BRD4 Inhibitor JQ1 Alleviates Acute On Chronic Liver Failure Through Modulating Monocytic MDSC And Its Mechanism Investigation

BackgroundACLF is a serious medical condition characterized by the rapid deterioration of liver function due to various precipitating factors on the basis of chronic liver disease.Unfortunately,there is currently no effective treatment for ACLF,and the mortality rate is high.One of the key features of ACLF is the presence of severe systemic inflammatory responses,which are positively correlated with short-term mortality in patients.Additionally,patients with ACLF often experience immunosuppression,creating a unique immune profile that requires specialized treatment strategies.Recent studies have revealed that immune cells with negative regulation of immune response,such as Treg and MDSC,infiltrate the liver of ACLF patients.Modulating these immunosuppressive cells represents a promising and new therapy for ACLF,as it can reduce liver injury by suppressing excessive local inflammatory responses.BRD4 is an epigenetic recognition protein that plays a critical role in pathways such as NFκB,which mediates the innate immune response and plays a pro-inflammatory role in autoimmune diseases and sepsis.JQ1 is the first publicly available BRD4 inhibitor and is considered a new generation of anti-inflammatory drug.It exerts immunomodulatory effects by regulating the secretion of immune cells(T cells,DCs,macrophages,etc.)and cytokines,making it a promising candidate for the treatment of ACLF.AimIn this study,we utilized sc RNA-seq to comprehensively map the complex immune cell profile in the liver during ACLF.Through this analysis,we identified specific immune cell subsets that are implicated in the disease pathogenesis of ACLF.Building upon these findings,we investigate the immunological mechanism of JQ1 in inducing M-MDSC to attenuate ALF in a mouse model.Our study provides a foundation for exploring novel targets and approaches for treating ACLF.Methods1.Ten human liver tissue samples(including control(n=2),cirrhosis(n=3)and ACLF(n=5)groups)were collected,and non-parenchymal cells were extracted from the liver.Single-cell transcriptome sequencing was performed,followed by functional annotation using GO and KEGG pathway enrichment analysis to identify differentially expressed genes and pathways between the groups.2.Monocyte/macrophage infiltration was determined by immunofluorescence staining of liver tissue with CD68 and TREM2.Cytokines and chemokines in both liver tissue and serum were quantified using protein microarray technology,including IL-1α,IL-1β,IL-4,IL-6,IL-8,IL-10,IL-13,MCP-1,IFN-γ,and TNF-α.3.The study utilized a mouse model of ALF induced by LPS/D-Gal N injection to examine the immune cell composition in both liver and blood.The immune cells examined by multicolor flow cytometry included M-MDSC,neutrophils,M1 macrophages,M2 macrophages,CD3~+T cells,CD4~+T cells,CD8~+T cells,NKT cells,NK cells,and B cells.4.The 24-hour survival rates of the JQ1 pretreatment group and the model group were observed,and differences in transaminases,liver pathology,and apoptosis were compared.Immune cells between liver and blood were analyzed by flow cytometry.Serum cytokines,including TNF-α,IL-6,IL-10,and TGF-β,were quantified using ELISA.Additionally,the differences in cytokine levels(TNF-α,IL-6,IL-10,TGF-β,Arg1,and i NOS)in the liver were compared using q PCR.Finally,Gr-1 immunohistochemical staining of liver tissue was performed to compare the differences in MDSCs between the JQ1 and control groups.5.MDSCs were induced in vitro from primary cells of mouse bone marrow,and the ratio of M-MDSCs and PMN-MDSCs in the JQ1 and control groups were measured by flow cytometry.Transcriptomic sequencing was performed,followed by functional annotation using GO and KEGG pathway enrichment analysis to identify differentially expressed genes and pathways between the groups.Ten core differential genes derived from PPI analysis were validated by q PCR.Results1.The study performed immune cell mapping of the liver in ACLF and identified an increased proportion of liver-infiltrating monocytes/macrophages,particularly the Mono1 subgroup that has immunosuppressive functions.This finding suggests that Mono1 cells,which are similar to M-MDSC cells,play a role in shaping the immunosuppressive hepatic microenvironment of ACLF.2.The study induced an ALF mouse model using LPS/D-Gal N and found that the immune status of the mice was altered during the acute injury period,with increased proportions of M-MDSC,neutrophils,M1and M2 macrophages in the liver,and decreased proportions of CD4~+T cells,NKT cells,and B cells.The peripheral blood also showed similar changes,indicating the potential involvement of these immune cells in the pathogenesis of ALF.3.The study found that JQ1 pretreatment significantly improved the survival of ALF mice and reduced liver injury,likely through regulating immune cell infiltration and the inflammatory response.Specifically,JQ1reduced the percentage of liver-infiltrated M-MDSC and the levels of TNF-αand IL-6,while increasing the percentages of NKT cells and B cells in the peripheral blood.JQ1 also decreased the local inflammatory response in the liver and enhanced the immunosuppressive function of MDSC.4.induced an ALF mouse model using LPS/D-Gal N and found that the immune status of the mice was altered during the acute injury period,with increased proportions of M-MDSC,neutrophils,M1 and M2macrophages in the liver,and decreased proportions of CD4~+T cells,NKT cells,and B cells.The peripheral blood also showed similar changes,indicating the potential involvement of these immune cells in the pathogenesis of ALF.5.The study identified potential mechanisms by which JQ1 exerts its effects,such as the activation of TNF signaling pathway and cytokine by ligand interaction signaling pathway.In vitro studies showed that JQ1induced the proliferation of M-MDSC and upregulated the expression of CCL2,a chemokine that promotes the recruitment of immune cells to the site of inflammation.Conclusion1.The immune profile of ACLF liver is characterized by massive infiltration of monocytes/macrophages and the involvement of M-MDSC-like cells in shaping the suppressive immune microenvironment in the liver,which plays an important role in the disease process of ACLF.2.JQ1 has a protective effect on the mouse model of ALF by promoting the expansion of liver-infiltrating M-MDSC and attenuating the local inflammatory response in the liver,providing a theoretical basis and a potential new therapeutic target for the treatment of ACLF.