| Objective:HBV-Acute-on-chronic liver failure（HBV-ACLF）is a type of liver failure with high short-term mortality and poor prognosis.China is a country where hepatitis B virus（HBV）is widely prevalent,and there are many patients with HBV-related chronic liver disease（CLD）.Various factors can lead to acute deterioration of CLD and liver failure.Due to the obvious changes in the immune cells and molecules of the disease,but the pathogenesis is unclear,which limits the research progress of immunological targeted therapy.The newly discovered myeloid derived suppressor cells（MDSCs）mainly secrete arginase-1（Arg-1）and transforming growth factor-β（TGF-β）,play an immunosuppressive role in tumor diseases,and play a variety of immune response effects in autoimmune diseases,allergic diseases and infectious diseases.However,the relevant mechanism of MDSCs in the progression of ACLF is still unclear.Therefore,this study will explore the characteristics of changes in MDSCs and related cytokines Arg-1 and TGF-βin ACLF,so as to further analyze the pathogenesis of ACLF.Methods:1)According to the inclusion and exclusion criteria of the research objects,62HBV-ACLF patients were collected as the HBV-ACLF group,55 CHB patients were included as the CHB group,and 60 healthy volunteers without any history of liver disease were included as the HC group.R software“limma”was used to screen the differentially expressed genes（DEGs）in peripheral blood PBMCs of HBV-ACLF group,CHB group,LC group and NC group for GO analysis and KEGG analysis.The relative levels of 28immune cell types in HBV-ACLF transcriptome data were quantified using the ss GSEA algorithm.The flow cytometry（FCM）was used to detect the changes in the number and function of peripheral blood MDSCs,Th1,Th2,and Th17 cells of the research subjects in the HC,CHB,and HBV-ACLF groups;The cytometric bead array（CBA）was used to detect the expression levels of inflammatory cytokines IFN-γ,TNF-α,IL-1,IL-4,IL-6,IL-9,IL-10 and IL-17 and MDSCs-related factors Arg-1 and TGF-β;The mRNA expression levels of T cell subset-specific transcription factors,cytokines and Notch pathway-related molecules were detected by real-time fluorescent quantitative PCR（RT-q PCR）,and finally analyzed the correlation between inflammatory factors and liver function indicators,Hes-1/Arg-1/TGF-βand CD4+T cell subset cytokines;2)Three groups of MDSCs cells were isolated and stimulated in vitro,and enzyme-linked immunosorbent assay（ELISA）method and RT-q PCR method was used to detect the expression levels of Arg-1 and TGF-β;the Western Blot method was used to detect the expression of Notch signaling pathway proteins NICD and Hes1 in MDSCs of patients with HBV-ACLF;3)The MDSCs of the three groups and CD4+T cells of the subjects in the HC group were isolated and co-cultured,and the MTT method was used to detect the proliferation level of CD4+T cells;the RT-q PCR method was used to detect the expression levels of CD4+T cell-specific transcription factor T-bet,GATA-3 and RORγt;the expression levels of cytokines IFN-γ,IL-4 and IL-17 in the supernatant were detected by ELISA method;the enzyme-linked immunospot assay（ELISPOT）method was used to detect the number of IFN-γ,IL-4 and IL-17 secreting cells;4)In vitro intervention experiment:The MDSCs from patients with HBV-ACLF were isolated and the Notch signaling pathway stimulator Jag-1-Fc was used to conduct intervention,and another group added Jag-1-Fc and DAPT（Notch pathway blocker）to conduct intervention;the Western Blot method was used to detect the activation level of Notch signaling pathway in MDSCs after intervention;5)After the intervention,the MDSCs were co-cultured with the CD4+T cells of the HC group,and the numbers of IFN-γ,IL-4 and IL-17 secreting cells were detected by FCM and ELISPOT methods;6)The ACLF mouse models were established,one group is ACLF group（give CCl4+bacterial infection）,and one group is chronic liver injury（CLI）group（give with CCl4）,and the other group was the control group（give delivery vehicle）.At the same time,the ACLF mice were intervened in vivo,and they were divided into an activation group（give Jag-1-Fc）and a control group（give PBS）.The mouse liver tissue sections were made,and the pathological changes of the liver tissue were observed by Hematoxylin Eosin（HE）,Masson and Sirius red staining;7)The mouse serum,liver,and spleen were collected,and the ELISA method was used to detect changes in serum-related cytokines;the Western Blot method was used to detect molecular changes in the Notch signaling pathway in mouse liver;the liver and spleen were grinded and homogenized to separate mononuclear cells,and the FCM method was used to detect small Changes in rat liver/spleen MDSCs,Th1,Th2 and Th17 cells.Results:1)This study found that there were 886 mRNA expression differences in DEGs at the intersection of HC group,CHB group,and HBV-ACLF group based on mRNA expression profile data（PRJNA548207）,and the Notch signaling pathway was significantly increased in HBV-ACLF;Among the 28 types of immune cells calculated by the data module,the number of active DCs,macrophages,and MDSCs in innate immune cells was upregulated,while the number of T cell subsets such as Th17 and CD4+Tem in adaptive immune cells was upregulated,while the number of CD8+Tem decreased.In the HBV-ACLF group,inflammatory factor IFN-γ,TNF-α,IL-1β,IL-4,IL-6,IL-9 and IL-17 were significantly higher than those in HC and CHB groups（P<0.05）,while IL-10 was significantly reduced（P<0.05）.There was a significant difference in the percentage of CD4+T cell in peripheral blood between the HC,CHB,and HBV-ACLF groups.The percentage of CD4+T cells in the HBV-ACLF group was 50.24±15.19%,which was higher than that in the CHB group（41.01±15.89%）and the HC group（42.63±15.91%）（P<0.05）.The percentages of Th1,Th2,and Th17 in peripheral blood of HBV-ACLF patients were significantly increased.There was no significant difference in the percentages of Th1,Th2,and Th17 between the HC group and CHB group.The percentage of Th1 in HBV-ACLF group was 32.89±14.59%,significantly higher than that in CHB group（22.69±8.29%）and HC group（22.93±7.63%）（P<0.05）.The Th2 percentage in the HBV-ACLF group was45.22±10.76%,significantly higher than that in the CHB group（33.35±10.17%）and HC group（31.67±10.66%）（P<0.05）.The Th17 percentage in the HBV-ACLF group was（22.93±7.67%）,significantly higher than that in the CHB group（7.73±3.19%）and HC group（10.88±5.19%）（P<0.05）.The percentage of MDSCs cells in the peripheral blood of HBV-ACLF patients was 25.12±8.27%,significantly higher than that of the CHB group（13.53±5.57%）and the HC group（7.53±3.62%）（P<0.05）.The relative expression level of Notch1 mRNA in PBMCs of HBV-ACLF patients was 22.73±5.21,significantly higher than Notch2,Notch3,and Notch4（P<0.05）.The levels of Hes1,Arg-1 and TGF-βin patients with HBV-ACLF were negatively correlated with the levels of IFN-γ,IL-4 and IL-17,all of which were statistically significant（P<0.05）;2)The Arg-1 level in the culture supernatant of MDSCs from HBV-ACLF patients was 13.35±5.34pg/m L,which was significantly lower than that in CHB group（32.21±7.17pg/m L）and HC group（24.13±6.43pg/m L）（P<0.05）,the TGF-βlevel in supernatant（7.58±2.04pg/m L）was significantly lower than CHB group（16.52±4.86pg/m L）and HC group（6.22±3.00pg/m L）（P<0.05）.After co-culture of MDSCs and CD4+T cells in the HC group,the level of IFN-γin the supernatant was 5.57±2.28pg/m L,which was significantly lower than that of the group without MDSCs（16.93±3.76pg/m L）（P<0.05）;IL-4 level was 5.65±1.41pg/m L,which was significantly lower than that in the group without MDSCs（17.6±1.41pg/m L）（P<0.05）;the level of IL-17 was 14.80±1.68pg/m L,which was significantly lower than that in the group without MDSCs（6.16±1.77pg/m L）（P<0.05）.After culture of MDSCs and CD4+T cells in different populations,compared with HC group,IFN-γ+CD4+T cells,IL-4+CD4+T cells and IL-17+CD4+T cells in CHB group and HBV-ACLF group were all increased,the difference was statistically significant（P<0.001）;3)Through in vitro experiments to activate the Notch signaling pathway of MDSCs from patients with HBV-ACLF,it was found that after stimulating MDSCs with Jag-1 agonists,Western Blot showed that the expression of NICD and Hes1 proteins was upregulated.Compared with DMSO group,the numbers of IFN-γ+CD4+T,IL-17+CD4+T and IL-4+CD4+T in culture system of Jag-1 group decreased significantly（P<0.05）,while there was no significant difference in cell level in culture system of Jag-1+DAPT group（P>0.05）.At the same time,compared with the DMSO group,the secretion levels of IFN-γ,IL-17 and IL-4 in the culture system of the Jag-1 group were significantly decreased（P<0.05）,while there was no significant difference in the secretion level of cytokines in culture system of Jag-1+DAPT group（P>0.05）;4)The ACLF mouse model showed significant pathological changes,with liver coefficient,pathological degree,liver function index levels,and fibrosis degree significantly higher than those of the CLI and Control group mice.After testing,it was found that the proportion of liver MDSCs cells in ACLF group mice（58.18±8.41）and absolute count（1965.00±270.29 cells/liver）were significantly higher than those in Control group mice（45.28±4.70）and absolute count（1367.17±161.64cells/liver）,with statistical significance（P<0.05）.Meanwhile,the proportion of MDSCs cells in the spleen of mice in the ACLF group（50.88±4.66）and absolute count（1404.88±184.09 cells/liver）were significantly higher than those in the Control group（38.12±3.52）and absolute count（939.63±157.26 cells/liver）,with statistical significance（P<0.05）.In addition,the level of serum MDSCs related cytokines Arg-1（57.31±10.19pg/m L）and TGF-β（46.62±4.31pg/m L）in ACLF group mice was significantly higher than that of Arg-1（31.64±4.47pg/m L）and TGF-β（25.77±3.61pg/m L）in control group mice;5)After intervention with the Jagged1 agonist,compared with the PBS group mice,the pathological morphology of the liver tissue in the Jagged1 group mice was improved,and the levels of liver function indicators in the mice were significantly reduced（P<0.05）.In terms of Notch signaling pathway,the liver NICD and HES1 protein levels of Jagged1group mice were significantly increased（P<0.05）,and the signaling pathway was activated.Meanwhile,the proportion of liver MDSCs cells（67.17±6.11）and absolute number（2210.00±308.59 cells/liver）in the Jagged1 group mice were significantly higher than those in the PBS group mice（54.45±4.75）and absolute number（1659.67±134.34cells/liver）,with statistical significance（P<0.05）.The levels of serum MDSCs related cytokines Arg-1（89.63±16.38pg/m L）and TGF-β（77.56±6.41pg/m L）in Jagged1 group mice was significantly higher than that of Arg-1 and TGF-βin PBS group mice Horizontal.In addition,Jagged1 significantly downregulated IL-1β（52.67±7.69pg/m L）,IL-6（31.43±6.24pg/m L）,and TNF-α（38.75±2.21pg/m L）level（P<0.05）.And the proportion of the liver of Jagged1 group mice IFN-γ+CD4+T cells,IL-4+CD4+T cells,IL-17+CD4+T cells,IFN-γ+CD8+T cells and the absolute number of these cells were significantly lower than those of PBS group mice,with statistical significance（P<0.05）.Conclusion:1)Peripheral blood PBMCs transcriptome data found that the increase of Notch signaling pathway in CHB and HBV-ACLF groups was related to the activation of Th17.Among the 28 different immune cells,innate immune cells were up-regulated,and MDSCs increased significantly,which may be involved in the occurrence of HBV-ACLF;2)The levels of CD4+T cell and inflammatory cytokines IFN-γ,TNF-α,IL-1β,IL-4,IL-6,IL-8,IL-9,IL-17 were increased in the peripheral blood of patients with HBV-ACLF,suggesting that T cell subsets and functional changes were involved in the process of HBV-ACLF;3)The number of MDSCs in the peripheral blood of patients with HBV-ACLF increased significantly,but the levels of functional factors Arg-1 and TGF-βdid not increase synchronously,indicating that MDSCs were dysfunctional during the process of HBV-ACLF,and could not effectively exert their functions of inhibiting T cells and the effect of resisting the inflammation reaction;The Arg-1 and TGF-βof MDSCs in patients with HBV-ACLF are negatively correlated with IFN-γ,IL-4 and IL-17,suggesting that the MDSCs are dysfunctional and fail to exert inhibitory effects,Patients not only have MDSCs function decline,but also the immune response dominated by T cells plays an important role in HBV-ACLF;4)In vitro experiments,the levels of Notch1 signaling pathway molecules NICD and Hes1 in MDSCs of HBV-ACLF were reduced,and the Notch1 signaling pathway was not fully activated,resulting in a weakened inhibition of T cell function by MDSCs.This suggests that the Notch1 signaling pathway may participate in the process of HBV-ACLF by regulating abnormal changes in MDSCs’function;5)After activating the Notch1 signaling pathway with agonists,the levels of NICD and Hes1increased,the inhibitory effect of MDSCs on T cells was enhanced,and the levels of cytokines IFN-γ,IL-4,and IL-17 decreased.If the Notch1 signaling pathway is blocked at the same time,the expression of Arg-1 and TGF-βsecreted by MDSCs is down-regulated,and its inhibitory function is significantly weakened.It is clear that the activation or inhibition of Notch signaling pathway in HBV-ACLF can regulate the secretion of T cells by MDSCs Inhibition of cytokines;6)The mouse model induced organ failure by a three-hit scheme（chronic CCl4+acute CCl4+bacterial load）,which produced a variety of cytokines that interacted with immune cells to form a complex cell-cytokine network system in ACLF.MDSCs in ACLF mice increased significantly,and effector T cells and their factors IFN-γ,IL-4 and IL-17 also increased at the same time,and the production of MDSCs failed to exert the ability to inhibit the inflammatory response;7)Injection of Notch1 signaling pathway agonists can alleviate the pathological changes and fibrosis of the mouse liver,accompanied by the enhancement of MDSCs’inhibition of T cells.If blockers are used at the same time,the results are opposite.It can be seen that the activation of Notch1 signaling pathway can regulate the production of Arg-1 by MDSCs and TGF-βplay a role in suppressing T cells.Activating the Notch1 signaling pathway in ACLF can improve the inhibitory effect of MDSCs on effector T cells,slow down the inflammatory response,reduce the liver immunopathological damage and slow down the process of liver fibrosis,which is beneficial to prevent the occurrence of ACLF,which is a good way to target this pathway in the future.The research and development of point ACLF treatment provides new immunological ideas. |
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