Neutrophil extrusion of neutrophil extracellular traps (NETs) and concomitant cell death (NETosis) provides host defense against extracellular pathogens, whereas macrophage death by pyroptosis enables defense against intracellular pathogens. We report the unexpected discovery that gasdermin D (GSDMD) connects these cell death modalities. We show that neutrophil exposure to cytosolic lipopolysaccharide or cytosolic Gram-negative bacteria ( Δ and ) activates noncanonical (caspase-4/11) inflammasome signaling and triggers GSDMD-dependent neutrophil death. GSDMD-dependent death induces neutrophils to extrude antimicrobial NETs. Caspase-11 and GSDMD are required for neutrophil plasma membrane rupture during the final stage of NET extrusion. Unexpectedly, caspase-11 and GSDMD are also required for early features of NETosis, including nuclear delobulation and DNA expansion; this is mediated by the coordinate actions of caspase-11 and GSDMD in mediating nuclear membrane permeabilization and histone degradation. In vivo application of deoxyribonuclease I to dissolve NETs during murine Δ challenge increases bacterial burden in wild-type but not in and mice. Our studies reveal that neutrophils use an inflammasome- and GSDMD-dependent mechanism to activate NETosis as a defense response against cytosolic bacteria.

IL-1β requires processing by caspase-1 to generate the active, pro-inflammatory cytokine. Acute IL-1β secretion from inflammasome-activated macrophages requires caspase-1-dependent GSDMD cleavage, which also induces pyroptosis. Mechanisms of IL-1β secretion by pyroptotic and non-pyroptotic cells, and the precise functions of caspase-1 and GSDMD therein, are unresolved. Here, we show that, while efficient early secretion of endogenous IL-1β from primary non-pyroptotic myeloid cells in vitro requires GSDMD, later IL-1β release in vitro and in vivo proceeds independently of GSDMD. IL-1β maturation is sufficient for slow, caspase-1/GSDMD-independent secretion of ectopic IL-1β from resting, non-pyroptotic macrophages, but the speed of IL-1β release is boosted by inflammasome activation, via caspase-1 and GSDMD. IL-1β cleavage induces IL-1β enrichment at PIP2-enriched plasma membrane ruffles, and this is a prerequisite for IL-1β secretion and is mediated by a polybasic motif within the cytokine. We thus reveal a mechanism in which maturation-induced IL-1β trafficking facilitates its unconventional secretion.

Highlights d The class IIa HDAC, HDAC7, drives TLR4-inducible glycolysis in macrophages d An interaction between HDAC7 and PKM2 drives macrophage immunometabolic responses d Deacetylation of PKM2 at K433 licenses it to drive proinflammatory IL-1b expression d Multiple class IIa HDACs can engage the PKM2 immunometabolic signaling hub

This study provides a detailed molecular mechanism for caspase-11 activation within the non-canonical inflammasome, giving new insight into host defence against cytosolic bacterial infection.

Inflammasomes are inflammatory signaling complexes that provide molecular platforms to activate the protease function of inflammatory caspases. Caspases-1, -4, -5, and -11 are inflammatory caspases activated by inflammasomes to drive lytic cell death and inflammatory mediator production, thereby activating host-protective and pathological immune responses. Here, we comprehensively review the mechanisms that govern the activity of inflammatory caspases. We discuss inflammatory caspase activation and deactivation mechanisms, alongside the physiological importance of caspase activity kinetics. We also examine mechanisms of caspase substrate selection and how inflammasome and cell identities influence caspase activity and resultant inflammatory and pyroptotic cellular programs. Understanding how inflammatory caspases are regulated may offer new strategies for treating infection and inflammasome-driven disease. Expected final online publication date for the Annual Review of Immunology, Volume 40 is April 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

The mammalian inhibitor of apoptosis proteins (IAPs) are key regulators of cell death and inflammation. A major function of IAPs is to block the formation of a cell death-inducing complex, termed the ripoptosome, which can trigger caspase-8-dependent apoptosis or caspase-independent necroptosis. Recent studies report that upon TLR4 or TNF receptor 1 (TNFR1) signaling in macrophages, the ripoptosome can also induce NLRP3 inflammasome formation and IL-1β maturation. Whether neutrophils have the capacity to assemble a ripoptosome to induce cell death and inflammasome activation during TLR4 and TNFR1 signaling is unclear. In this study, we demonstrate that murine neutrophils can signal via TNFR1-driven ripoptosome assembly to induce both cell death and IL-1β maturation. However, unlike macrophages, neutrophils suppress TLR4-dependent cell death and NLRP3 inflammasome activation during IAP inhibition via deficiencies in the CD14/TRIF arm of TLR4 signaling.

Innate immune cells such as macrophages and neutrophils initiate protective inflammatory responses and engage antimicrobial responses to provide frontline defence against invading pathogens. These cells can both restrict the availability of certain transition metals that are essential for microbial growth and direct toxic concentrations of metals towards pathogens as antimicrobial responses. Zinc is important for the structure and function of many proteins, however excess zinc can be cytotoxic. In recent years, several studies have revealed that innate immune cells can deliver toxic concentrations of zinc to intracellular pathogens. In this review, we discuss the importance of zinc status during infectious disease and the evidence for zinc intoxication as an innate immune antimicrobial response. Evidence for pathogen subversion of this response is also examined. The likely mechanisms, including the involvement of specific zinc transporters that facilitate delivery of zinc by innate immune cells for metal ion poisoning of pathogens are also considered. Precise mechanisms by which excess levels of zinc can be toxic to microorganisms are then discussed, particularly in the context of synergy with other antimicrobial responses. Finally, we highlight key unanswered questions in this emerging field, which may offer new opportunities for exploiting innate immune responses for anti-infective development.

TLR-inducible zinc toxicity is an antimicrobial mechanism utilized by macrophages, however knowledge of molecular mechanisms mediating this response is limited. Here, we show that E. coli exposed to zinc stress within primary human macrophages reside in membrane-bound vesicular compartments. Since SLC30A zinc exporters can deliver zinc into the lumen of vesicles, we examined LPS-regulated mRNA expression of Slc30a/SLC30A family members in primary mouse and human macrophages. A number of these transporters were dynamically regulated in both cell populations. In human monocyte-derived macrophages, LPS strongly up-regulated SLC30A1 mRNA and protein expression. In contrast, SLC30A1 was not LPS-inducible in macrophage-like PMA-differentiated THP-1 cells. We therefore ectopically expressed SLC30A1 in these cells, finding that this was sufficient to promote zinc-containing vesicle formation. The response was similar to that observed following LPS stimulation. Ectopically expressed SLC30A1 localized to both the plasma membrane and intracellular zinc-containing vesicles within LPS-stimulated THP-1 cells. Inducible overexpression of SLC30A1 in THP-1 cells infected with the Escherichia coli K-12 strain MG1655 augmented the zinc stress response of intracellular bacteria and promoted clearance. Furthermore, in THP-1 cells infected with an MG1655 zinc stress reporter strain, all bacteria contained within SLC30A1-positive compartments were subjected to zinc stress. Thus, SLC30A1 marks zinc-containing compartments associated with TLR-inducible zinc toxicity in human macrophages, and its ectopic over-expression is sufficient to initiate this antimicrobial pathway in these cells. Finally, SLC30A1 silencing did not compromise E. coli clearance by primary human macrophages, suggesting that other zinc exporters may also contribute to the zinc toxicity response.