Bacillus anthracis induces NLRP3 inflammasome activation and caspase-8–mediated apoptosis of macrophages to promote lethal anthrax

Lethal toxin (LeTx)-mediated killing of myeloid cells is essential for Bacillus anthracis, the causative agent of anthrax, to establish systemic infection and induce lethal anthrax. The “LeTx-sensitive” NLRP1b inflammasome of BALB/c and 129S macrophages swiftly responds to LeTx intoxication with pyroptosis and secretion of interleukin (IL)-1β. However, human NLRP1 is nonresponsive to LeTx, prompting us to investigate B. anthracis host–pathogen interactions in C57BL/6J (B6) macrophages and mice that also lack a LeTx-sensitive Nlrp1b allele. Unexpectedly, we found that LeTx intoxication and live B. anthracis infection of B6 macrophages elicited robust secretion of IL-1β, which critically relied on the NLRP3 inflammasome. TNF signaling through both TNF receptor 1 (TNF-R1) and TNF-R2 were required for B. anthracis-induced NLRP3 inflammasome activation, which was further controlled by RIPK1 kinase activity and LeTx-mediated proteolytic inactivation of MAP kinase signaling. In addition to activating the NLRP3 inflammasome, LeTx-induced MAPKK inactivation and TNF production sensitized B. anthracis-infected macrophages to robust RIPK1- and caspase-8–dependent apoptosis. In agreement, purified LeTx triggered RIPK1 kinase activity- and caspase-8–dependent apoptosis only in macrophages primed with TNF or following engagement of TRIF-dependent Toll-like receptors. Consistently, genetic and pharmacological inhibition of RIPK1 inhibited NLRP3 inflammasome activation and apoptosis of LeTx-intoxicated and B. anthracis-infected macrophages. Caspase-8/RIPK3-deficient mice were significantly protected from B. anthracis-induced lethality, demonstrating the in vivo pathophysiological relevance of this cytotoxic mechanism. Collectively, these results establish TNF- and RIPK1 kinase activity–dependent NLRP3 inflammasome activation and macrophage apoptosis as key host–pathogen mechanisms in lethal anthrax.

The bacterial pathogen Bacillus anthracis is a rare, but notoriously deadly pathogen in humans with mortality rates from anthrax varying from ∼20% for cutaneous anthrax to 80% and higher for inhalation anthrax. This encapsulated, spore-forming, gram-positive bacterial pathogen efficiently kills infected hosts through the systemic action of two secreted toxins (1). Edema toxin (EdTx) and lethal toxin (LeTx) share a receptor-binding protein named protective antigen (PA) that transfers the edema factor (EF) and lethal factor (LF) moieties into the cytosol of target cells, where the latter exert their cytopathic and cytotoxic effects (1, 2).Studies in macaques and mice identified LeTx as a major virulence factor driving systemic dispersion of vegetative bacteria, which ultimately may result in fatal anthrax (3, 4). LeTx internalization by macrophages drives macrophage cell death, which is a key early pathogenic event during spore infections that allows vegetative bacteria to establish systemic infection of its host (5). LF is a highly selective Zn²⁺-dependent metalloprotease that, once internalized, cleaves a subset of mitogen-activated protein kinase kinases (MAPKKs) to abolish downstream MAPK signaling in LeTx-intoxicated macrophages (6). In addition, macrophages of BALB/c and 129S mice express a LeTx-sensitive Nlrp1b allele that responds to LF-mediated cleavage with NLRP1b inflammasome activation and pyroptosis (7–9). However, human NLRP1 and the Nlrp1b allele of C57BL/6J (B6) macrophages are nonresponsive to LeTx, suggesting that B. anthracis may induce macrophage cell death through alternative mechanisms that are poorly understood.Here, we show that B. anthracis infection induces NLRP3 inflammasome activation and caspase-8–mediated apoptosis of B6 macrophages. Notably, B. anthracis sensitizes macrophages by promoting TNF production concomitantly with LeTx-mediated inactivation of p38 MAPK signaling. LeTx intoxication of TLR3/4- or TNF-activated macrophages similarly sensitized macrophages to TNF- and RIPK1 kinase activity–dependent NLRP3 inflammasome activation and cell death induction. Caspase-8/RIPK3-deficient mice were significantly protected from B. anthracis-induced lethality, demonstrating the in vivo pathophysiological relevance of this cytotoxic mechanism in lethal anthrax.