No abstract

Multiple regulated neutrophil cell death programs contribute to host defense against infections. However, despite expressing all necessary inflammasome components, neutrophils are thought to be generally defective in Caspase-1-dependent pyroptosis. By screening different bacterial species, we found that several Pseudomonas aeruginosa (P. aeruginosa) strains trigger Caspase-1-dependent pyroptosis in human and murine neutrophils. Notably, deletion of Exotoxins U or S in P. aeruginosa enhanced neutrophil death to Caspase-1-dependent pyroptosis, suggesting that these exotoxins interfere with this pathway. Mechanistically, P. aeruginosa Flagellin activates the NLRC4 inflammasome, which supports Caspase-1-driven interleukin (IL)-1β secretion and Gasdermin D (GSDMD)-dependent neutrophil pyroptosis. Furthermore, P. aeruginosa-induced GSDMD activation triggers Calcium-dependent and Peptidyl Arginine Deaminase-4-driven histone citrullination and translocation of neutrophil DNA into the cell cytosol without inducing extracellular Neutrophil Extracellular Traps. Finally, we show that neutrophil Caspase-1 contributes to IL-1β production and susceptibility to pyroptosis-inducing P. aeruginosa strains in vivo. Overall, we demonstrate that neutrophils are not universally resistant for Caspase-1-dependent pyroptosis.

Neutrophils are the most prevalent immune cells in circulation, but the repertoire of canonical inflammasomes in neutrophils and their respective involvement in neutrophil IL-1b secretion and neutrophil cell death remain unclear. Here, we show that neutrophiltargeted expression of the disease-associated gain-of-function Nlr-p3 A350V mutant suffices for systemic autoinflammatory disease and tissue pathology in vivo. We confirm the activity of the canonical NLRP3 and NLRC4 inflammasomes in neutrophils, and further show that the NLRP1b, Pyrin and AIM2 inflammasomes also promote maturation and secretion of interleukin (IL)-1b in cultured bone marrow neutrophils. Notably, all tested canonical inflammasomes promote GSDMD cleavage in neutrophils, and canonical inflammasome-induced pyroptosis and secretion of mature IL-1b are blunted in GSDMD-knockout neutrophils. In contrast, GSDMD is dispensable for PMA-induced NETosis. We also show that Salmonella Typhimurium-induced pyroptosis is markedly increased in Nox2/Gp91 Phox -deficient neutrophils that lack NADPH oxidase activity and are defective in PMA-induced NETosis. In conclusion, we establish the canonical inflammasome repertoire in neutrophils and identify differential roles for GSDMD and the NADPH complex in canonical inflammasome-induced neutrophil pyroptosis and mitogen-induced NETosis, respectively.

Inflammatory caspase‐11 (rodent) and caspases‐4/5 (humans) detect the Gram‐negative bacterial component LPS within the host cell cytosol, promoting activation of the non‐canonical inflammasome. Although non‐canonical inflammasome‐induced pyroptosis and IL‐1‐related cytokine release are crucial to mount an efficient immune response against various bacteria, their unrestrained activation drives sepsis. This suggests that cellular components tightly control the threshold level of the non‐canonical inflammasome in order to ensure efficient but non‐deleterious inflammatory responses. Here, we show that the IFN‐inducible protein Irgm2 and the ATG8 family member Gate‐16 cooperatively counteract Gram‐negative bacteria‐induced non‐canonical inflammasome activation, both in cultured macrophages and in vivo. Specifically, the Irgm2/Gate‐16 axis dampens caspase‐11 targeting to intracellular bacteria, which lowers caspase‐11‐mediated pyroptosis and cytokine release. Deficiency in Irgm2 or Gate16 induces both guanylate binding protein (GBP)‐dependent and GBP‐independent routes for caspase‐11 targeting to intracellular bacteria. Our findings identify molecular effectors that fine‐tune bacteria‐activated non‐canonical inflammasome responses and shed light on the understanding of the immune pathways they control.

Regulated cell necrosis supports immune and anti-infectious strategies of the body; however, dysregulation of these processes drives pathological organ damage. Pseudomonas aeruginosa expresses a phospholipase, ExoU that triggers pathological host cell necrosis through a poorly characterized pathway. Here, we investigated the molecular and cellular mechanisms of ExoU-mediated necrosis. We show that cellular peroxidised phospholipids enhance ExoU phospholipase activity, which drives necrosis of immune and non-immune cells. Conversely, both the endogenous lipid peroxidation regulator GPX4 and the pharmacological inhibition of lipid peroxidation delay ExoU-dependent cell necrosis and improve bacterial elimination in vitro and in vivo. Our findings also pertain to the ExoU-related phospholipase from the bacterial pathogen Burkholderia thailandensis, suggesting that exploitation of peroxidised phospholipids might be a conserved virulence mechanism among various microbial phospholipases. Overall, our results identify an original lipid peroxidation-based virulence mechanism as a strong contributor of microbial phospholipase-driven pathology.

Regulated cell necrosis supports immune and anti-infectious strategies of the body; however, dysregulation of these processes drives pathological organ damage. Pseudomonas aeruginosa expresses a phospholipase, ExoU that triggers pathological host cell necrosis through a poorly characterized pathway. Here, we investigated the molecular and cellular mechanisms of ExoU-mediated necrosis. We show that cellular peroxidised phospholipids enhance ExoU phospholipase activity, which drives necrosis of immune and non-immune cells. Conversely, both the endogenous lipid peroxidation regulator GPX4 and the pharmacological inhibition of lipid peroxidation delay ExoU-dependent cell necrosis and improve bacterial elimination in vitro and in vivo. Our findings also pertain to the ExoU-related phospholipase from the bacterial pathogen Burkholderia thailandensis, suggesting that exploitation of peroxidised phospholipids might be a conserved virulence mechanism among various microbial phospholipases. Overall, our results identify an original lipid peroxidation-based virulence mechanism as a strong contributor of microbial phospholipase-driven pathology.

No abstract

27Inflammatory caspase-11 (rodent) and caspases-4 and -5 (human) detect gram-28 negative bacterial component LPS in the host cell cytosol, which promotes activation 29 of the non-canonical inflammasome. Although non-canonical inflammasome-induced 30 pyroptosis and IL-1 related cytokine release is of importance to mount an efficient 31 immune response against various bacteria, its unrestrained activation drives sepsis. 32 This suggests that cellular components might tightly control the threshold level of the 33 non-canonical inflammasome in order to ensure efficient but not deleterious 34 inflammatory response. Here we show that the IFN-inducible protein Irgm2 and the 35 ATG8 family member Gate-16 cooperatively slow down non-canonical inflammasome 36 activation both in macrophages and in vivo. Specifically, the Irgm2/Gate-16 axis 37 dampens caspase-11 targeting to intracellular bacteria, which lower caspase-11-38 mediated pyroptosis and cytokine release. Specifically, deficiency in Irgm2 or Gate16 39 opens an alternative road for caspase-11 targeting to intracellular bacteria, 40 independently of the classical pathway driven by the Guanylate Binding Proteins 41 (GBPs). Thus, our findings provide new molecular effectors involved at fine-tuning the 42 optimal non-canonical inflammasome response and add novel insights in the 43 understanding of the immune pathways they control.44 45