Alterations And Mechanisms Of B Lymphocytes In Immunosuppressive Phase Of Septic Shock

Part 1 Immune System Alterations in Septic Shock Patients:A Clinical Retrospective AnalysisObjectives:The purpose of this study is to explore the alterations of the immune system in septic shock patients by analyzing the data of immunology-related laboratory tests,to provide a scientific basis for immunomodulatory therapy.Methods:The data of 243 septic shock patients were collected retrospectively.General data included gender,age,medical history,Sepsis-related Organ Failure Assessment(SOFA)score,Acute Physiology and Chronic Health Evaluation(APACHE)II score,and so on.Laboratory data included blood routine results(neutrophil,lymphocyte,and monocyte counts),infection biomarker results(C-reactive protein,ferritin,and procalcitonin levels),serum cytokine(IL-1?,IL-2R,IL-6,IL-8,IL-10,and TNF-?)levels,serum immunoglobulin(Ig A,Ig G,and Ig M)levels,serum complement protein(C3 and C4)levels,lymphocyte subset counts(the counts of T cells,CD4~+T cells,CD8~+T cells,B cells,and NK cells,as well as the ratios of CD4~+/CD8~+T cells),and lymphocyte subset functions(the proportions of PMA/ionomycin-stimulated IFN-?positive cells in CD4~+,CD8~+T cells,and NK cells).All data were obtained within 5-7days of diagnosis of septic shock.According to the prognosis,the patients were divided into survival(101 cases)and death group(142 cases).A control group of 15 healthy volunteers was recruited to test the above-mentioned laboratory parameters of these septic shock patients in the clinical laboratory.The results were compared among the three groups.Univariate and multivariate Logistic regression analysis was used to determine the risk factors associated with mortality.Results:Compared with healthy controls,the counts of neutrophil,the levels of C-reactive protein,ferritin,and procalcitonin,as well as the levels of IL-1?,IL-2R,IL-6,IL-8,IL-10,and TNF-?increased in septic shock patients,while the counts of lymphocyte,T cells,CD4~+T cells,CD8~+T cells,B cells,and NK cells,the levels of Ig M,C3,and C4,and the percentages of IFN-?positive cells in CD4~+T cells decreased.Compared with survivors,nonsurvivors had higher IL-6,IL-8,and IL-10 levels,while lower Ig M,C3,and C4 levels and lower lymphocyte,T-cell,CD4~+T-cell,CD8~+T-cell,and B-cell counts.Low total lymphocyte and CD4~+T-cell counts,and low Ig M and C3levels were risk factors related to mortality.Conclusions:The immune function of septic shock patients has undergone significant changes,inflammatory stimulation and immunosuppression exist at the same time.These changes should be taken into account in immunomodulatory treatment.Part 2 Alterations of B Cells in Immunosuppressive Phase of Septic Shock PatientsObjectives: Current knowledge about B-cell status in the immunosuppressive phase of septic shock is sparse.This study aimed to investigate the changes in the count and function of B cells in the immunosuppressive phase of septic shock.Methods: The basic information,medical history,Sepsis-related Organ Failure Assessment(SOFA)score,Acute Physiology and Chronic Health Evaluation(APACHE)II score,and the levels of C-reactive protein and procalcitonin were collected from the clinical electronic medical record system.The absolute counts of lymphocytes and B cells of 81 septic shock patients and 13 healthy controls,and serum immunoglobulin levels of 64 septic shock patients and 10 healthy controls were determined by the clinical laboratory.In the field of sepsis,a novel classification method of B cell subpopulation was used to divide peripheral blood B cells into 5 subsets.The percentages and counts of B-cell subsets of 33 septic shock patients and 10 healthy controls and the immunoglobulin(Ig)M expression on B-cell subsets of 20 septic shock patients and five healthy controls were quantified by flow cytometry.Immunoglobulin levels produced by B cells after stimulation in vitro of 20 septic shock patients and five healthy controls were tested by enzyme-linked immunosorbent assay.According to the 60-day prognosis,septic shock patients were divided into survival group and nonsurvival group,and compared with the control group composed of healthy volunteers.Spearman rank correlation coefficient was used to evaluate the correlation between serum Ig M level and the B-cell subset counts of septic shock patients.The receiver operating characteristic(ROC)curve was plotted to evaluate the predictive value of each index for 60-day mortality.Results: Septic shock nonsurvivors were older and had higher APACHE II and MaxSOFA scores than survivors.Lymphopenia was common in septic shock patients,witha greater reduction in nonsurvivors than in survivors.B-cell counts also decreased in septic shock patients,and nonsurvivors had significantly lower B-cell counts on day 7 than survivors.Redistribution and selective depletion of B-cell subsets in septic shock patients were discovered,the percentage of activated and tissue-like memory B cells increase was probably due to the decrease in the absolute counts of immature/transitional B cells,naive B cells,and resting memory B cells.The decrease of serum Ig M level in septic shock patients was not the result of immunoglobulin class switch but may be related to the decrease of resting memory B-cell counts.Alterations in the counts and function of B cell subsets were more pronounced in the nonsurvivors than in the survivors.Additionally,ROC curve analysis showed that the data of B-cell subsets had a good predictive value for 60-day mortality risk.Conclusions: Severe abnormalities of B-cell count and function are present in the immunosuppressive phase of septic shock and are associated with prognosis.The mechanism of selective depletion of B cell subsets is worthy of further study.Part 3 Mechanism of Selective Depletion of B Cell Subsets in Immunosuppressive Phase of Septic Shock PatientsObjectives: This study aimed to investigate the mechanism of selective depletion of B cell subsets in septic shock patients during the immunosuppressive phase.Methods: The basic information,medical history,Sepsis-related Organ Failure Assessment(SOFA)score,and Acute Physiology and Chronic Health Evaluation(APACHE)II score were collected from the clinical electronic medical record system.Thirty-two septic shock patients were recruited as the septic shock group and 10 healthy volunteers as the control group.Absolute counts of peripheral blood B cell subsets were measured by Flow cytometry in septic shock patients and healthy volunteers,and were compared between the two groups.The expression of CD95 on the surface of B cell subsets,and the expression of Bcl-2,Cleaved Caspase-8,Cleaved Caspase-9,Caspase-3,and Caspase-1 in B cell subsets were measured by Flow cytometry in septic shock patients and healthy volunteers,and were compared within and between groups.Results: There was selective depletion of B cell subsets in septic shock patients.The counts of immature/transitional B cells,naive B cells,resting memory B cells,and activated memory B cells in the septic shock group were significantly lower than those in the healthy control group but no significant difference in the counts of tissue-like memory B cells between the two groups.When comparing within the group,the expression levels of CD95,Bcl-2,Cleaved Caspase-8,Cleaved Caspase-9,Caspase-3,and Caspase-1 in different B cell subsets were not consistent in both the healthy control group and septic shock group.When comparing between groups,the level of Bcl-2 in immature/transitional B cells of the septic shock group was lower than the healthy control group,and the level of cleaved caspase-9 was higher;the level of CD95 in immature/transitional B cells,naive B cells,resting memory B cells,and activated memory B cells,and the level of Cleaved Caspase-8 in immature/transitional B cells, resting memory B cells,and activated memory B cells was higher;The levels of Caspase-3 and Caspase-1 in immature/transitional B cells,resting memory B cells,and activated memory B cells were also higher.Besides,the level of Caspase-1 in immature/transitional B cells of septic shock patients was higher than that of Caspase-3,while the level of Caspase-3 in activated memory B cells was higher than that of Caspase-1.Conclusions: Whether in healthy volunteers or septic shock patients,the sensitivity of B cell subsets to apoptosis(intrinsic or extrinsic)and pyroptosis was different.The depletion of immature/transitional B cells in septic shock patients is mainly caused by pyroptosis accompanied by both intrinsic and extrinsic apoptosis;the depletion of activated memory B cells is mainly caused by intrinsic apoptosis accompanied by pyroptosis;extrinsic apoptosis and pyroptosis play a similar role in the depletion of resting memory B cells;the depletion of naive B cells may not be related to apoptosis and pyroptosis;although tissue-like memory B cells express high levels of Caspase-3 and Caspase-1,it may be related to their population characteristics,and they do not show obvious depletion.