白念珠菌诱导小鼠骨髓来源巨噬细胞焦亡的初步研究

A preliminary study on Candida albicans-induced pyroptosis of murine bone marrow-derived macrophages

目的探讨白念珠菌对小鼠骨髓来源巨噬细胞(BMDM)焦亡的影响。方法通过活细胞工作站实时观察白念珠菌[感染复数(MOI)=50,下同]体外诱导BMDM后是否发生焦亡;将BMDM分为对照组、白念珠菌组,分别用磷酸盐缓冲液和白念珠菌酵母诱导BMDM 6 h,实时荧光定量PCR检测NOD样受体热蛋白结构域相关蛋白3(NLRP3)、白细胞介素1β(IL-1β)、IL-18 mRNA表达水平,Western印迹法检测NLRP3、胱天蛋白酶1(Caspase-1)、Gasdermin D(GSDMD)的表达及剪切水平。用白念珠菌诱导BMDM不同时间(0、10、15、20及25 h)后,酶联免疫吸附试验检测IL-1β、IL-18分泌水平。白念珠菌体外诱导野生型(WT)BMDM及GSDMD敲除(KO)BMDM 15 min,采用流式细胞仪比较WT、GSDMD KO BMDM对白念珠菌的吞噬率;诱导6 h后,采用流式细胞仪及乳酸脱氢酶(LDH)释放法分别检测WT、GSDMD KO BMDM的死亡率。分别设置空白对照组、对照组、样品最大酶活性对照孔组、IL-1β组、白念珠菌组、IL-1β+白念珠菌组,用IL-1β和/或白念珠菌酵母诱导BMDM后用LDH释放法分析WT及GSDMD KO BMDM死亡率。采用非配对t检验、Kruskal-Wallis检验、方差分析等统计学方法进行统计分析。结果白念珠菌体外诱导BMDM后细胞可出现气球样变及出泡现象,证实焦亡发生;与对照组相比,白念珠菌组诱导BMDM 6 h后NLRP3、IL-1β的mRNA表达水平明显升高(t=13.02、17.51,P=或<0.001),而IL-18 mRNA表达水平无明显变化(P=0.486),且白念珠菌诱导可引起炎症小体NLRP3表达增多及Caspase-1、GSDMD剪切。白念珠菌诱导BMDM不同时间(0、10、15、20及25 h)后IL-1β的分泌水平随时间逐渐升高(H=12.90,P=0.012),而IL-18的分泌水平无明显变化(F=0.48,P=0.753),且GSDMD KO组BMDM的IL-1β分泌水平低于WT BMDM组(F=24.22,P=0.008)。白念珠菌体外诱导15 min后,GSDMD KO BMDM[(50.3±1.10)%]对白念珠菌的吞噬率低于WT BMDM[(58.53±1.19)%,t=5.09,P=0.007];白念珠菌体外诱导6 h后,流式细胞仪检测显示GSDMD KO组BMDM的死亡率(38.40%±0.50%)高于WT组BMDM(34.37%±0.52%),t=4.72,P=0.009;LDH检测同样显示GSDMD KO BMDM的死亡率(22.52%±0.18%)高于WT BMDM(12.48%±0.15%),t=42.36,P<0.001;IL-1β预处理后予白念珠菌体外诱导的WT BMDM及GSDMD KO BMDM的细胞死亡率均较单纯白念珠菌诱导组降低(均P<0.001)。结论白念珠菌可诱导BMDM发生焦亡,焦亡的发生可促进IL-1β释放,并通过提高巨噬细胞的免疫活性,降低巨噬细胞死亡率。

Objective To investigate the effect of Candida albicans(C.albicans)on pyroptosis of murine bone marrow-derived macrophages(BMDMs).Methods Live-cell imaging was used to observe morphologic changes of in vitro C.albicans-infected BMDMs(multiplicity of infection[MOI]=50)so as to evaluate whether pyroptosis occurred.Cultured BMDMs were divided into a control group and a C.albicans group,which were treated with phosphate-buffered saline and C.albicans suspensions respectively for 6 hours;then,real-time fluorescence-based quantitative PCR was performed to determine the mRNA expression of NOD-like receptor pyrin domain containing 3(NLRP3),interleukin(IL)-1βand IL-18,and Western blot analysis to determine the protein expression and cleavage levels of NLRP3,caspase-1 and gasdermin D(GSDMD).BMDMs were cultured with C.albicans suspensions for different durations(0,10,15,20,and 25 hours),and enzyme-linked immunosorbent assay was conducted to detect secretion levels of IL-1βand IL-18.Cultured wild-type BMDMs and GSDMD-knockout BMDMs were treated with C.albicans suspensions for 15 minutes,and then rates of phagocytosis of C.albicans by wild-type BMDMs and GSDMD-knockout BMDMs were estimated by flow cytometry;after 6-hour treatment with C.albicans,flow cytometry and lactate dehydrogenase(LDH)release assay were performed to assess mortality rates of wild-type BMDMs and GSDMD-knockout BMDMs.In addition,some wild-type BMDMs and GSDMD-knockout BMDMs were separately divided into blank control group,control group,maximum enzyme activity-sample control group,IL-1βalone group,C.albicans alone group,and IL-1β+C.albicans group,and cell mortality rates were detected by the LDH release assay after treatment with IL-1βand/or C.albicans.Statistical analysis was carried out by using unpaired t test,Kruskal-Wallis test,analysis of variance,and other statistical methods.Results After in vitro treatment with C.albicans,swelling and ballooning with large bubbles blowing from the plasma membrane occurred in BMDMs,suggesting the occurrence of cell pyroptosis;compared with the control group,the C.albicans group showed significantly increased mRNA expression levels of NLRP3 and IL-1βafter 6-hour treatment with C.albicans(t=13.02,17.51,respectively,P=or<0.001),but no significant change in the IL-18 mRNA expression level(P=0.486),and Western blot analysis showed that C.albicans could increase the expression of NLRP3 inflammasomes,as well as cleaved caspase-1 and GSDMD.After the treatment with C.albicans for different durations(0,10,15,20,and 25 hours),the secretion level of IL-1βby BMDMs gradually increased over time(H=12.90,P=0.012),while the secretion level of IL-18 did not significantly change(F=0.48,P=0.753),and the secretion level of IL-1βwas significantly lower in the GSDMD-knockout BMDM group than in the wild-type BMDM group(F=24.22,P=0.008).After 15-minute in vitro treatment with C.albicans,the phagocytosis rate of C.albicans was significantly lower in the GSDMD-knockout BMDM group(50.3%±1.10%)than in the wild-type BMDM group(58.53%±1.19%,t=5.09,P=0.007);after 6-hour treatment with C.albicans,the cell mortality rate was significantly higher in the GSDMD-knockout BMDM group than in the wild-type BMDM group(flow cytometry:38.40%±0.50%vs.34.37%±0.52%,t=4.72,P=0.009;LDH release assay:22.52%±0.18%vs.12.48%±0.15%,t=42.36,P<0.001);the cell mortality rates of wild-type BMDMs and GSDMD-knockout BMDMs both significantly decreased in the IL-1β+C.albicans groups compared with the C.albicans groups(both P<0.001).Conclusion Pyroptosis could be induced in murine BMDMs after C.albicans infection,which promotes the release of IL-1βand may reduce the mortality rate of macrophages by improving their immune activity.