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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.

Congenital anemias include a broad range of disorders marked by inherent abnormalities in red blood cells. These abnormalities include enzymatic, membrane, and congenital defects in erythropoiesis, as well as hemoglobinopathies such as sickle cell disease and thalassemia. These conditions range in presentation from asymptomatic cases to those requiring frequent blood transfusions, exhibiting phenotypic heterogeneity and different degrees of severity. Despite understanding their different etiologies, all of them have a common pathophysiological origin with congenital defects of erythropoiesis. We can find different types, from congenital sideroblastic anemia (CSA), which is a bone marrow failure anemia, to hemoglobinopathies as sickle cell disease and thalassemia, with a higher prevalence and clinical impact. Recent efforts have focused on understanding erythropoiesis dysfunction in these anemias but, so far, deep gene sequencing analysis comparing all of them has not been performed. Our study used Quant 3′ mRNA-Sequencing to compare transcriptomic profiles of four sickle cell disease patients, ten thalassemia patients, and one rare case of SLC25A38 CSA. Our results showed clear differentiated gene map expressions in all of them with respect to healthy controls. Our study reveals that genes related to metabolic processes, membrane genes, and erythropoiesis are upregulated with respect to healthy controls in all pathologies studied except in the SLC25A38 CSA patient, who shows a unique gene expression pattern compared to the rest of the congenital anemias studied. Our analysis is the first that compares gene expression patterns across different congenital anemias to provide a broad spectrum of genes that could have clinical relevance in these pathologies.

In 2020–2021, a “mysterious illness” struck Senegalese fishermen, causing severe acute dermatitis in over one thousand individuals following exposure through drift-net fishing activity. Here, by performing deep analysis of the environmental samples we reveal the presence of the marine dinoflagellate Vulcanodinium rugosum and its associated cyclic imine toxins. Specifically, we show that the toxin PortimineA, strongly enriched in environmental samples, impedes ribosome function in human keratinocytes, which subsequently activates the stress kinases ZAKα and P38 and promotes the nucleation of the human NLRP1 inflammasome, leading to the release of IL-1β/IL-18 pro-inflammatory cytokines and cell death. Furthermore, cell-based models highlight that naturally occurring mutations in the P38-targeted sites of human NLRP1 are unable to respond to PortimineA exposure. Finally, the development and use of human organotypic skins and zebrafish models of PortimineA exposure demonstrate that the ZAKα-NLRP1 axis drives skin necrosis and inflammation. Our results exemplify the threats to human health caused by emerging environmental toxins and identify ZAKα and NRLP1 as important pharmacological targets to mitigate PortimineA toxicity. Synopsis In 2020-2021, a mysterious skin disease affected fishermen in Senegal. High levels of Portimine A toxin, produced by the dinoflagellate Vulcanodinium rugosum , were found in Senegal’s fishing zones. Portimine A induced a strong inflammatory response in human skin epithelial cells. Portimine A-inhibited translation supported a ribotoxic stress response mediated by ZAKα kinase. ZAKα promoted NLRP1 inflammasome-dependent skin inflammation in response to Portimine A in human skin models and in zebrafish models. In 2020-2021, a mysterious skin disease affected fishermen in Senegal.

Pyroptosis is a lytic cell death triggered by the cleavage of gasdermin (GSDM) proteins and subsequent pore formation by the N-terminal domain oligomerization in the plasma membrane. GSDMD is cleaved by caspase-1/-4/-5/-11 upon inflammasome activation and mediates IL-1beta and IL-18 release. GSDMD pores favors ninjurin-1 (NINJ1) induced plasma membrane rupture and cell death. Here, we demonstrate that GSDMD mediates early ATP release upon NLRP3 inflammasome activation independently of NINJ1, occurring before IL-1beta release and cell death and constituting an early danger signal. The release of ATP is a transient signal terminated before the cells continue to permeabilize and die. The different N-terminal of GSDMA to E are also able to release ATP and induce monocyte migration towards pyroptotic cells. This study reveals ATP release as an early, and transient danger signal depending on GSDMD plasma membrane permeabilization, independently of the late stages of lytic cell death.Competing Interest StatementPP declares that he is an inventor in a patent filled on March 2020 by the Fundacion para la Formacion e Investigacion Sanitaria de la Region de Murcia (PCT/EP2020/056729) for a method to identify patients with sepsis and NLRP3-disfunction, being consultant of Viva in vitro diagnostics SL. PP, LH-N and DA-B are co-founders of Viva in vitro diagnostics SL, but declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The remaining authors declare no competing interests.

Nigericin, an ionophore derived from Streptomyces hygroscopicus, is arguably the most commonly used tool compound to study the NLRP3 inflammasome. Recent findings, however, showed that nigericin also activates the NLRP1 inflammasome in human keratinocytes. In this study, we resolve the mechanistic basis of nigericin-driven NLRP1 inflammasome activation. In multiple non-hematopoietic cell types, nigericin rapidly and specifically inhibits the elongation stage of the ribosome cycle by depleting cytosolic potassium ions. This activates the ribotoxic stress response (RSR) sensor kinase ZAKɑ, p38 and JNK, as well as the hyperphosphorylation of the NLRP1 linker domain. As a result, nigericin-induced pyroptosis in human keratinocytes is blocked by extracellular potassium supplementation, ZAKɑ knockout or pharmacologic inhibitors of ZAKɑ and p38 kinase activities. By surveying a diverse panel of ionophores, we show that the electroneutrality of potassium efflux is essential to activate ZAKɑ-driven RSR, likely because a greater extent of K+ depletion is necessary to activate ZAKɑ-NLRP1 than NLRP3. These findings resolve the mechanism by which nigericin activates NLRP1 in nonhematopoietic cell types and demonstrate an unexpected connection between RSR, perturbations of potassium ion flux and innate immunity. Nigericin is familiar to the inflammasome field as the most robust and commonly used NLRP3 inducer. It has enabled numerous breakthroughs in the field linking NLRP3 activation to potassium efflux. In this manuscript, we report that nigericin activates an alternate inflammasome sensor, NLRP1 in primary human skin, nasal and corneal epithelial cells. NLRP1 activation by nigericin requires K+ efflux-driven ribosome stalling and the ribotoxic stress response (RSR) sensor MAP3K, ZAKɑ. We further identify the key biophysical principles that explain why only a subset of K+ ionophores, exemplified by nigericin, function as ‘super’ inflammasome agonists that can activate either NLRP1 or NLRP3, depending on cell type. These results reveal an unexpected connection between RSR, potassium ion flux and innate immunity.

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

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most prevalent cause of liver disease worldwide. This progressive condition ranges from simple steatosis to a more advanced stage, known as metabolic dysfunction-associated steatohepatitis (MASH), which is characterized by inflammation, hepatocellular ballooning, and hepatic steatosis. Several studies have demonstrated the involvement of inflammasomes in MASH development. Recently, the NLRP1 inflammasome has gained attention as an important sensor in various human inflammatory conditions, though its role in metabolic diseases like MASLD remains unclear. In this study, we identified significantly higher mRNA and protein levels of NLRP1 in liver samples from patients with MASH compared to those with normal or steatotic livers. Furthermore, NLRP1 mRNA levels correlated with hepatic palmitic acid (PA) levels. We also showed that NLRP1 inflammasome expression is mediated by PA in both human HepG2 cells and human liver organoids. Importantly, we found that NLRP1 was activated by PA, but not by other saturated fatty acids like myristic acid, and that PA-induced NLRP1 activation was inhibited by oleic acid. These findings uncover a previously unknown role of the hepatic NLRP1 inflammasome in the human liver. However, further research is needed to fully understand the complex interactions between NLRP1, inflammation, and metabolic processes in the development of MASLD.Competing Interest StatementThe authors have declared no competing interest.

The intracellular inflammasome complex have been implicated in the maladaptive tissue damage and inflammation observed in chronic Pseudomonas aeruginosa infection. Human airway and corneal epithelial cells, which are critically altered during chronic infections mediated by P. aeruginosa, specifically express the inflammasome sensor NLRP1. Here, together with a companion study, we report that the NLRP1 inflammasome detects Exotoxin A (EXOA), a ribotoxin released by P. aeruginosa Type 2 Secretion System (T2SS) during chronic infection. Mechanistically, EXOA-driven Eukaryotic Elongation Factor 2 (EEF2) ribosylation and covalent inactivation promotes ribotoxic stress and subsequent NLRP1 inflammasome activation, a process shared with other EEF2-inactivating toxins, Diphtheria Toxin and Cholix Toxin. Biochemically, irreversible EEF2 inactivation triggers ribosome stress-associated kinases ZAKα- and P38-dependent NLRP1 phosphorylation and subsequent proteasome-driven functional degradation. Finally, Cystic Fibrosis cells from patients exhibit exacerbated P38 activity and hypersensitivity to EXOA-induced ribotoxic stress-dependent NLRP1 inflammasome activation, a process inhibited by the use of ZAKα inhibitors. Altogether, our results show the importance of P. aeruginosa virulence factor EXOA at promoting NLRP1-dependent epithelial damage and identify ZAKα as a critical sensor of virulence-inactivated EEF2. P. aeruginosa induces NLRP1-dependent pyroptosis in human corneal and nasal epithelial cells P. aeruginosa Exotoxin A (EXOA) and other EEF2-inactivating bacterial exotoxins activate the human NLRP1 inflammasome EEF2 inactivation promotes ribotoxic stress response and ZAKα kinase-dependent NLRP1 inflammasome activation. Bronchial epithelial cells from Cystic Fibrosis patients show extreme sensitivity to ribotoxic stress-dependent NLRP1 inflammasome activation in response to Exotoxin A P38 and ZAKα inhibition protects Cystic Fibrosis epithelial cell from EXOA-induced pyroptosis

Multiple neutrophil death programs contribute to host defense against infections. Although expressing all necessary components, neutrophils specifically fail to undergo pyroptosis, a lytic form of cell death triggered by the activation of the pro-inflammatory complex inflammasome. In the light of the arm race, we hypothesized that intrinsic neutrophil pyroptosis resistance might be bypassed in response to specific microbial species. We show that Pseudomonas aeruginosa (P. aeruginosa) stimulates Caspase-1-dependent pyroptosis in human and murine neutrophils. Mechanistically, activated NLRC4 inflammasome supports Caspase-1-driven Gasdermin-D (GSDMD) activation, IL-1β cytokine release and neutrophil pyroptosis. Furthermore, GSDMD activates Peptidyl Arginine Deaminase-4 which drives an incomplete NETosis where neutrophil DNA fills the cell cytosol but fails crossing plasma membrane. Finally, we show that neutrophil Caspase-1 account for IL-1beta production and contributes to various P. aeruginosa strains spread in mice. Overall, we demonstrate that neutrophils are fully competent for Caspase-1-dependent pyroptosis, which drives an unsuspected incomplete NETosis. Competing Interest Statement The authors have declared no competing interest.

Summary 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. Competing Interest Statement The authors have declared no competing interest.