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