liver failure.No significant difference was observed in the rate of liver failure in the baseline peripheral blood of PMN-MDSCs>46.2%（compared with≤46.2%,P>0.05）.Thus,the expansion of MDSCs and M-MDSCs was positively associated with the incidence of liver failure in the AD cohort.Further multiple regression analysis showed that gender,alcohol etiology,bacterial infection and the proportion of M-MDSCs in MDSCs were independent risk factors for poor prognosis of liver failure in AD,and the OR value of correlation analysis between M-MDSCs and liver failure in AD was 1.093（95%CI:1.036-1.152,P=0.001）.Conclusion:MDSCs,PMN-MDSCs and M-MDSCs subsets in peripheral blood of patients with AD were significantly expanded.MDSCs are independent risk factors for the development of AD.Expansion of the M-MDSCs subset,but not PMN-MDSCs subset,may increase the risk of developing liver failure with an adverse prognosis of AD.M-MDSCs may be a promising target for AD immunotherapy.2.The single cell RNA-sequencing analysis of mononuclear myeloid suppressor cells in acute decompensated cirrhosisTo investigate the heterogeneity and gene expression profile of circulating M-MDSCs immune cells in the progression of liver failure in patients with AD.This part of the study included 3 patients with AD with liver failure phenotype,including 2patients with Pre-ACLF（ACLF1,ACLF2）and 1 patient with AD-ACLF（ACLF3）,and3 healthy controls.Peripheral anticoagulant whole blood samples were collected.PBMCs were separated from peripheral blood samples by density gradient separation method,CD14⁺cell cluster were selected by magnetic bead enrichment,and sc RNA-seq was performed by BD Rhapsody and i1lumina Nova Seq 6000 platforms.Dimension reduction,clustering and visual analysis using the Seurat R software package.Annotate the cell type of each cluster unbiased using Single R software.The Find Markers software package was used to identify differential expressed genes（DEGs）between different populations of cells.The functional differences of DEGs were analyzed using the Gene Onotology Consortium（GO）annotation analysis and the Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway.The results showed that 74654 high-quality CD14⁺cells were successfully obtained by single cell capture technology.15680,15837 and 14499 high-quality cells were obtained in ACLF1,2 and 3 respectively.9417,10332 and 8889 high-quality CD14⁺cells were obtained in the control group respectively.After integrating the data of dimensionality reduction,10 subgroups were obtained by clustering and cell type annotation was carried out by analyzing cell markers of each subset.The results showed that subsets 1,2,and 9 were definded as CD11b⁺CD33⁺HLA-DR^low/-CD14⁺CD15^-cell clusters,annotated as M-MDSCs clusters.Further analysis showed that the proportion of M-MDSCs clusters in CD14⁺cells in ACLF 1,2 and 3 accounted for 40.6%,30.38%and 58.93%respectively,and the proportion in control 1,2 and 3 accounted for 11.44%,10.9%and 11.04%respectively.M-MDSCs in peripheral blood CD14⁺cells of ACLF group were significantly expanded,and a higher proportion in AD-ACLF was detected.According to the cell type annotation,we further analyzed the characteristics of the subsets of M-MDSCs.The results showed that subet 1 and 2 had similar characteristic genes.Subset 9 not only had the characteristic genes of subset 1 and 2 but also the high and specific expression of marker of proliferation Ki-67（MKI67）.Thus,we defined subset 9 as a new M-MDSCs subtype,MKI67⁺M-MDSCs,namely the proliferative M-MDSCs.Subset 9 was significantly amplified in AD-ACLF,but not in Pre-ACLF.Further analysis of differential genes between groups showed that 293 up-regulated genes and 311 down-regulated genes were detected in ACLF group compared with control group in subset 1,307 up-regulated genes and 252 down-regulated genes were detected in subset 2,and 309 up-regulated genes and 749 down-regulated genes were detected in subset 9.GO analysis and KEGG enrichment analysis showed that significantly upregulated differential genes such as CLU,PLAC8,S100A8,S100A9,S100A12,CR1,MKI67,RNASE2,HMGN1,HP were involved in the regulation of immune response,cell activation,cell death,cell proliferation,apoptosis,intracellular signal transduction and other processes.CLU was the gene with the highest differential multiple and high specific expression among the three subsets of M-MDSCs.Significantly down-regulated differential genes including CCL3,CCL4,CCL3L1,CXCL8,HLA-DRB1,HLA-DPA1,HLA-DRA,HLA-DPB1,HLA-DMB,ECR1,IER3were involved in the modulation of monocyte/macrophage chemotactic function,antigen processing and presentation,cell proliferation and apoptosis.The results also showed that the pro-apoptotic signaling pathways including JNK,Fas-FADD and P53 pathways,and anti-apoptotic signaling pathways including NF-Kβand PI3K-AKT pathways were all involved in the regulation of M-MDSCs apoptosis signals.Differential gene expression profile showed that the difference ratio of c-JUN and c-FOS genes related to JNK signaling pathway was the highest.Conclusion:The number and proportion of M-MDSCs in peripheral blood CD14⁺cells of AD patients increased significantly in the progression of liver failure.The number and proportion of M-MDSCs in peripheral blood of patients with AD-ACLF increased significantly.We annotated a new subset of MKI67⁺M-MDSCs,namely proliferative M-MDSCs,which may serve as a characteristic subset in patients with AD-ACLF.CLU is specifically highly expressed in M-MDSCs and can be used as a diagnostic and therapeutic target in the progression of liver failure in patients with AD.The up-regulation of anti-apoptotic pathway related genes and down-regulation of pro-apoptotic pathway related genes may be factors in the expansion of M-MDSCs,and the pro-apoptotic JNK signaling pathway may be the target of M-MDSCs immunotherapy intervention.