Abstract: 【Abstract】 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）-1β and 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-1β and 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-1β alone 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-1β and/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-1β after 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-1β by 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-1β was 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-1β and may reduce the mortality rate of macrophages by improving their immune activity.

Key words: Candida albicans, Macrophages, Pyroptosis, Interleukin-1beta, Interleukin-18, GSDMD