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Inflammatory caspases sensing lipopolysaccharide (LPS) to drive gasdermin (GSDM)-mediated pyroptosis is an important immune response mechanism for anti-infection defense in mammals. In this work, we resolved an LPS-induced and GSDM-gated pyroptosis signaling cascade in Cnidarians. Initially, we identified a functional GSDM protein, HyGSDME, in Hydra , executing cytosolic LPS-induced pyroptosis in a caspase-dependent manner. Further, we identified a proinflammatory caspase, HyCaspA , capable of sensing cytosolic LPS by an uncharacterized N-terminal domain relying on its unique hydrophobic property, thereby triggering its oligomerization and self-activation. Subsequently, the LPS-activated HyCaspA cleaved an apoptotic caspase, HyCARD2, to trigger HyGSDME-gated pyroptosis. Last, HyGSDME exhibited an enriched distribution on the ectodermal layer of Hydra polyps, exerting a canonical immune defense function against surface-invading bacteria. Collectively, our work resolved an ancient pyroptosis signaling cascade in Hydra , suggesting that inflammatory caspases sensing cytosolic LPS to initiate GSDM-gated pyroptosis are a conserved immune defense mechanism from Cnidarians to mammals.

As the executor of pyroptosis known to date, gasdermins (GSDMs), consists of GSDMA, GSDMB, GSDMC, GSDMD, GSDME and pejvakin, might play critical roles in anti-bacterial infection as well as inflammatory diseases. However, zebrafish only harbors a pair of Gsdme (Gsdmea/b), and their activation mechanisms remain largely unknown. Herein, we investigate the activation mechanism of Gsdmea/b cleaved by inflammatory and apoptotic caspases in zebrafish，and found that Gsdmea/b are equally cleaved by Caspase 19b, a sister of Caspy2, but not Caspy. Moreover, the zebrafish apoptotic effector caspases, including Caspase 3a/b and Caspase 7, also can cleave Gsdmea/b at the same sites as inflammatory caspases recognized. Importantly, our results reveal that Caspase 8a/b can cleave Gsdmeb, but only Caspase 8a can cleave Gsdmea. Taken together, these findings suggest that zebrafish Gsdmea/b can concurrently function as GSDMD and GSDME in mammals, which will contribute to better understanding the mechanism of pyroptosis activation in teleost, as well as provide a clue for drug screening model against inflammatory diseases., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Host protective inflammatory caspase activity must be tightly regulated to prevent pathogens infection, however, the inflammatory caspase-engaged inflammasome activation in teleost fish remains largely unknown. In this study, we reveal a bifurcated evolutionary role of the inflammatory caspase in mediating both non-canonical and canonical inflammasome pathways in teleost fish. Through characterization of a unique inflammatory SmCaspase from the teleost Scophthalmus maximus (turbot), we found it can directly recognize cytosolic lipopolysaccharide (LPS) via its N-terminal CARD domain, resulting in caspase-5-like proteolytic enzyme activity-mediated pyroptosis in Turbot Muscle Fibroblasts. Interestingly, we also found that this inflammatory caspase can be recruited to SmNLRP3-SmASC to form the NLRP3 inflammasome complex, engaging the SmIL-1β release in Head Kidney-derived Macrophages. Consequently, the SmCaspase activation can recognize and cleave the SmGSDMEb to release its N-terminal domain, mediating both pyroptosis and bactericidal activities. Furthermore, the SmCaspase-SmGSDMEb axis-gated pyroptosis governs the bacterial clearance and epithelial desquamation in fish gill filaments in vivo. To our knowledge, this study is the first to identify an inflammatory caspase acting as a central coordinator in NLRP3 inflammasome, as well as a cytosolic LPS receptor; thus uncovering a previously unrecognized function of inflammatory caspase in turbot innate immunity., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The noncanonical inflammasome is critical for cytosolic sensing of Gram-negative pathogens. Here, we show that bacterial infection induces caspy2 activation in zebrafish fibroblasts, which mediates pyroptosis via a caspase-5-like activity. Zebrafish caspy2 binds directly to lipopolysaccharide via the N-terminal pyrin death domain, resulting in caspy2 oligomerization, which is critical for pyroptosis. Furthermore, we show that caspy2 is highly expressed in the zebrafish gut and is activated during infection. Knockdown of caspy2 expression impairs the ability of zebrafish to restrict bacterial invasion in vivo, and protects larvae from lethal sepsis. Collectively, our results identify a crucial event in the evolution of pattern recognition into the death domain superfamily-mediated intracellular lipopolysaccharide-sensing pathway in innate immunity.

The noncanonical inflammasome induced by intracellular lipopolysaccharide (LPS) leads to caspase-11-dependent pyroptosis, which is critical for induction of endotoxic shock in mice. However, the signaling pathway downstream of caspase-11 is unknown. We found that cytosolic LPS stimulation induced caspase-11-dependent cleavage of the pannexin-1 channel followed up by ATP release, which in turn activated the purinergic P2X7 receptor to mediate cytotoxicity. In the absence of P2X7 or pannexin-1, pyroptosis induced by cytosolic LPS was abrogated. Cleavage of pannexin-1 required the catalytic activity of caspase-11 and was essential for ATP release and P2X7-mediated pyroptosis. Priming the caspase-11 pathway in vivo with LPS or Toll-like receptor-3 (TLR3) agonist resulted in high mortality in wild-type mice after secondary LPS challenge, but not in Casp11(-/-), Panx1(-/-), or P2x7(-/-) mice. These results reveal a critical role for pannexin-1 and P2X7 downstream of caspase-11 for pyroptosis and susceptibility to sepsis induced by the noncanonical inflammasome., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Summary The activator and composition of the NLRP6 inflammasome remain poorly understood. We find that lipoteichoic acid (LTA), a molecule produced by Gram-positive bacteria, binds and activates NLRP6. In response to cytosolic LTA or infection with Listeria monocytogenes , NLRP6 recruited caspase-11 and caspase-1 via the adaptor ASC. NLRP6 activation by LTA induced processing of caspase-11, which promoted caspase-1 activation and interleukin-1β (IL-1β)/IL-18 maturation in macrophages. Nlrp6 −/− and Casp11 −/− mice were less susceptible to L. monocytogenes infection, which was associated with reduced pathogen loads and impaired IL-18 production. Administration of IL-18 to Nlrp6 −/− or Casp11 −/− mice restored the susceptibility of mutant mice to L. monocytogenes infection. These results reveal a previously unrecognized innate immunity pathway triggered by cytosolic LTA that is sensed by NLRP6 and exacerbates systemic Gram-positive pathogen infection via the production of IL-18. Graphical Abstract Highlights • LTA from Gram-positive bacteria binds and activates NLRP6 • Listeria and cytosolic LTA induce caspase-11 processing via NLRP6 and ASC • Processed caspase-11 promotes caspase-1 activation and IL-18 secretion • NLRP6 and caspase-11 exacerbate Gram-positive pathogen infection in vivo Lipoteichoic acid produced by Gram-positive bacteria is sensed by the NLRP6 inflammasome and leads to the processing of both caspase-1 and caspase-11, exacerbating infection. [ABSTRACT FROM AUTHOR]