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The innate immune system is critical for host defense against microbial pathogens, yet many pathogens express virulence factors that impair immune function. Here, we used the bacterial pathogen Legionella pneumophila to understand how the immune system successfully overcomes pathogen subversion mechanisms. L. pneumophila replicates within macrophages by using a type IV secretion system to translocate bacterial effectors into the host cell cytosol. As a consequence of effector delivery, host protein synthesis is blocked at several steps, including translation initiation and elongation. Despite this translation block, infected cells robustly produce proinflammatory cytokines, but the basis for this is poorly understood. By using a reporter system that specifically discriminates between infected and uninfected cells within a population, we demonstrate here that infected macrophages produced IL-1α and IL-1β, but were poor producers of IL-6, TNF, and IL-12, which are critical mediators of host protection. Uninfected bystander cells robustly produced IL-6, TNF, and IL-12, and this bystander response required IL-1 receptor (IL-1R) signaling during early pulmonary infection. Our data demonstrate functional heterogeneity in production of critical protective cytokines and suggest that collaboration between infected and uninfected cells enables the immune system to bypass pathogen-mediated translation inhibition to generate an effective immune response.

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.

Toll-like receptor signaling and subsequent activation of NF-κB- and MAPK-dependent genes during infection play an important role in antimicrobial host defense. The YopJ protein of pathogenic Yersinia species inhibits NF-κB and MAPK signaling, resulting in blockade of NF-κB-dependent cytokine production and target cell death. Nevertheless, Yersinia infection induces inflammatory responses in vivo. Moreover, increasing the extent of YopJ-dependent cytotoxicity induced by Yersinia pestis and Yersinia pseudotuberculosis paradoxically leads to decreased virulence in vivo, suggesting that cell death promotes anti-Yersinia host defense. However, the specific pathways responsible for YopJ-induced cell death and how this cell death mediates immune defense against Yersinia remain poorly defined. YopJ activity induces processing of multiple caspases, including caspase-1, independently of inflammasome components or the adaptor protein ASC. Unexpectedly, caspase-1 activation in response to the activity of YopJ required caspase-8, receptor-interacting serine/threonine kinase 1 (RIPK1), and Fas-associated death domain (FADD), but not RIPK3. Furthermore, whereas RIPK3 deficiency did not affect YopJ-induced cell death or caspase-1 activation, deficiency of both RIPK3 and caspase-8 or FADD completely abrogated Yersinia-induced cell death and caspase-1 activation. Mice lacking RIPK3 and caspase-8 in their hematopoietic compartment showed extreme susceptibility to Yersinia and were deficient in monocyte and neutrophil-derived production of proinflammatory cytokines. Our data demonstrate for the first time to our knowledge that RIPK1, FADD, and caspase-8 are required for YopJ-induced cell death and caspase-1 activation and suggest that caspase-8-mediated cell death overrides blockade of immune signaling by YopJ to promote anti-Yersinia immune defense.