研究背景

侵袭性真菌感染主要包括侵袭性念珠菌病、侵袭性烟曲霉病等，在免疫缺陷患者中具有高发病率和死亡率，严重危害患者的生命安全。其中，白念珠菌和烟曲霉是侵袭性真菌感染中非常重要的两种真菌病原体。宿主巨噬细胞在抗真菌感染中发挥重要作用，对巨噬细胞的抗真菌免疫机制研究非常重要。

自噬是一种保守的细胞应激反应，在抗感染免疫反应中发挥重要作用。真菌感染可以诱导巨噬细胞自噬，有助于胞内真菌清除，从而消除感染。自噬过程包括吞噬泡形成、自噬体形成、自噬体与溶酶体融合形成自噬溶酶体、自噬溶酶体降解等4个步骤；自噬流是这些步骤在细胞内连续出现的动态过程，常用来评价细胞自噬是否行使正常功能。目前有研究证实烟曲霉可诱导巨噬细胞自噬，但是关于烟曲霉对巨噬细胞基础自噬流的影响尚无定论。

焦亡是一种比较新的程序性死亡方式，近些年来焦亡已成为感染领域的研究热点。既往关于细胞焦亡的研究主要是关注细菌和病毒感染，但越来越多的证据表明，焦亡在真菌感染中也发挥着关键作用。目前尚关于无烟曲霉及白念珠菌诱导巨噬细胞焦亡的直接证据，关于烟曲霉及白念珠菌能否直接诱导巨噬细胞焦亡以及焦亡发生的生物学意义仍不明确。

研究目的

本研究的主要目的是，探索烟曲霉对小鼠巨噬细胞自噬流的影响；通过体外及体内实验对烟曲霉能否诱导巨噬细胞焦亡进行初步研究，并初步探索巨噬细胞焦亡在烟曲霉系统感染中发挥的作用以及焦亡的发生机制；通过体外实验对白念珠菌诱导小鼠巨噬细胞焦亡进行初步研究。

研究方法

1. 烟曲霉体外刺激小鼠骨髓来源巨噬细胞（BMDMs）不同时间，免疫印迹法（WB）检测自噬关键蛋白微管相关蛋白1轻链3（LC3）⁃Ⅰ型/Ⅱ型的转换及磷酸化雷帕霉素靶蛋白（p⁃mTOR）Ser2481的蛋白水平；烟曲霉和溶酶体阻断剂单独或联合体外刺激BMDMs不同时间后，WB检测LC3⁃Ⅱ蛋白水平。

2. 烟曲霉体外刺激BMDMs后，活细胞工作站观察细胞焦亡样形态；实时荧光定量PCR（qRT PCR）检测NOD样受体热蛋白结构域相关蛋白（NLRP3）、白细胞介素-1β（IL⁃1β）、白细胞介素-18（IL⁃18）mRNA表达水平；WB检测NLRP3、胱天蛋白酶-1（caspase-1）、Gasdermin D（GSDMD）的表达水平；酶联免疫吸附试验（ELISA）检测IL⁃1β、IL⁃18分泌水平；敲除GSDMD后，检测焦亡指标的变化。

3. 构建烟曲霉系统感染模型，比较WT小鼠及GSDMD KO小鼠的生存时长、体重变化、脏器菌载量，WB检测WT小鼠各脏器的GSDMD表达水平，WB检测WT小鼠及GSDMD KO小鼠脾脏巨噬细胞的NLRP3、caspase-1、GSDMD表达水平，ELISA检测WT小鼠血液及脾脏匀浆的IL-1β、IL-18水平。

4. 分别抑制THP-1巨噬样细胞的NLRP3、caspase-1、树突细胞相关性C型凝集素1（Dectin-1）、膜结合Toll样受体（TLR）2、TLR4、核转录因子kappaB（NF-κB）、胞外信号调节激酶（ERK 1/2），再行烟曲霉体外刺激THP-1巨噬样细胞，通过WB检测GSDMD表达水平。

5. 白念珠菌体外刺激BMDMs后，活细胞工作站观察细胞焦亡样形态，qRT PCR检测NLRP3、IL⁃1β、IL⁃18 mRNA表达水平，WB检测NLRP3、caspase⁃1、GSDMD的表达水平，ELISA检测IL⁃1β、IL⁃18分泌水平，流式细胞术比较WT及GSDMD KO BMDMs对白念珠菌的吞噬率，流式细胞术及LDH释放法分别比较WT及GSDMD KO BMDMs的死亡率，IL-1β预处理BMDMs后再行白念珠菌刺激，观测BMDMs死亡率是否较单纯白念珠菌感染组下降。

研究结果

1. 烟曲霉体外刺激可引起小鼠巨噬细胞LC3⁃Ⅱ表达水平随时间逐渐升高，各溶酶体阻断剂处理后可使LC3⁃Ⅱ水平进一步升高。

2. 体外实验证明烟曲霉感染巨噬细胞后，巨噬细胞可以出现焦亡特征性表现，而敲除GSDMD后，无法诱导焦亡发生。

3. 在体实验中，小鼠系统感染烟曲霉后，与GSDMD KO小鼠相比，WT小鼠存活率更高、脏器菌载量更低；WT小鼠多个脏器均发生焦亡；WT小鼠脾脏巨噬细胞发生炎症小体活化及焦亡，而GSDMD敲除小鼠未发生上述变化。

4. 分别抑制NLRP3、caspase-1、Dectin-1、TLR2、TLR4、NF-κB、ERK 1/2后，再行烟曲霉体外刺激THP-1巨噬样细胞，发现焦亡被抑制。

5. 白念珠菌体外刺激可引起小鼠巨噬细胞焦亡，而敲除GSDMD后，无法诱导焦亡发生；与WT组相比，GSDMD KO组巨噬细胞对白念珠菌的吞噬率更低，死亡率更高；IL-1β预处理巨噬细胞后再行白念珠菌刺激，可使巨噬细胞死亡率降低。

研究结论

1. 烟曲霉体外刺激可增加小鼠巨噬细胞基础自噬流。

2. 烟曲霉可以诱导巨噬细胞发生焦亡，敲除GSDMD后，无法诱导焦亡发生；烟曲霉系统感染可以诱导小鼠焦亡发生，且焦亡可能发挥了抗感染保护作用。

3. NLRP3-caspase-1-GSDMD是烟曲霉诱导巨噬细胞焦亡的炎症小体通路，烟曲霉诱导巨噬细胞焦亡可能通过Dectin-1/TLR2/TLR4受体激活巨噬细胞，并传递信号，激活其下游NF-κB通路、ERK 1/2丝分裂原活化蛋白激酶（MAPK）通路蛋白，从而激活焦亡。

4. 白念珠菌可诱导巨噬细胞发生焦亡；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。

Background and objectives

Invasive fungal infections mainly include invasive candidiasis, invasive aspergillosis, etc., which have high morbidity and mortality in immunodeficient patients, and seriously endanger the life safety of patients. Candida albicans (C. albicans) and Aspergillus fumigatus (A. fumigatus) are two very important fungal pathogens in invasive fungal infections. Macrophages play an important role in antifungal infection and they are very important for the study of anti-fungal immune mechanisms of macrophages. Autophagy is a conservative cellular stress response, which plays an important role in the anti-infective immune response. Autophagy can be induced by fungal infections and contribute to the elimination of intracellular fungi, thus eliminating infection. The process of autophagy includes four steps: the formation of phagophores, the formation of autophagosomes, the fusion of autophagosomes and lysosomes, and the degradation of autophagy lysosomes. Autophagy flux is a dynamic process in which these steps occur continuously. It is often used to evaluate whether autophagy performs normal function. At present, some studies have confirmed that A. fumigatus can induce autophagy of macrophages, but the effect of A. fumigatus on the basic autophagy flux of macrophages has not been determined.

Pyroptosis is a relatively new form of programmed death. In recent years, pyroptosis has become a research hotspot in the field of infection. Previous studies on pyroptosis mainly focused on bacterial and viral infections, but there is growing evidences that pyroptosis also plays a key role in fungal infections. At present, there is no strong evidence that A. fumigatus and C. albicans can induce pyroptosis of macrophages, and its biological significance is still unclear.

Our study was conducted on the autophagy flux and pyroptosis of macrophages induced by A. fumigatus, and pyroptosis of macrophages induced by C. albicans.

Materials and methods

1. Murine bone marrow-derived macrophages (BMDMs) were stimulated by A. fumigatus for different times, and the protein level of autophagy key protein microtubule-associated protein 1 light chain 3 (LC3)-Ⅰ/Ⅱ and phosphorylated mechanistic target of rapamycin (p-mTOR) Ser2481 were detected by Western blotting (WB). After BMDMs were stimulated with A. fumigatus and lysosome blockers alone or in combination for different times, the levels of LC3-Ⅱ were detected by WB.

2. After stimulation of macrophages by A. fumigatus, the morphology of macrophage pyroptosis was observed by live⁃cell imaging, the mRNA expression levels of nucleotide binding oligomerization domain-like receptors (NLRP)3, IL-1β and IL-18 were detected by real-time fluorescence quantitative PCR (qRT PCR), and the expression levels of NLRP3, caspase-1 and GasderminD (GSDMD) were detected by WB. The secretion levels of IL-1β and IL-18 were detected by enzyme-linked immunosorbent assay (ELISA). After knocking out GSDMD, changes of pyroptosis indicators were detected.

3. After A. fumigatus system infection, the survival time, body weight and organ fungal loads of WT mice and GSDMD KO mice were compared. The expression levels of GSDMD in different organs of WT mice were detected by WB. The expression levels of NLRP3, caspase-1 and GSDMD in splenic macrophages of WT mice and GSDMD KO mice were detected by WB. The levels of IL-1β and IL-18 in blood and spleen homogenates of WT mice were detected by ELISA.

4. THP-1-derived macrophages were pretreated with inhibitors of NLRP3,caspase-1,dendritic cell-associated type C lectin 1 (Dectin-1),Toll-like receptor (TLR) 2,TLR4,Nuclear factor-κappa banding (NF-κB) and extracellular-signal-regulated kinases (ERK1/2) respectively, then stimulated by A. fumigatus, and the expression of GSDMD was detected by WB.

5. After BMDMs were stimulated by C. albicans in vitro, the morphology of macrophage pyroptosis was observed by live⁃cell imaging, the mRNA expression levels of NLRP3, IL-1β and IL-18 were detected by qRT PCR, the expression levels of NLRP3, caspase-1 and GSDMD were detected by WB, the secretion levels of IL-1β and IL-18 were detected by ELISA; the phagocytosis rates of WT and GSDMD KO BMDMs on C. albicans were compared by flow cytometry, the mortality of WT and GSDMD KO BMDMs were compared by flow cytometry and LDH. After IL-1β pretreatment, BMDMs were stimulated by C. albicans to observe the change of mortality of BMDMs.

1. The expression of LC3-II in murine macrophages induced by A. fumigatus increased, LC3-II was further increased when treated with a combination of lysosomal inhibitors and A. fumigatus.

2. In vitro experiments showed that A. fumigatus could induce pyroptosis of macrophages, but could not induce pyroptosis after knocking out GSDMD.

3. In vivo experiments, after systemic infection with A. fumigatus, WT mice had higher survival rate and lower organ fungal loads than GSDMD KO mice; pyroptosis occurred in multiple organs and blood of WT mice; and pyroptosis occurred in spleen macrophages of WT mice, but not in GSDMD knockout mice.

4. After inhibition of NLRP3, caspase-1, Dectin-1, TLR2, TLR4, NF-κB or ERK 1/2 respectively, the cleavage of GSDMD in THP-1-derived macrophages infected with A. fumigatus decreased.

5. In vitro stimulation of C. albicans could cause pyroptosis of in murine macrophages, but it could not be induced after knockout of GSDMD. Compared with WT group, the phagocytosis rate of macrophages in GSDMD KO group was lower and the mortality was higher. Macrophages stimulated by C. albicans after pretreatment with IL-1β could reduce the mortality of macrophages.

Conclusions

1. A. fumigatus could increase the basic autophagy flux in murine macrophages in vitro.

2. A. fumigatus could induce macrophage pyroptosis, but could not induce pyroptosis after knocking out GSDMD. A. fumigatus systemic infection could induce pyroptosis in mice, and pyroptosis might play a protective role against infection.

3. NLRP3-caspase-1-GSDMD was the inflammasome pathway of macrophage pyroptosis induced by A. fumigatus. Macrophage pyroptosis induced by A. fumigatus may be activated through Dectin-1/TLR2/TLR4 receptors and subsequent downstream of NF-κB and ERK 1/2 MAPK pathways.

4. C. albicans could induce macrophage pyroptosis, which could promote the release of IL-β and reduce the mortality of macrophages by increasing the immune activity of macrophages.