Immune Endotype Analysis And Investigation Of The Mechanism Of Immunosuppression Mediated By M-MDSCs In Sepsis

Objective:Sepsis is an infectious disease with high morbidity and mortality,and host immunosuppression induced by infection is the main reason of high mortality.Due to the high heterogeneity of sepsis patients,the classification of immune endotypes is the key to improve the prognosis.Therefore,the present study investigated the immune endotypes and the mechanism of monocytic-myeloid-derived suppressor cells(M-MDSCs)-mediated immunosuppression involved in sepsis.Methods:In the current study,intracellular and surface proteins of immune cells were measured by flow cytometry.Cytokines were detected by flow fluorescence lattice technique.Unsupervised k-means cluster analysis and random forest approach were applied to build a classification model of sepsis endotypes.RNA sequencing was applied to compare the transcriptomic difference between M-MDSCs from sepsis patients and monocytes from healthy controls(HCs).Results:This study comprehensively described the immune landscape of sepsis patients.The percentage of MDSCs,inflammatory factors such as IL-6 and anti-inflammatory factors such as IL-1ra exhibited an increasing trend from HCs to patients with bacteremia and sepsis.On contrast,the percentage of myeloid dendritic cells(mDCs),CD4~+T cell count and CD8~+T cell count showed a decreasing trend from HCs to patients with bacteremia and sepsis.Furthermore,the study compared the difference of immune landscape between the survived and deceased patients with sepsis.The results indicated that the increase of M-MDSCs was an independent risk factor for the death of sepsis patients.In this study,sepsis was classified into three endotypes.High level of HLA-DR was expressed on CD4~+and CD8~+T cells in patients with endotype 1,which is called“effector type”.And sepsis patients of endotype 1 also had higher the percentage of effector memory(EM)CD4~+T cells.Sepsis patients of endotype 2 had significantly higher level of immune potential indicators such as CD4~+T cell number,na?ve CD8~+T cell percentage and CD28~+CD8~+T cell percentage than those in the other two groups,which is called“potential type”.Sepsis patients of endotype 3 had high level of proinflammatory cytokines such as GM-CSF,anti-inflammatory cytokines such as IL-1ra,and suppressor cells such as M-MDSCs,which is called"dysregulation type".The survival rates of patients with endotype 1,endotype 2 and endotype 3 were 83.87%,75.76%and 57.69%,respectively.The survival rate of endotype 2 was significantly higher than endotype 3(P<0.05).In addition,this study established a classification model of sepsis endotypes based on random forest analysis.Compared with monocytes of HCs,M-MDSCs of sepsis patients expressed significantly higher inhibitory molecules,including programmed cell death-1(PD-1),CD73,programmed cell death 1 ligand 1(PD-L1),cytotoxic T lymphocyte associated protein-4(CTLA-4),and T-cell immunoreceptors with immunoglobulin and ITIM domains(TIGIT).M-MDSCs significantly inhibit the proliferation of CD4~+T cells,and the expression of HLA-DR and CD28 on CD4~+T cells.Meanwhile,M-MDSCs could also significantly inhibit the expression of NKp30,NKp46 and NKG2D on natural killer(NK)cells,as well as perforin and granzyme B.M-MDSCs promoted the proliferation of regulatory T(Treg)cells,and increased the expression of T-cell immunoglobulin and mucin domain-3(Tim-3),PD-1,CTLA-4,CD39on Treg cells.In addition,this study found that the relative expression of V-set and immunoglobulin domain-containing 4(VSIG4)m RNA in M-MDSCs of sepsis patients was significantly higher when comparing with monocytes of HCs,while M-MDSCs also expressed higher VSIG4 protein on the surface.It was observed that the concentrations of inflammatory cytokines including IL-1β,IL-6 and TNF-αobviously increased in culture supernatant of THP-1 cell well with VSIG4 knockdown.The proliferation of CD4~+T cells and the expression of HLA-DR and CD28 were significantly increased with the addition of the VSIG4 blocking protein compared with the solvent control.Conclusions:This study found the impairment of innate and adaptive immunity accompanying immune dysregulation in patients with sepsis,classified sepsis into three immune endotypes,and found the prognosis of sepsis patients was related to immune endotypes.In addition,this study found that M-MDSCs played an important role in the immunosuppression of sepsis patients,and VSIG4 was an important molecule that regulated the immunosuppression of M-MDSCs.