Azurophil granule proteins constitute the major mycobactericidal proteins in human neutrophils and enhance the killing of mycobacteria in macrophages.

Prajna Jena,Thomas Lindstrøm,Steffen Stenger,Soumitra Mohanty,Ole E Sørensen,Avinash Sonawane,Niels Borregaard,Stephanie Kallert,Tirthankar Mohanty,Matthias Morgelin,Niels Bovenschen

doi:10.1371/journal.pone.0050345

Prajna Jena, Thomas Lindstrøm + Show 9 more

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https://doi.org/10.1371/journal.pone.0050345

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Abstract

Pathogenic mycobacteria reside in, and are in turn controlled by, macrophages. However, emerging data suggest that neutrophils also play a critical role in innate immunity to tuberculosis, presumably by their different antibacterial granule proteins. In this study, we purified neutrophil azurophil and specific granules and systematically analyzed the antimycobacterial activity of some purified azurophil and specific granule proteins against M. smegmatis, M. bovis-BCG and M. tuberculosis H37Rv. Using gel overlay and colony forming unit assays we showed that the defensin-depleted azurophil granule proteins (AZP) were more active against mycobacteria compared to other granule proteins and cytosolic proteins. The proteins showing antimycobacterial activity were identified by MALDI-TOF mass spectrometry. Electron microscopic studies demonstrate that the AZP disintegrate bacterial cell membrane resulting in killing of mycobacteria. Exogenous addition of AZP to murine macrophage RAW 264.7, THP-1 and peripheral blood monocyte-derived macrophages significantly reduced the intracellular survival of mycobacteria without exhibiting cytotoxic activity on macrophages. Immunofluorescence studies showed that macrophages actively endocytose neutrophil granular proteins. Treatment with AZP resulted in increase in co-localization of BCG containing phagosomes with lysosomes but not in increase of autophagy. These data demonstrate that neutrophil azurophil proteins may play an important role in controlling intracellular survival of mycobacteria in macrophages.

Highlights

The immune responses to Mycobacterium tuberculosis (Mtb) are complex and poorly understood
Exponentiallygrown M. smegmatis and M. bovis BCG representing fast and slow-growing mycobacteria, respectively, were incubated with LPS-stimulated neutrophils and the number of colony forming unit (CFU) was analyzed by harvesting bacteria at different time points by plating serial dilutions and surviving colonies were counted after 3 days and 3 weeks for M. smegmatis and BCG, respectively
Neutrophils contain many antimicrobial proteins stored in different granules, but no systematic and comparative studies have been done to identify the importance of different granules or granule proteins in killing of mycobacteria

Summary

Introduction

The immune responses to Mycobacterium tuberculosis (Mtb) are complex and poorly understood. The early host response to Mtb infection involves primarily resident alveolar macrophages and infiltrated neutrophils [1]. Evidences for the role of neutrophils in innate immunity to tuberculosis (TB) include the observations that during the early stages of Mtb infection polymorphonuclear leukocytes (PMN) migrate and accumulate at the site of infection [8,9], the risk for TB infection diminish with increased neutrophil count, and killing of M. bovis BCG in a whole blood was significantly impaired by neutrophil depletion [10] and in neutrophil serine proteases cathepsin G and neutrophil elastase deficient mice [11]. Innate immune responses to Mtb in RAG-deficient mice showed a compensatory function for neutrophils in controlling the bacterial burden in the absence of IFN-c from T cells [12]. The role of neutrophils in TB is conceivable from the study showing that no granuloma formation was observed in PMN depleted mice up to 60 days of post infection with Mtb [13]

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