SUMO化缺失对脓毒症小鼠树突状细胞功能的影响及其在脓毒症中的作用

Effect of deletion of SUMOylation on dendritic cell function in septic mice and its role in sepsis

目的:探讨类小泛素化修饰(small ubiquitin-like modifier,SUMO)缺失对脓毒症小鼠树突状细胞(dendritic cells,DC)功能的影响及其在脓毒症中的作用。方法:建立DC特异性的泛素结合酶9(ubiquitin-conjugating enzyme 9,UBC9)缺陷(UBC9^ΔDC)小鼠和野生型(wild type,WT)小鼠的阑尾结扎穿孔(cecal ligation and puncture,CLP)脓毒症模型。计算并比较小鼠的7 d病死率;检测CLP术后24 h血液、肝和脾的细菌负荷;采用ELISA法检测CLP术后48 h小鼠血浆和骨髓来源树突状细胞(bone marrow-derived dendritic cells,BMDC)培养上清液中IL-1β,IL-6,IL-18和TNF-α水平以及小鼠脾单个核细胞培养上清液中IL-4和IFN-γ的水平;采用流式细胞术分析CLP术后48 h脾DC表面分子MHC Ⅱ,CD54和CD80的表达和脾单个核细胞中Th1,Th2细胞亚群的比例。结果:CLP术后,与WT脓毒症小鼠相比,UBC9^ΔDC脓毒症小鼠7 d病死率增加(P<0.05),血液(P<0.01)、肝(P<0.01)和脾(P<0.05)的细菌负荷增加;血浆和BMDC培养上清液中IL-1β和IL-18水平显著升高(均P<0.01);DC的数量、表面分子表达差异无统计学意义(均P>0.05);脾Th2细胞数量显著增加(P<0.05),Th1/Th2比值下调但差异无统计学意义(P>0.05),IL-4及IFN-γ水平均有升高且IFN-γ/IL-4比值显著下调(均P<0.05)。结论:SUMO化缺失可能通过DC调控炎症因子释放,异常活化T细胞,从而导致脓毒症小鼠死亡。

Objective: To investigate the effect of deletion of small ubiquitin-like modifier(SUMO)on the function of dendritic cells(DC) in septic mice and its role in sepsis.Methods: Septic models of DC-specific ubiquitin-conjugating enzyme 9(UBC9) deficient(UBC9^ΔDC) mice and wild type(WT) mice with cecal ligation and puncture(CLP) were established. The differences in 7-day mortality of the mice were analyzed. Bacteria loads of blood, liver, and spleen were tested. ELISA was used to detect the levels of IL-1β, IL-6, IL-18, and TNF-α in plasma and culture medium of bone marrow-derived dendritic cells(BMDC). The levels of cytokine IFN-γ and IL-4 in supernatant of spleen mononuclear cells were detected by ELISA.The expressions of MHC Ⅱ, CD54, and CD80 on the cell surface of DC were analyzed by flow cytometry. The percentages of Th1, and Th2 cells in spleen mononuclear cells were analyzed by flow cytometry.Results: Compared with the WT septic mice, the 7-day mortality of UBC9^ΔDCseptic mice was higher(P<0.05). Bacterial loads in blood(P<0.01), liver(P<0.01), and spleen(P<0.05) were significantly increased in UBC9^ΔDC septic mice. Levels of IL-1β and IL-18 in plasma and culture supernatant of BMDC were also significantly increased in UBC9^ΔDC septic mice(all P<0.01). There was no significant difference in the number of DC and the expression of cell surface molecules in DC of UBC9^ΔDCseptic mice(all P>0.05). The percentage of Th2 cells was significantly increased in UBC9^ΔDC septic mice(P<0.05). The ratio of Th1 to Th2 was decreased in UBC9^ΔDC septic mice but the difference was not significant(P>0.05). Levels of IFN-γ and IL-4 were increased in UBC9^ΔDC septic mice, and the ratio of IFN-γ to IL-4 were significantly decreased in UBC9^ΔDC septic mice(all P<0.05).Conclusion: Deletion of SUMOylation may increase the mortality of mice with sepsis through regulating the release of inflammatory factors from DC and abnormal activation of T cells by DC.