Deficient Complement Opsonization Impairs Mycobacterium avium Killing by Neutrophils in Cystic Fibrosis

Abstract Nontuberculous mycobacteria (NTM), including Mycobacterium avium, are clinically important pathogens in cystic fibrosis (CF). The innate immune response to M. avium remains incompletely understood. We evaluated the role of complement opsonization in neutrophil-mediated killing of M. avium. Killing assays were performed using neutrophils from healthy donors (HD) and persons with CF (pwCF). Clinical isolates of M. avium were opsonized with plasma from HD or pwCF, which was intact or heat-treated to inactivate complement. HD neutrophils had killing activity against M. avium opsonized with intact HD plasma and killing was significantly reduced when M. avium was opsonized with heat-inactivated HD plasma. When opsonized with HD plasma, CF neutrophils had killing activity against M. avium that was not different than HD neutrophils. When opsonized with intact plasma from pwCF, HD neutrophil killing of M. avium was significantly reduced. Opsonization of M. avium with C3-depleted serum or IgM-depleted plasma resulted in significantly reduced killing. Plasma C3 levels were elevated in pwCF with NTM infection compared to pwCF without NTM infection. These studies demonstrate that human neutrophils efficiently kill M. avium when opsonized in the presence of plasma factors from HD that include C3 and IgM. Killing efficiency is significantly lower when the bacteria are opsonized with plasma from pwCF. This indicates a novel role for opsonization in neutrophil killing of M. avium, and a deficiency in complement opsonization as a mechanism of impaired M. avium killing in CF. Original Data Fig1-4,6: Original data used to generate Fig 1A & B (HD and CF neutrophil killing of M. avium), Fig 2A (quantitation of HD and CF neutrophil + FITC-Mav confocal microscopy), Fig 2B (HD neutrophil killing of M. avium in the presence of inhibitors), Fig 3 (HD neutrophil killing of M. avium opsonized with CF plasma), Fig 4A-C (HD neutrophil killing of M. avium opsonized with complement depleted sera and antibody depleted plasma), and Fig 6A (levels of complement proteins in plasma from HD, CF without NTM, and CF with NTM). Fig 2A Confocal Images: Original uncropped confocal images used for Fig 2A, HD and CF neutrophil + FITC-Mav confocal microscopy Fig 5 Western Blots: Original uncropped western blot images used in Fig 5, comparison of protein levels of C3, C1q, Ig, IgA, and IgM bound to M. avium opsonized with WP compared to WP alone via western blot.

摘要 非结核分枝杆菌（Nontuberculous mycobacteria, NTM，含鸟分枝杆菌）是囊性纤维化（cystic fibrosis, CF）患者临床中重要的致病菌。目前针对鸟分枝杆菌的固有免疫应答机制仍未完全阐明。本研究评估了补体调理作用在中性粒细胞介导的鸟分枝杆菌杀伤过程中的作用。实验采用健康供者（healthy donors, HD）与囊性纤维化患者（persons with CF, pwCF）的中性粒细胞开展杀伤实验。将鸟分枝杆菌临床分离株用健康供者或囊性纤维化患者的血浆进行调理处理，部分血浆经热处理以灭活补体。结果显示，健康供者中性粒细胞对经完整健康供者血浆调理的鸟分枝杆菌具有杀伤活性；当鸟分枝杆菌经热处理灭活补体的健康供者血浆调理后，其杀伤活性显著降低。经健康供者血浆调理后，囊性纤维化患者的中性粒细胞对鸟分枝杆菌的杀伤活性与健康供者中性粒细胞无显著差异。而当用囊性纤维化患者的完整血浆调理鸟分枝杆菌时，健康供者中性粒细胞的杀伤活性显著降低。使用C3缺失血清或IgM缺失血浆对鸟分枝杆菌进行调理，可显著降低中性粒细胞的杀伤活性。相较于未感染非结核分枝杆菌的囊性纤维化患者，合并非结核分枝杆菌感染的囊性纤维化患者血浆中C3水平升高。本研究证实，当鸟分枝杆菌经健康供者血浆中包含C3与IgM的血浆因子调理后，人类中性粒细胞可高效杀伤该菌；而当细菌经囊性纤维化患者血浆调理后，杀伤效率显著下降。这表明调理作用在中性粒细胞杀伤鸟分枝杆菌过程中具有新的调控作用，且补体调理功能缺陷是囊性纤维化患者体内鸟分枝杆菌杀伤能力受损的潜在机制。

原始数据 图1、4、6：用于生成图1A、B（健康供者与囊性纤维化患者中性粒细胞对鸟分枝杆菌的杀伤实验）、图2A（健康供者与囊性纤维化患者中性粒细胞+异硫氰酸荧光素标记鸟分枝杆菌共聚焦成像定量分析）、图2B（抑制剂存在下健康供者中性粒细胞对鸟分枝杆菌的杀伤实验）、图3（经囊性纤维化患者血浆调理的鸟分枝杆菌的健康供者中性粒细胞杀伤实验）、图4A-C（经补体缺失血清与抗体缺失血浆调理的鸟分枝杆菌的健康供者中性粒细胞杀伤实验）以及图6A（健康供者、未感染非结核分枝杆菌的囊性纤维化患者、合并非结核分枝杆菌感染的囊性纤维化患者血浆中补体蛋白水平）的原始数据。
图2A共聚焦成像：用于图2A的原始未裁剪共聚焦图像，即健康供者与囊性纤维化患者中性粒细胞+异硫氰酸荧光素标记鸟分枝杆菌共聚焦成像。
图5蛋白质免疫印迹（Western Blot）：用于图5的原始未裁剪蛋白质免疫印迹图像，即对比经WP调理与未经调理的鸟分枝杆菌结合的C3、C1q、免疫球蛋白、IgA及IgM蛋白水平的蛋白质免疫印迹实验。