ProBDNF/p75^(NTR)在脓毒症患者外周血淋巴细胞中的表达变化及对淋巴细胞分化的影响

Expression of proBDNF/p75^(NTR) in peripheral blood lymphocytes of patients with sepsis and its impact on lymphocyte differentiation

目的:脓毒症是宿主对感染的反应失调引起的危及生命的器官功能障碍。由于缺乏有效的治疗手段,脓毒症一直是临床治疗的难点和挑战。研究表明脑源性神经营养因子前体(pro-brain-derived neurotrophic factor,proBDNF)可通过结合其高亲和力受体p75神经营养因子受体(p75 neurotrophin receptor,p75^(NTR))激活下游信号通路,扰乱免疫炎症微环境,在脓毒症病程的进展中发挥重要作用。本研究主要探讨淋巴细胞来源的proBDNF/p75^(NTR)在脓毒症患者中的表达变化及其对淋巴细胞分化的影响。方法:收集健康志愿者(对照组,n=40)和初次入院的脓毒症患者(脓毒症组,n=40)外周血样本,进行血常规临床指标检测,采用流式细胞术检测淋巴细胞亚群及其proBDNF/p75^(NTR)的表达变化;体外分选对照组外周血淋巴细胞并采用脂多糖(lipopolysaccharide,LPS)刺激培养,流式细胞分析技术检测LPS刺激对淋巴细胞亚群proBDNF/p75^(NTR)的表达影响;随后采用p75^(NTR)的拮抗剂(p75^(ECD)-Fc)抑制p75^(NTR)观察对淋巴细胞分化的影响。结果:与对照组相比,脓毒症组患者入院时白细胞计数、中性粒细胞计数和中性粒细胞百分比均显著升高,淋巴细胞计数与淋巴细胞百分比均显著降低(均P<0.001),中性粒细胞与淋巴细胞比值、单核细胞与淋巴细胞比值均显著升高(均P<0.05)。与对照组相比,proBDNF在脓毒症组外周血中CD19^(+)B细胞的表达上调(P<0.05),而p75^(NTR)在CD19^(+)B细胞、CD4^(+)T细胞和CD8^(+)T细胞中的表达均上调(均P<0.05)。体外采用LPS刺激可诱导对照组外周血淋巴细胞的proBDNF/p75^(NTR)表达上调(均P<0.05),趋势与在脓毒症组外周淋巴细胞的表达变化基本一致。抑制p75^(NTR)可增加CD4^(+)T细胞和CD19^(+)B细胞的百分比,促进细胞因子IL-4和IL-10的表达,并降低IL-1β和IL-6的产生(均P<0.05)。结论:脓毒症患者入院时即处于以淋巴细胞数量减少为特征的免疫抑制阶段,同时伴有中性粒细胞占比增加。ProBDNF/p75^(NTR)在脓毒症患者外周血淋巴细胞表达增加,抑制p75^(NTR)可能通过调节淋巴细胞的分化,参与脓毒症进展。

Objective:Sepsis is a life-threatening organ dysfunction caused by the host’s imbalanced response to infection.Due to lack of effective treatments,it has always been the difficulty and focus of clinical treatment of sepsis.Studies have shown that pro-brain-derived neurotrophic factor(proBDNF)binds to the high-affinity total neurotrophic factor p75 neurotrophin receptor(p75^(NTR)),which activates downstream signaling cascades and disrupts immunological inflammation and plays an important role in the progression of sepsis.This study aims to explore the expression changes of lymphocyte-derived proBDNF/p75^(NTR) in patients with sepsis and its effect on lymphocyte differentiation.Methods:From the healthy donors(control group,n=40)and sepsis patients(sepsis group,n=40)admitted to the hospital for the first time,peripheral blood samples and blood routine clinical detection indicators were obtained.By using flow cytometry,the proportion of lymphocyte subsets and their expression of proBDNF/p75^(NTR) were examined.The peripheral blood lymphocytes were isolated from the control group and incubated with lipopolysaccharide(LPS).Flow cytometry analysis technology was used to detect the expression of proBDNF/p75^(NTR) on LPS-treated lymphocyte subsets.On this basis,we investigated the effects on lymphocyte differentiation by inhibiting p75^(NTR).Results:White blood cell count,neutrophil count,and neutrophil percentage of the patients in the sepsis group at admission were significantly higher than those in the control group;on the contrary,lymphocyte count and lymphocyte percentage in the sepsis group were lower than those in the control group(all P<0.001).The patients in the sepsis group had considerably greater neutrophil/lymphocyte and monocyte/lymphocyte ratios than those in the control group(both P<0.05).In the peripheral blood of sepsis patients,proBDNF expression was upregulated on CD19^(+)B cells,whereas p75^(NTR) expression was elevated on B cells,CD4^(+)T cells,and CD8^(+)T cells(all P<0.05).ProBDNF/p75^(NTR) expression was upregulated by LPS stimulation in vitro in peripheral blood cells of the control group(P<0.05),and this tendency was similar to the expression alterations in peripheral lymphocytes of the sepsis group.Inhibition of p75^(NTR) increased CD4^(+)T cell and CD19^(+)B cell percentages,cytokine expression of IL-4 and IL-10,and reduced IL-1βand IL-6 production(all P<0.05).Conclusion:The immunosuppressive state of sepsis patients is indicated by a reduction in lymphocyte count and an increase in the proportion of inactive neutrophils.ProBDNF/p75^(NTR) expression is upregulated in the peripheral blood lymphocytes of sepsis patients,and p75^(NTR) inhibition may control lymphocyte differentiation involved in sepsis progression.