Azurophil Granule Proteins Constitute the Major Mycobactericidal Proteins in Human Neutrophils and Enhance the Killing of Mycobacteria in Macrophages

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.

Correlates between Models of Virulence for Mycobacterium tuberculosis among Isolates of the Central Asian Lineage: a Case for Lysozyme Resistance Testing?

“…Consistent with the trends observed in the guinea pig model and the data on MDMs, C72 was found to be the most sensitive to lysozyme in vitro, although statistical significance was not reached between C72 and C40, suggesting that there are additional determinants of lysozyme sensitivity in CAS strains. Nevertheless, our data support the results of previous studies demonstrating lysozyme sensitivity for H37Rv in vitro (57,58,60) and for M. smegmatis ex vivo (67). We now also demonstrate a broad range of lysozyme sensitivities in M. tuberculosis isolates across different lineages (P Ͻ 0.001).…”

Modulation of neutrophil NETosis: interplay between infectious agents and underlying host physiology

“…tuberculosis bacilli and members of the innate immune system may, however, be more complex than previously thought, a change in concept supported by a spate of recent publications [18, 19, 25]. In this context, it has been observed that macrophages are able to ingest neutrophil azurophil granular proteins and use them to kill invasive mycobacteria [26, 27]. In the first of these reports, Steinwede et al examined the role of cathepsin G (CG) and neutrophil elastase (NE) in the alveolar clearance of mycobacterial infections, in particular M .…”

Tobacco exposure and susceptibility to tuberculosis: Is there a smoking gun?

“…While efferocytosis of non-infected neutrophils leads to deactivation of macrophages through release of TGFb, efferocytosis of MTB-infected neutrophils leads to macrophage activation through the actions of tumor necrosis factor-alpha (TNFa) and heat shock proteins [16]. Another mechanism by which efferocytosis can help clear MTB is macrophage uptake of neutrophil azurophilic granules, shown to enhance P-L fusion in macrophages [17].…”

“…One possible mechanism is CS-induced expression of PD-L1 on neutrophil surfaces, compromising their function; although this hypothesis is unproven, PD-L1 is increased on neutrophils of active TB patients [43]. Another potential mechanism is the ability of CS to inhibit endogenous oxidative burst of neutrophils [44] although human neutrophils kill mycobacteria principally through oxygen-independent mechanisms such as antimicrobial peptides [17,45]. A third possibility is that LPS e found abundantly in CS e can induce expression of immunosuppressive (N2) neutrophils (CD16 hi CD62L lo CD11b hi CD54 hi ), capable of inhibiting T cell proliferation [39].…”