CR3/SYK通过NUPR1调控经典或非经典自噬介导固有免疫细胞抵御白念珠菌感染的分子机制研究

Innate immune cells utilize pattern-recognition receptors (PRRs) to identify pathogens and trigger immune responses, autophagy and LC3-associated phagocytosis (LAP, noncanonical autophagy) have been confirmed facilitating in killing extracellular pathogens. However, the role of autophagy and LAP during Candida infections have not been distinguished, the antifungal activities of them are still controversy and need further studies. Our previous study found that the killing capability to Candida albicans of human neutrophils has been showed a positive linkage to the level of NUPR1 and ratio of LC3B-II/I (both of them mediated by CR3/SYK), which indicated that the killing capability of neutrophils might be monitored by autophagy (or LAP) via CR3/SYK/NURP1. The present study plan to choose primary and subcultured cells, NUPR1 gene knockout mice, and utilize lentiviral transfection, flow cytometry, fluorescence confocal microscopy and etc. to confirm the hypothesis mentioned above by the followings, 1. Whether the killing capability to C. albicans of innate immune cells is monitored by autophagy (or LAP) via CR3/SYK/NUPR1, and which one plays the leading role (autophagy or LAP). 2. To study the antifungal role of NUPR1 in vivo, and investigate the therapeutic strategy based on NUPR1. Finally, the study will describe the signal transduction pathway “ CR3/SYK—NUPR1—autophagy (or LAP)—fungal killing”, which may provide new strategies for antifungal therapies.

固有免疫细胞经模式识别受体（PRRs）识别病原菌，触发胞内信号通路产生杀菌效应，其中自噬及LC3相关吞噬（LAP，非经典自噬）被证实能有效清除病原菌，但在念珠菌感染中，二者没有被很好的区分，相关分子机制研究有待深入；我们发现，人中性粒细胞对白念珠菌杀菌率与CR3/SYK介导的NUPR1及LC3B-II/I改变相关，推测，CR3/SYK可能通过NUPR1调控细胞自噬或LAP，进而影响了其对白念珠菌的杀菌率；本项目拟借助中性粒细胞及巨噬细胞（原代细胞、细胞系）、基因缺失小鼠，采用转染、荧光共聚焦等技术，从以下两方面研究：1.CR3/SYK是否通过NUPR1调控细胞清除白念珠菌，其中自噬及LAP哪一种起主导作用，及相应分子机制；2.体内证明NUPR1对抵御白念珠菌感染的重要性，及以此为靶点进行治疗的可行性；最终绘出“CR3/SYK-NUPR1-自噬（或LAP）-杀菌”机制图，为进一步研究做铺垫。

侵袭性念珠菌感染易发生在肿瘤、免疫受损等重症患者中，发病率及死亡率长期居高不下，因此，念珠菌的感染免疫分子机制值得深入研究，以获得可能的治疗靶点，提高治愈率，改善患者预后；经典自噬及LC3相关吞噬（LAP，非经典自噬）被发现能够帮助固有免疫细胞清除病原菌，尤其是LAP途径，在特定条件下能够为一些病毒提供复制场所，具有多种作用，但在念珠菌感染免疫中对二者的功能还知之甚少，值得进一步研究。我们在体外成功诱导THP-1细胞成为THP-1源性巨噬细胞，利用此细胞及其它种属来源巨噬细胞、白念珠菌及白念珠菌胞壁葡聚糖颗粒（Whole glucan patical, WGP），通过 qPCR、western blot、沉默目的基因、流式细胞技术、免疫荧光等分子生物学手段分别对经典自噬及LAP途径的相关分子进行了检测。研究发现，经典自噬不参与巨噬细胞清除白念珠菌；白念珠菌胞壁β-glucan能够刺激巨噬细胞Rubicon升高，引起ROS增加，引发LAP途径，但Rubicon升高并不能增加巨噬细胞杀菌率，相反，沉默Rubicon后巨噬细胞对白念珠菌杀菌率明显增高；进一步的研究发现，巨噬细胞清除白念珠菌的过程中，虽然LAP途径被激活，但LAPosome与溶酶体融合受阻，抑制了溶酶体的降解作用；此外，沉默Rubicon前后，经WGP处理的巨噬细胞中溶酶体活性均有明显上升，且二者之间没有明显差异，但沉默Rubicon后杀菌率明显增加，此时抑制溶酶体活性，杀菌率重新出现降低，提示溶酶体能够直接对含有白念珠菌的吞噬小体进行降解，并不依赖经典自噬或LAP途径，相反，LAP途径激活产生的LAPosome不能与溶酶体融合，可能为白念珠菌提供了庇护所，从而降低了杀菌率。综上，该研究发现LAP途径、Rubicon是调节固有免疫细胞抵御白念珠菌感染的关键，这有助于为侵袭性念珠菌感染的治疗，及新的抗真菌药物研发提供理论依据。