(468) Complement Opsonization Promotes Efficient Mycobacterium avium Killing by Human Neutrophils

Background: Nontuberculous mycobacteria (NTM), including Mycobacterium avium, are clinically important pathogens in cystic fibrosis (CF) with long-term prevalence rates of 20% in the CF population. Treatment regimens for NTM are prolonged and highly burdensome with suboptimal cure rates. The innate immune response to M. avium, the most commonly recovered NTM in the US CF population, remains incompletely understood. Neutrophils, which are found in vast quantities in the CF airway, have killing capability against M. abscessus, but limited data exists regarding their response to M. avium. Hypothesis: Complement-mediated opsonization is required for effective neutrophil-mediated killing of M. avium.

Methods: Peripheral neutrophils were isolated from healthy donors (HD) and persons with CF (pwCF). M. avium isolates were obtained from CF donor sputum. The isolates had a smooth morphology and were recovered from the subjects enrolled in the PREDICT trial (PRospective Evaluation of NTM Disease In CysTic Fibrosis, NCT02073409). M. avium was opsonized with plasma from HD or pwCF, which was either intact or heat-treated to inactivate complement. Opsonization of M. avium with antibody-depleted plasma, HD serum, and complement C3-depleted serum was also tested. Neutrophils were incubated with opsonized M. avium at 37C for 0-120 min, dispersed, and plated. Colony forming units and percent killing was calculated relative to time=0 for each condition.

Results: HD neutrophils had killing activity against M. avium opsonized with intact HD plasma (69% at 60 min) and killing was significantly reduced when M. avium was opsonized with heat-inactivated HD plasma (28%, p=< 0.0001). Killing activity was minimal against M. avium in the absence of plasma components. When opsonized with HD plasma, CF neutrophils had killing activity against M. avium that was not different than HD neutrophils (heat-inactivated HD plasma opsonization = 29%, p=0.87 and with intact plasma opsonization = 71%, p=0.89). When opsonized with intact plasma from pwCF, M. avium killing by HD neutrophils was significantly reduced (34%, p=0.01). Opsonization of M. avium with C3-depleted serum resulted in significantly reduced killing (19% at 60 min) compared to intact serum (53%, p=0.0013). Opsonization of M. avium with antibody-depleted plasma resulted in decreased killing (17% at 60 min) compared to intact plasma (57%, p< 0.001).

Conclusions: Human neutrophils efficiently kill M. avium when the bacteria are opsonized with intact plasma from healthy donors, but killing efficiency is significantly lower when the bacteria are opsonized with plasma from pwCF or with serum that is depleted of complement C3 or antibody components. This indicates a novel role for opsonization in neutrophil killing of M. avium which is likely dependent upon opsonophagocytosis, a process that may be deficient in cystic fibrosis.

Acknowledgements: This work was supported by the CFF (LENHAR20D0).

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