Inactivation Of OX40-OX40L Signaling Pathway Decreases T Follicular Helper Cells Generation And Contributes To Impaired B-cell Maturation During Sepsis

Background:Sepsis is defined as life-threatening organ dysfunction due to a dysregulated host response to infection and is the disease with the highest mortality rate in surgical intensive care units.Sepsis is the result of persistent uncontrollable infection,today many individuals with septic shock,who would previously have died,will survive a reflection of improved treatment of infection and better and safer organ support(Continuous renal replacement therapy,Extracorporeal Membrane Oxygenation etc.),but long-term mortality is also high.Over the last years,we still lack specific therapies to directly treat the dysregulated host response and immunotherapy may be the dawn of septic patients.Sepsis can be considered a race to the death between pathogens and the host immune system,the immune status of the patients is the most critical factor for the prognosis of sepsis,especially refractory sepsis.During sepsis,a large number of pathogenic bacteria invade the body,and the immune system kills and swallows them leading to hyper-inflammatory phase:hyperthermia and shock.At the same time,a large number of pathogenic bacteria can inhibit the generation and maturation of immune cells,promote immune cell death or damage immune cell function leading to immunosuppression.Therefore,both proinflammatory and anti-inflammatory responses occur early and simultaneously in sepsis.Although every septic patient in surgical intensive care units receives treatments to destroy pathogens and restore immune function,immunosuppression remains widespread.Immunosuppression is an important cause of a poor sepsis prognosis,because it can lead to a failure to eradicate primary infections and the acquisition of lethal secondary infections(bacterial,fungal or viral).Exploring the mechanism of immunosuppression during sepsis is an important means to improve the prognosis of sepsis.In clinical work,we found lymphocytes in peripheral blood were low in patients with sepsis.Lymphopenia,including B lymphopenia and T lymphopenia,is a main cause of immunosuppression during sepsis.In the past,some studies emphasize the importance of T lymphopenia in sepsis and attempt to reveal its mechanism,but few studies focus on B lymphopenia.Here,we investigated the mechanism of reduced B cell numbers during sepsis through retrospective clinical research,prospective clinical research and animal experiments.Methods:To identify the exact lymphocyte counts that affect the prognosis of sepsis,we firstly conducted a retrospective study.In a retrospective clinical study,we compared the lymphocyte counts in peripheral blood between 28-day survivors and 28-day non-survivors at sepsis onset and24 h after sepsis onset.Cox regression was used to analyze risk factors of poor sepsis prognosis.Kaplan-Meier was used for survival analysis.Then we conducted a prospective observational clinical experiment,differences in B-cell maturation,B cell death and numbers of circulating Tfh(c Tfh)cell were compared between 28-day survivors and 28-day non-survivors,mainly by flow cytometry and enzyme-linked immunosorbent assay(ELISA).Finally,in animal experiments,cecal ligation and puncture(CLP)model was used.Flow cytometry,ELISA and cell re-transfusion were mainly used to explore the mechanism of B lymphocyte maturation and its related factors during sepsis.Results:In retrospective study(n=123),we found that 28-day non-survivors had lower lymphocyte counts than 28-day survivors both at sepsis onset:0.43±0.22×10~9/L vs.0.55±0.27×10~9/L,and 24 h after sepsis onset:0.40±0.25×10~9/L vs.0.62±0.39×10~9/L.And patients with lymphocyte counts less than 0.4×10~9 cells/L had higher mortality than patients with lymphocyte counts above 0.4×10~9 cells/L both at sepsis onset(Log-Rand p<0.0001)and 24 h after sepsis onset(Log-Rank p<0.0001).In observational prospective cohort study(n=40),compared to survivors(n=23),non-survivors(n=17)had fewer numbers of mature B cell and c Tfh cell(sepsis onset:memory B cells:3.44%vs.4.48%,antibody-secreting cells:4.53%vs.6.30%,c Tfh cells:3.57%vs.4.49%;24 h after sepsis onset:memory B cells:4.05%vs.7.20%,antibody-secreting cells:5.25%vs.8.78%,c Tfh cells:3.98%vs.6.15%),while there were no differences in numbers of na?ve B cell and cell death of mature B cells between them.We further noticed the numbers of c Tfh cell positively correlated with the numbers of mature B cell and immunoglobulin concentrations,but not na?ve B lymphocytes.In animal experiments,we found the worse germinal center(GC)reaction was seen in the mouse with more serious sepsis,which was caused by invalid treatment.Tfh cell re-transfusion could rescue B lymphocyte maturation and GC reactions and,therefore,improve the survival rate.Inactivation of OX40-OX40L signal pathway inhibited Tfh cell production and leaded to B lymphocyte maturation disorder,and when it was activated the production of Tfh cell,GC reaction and B lymphocyte maturation and survival were improved.Conclusion:Impaired B lymphocyte maturation mainly contributes to B lymphopenia.Inadequate of Tfh cells in spleen limited to GC reaction and leaded to impaired B lymphocyte maturation.Activation of OX40-OX40L signal pathway could improve the numbers of Tfh cells and GC reactions and survival.Tfh cells,GC reactions and OX40-OX40L signal pathway may be a new therapeutic target for treating sepsis.