The Study On The Mechanism Of IL-6R Antibody Alleviating Immune Disorders In Sepsis

BackgroundSepsis refers to organ dysfunction syndrome caused by dysregulation of the response to infection,and its pathogenic mechanism focuses not only on systemic inflammatory response syndrome(SIRS),but also on immune system regulation disorders,that is,from initial excessive immune activation to extensive immunosuppression at a later stage.According to the latest epidemiological survey of sepsis,there are about 20 million patients with sepsis each year worldwide,of which more than 6 million patients die,with a mortality rate of more than 25%,and about half of the surviving patients have sepsis sequelae.Disturbed regulation of the immune system is one of the key mechanisms in the development of sepsis.Pathogens are recognized by the innate immune system after invading the body and produce innate immune responses;however,overactivated inflammatory responses can lead to a series of pathophysiological changes such as vascular endothelial cell injury,leukocyte infiltration,immune cell recruitment,coagulation system activation,and rapid consumption of coagulation-related factors,leading to multiple organ dysfunction and even death.Therefore,improving immune disorders in sepsis has become a new direction for the treatment of sepsis.Combined with our previous research results,we consider that the efficacy of IL-6R antibody may be related to the immune status of the body when the antibody is used.In the early stage of sepsis,when the body is in the immune enhancement stage,the use of IL-6R antibody can reduce the degree of cytokine storm and improve the prognosis.Secondary infection is aggravated when the body is in an immunosuppressive phase.The heterogeneity of IL-6R antibody efficacy may be related to its different mechanism of action on adaptive immune cells(mainly T lymphocytes)in different immune states of sepsis.We intend to explore the timing of IL-6R antibody use in the clinical treatment of sepsis by studying the regulation of IL-6R antibody on lymphocytes in different immune states of sepsis and provide a reference basis for targeted therapy of sepsis.Methods1.Peripheral blood mononuclear cells(PBMC)were isolated from peripheral blood of 3patients with sepsis and 3 healthy subjects and analyzed by single cell sequencing data integration.The differences of PBMC between patients with sepsis and healthy subjects were compared by UMAP algorithm,GSVA analysis,GSEA enrichment analysis,and cell timed analysis,and the regularity of immune system disorders in the early stage of sepsis was analyzed.2.A mouse model of sepsis was established by cecal ligation and perforation(CLP)to observe the changes in vital signs,morphology,and body weight of the mice after modeling.The mice were divided into IL-6KO mice group and WT mice group,and scored using the Clinical Disease Score(CDS)and Sepsis Mouse Score(MSS)systems after CLP;serum and bronchoalveolar lavage fluid inflammatory cytokines interleukin-1β(IL-1β),interleukin-6(IL-6),interleukin-10(IL-10),and tumor necrosis factor-α(TNF-α)were measured by enzyme-linked immunosorbent assay(ELISA)in both groups,i.e.TNF-α)levels;changes in organs and immune cells were observed by immunofluorescence staining(IFC)and Terminal deoxynucleotidyl transferase-mediated d UTP Nick End Labeling(TUNEL)staining.Mice were divided into sham operation group,CLP group and TCZ +CLP group,and the survival rate,clinical disease score and sepsis score of mice were observed at 72 hours.After CLP was successfully modeled,splenic lymphocytes were extracted from IL-6KO mice and mice treated with tocilizumab antibodies after sacrifice,and apoptosis of lymphocytes was detected by flow cytometry.3.RAW264.7 macrophages were stimulated using lipopolysaccharide(LPS)to detect inflammatory factor m RNA expression levels at different time points.RAW264.7 cells were transfected with IL-6R si RNA,and the transferred cells were stimulated with LPS.The expression levels of inflammatory cytokines IL-1β,TNF-α and IL-6 were detected by ELISA;the polarization level of macrophages was detected by flow cytometry;mouse splenic lymphocytes were extracted with a mouse splenic lymphocyte extraction kit,Raw264.7 cells were stimulated with LPS and co-cultured with lymphocytes,and lymphocyte proliferation was detected by CCK8 assay;the secretion of inflammatory factors in the cell supernatant was detected by ELISA kit,and the apoptosis of lymphocytes was detected by flow cytometry and Western blot techniques.Results1.All cell clusters were divided into 14 clusters,which were CD8 + T cells,CD4 + T cells,B cells,CD14 + monocytes,FCGR3 A + monocytes,NK cells and DC cells.The proportion of CD8 + T cells and B cells was significantly increased,and the proportion of monocytes and CD4 + T cells was significantly reduced in PBMCs of septic patients.GO enrichment analysis of differential gene function in CD14 monocytes revealed that inflammatory factor-mediated,immune response regulation,and response to IFN-γ were closely related to their function.CD14,CD16 monocytes are also involved in the positive regulation of the apoptotic process,indicating that apoptosis is essential during the early development of sepsis.GSVA analysis showed activation of monocytes in the IL-6-JAKSTAT3 signaling pathway,indicating that IL-6 plays an important role in the early pathogenesis of sepsis.Subsequently,T cells were reclustered and divided into 16 clusters,and the proportion of gd T cells,CD8 + TEM cells,and NK cells was decreased;the proportion of CD4 + TCM cells,CD4 + Treg cells,and CD4 blasts was increased in the sepsis group compared with the healthy control group.Many upregulated genes regulating T cells are involved in TLR signaling pathways and are associated with primordial immune responses.While many down-regulated genes are involved in the transduction of the c AMP signaling pathway,this pathway is associated with T cell receptor-mediated immune activation.Recognizing the signal changes between healthy and septic groups in specific cell populations,it was found that RESISTIN signaling mainly changed dramatically in CD14 and CD16 monocyte populations,and it was speculated that this signaling played an important role in the pathogenesis of sepsis.2.A mouse model of sepsis was established by CLP.First,the survival,expression of inflammatory factors,organ(especially lung)injury and apoptosis of IL-6KO mice and WT mice after CLP were observed.The results showed that complete knockout of IL-6decreased the survival rate of septic mice,aggravated organ injury in septic mice,and aggravated cytokine storm caused by inflammation.We conclude that IL-6 is involved in pathogen clearance in the early phase of sepsis,and complete lack of IL-6 leads to ineffective pathogen clearance and aggravates inflammatory responses and organ damage.Subsequently,we injected TCZ into septic mice at different time points to observe the survival and organ injury of mice.The results showed that the use of IL-6R antibody in the early stage of sepsis increased the survival rate of septic mice and alleviated organ injury in septic mice.3.Macrophages were transfected with IL-6R si RNA to observe the changes of inflammatory factors and macrophage polarization specific markers before and after transfection,and it was found that IL-6R si RNA could reduce the inflammatory response and promote M2 macrophage polarization.Macrophages were transfected with IL-6R si RNA and co-cultured with macrophages and lymphocytes to detect the expression of inflammatory factors and apoptosis in cell supernatants and lymphocytes,and it was found that IL-6R si RNA could reduce lymphocyte inflammatory response and lymphocyte apoptosis.Conclusion In the early stage of sepsis,the proportion of CD8 + T cells and B cells increased significantly,and the proportion of monocytes and CD4 + T cells decreased significantly.Inflammatory factor-mediated,immune response regulation,and response to IFN-γ are involved in monocyte responses.Many upregulated genes regulating T cells are involved in TLR signaling pathways and are associated with primordial immune responses.While many down-regulated genes are involved in the transduction of the c AMP signaling pathway,this pathway is associated with T cell receptor-mediated immune activation.Recognizing the signal changes between healthy and septic groups in specific cell populations,it was found that RESISTIN signaling mainly changed dramatically in CD14 and CD16 monocyte populations,and it was speculated that this signaling played an important role in the pathogenesis of sepsis.IL-6 plays an important role in sepsis.After complete knockdown of IL-6,the body cannot effectively remove the invading pathogens,the inflammatory response is more obvious,and the organ injury is more serious.The use of TCZ,an IL-6R antibody,as soon as possible in the early stages of sepsis can effectively reduce the inflammatory response,promote macrophage transformation to M2 type and reduce lymphocyte apoptosis.