CARD9缺陷的暗色丝孢霉病患者中性粒细胞抗白念珠菌免疫功能的研究

摘要

Caspase recruitment domain⁃containing protein 9 ( CARD9) is an adaptor molecule that is critical for NF⁃κB activa⁃tion,and forms a complex with B⁃cell lymphoma 10 ( BCL10) and mucosa⁃associated lymphoid tissue lymphoma translocation gene 1 ( MALT1) that mediates C⁃type lectin receptors ( CLRs)⁃triggered intracellular signaling during antifungal immunity. In the present study,we investigated the functions of polymorphonuclear neutrophils ( PMNs) from phaeohyphomycosis patients with CARD9 defi⁃ciencies against Candida albicans (C.albicans).PMNs were isolated from patients and healthy blood donors and subsequently chal⁃lenged with C.albicans .By calculating colony⁃forming units and MTT assay,the killing of C.albicans by CARD9⁃deficient neutrophils was detected, and the ROS production and phagocytotic ability of CARD9⁃deficient neutrophils was assayed by Flow CytoMetry (FCM).The phagolysosome ultrastructure and the secretion of IL⁃8 were analyzed separately by EM and CBA kit.As a result,we demonstrated that,compared with healthy donors,CARD9⁃deficient PMNs exhibited defects in C. albicans killing and IL⁃8 produc⁃tions,which can be rescued in the presence of serum;however,the CARD9⁃deficient PMNs exhibited normal reactive oxygen species generation,phagocytotic ability and phagolysosome ultrastructure.In conclusion,our results indicate that CARD9 is indispensable for C.albicans killing by PMNs,and serum⁃opsonization acts as a CARD9⁃independent way,which could be a promising immunotherapy in the future.%目的：探讨CARD9缺陷的暗色丝孢霉病患者中性粒细胞抗白念珠菌的免疫功能。方法提取CARD9缺陷患者及正常人外周血中性粒细胞，并使其与白念珠菌进行孵育。采用菌落计数法及MTT法检测中性粒细胞对白念珠菌的杀伤，流式细胞术检测中性粒细胞活性氧的产生及吞噬能力，电镜观察吞噬溶酶体的超微结构，流式微球检测试剂盒检测培养上清中IL⁃8的分泌情况。结果与对照组比较， CARD9缺陷患者中性粒细胞对白念珠菌孢子的杀伤及IL⁃8的表达存在缺陷，菌丝杀伤能力、活性氧的产生、吞噬能力以及吞噬溶酶体的结构无明显异常，且血清调理后杀伤能力以及IL⁃8的表达得到恢复。结论 CARD9蛋白在中性粒细胞杀伤白念珠菌孢子免疫过程中发挥着关键作用，血清调理可恢复该缺陷。