Up-regulation Of CXCR4 On Myeloid-derived Suppressor Neutrophils Subgroups May Be Involved In Immunosuppression Of Sepsis Of The Elderly Patient

Background Sepsis is a prevalent,serious medical condition with substantial mortality and a significant consumption of health-care resources.Its incidence has increased around9% annually in general population over the last years and specially in aged patients group.There is a also a concern about to admit patients in the intensive care units taking into account that the outcome of elderly patients is poorer compared to younger people.Nevertheless,the management of septic elderly patients does not differ substantially from younger people.In addition,the quality of life in septic elderly survivors is also lower than in younger people.Therefore,study of the pathogenesis of sepsis in the elderly patient is the foundation to solve the current limitations of sepsis treatment.It is known that sepsis is a complex response involving a variety of cells and mediators.More and more studies have shown that neutrophils are involved in the inflammatory response of sepsis and play a synergistic effect.Neutrophils are the key effector cells of the innate immune system and are vital in the hosts response to infection.Neutrophils possess a broad arsenal of defensive strategies: reactive oxygen species production,phagocytosis,release of antimicrobial granule contents.These functions serve to protect against invading Differentiation of immune cells into different functional phenotypes has been well described for macrophages and dendritic cells.More recently there have been several reports indicating that similar distinct functional neutrophil phenotypes exist in the circulation.characterized neutrophil subsets in experimental studies based on CD16（FcγRIII receptor）and CD62L（L-selectin）expression.Besides mature neutrophils(CD16^high CD62Lhigh),two other distinct neutrophil subsets: immature neutrophils（CD16low CD62Lhigh）and suppressive neutrophils(CD16^high CD62^Llow).Immature neutrophils may arise after depletion of mature neutrophils from the bone marrow and are deemed incompetent in anti-microbial immune functions.The suppressive neutrophils show a hypersegmented nucleus which implies increased maturation compared to mature neutrophils.Interestingly,the suppressive neutrophils subset was described to be capable of T cell suppression and could play an important role in the dampening of severe inflammatory responses.However,they could also cause immune paralysis,limiting the effectivity of the immune system against invading pathogens.In recent years,Chemokines are a small molecule inflammatory cytokine with a molecular weight of 8000 to 15000,which plays an important role in the activation and migration of inflammatory cells by binding to the relevant receptor on the cell surface.Based on the number and location of N-terminal cysteine residues,chemokines can be divided into four subfamilies,CXC,CC,C and CX3 C.According to the different binding ligand,chemokine receptors can also be divided into four categories,namely,CXCR,CCR,XCR and CX3 CR.Stromal cell derived factor-1（SDF-1）is an inflammatory chemokine that is secreted by bone marrow stromal cells.The system is named CXCL12（CXC chemokine ligand 12）.SDF-1 is the only natural chemokine that binds to CXC chemokine by CXC chemokine receptor（CXCR）4,and SDF-1 and CXCR4 are widely expressed in a variety of cells and tissues and are mobilized in cells,Migration,proliferation and survival play a vital role.Based on the presence of neutrophil activation in the inflammatory environment in vivo,we investigated the role of inhibitory neutrophil subpopulations in immunosuppression in patients with sepsis by studying the function of different subgroups of neutrophils Mechanism for the study of sepsis to provide new research targets.In this study,the different features of neutrophil subpopulations were described in the sepsis elderly patients and aged sepsis mice.To study the correlation between CD16 high CD62^Llow CXCR4 highneutrophil subgroup and disease severity in patients with sepsis.Discuss the mechanisms of CD16 high CD62^Llow neutrophil subpopulation on Treg cell regulation by CXCR4 in immunosuppression of aged sepsis.Finally,the protective effect of CD11 bhigh CD62^Llow Ly6 Clow CXCR4high neutrophils on the organs of aged sepsis mice was explored from the perspective of steady state,release,recruitment and recovery of neutrophil subpopulations.To explore the role of neutrophil subpopulation in the formation of neutrophils in patients with sepsis and immune mechanism for the clinical diagnosis and treatment of sepsis to provide new targets and ideas.Materials and MethodsPart 11.The peripheral blood neutrophils of elderly healthy volunteers were sorted by gradient centrifugation and identified by immunofluorescence and flow cytometry.2.The changes of neutrophil phenotype after sepsis in peripheral blood of neutrophils for elderly patients and elderly healthy volunteers were detected by flow cytometry.3.Analysis of the subgroups of CD15 ⁺ CD16 ⁺ neutrophils in peripheral blood of elderly patients with sepsis.4.To analyze the expression of CD11 b and CD64 in peripheral blood neutrophils subgroups of patients with sepsis.5.The correlation between CD16 high CD62^Llow CXCR4 highneutrophil subgroup and and the severity in patients with sepsis.Part 21.The CD16 high CD62^Llow neutrophil subgroups of elderly patients with sepsis were sorted by flow cytometry.The peripheral blood mononuclear cells of healthy elderly volunteers were co-cultured with CD16 high CD62^Llow neutrophil subgroups in different proportions and analyzed for T lymphocytes Proliferation inhibitory.2.The levels of IFN-γ,IL-6 cytokines were detected by ELISA in the co-culture system.3.The CD16 high CD62^Llow neutrophil subgroups in elderly patients with sepsis were co-cultured with peripheral blood mononuclear cells（PBMCs）from healthy volunteers.The flow cytometry was used to analyze the different subgroups of Treg cells in co-culture system4.The inhibitory effect of CD16 high CD62^Llow neutrophil subgroup on the proliferation of T lymphocytes were analyzed by flow cytometry when treated with different inhibitors.Part 31.The function of neutrophils subsets in peripheral blood of aged sepsis mice was analyzed by flow cytometry,and the expression of CD11 bhigh CD62^Llow Ly6 Clow CXCR4high was observed in aged mice with severe sepsis.2.Flow cytometry was used to detect the changes of inhibitory neutrophil subsets in the blood,bone marrow,spleen,lung,liver and kidney of the aged mice before and after the establishment of the severe sepsis model.3.Flow cytometry was used to analyze the changes of inhibitory neutrophil subsets in blood,bone marrow,spleen,lung,liver and kidney of severe septic mice compared with untreated group after treatment with CXCR4 inhibition.4.To investigate the effect of blocking CXCR4 on 7-day survival in severe sepsis mice.Resultspart 11.The expression of CD16 high CD62^Llow and immature neutrophil subgroup CD16 low CD62Lhigh in peripheral blood of elderly patients with sepsis were significantly higher than that of normal healthy elderly controls.There was no significant difference in CD16 high CD62Lhigh and CD16 low CD62^Llow expression.2.The percentage of neutrophils CD16 high CD62^Llow and CD16 low CD62Lhigh neutrophils in LPS-stimulated elderly healthy volunteers were also significantly increased,and there was no significant difference in CD16 high CD62Lhigh and CD16 low CD62^Llow expression.3.CD16 low CD62^Llow subset,CD16 low CD62^Llow subset,CD16 low CD62Lneg subgroup and CD16 high CD62Lneg subset accounted for a higher proportion of CD16⁺ subgroups,and the CD16 high CD62Lhigh subsets in sepsis patients Accounting for the lowest column.4.The expression of CD11 b and CD64 in CD16 ⁺ subgroup of whole blood neutrophils in elderly septic patients showed that: CD16 high CD62^Llow subgroups have the highest CD11 b and CD64 expressions.CD16 low CD62Lhigh subgroup and CD16 high CD62^Llow subgroup were positively correlated with APACHE Ⅱ score.5.The level of APACHE Ⅱ score,blood lactate and CD16 high CD62^Llow CXCR4 high expression in the dead groups were higher than those in the survival groups.the septic shock groups have higher APACHE Ⅱ score,blood lactate levels than the sepsis group.The higher expression of CD16 high CD62^Llow CXCR4 high in septic groups indicate higher mortality,all the differences were significant（p <0.05）.Part 21.Different proportions of CD16 high CD62^Llow neutrophil subgroups in elderly septic patient co-cultured with peripheral blood mononuclear cells（PBMCs）in elderly healthy volunteers,the results showed the concentration-dependent inhibition for T lymphocyte proliferation with statistically significant（p <0.05）.2.The CD16 high CD62^Llow neutrophil subgroup in elderly septic patients and the peripheral blood mononuclear cells of elderly healthy volunteers were co-cultured at 2: 1 ratio.Compared with the group of individual cultured peripheral blood mononuclear cells,the levels of IFN-γ and IL-6 were significantly lower in co-cultured system with statistically significant（P <0.05）.3.The CD16 high CD62^Llow neutrophil subgroup in elderly septic patients and the peripheral blood mononuclear cells of elderly healthy volunteers were co-cultured at 2: 1 ratio.Compared with the group of individual cultured peripheral blood mononuclear cells,the expression of Treg cells were increased,especially the effector Treg cells were biggest increased.4.The regulation of CD16 high CD62^Llow neutrophil subgroups in elderly septic patient mediated T lymphocytes proliferation through CXCR4.Part 31.The CD11bhighCD62^LlowLy6 Clowand CD11blowCD62LhighLy6Chigh gradually increased by lengthened disease duration and the expression of CD11 bhigh CD62^Llow Ly6 Clow CXCR4high was gradually increased with the severity of sepsis in mice,all the differences were significant（p <0.05）.2.Compared with control aged animals,endotoxemia caused the accumulation of CD11 bhigh CD62^Llow in the blood,liver,lung and kidney of mice,the inhibitory neutrophils CD11 bhigh CD62^Llow were significantly decreased,the differences were statistically significant（p <0.05）.The numbers of CD11 bhigh CD62^Llow in the inhibitory neutrophils accumulated in the blood,liver,lung and kidney of the aged mice were significantly lower before inhibition of CXCR4 in septic mice than that of in endotoxemia mice only,the difference was statistically significant（p <0.05）.3.Blocking CXCR4 signal can significantly improve the survival rate of aged severe sepsis mice within 7 days.Conclusions1.The peripheral blood neutrophil CD16 high CD62^Llow CXCR4 high in elderly patients with sepsis is positively correlated with the severity and survival of sepsis.2.CD16 high CD62^Llow neutrophil subgroups regulated the steady state of Treg cells through CXCR4 in co-culture system,the function of Treg cells was enhanced.3.Blocking the CXCR4 signal can mediate the homing effect of suppressive neutrophil subgroups in aged septic mice.At the same time improve the survival rate of aged septic mice in 7 days.