Hypoxia increases neutrophil-driven matrix destruction after exposure to Mycobacterium tuberculosis

Abstract

The importance of neutrophils in the pathology of tuberculosis (TB) has been recently established. We demonstrated that TB lesions in man are hypoxic, but how neutrophils in hypoxia influence lung tissue damage is unknown. We investigated the effect of hypoxia on neutrophil-derived enzymes and tissue destruction in TB. Human neutrophils were stimulated with M. tuberculosis (M.tb) or conditioned media from M.tb-infected monocytes (CoMTB). Neutrophil matrix metalloproteinase-8/-9 and elastase secretion were analysed by luminex array and gelatin zymography, gene expression by qPCR and cell viability by flow cytometry. Matrix destruction was investigated by confocal microscopy and functional assays and neutrophil extracellular traps (NETs) by fluorescence assay. In hypoxia, neutrophil MMP-8 secretion and gene expression were up-regulated by CoMTB. MMP-9 activity and neutrophil elastase (NE) secretion were also increased in hypoxia. Hypoxia inhibited NET formation and both neutrophil apoptosis and necrosis after direct stimulation by M.tb. Hypoxia increased TB-dependent neutrophil-mediated matrix destruction of Type I collagen, gelatin and elastin, the main structural proteins of the human lung. Dimethyloxalylglycin (DMOG), which stabilizes hypoxia-inducible factor-1α, increased neutrophil MMP-8 and -9 secretion. Hypoxia in our cellular model of TB up-regulated pathways that increase neutrophil secretion of MMPs that are implicated in matrix destruction.

The Impact of Hypoxia on Neutrophil Degranulation and Consequences for the Host

“…Neutrophils isolated from healthy volunteers subjected to acute hypoxia showed increased neutrophil elastase (NE) release ex vivo [23], although these cells experienced ambient oxygen during isolation and on subsequent culture ("hypoxia-re-oxygenation"). Consistent with these results; however, isolated human neutrophils incubated under hypoxia released more NE, MPO, lactoferrin, MMP-8, and MMP-9 when compared with normoxia, representing increased secretion of azurophil, specific and gelatinase granules [24,25]. McGovern et al and Hoenderdos et al found that this hypoxia-augmented degranulation occurred rapidly (within 2 h), was not prevented by inhibition of protein translation, and could not be recapitulated by pharmacological HIF-1α stabilisation [24,32].…”

“…It should be noted that the degree of neutrophil apoptosis under normoxia versus hypoxia begins to diverge after 5 h in culture, with significantly improved cell survival under hypoxia beyond 20 h, which may contribute to observed changes in protein secretion at later time points [95]. However, in this study, the authors demonstrated no significant difference in cell viability between normoxic versus hypoxic neutrophils exposed to media from tuberculosis (TB)-infected monocytes, as stimulated cells exhibited prolonged survival, which was not further augmented by hypoxia [25]. Consistent with this work, three further studies have suggested HIF-dependent changes in mRNA or protein content of selected neutrophil granule proteins (Figure 1).…”

“…Consistent with this work, three further studies have suggested HIF-dependent changes in mRNA or protein content of selected neutrophil granule proteins (Figure 1). The same group showed an increase in MMP-8 gene expression under hypoxia after 24 h, although MMP-9 and NE gene expression were unchanged [25], despite putative HREs being identified in the promotor regions of both genes [24]. Peyssonnaux et al showed an increase in murine neutrophil cathelicidin-related antimicrobial peptide (CRAMP, analogous to the human-specific granule protein cathelicidin) mRNA and protein content under hypoxia [26].…”

“…As frontline effectors of host defence, neutrophils need to function effectively in hypoxic environments; to enable this, they have the ability to sense surrounding oxygen tensions and respond by modulation of several key functions. One such adaptation is a heightened degranulation response under hypoxic conditions [23][24][25][26], which is the focus of this review. Neutrophils rely on oxygen sensing by prolyl and asparaginyl hydroxylase enzymes, which control the expression of the hypoxia-inducible factors (HIFs).…”

“…Examining a later degranulation response, Ong et al demonstrated an increase in MMP-8 secretion from purified neutrophils after 24 h, both when cells were incubated under true hypoxia and when HIF-1α was stabilised pharmacologically [25], suggesting that this delayed response is HIF-dependent. It should be noted that the degree of neutrophil apoptosis under normoxia versus hypoxia begins to diverge after 5 h in culture, with significantly improved cell survival under hypoxia beyond 20 h, which may contribute to observed changes in protein secretion at later time points [95].…”