| BackgroundSepsis is a life-threatening condition that arises when the body’s response to infection causes injury to its own tissues and organs.It was caused by pathogens that evade the host’s defense mechanism,continuously stimulate and damage host cells,and ultimately lead to the inability of host immune function to maintain homeostasis.Sepsis is one of the major causes of death in hospitalized patients.Approximately 11 million patients died from sepsis globally in 2017,accounting for nearly 20%of all deaths.Although early identification of sepsis and intervention can improve the prognosis of patients,there still is a long way.The early mortality of sepsis has decreased through treatment bundle strategies such as elimination infection source,promptly and effective application of antibiotics,adequate fluid resuscitation and vital organ support,however,many patients still died from secondary infection.To effectively treat sepsis and reduce the high morbidity and mortality of sepsis,it is urgent to find effective therapeutic targets.Immunotherapy is the treatment or prevention of disease that involves the stimulation,enhancement,suppression,or desensitization of the immune system.Immunotherapy for some cancers has made significant progress,but sepsis is still undergoing exploration.T lymphocytes are one of the most important immune cells,with which decreased obviously prone to worsening infection and worse prognosis by clinical studies.T cells are activated by CD28 costimulatory molecule binds to CD80/CD86,a B7 family molecule on the surface of APC.In many diseases,the balance between T cell subtypes is regulated by CD28.Helper Th17 cells(Th17)and regulatory T cells(Treg)are important T cell subtypes,and the balance between Th17 cells and Treg cells is a key checkpoint of sepsis.This study intends to improve the prognosis of sepsis by regulating the imbalance of Th17/Treg cells in sepsis by blocking CD28.ObjectiveTo evaluate the correlation of the level of CD28 expression in T cell,the ratio of Th17 to Treg cells and clinical prognosis in patients with early sepsis.And to understand the effects and mechanism of CD28 on regulating Th17/Treg cell ratio in septic mice.Methods1.To explore the effect of Th17/Treg cells level on clinical prognosis in patients with sepsis.Th17/Treg cell ratio and CD28 expression were evaluated between sepsis patients and healthy volunteers.A total of 65 patients with sepsis admitted to the Intensive Care Unit from January 2020 to October 2020 were enrolled.Their demographic characteristics,SOFA score,APACHE II score,length of ICU stay and prognosis were recorded.The expression levels of IL-6,TNF-α,IL-10 and IL-17A in peripheral venous blood were detected,and peripheral blood mononuclear cells(PBMC)were isolated.The expression levels of membrane antibodies(CD28,CD80,CD86,CTLA-4)and intracellular antibodies(FoxP3,IFN-γ,IL-4,IL-17A)were analyzed by flow cytometry.The predictive value of Th17/Treg cell ratio in the severity and prognosis of sepsis patients was analyzed.2.Th17 cells and Treg cells in different periods in septic mice.Mice were randomly divided into normal group and cecal ligation puncture group(CLP group).Mice in the CLP group were sampled for spleen Th17 and Treg cell levels and related cytokines levels at 24 h,72 h,5 d and 7 d after surgery,respectively.Therefore,to observe the effect of anti-mouse CD28 antibody on the survival rate,clinical score,the index of spleen and the levels of IL-6,TNF-α,IL-10 and IL-17A in serum of sepsis mice.3.Effects of anti-CD28 antibody on Th17/Treg cell ratio in septic mice and its potential mechanism.Mice were randomly divided into Sham group(Sham group),CLP group,CLP+anti-CD28 antibody group(CLP+anti-CD28)and CLP+isotype group(CLP+isotype).CLP groups were given an intraperitoneal injection of normal saline,anti-CD28 antibody and isotype IgG after the operation,respectively.Then the spleen was sampled to detect Th17 cell,Treg cell,the ratio of Th17/Treg cells,and the levels of Bcl-2 and Bax.Results1.Th17/Treg cell ratio and CD28 expression level in peripheral blood of patients in early sepsis were significantly higher than those of normal volunteers.The expression levels of Th17 cells,IL-17A and Th17/Treg cell ratio in sepsis patients with ICU stay longer than 14 days were significantly higher than those in patients with ICU stay less than 14 days.There was no difference in the expression levels of CD28,CTLA-4,CD80,CD86,Th1 cells,Th2 cells and Th1/Th2 cells ratio between the two groups.Th17/Treg cell ratio was positively correlated with SOFA score,APACHE Ⅱ score,PCT expression level and length of ICU stay of sepsis patients.Th17/Treg cell ratio and APACHE Ⅱ score combined to predict the area under the curve(AUC)of sepsis patients with ICU stay longer than 14 days was 0.800(95%CI:0.676-0.892).2.The expression of Th17 cells and Treg cells in the spleen of septic mice on days 1,3,5 and 7 after CLP was significantly higher than that of the normal group.On day 5 after CLP,Th17 cells and Treg cells in mouse spleen cells increased most significantly,while Th17/Treg cell ratio peaked at day 3 after CLP.Anti-CD28 antibody improved survival rate,clinical score,spleen index and spleen tissue injury in septic mice.The expression of pro-inflammatory cytokines IL-6,TNF-α and IL-17A decreased in CLP mice after 3 days of treatment with the anti-CD28 antibody.3.Anti-CD28 antibody can reduce the expression of Th17 cells and Treg cells and Th17/Treg cell ratio in the spleen of septic mice 3 days after surgery.The apoptosis of spleen cells in septic mice was reduced 3 days after the operation.With the TUNEL staining,the antiCD28 antibody could reduce the proportion of apoptosis in spleen cells,and increase the expression level of Bcl-2,decreasing the expression level of Bax.ConclusionsThe ratio of Th17/Treg cells can be used as a predictor of the length of ICU stay in patients with sepsis.Anti-CD28 antibody improves the prognosis of septic mice by modulating Th17/Treg ratio and inhibiting apoptosis in spleen cells. |
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