Abstract
The capacity of infected cells to undergo apoptosis upon insult with a pathogen is an ancient innate immune defense mechanism. Consequently, the ability of persisting, intracellular pathogens such as the human pathogen Mycobacterium tuberculosis (Mtb) to inhibit infection-induced apoptosis of macrophages is important for virulence. The nuoG gene of Mtb, which encodes the NuoG subunit of the type I NADH dehydrogenase, NDH-1, is important in Mtb-mediated inhibition of host macrophage apoptosis, but the molecular mechanism of this host pathogen interaction remains elusive. Here we show that the apoptogenic phenotype of MtbΔnuoG was significantly reduced in human macrophages treated with caspase-3 and -8 inhibitors, TNF-α-neutralizing antibodies, and also after infection of murine TNF−/− macrophages. Interestingly, incubation of macrophages with inhibitors of reactive oxygen species (ROS) reduced not only the apoptosis induced by the nuoG mutant, but also its capacity to increase macrophage TNF-α secretion. The MtbΔnuoG phagosomes showed increased ROS levels compared to Mtb phagosomes in primary murine and human alveolar macrophages. The increase in MtbΔnuoG induced ROS and apoptosis was abolished in NOX-2 deficient (gp91−/−) macrophages. These results suggest that Mtb, via a NuoG-dependent mechanism, can neutralize NOX2-derived ROS in order to inhibit TNF-α-mediated host cell apoptosis. Consistently, an Mtb mutant deficient in secreted catalase induced increases in phagosomal ROS and host cell apoptosis, both of which were dependent upon macrophage NOX-2 activity. In conclusion, these results serendipitously reveal a novel connection between NOX2 activity, phagosomal ROS, and TNF-α signaling during infection-induced apoptosis in macrophages. Furthermore, our study reveals a novel function of NOX2 activity in innate immunity beyond the initial respiratory burst, which is the sensing of persistent intracellular pathogens and subsequent induction of host cell apoptosis as a second line of defense.
Highlights
The phagocytic NADPH-oxidase (NOX2-complex or phox) resides on phagosomes and has been shown to be involved in microcidal activity in phagocytes
We demonstrate that an M. tuberculosis mutant deficient in NDH-1 accumulates reactive oxygen species (ROS) inside the macrophage which induces the secretion of an inflammatory cytokine (TNF-a) and subsequent host cell death
We propose that a novel function of the host cell NOX-2 complex is to allow sensing of intracellular pathogens by the host cell in order to commit suicide and limit bacterial survival
Summary
The phagocytic NADPH-oxidase (NOX2-complex or phox) resides on phagosomes and has been shown to be involved in microcidal activity in phagocytes. The multicomponent NOX2 complex consists of two transmembrane proteins, gp91phox and gp phox, and three cytosolic components, p40 phox p47 phox and p67 phox [1,2]. The gp91phox subunit, which is constitutively associated with gp phox, is a transmembrane redox chain that generates phagosomal superoxide by transferring electrons from cytosolic NADPH to phagosomal oxygen [1]. NOX2-generated superoxide can be converted into a multitude of microcidal oxidants, including hydrogen peroxide and hypochlorous acid, which are important components of the bactericidal activity of the macrophage phagosome [3]. Mice deficient in the NOX2 subunits are much more susceptible to infections with bacterial pathogens such as Salmonella typhimurium for example [3,5]. Some pathogens have developed strategies to counter the NOX2 response by either inhibiting NOX2 assembly on the phagosome, as is the case for S. typhimurium [3] and Helicobacter pylori [6], or reducing steady-state levels of NOX2 components as illustrated by Anaplasma phagocytophila [7] or Ehrlichia chaffeensis [8] (for review [9])
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