Human caspase-4 mediates noncanonical inflammasome activation against gram-negative bacterial pathogens

Inflammasomes are critical for host defense against bacterial pathogens. In murine macrophages infected by gram-negative bacteria, the canonical inflammasome activates caspase-1 to mediate pyroptotic cell death and release of IL-1 family cytokines. Additionally, a noncanonical inflammasome controlled by caspase-11 induces cell death and IL-1 release. However, humans do not encode caspase-11. Instead, humans encode two putative orthologs: caspase-4 and caspase-5. Whether either ortholog functions similar to caspase-11 is poorly defined. Therefore, we sought to define the inflammatory caspases in primary human macrophages that regulate inflammasome responses to gram-negative bacteria. We find that human macrophages activate inflammasomes specifically in response to diverse gram-negative bacterial pathogens that introduce bacterial products into the host cytosol using specialized secretion systems. In primary human macrophages, IL-1β secretion requires the caspase-1 inflammasome, whereas IL-1α release and cell death are caspase-1–independent. Instead, caspase-4 mediates IL-1α release and cell death. Our findings implicate human caspase-4 as a critical regulator of noncanonical inflammasome activation that initiates defense against bacterial pathogens in primary human macrophages.Pattern recognition receptors (PRRs) of the innate immune system are critical for promoting defense against bacterial pathogens (1). Cytosolic PRRs are key for discriminating between pathogenic and nonpathogenic bacteria, because many pathogens access the host cytosol, a compartment where microbial products are typically not found (2). Cytosolic PRRs respond to patterns of pathogenesis that are often associated with virulent bacteria, such as the use of pore-forming toxins or injection of effector molecules through specialized secretion systems (3). A subset of cytosolic PRRs induces the formation of multiprotein complexes known as inflammasomes (4). In mice, the canonical inflammasome activates caspase-1, an inflammatory caspase that mediates cell death and IL-1 family cytokine secretion (5, 6). Additionally, the noncanonical inflammasome activates caspase-11 in response to many gram-negative bacteria (7–14). The canonical and noncanonical inflammasomes differentially regulate release of IL-1α and IL-1β (7). Caspase-11 mediates LPS-induced septic shock in mice (7, 15), and caspase-11 responds to cytoplasmic LPS independent of Toll-like receptor 4 (16, 17).In addition to its pathologic role in septic shock, the noncanonical inflammasome is critical for host defense in mice (11, 18). However, in humans, it is unclear whether an analogous noncanonical inflammasome exists. Whereas mice encode caspase-11, humans encode two putative functional orthologs: caspase-4 and caspase-5 (19–21). All three inflammatory caspases bind directly to LPS in vitro (22). In murine macrophages, caspase-1 and caspase-11 have both distinct and overlapping roles in the release of IL-1α and IL-1β and the induction of cell death (7). However, the precise role of the human inflammatory caspases in the context of infection by bacterial pathogens remains unclear.To elucidate how human inflammasome activation is regulated, we investigated the contribution of inflammatory caspases to the response against gram-negative bacterial pathogens in human macrophages. Here, we show that both canonical caspase-1–dependent and noncanonical caspase-1–independent inflammasomes are activated in primary human macrophages and that caspase-4 mediates caspase-1–independent inflammasome responses against several bacterial pathogens, including Legionella pneumophila, Yersinia pseudotuberculosis, and Salmonella enterica serovar Typhimurium (S. Typhimurium). Importantly, noncanonical inflammasome activation in human macrophages is specific for virulent strains of these bacteria that translocate bacterial products into the host cytosol via the virulence-associated type III secretion system (T3SS) or type IV secretion system (T4SS). Thus, caspase-4 is critical for noncanonical inflammasome responses against virulent gram-negative bacteria in human macrophages.