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Pathogenesis of Pseudomonas aeruginosa infections can include bacterial survival inside epithelial cells. Previously, we showed that this involves multiple roles played by the type three secretion system (T3SS), and specifically the effector ExoS. This includes ExoS-dependent inhibition of a lytic host cell response that subsequently enables intracellular replication. Here, we studied the underlying cell death response to intracellular P. aeruginosa, comparing wild-type to T3SS mutants varying in capacity to induce cell death and that localize to different intracellular compartments. Results showed that corneal epithelial cell death induced by intracellular P. aeruginosa lacking the T3SS, which remains in vacuoles, correlated with the activation of nuclear factor-κB as measured by p65 relocalization and tumor necrosis factor alpha transcription and secretion. Deletion of caspase-4 through CRISPR-Cas9 mutagenesis delayed cell death caused by these intracellular T3SS mutants. Caspase-4 deletion also countered more rapid cell death caused by T3SS effector-null mutants still expressing the T3SS apparatus that traffic to the host cell cytoplasm, and in doing so rescued intracellular replication normally dependent on ExoS. While HeLa cells lacked a lytic death response to T3SS mutants, it was found to be enabled by interferon gamma treatment. Together, these results show that epithelial cells can activate the noncanonical inflammasome pathway to limit proliferation of intracellular P. aeruginosa, not fully dependent on bacterially driven vacuole escape. Since ExoS inhibits the lytic response, the data implicate targeting of caspase-4, an intracellular pattern recognition receptor, as another contributor to the role of ExoS in the intracellular lifestyle of P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa can exhibit an intracellular lifestyle within epithelial cells in vivo and in vitro. The type three secretion system (T3SS) effector ExoS contributes via multiple mechanisms, including extending the life of invaded host cells. Here, we aimed to understand the underlying cell death inhibited by ExoS when P. aeruginosa is intracellular. Results showed that intracellular P. aeruginosa lacking T3SS effectors could elicit rapid cell lysis via the noncanonical inflammasome pathway. Caspase-4 contributed to cell lysis even when the intracellular bacteria lacked the entire T33S and were consequently unable to escape vacuoles, representing a naturally occurring subpopulation during wild-type infection. Together, the data show the caspase-4 inflammasome as an epithelial cell defense against intracellular P. aeruginosa, and implicate its targeting as another mechanism by which ExoS preserves the host cell replicative niche.

Pyroptosis is a proinflammatory mode of lytic cell death mediated by accumulation of plasma membrane (PM) macropores composed of gasdermin-family (GSDM) proteins. It facilitates two major functions in innate immunity: (i) elimination of intracellular replicative niches for pathogenic bacteria; and (ii) non-classical secretion of IL-1 family cytokines that amplify host-beneficial inflammatory responses to microbial infection or tissue damage. Physiological roles for gasdermin D (GSDMD) in pyroptosis and IL-1β release during inflammasome signaling have been extensively characterized in macrophages. This involves cleavage of GSDMD by caspase-1 to generate GSDMD macropores that mediate IL-1β efflux and progression to pyroptotic lysis. Neutrophils, which rapidly accumulate in large numbers at sites of tissue infection or damage, become the predominant local source of IL-1β in coordination with their potent microbiocidal capacity. Similar to macrophages, neutrophils express GSDMD and utilize the same spectrum of diverse inflammasome platforms for caspase-1-mediated cleavage of GSDMD. Distinct from macrophages, neutrophils possess a remarkable capacity to resist progression to GSDMD-dependent pyroptotic lysis to preserve their viability for efficient microbial killing while maintaining GSDMD-dependent mechanisms for export of bioactive IL-1β. Rather, neutrophils employ cell-specific mechanisms to conditionally engage GSDMD-mediated pyroptosis in response to bacterial pathogens that use neutrophils as replicative niches. GSDMD and pyroptosis have also been mechanistically linked to induction of NETosis, a signature neutrophil pathway that expels decondensed nuclear DNA into extracellular compartments for immobilization and killing of microbial pathogens. This review summarizes a rapidly growing number of recent studies that have produced new insights, unexpected mechanistic nuances, and some controversies regarding the regulation of, and roles for, neutrophil inflammasomes, pyroptosis, and GSDMs in diverse innate immune responses.

Pseudomonas aeruginosa is one of the most virulent opportunistic Gram-negative bacterial pathogens in humans. It causes many acute and chronic infections with morbidity and mortality rates as high as 40%. P. aeruginosa owes its pathogenic versatility to a large arsenal of cell-associated and secreted virulence factors which enable this pathogen to colonize various niches within hosts and protect it from host innate immune defenses. Induction of cytotoxicity in target host cells is a major virulence strategy for P. aeruginosa during the course of infection. P. aeruginosa has invested heavily in this strategy, as manifested by a plethora of cytotoxins that can induce various forms of cell death in target host cells. In this review, we provide an in-depth review of P. aeruginosa cytotoxins based on their mechanisms of cytotoxicity and the possible consequences of their cytotoxicity on host immune responses.

While immunotherapy has emerged as a breakthrough cancer therapy, it is only effective in some patients, indicating the need of alternative therapeutic strategies. Induction of cancer immunogenic cell death (ICD) is one promising way to elicit potent adaptive immune responses against tumor-associated antigens. Type I interferon (IFN) is well known to play important roles in different aspects of immune responses, including modulating ICD in anti-tumor action. However, how to expand IFN effect in promoting ICD responses has not been addressed. Here we show that depletion of ubiquitin specific protease 18 (USP18), a negative regulator of IFN signaling, selectively induces cancer cell ICD. Lower USP18 expression correlates with better survival across human selected cancer types and delays cancer progression in mouse models. Mechanistically, nuclear USP18 controls the enhancer landscape of cancer cells and diminishes STAT2-mediated transcription complex binding to IFN-responsive elements. Consequently, USP18 suppression not only enhances expression of canonical IFN-stimulated genes (ISGs), but also activates the expression of a set of atypical ISGs and NF-κB target genes, including genes such as Polo like kinase 2 (PLK2), that induce cancer pyroptosis. These findings may support the use of targeting USP18 as a potential cancer immunotherapy.

Immunotherapy, the most promising strategy of cancer treatment, has achieved promising outcomes, but its clinical efficacy in pancreatic cancer is limited mainly due to the complicated tumor immunosuppressive microenvironment. As a highly inflammatory form of immunogenic cell death (ICD), pyroptosis provides a great opportunity to alleviate immunosuppression and promote systemic immune responses in solid tumors. Herein, membrane-targeted photosensitizer TBD-3C with aggregation-induced emission (AIE) feature to trigger pyroptosis-aroused cancer immunotherapy via photodynamic therapy (PDT) is applied. The results reveal that pyroptotic cells induced by TBD-3C could stimulate M1-polarization of macrophages, cause maturation of dendritic cells (DCs), and activation of CD8+ cytotoxic T-lymphocytes (CTLs). Pyroptosis-aroused immunological responses could convert immunosuppressive "cold" tumor microenvironment (TME) to immunogenic "hot" TME, which not only inhibits primary pancreatic cancer growth but also attacks the distant tumor. This work establishes a platform with high biocompatibility for light-controlled antitumor immunity and solid tumor immunotherapy aroused by cell pyroptosis.

Background & aimsAlthough Ikaros (IKZF1) is a well-established transcriptional regulator in leukocyte lymphopoiesis and differentiation, its role in myeloid innate immune responses remains unclear. Sirtuin 1 (SIRT1) is a histone/protein deacetylase involved in cellular senescence, inflammation, and stress resistance. Whether SIRT1 signaling is essential in myeloid cell activation remains uncertain, while the molecular communication between Ikaros and SIRT1, two major transcriptional regulators, has not been studied.MethodsWe undertook molecular and functional studies to interrogate the significance of the myeloid Ikaros-SIRT1 axis in innate immune activation and whether it may serve as a homeostatic sentinel in human liver transplant recipients (hepatic biopsies) and murine models of sterile hepatic inflammation (liver warm ischemia-reperfusion injury in wild-type, myeloid-specific Sirt1-knockout, and CD11b-DTR mice) as well as primary bone marrow-derived macrophage (BMM) cultures (Ikaros silencing vs. overexpression).ResultsIn our clinical study, we identified increased post-reperfusion hepatic Ikaros levels, accompanied by augmented inflammasome signaling yet depressed SIRT1, as a mechanism of hepatocellular damage in liver transplant recipients. In our experimental studies, we identified infiltrating macrophages as the major source of Ikaros in IR-stressed mouse livers. Then, we demonstrated that Ikaros-regulated pyroptosis - induced by canonical inflammasome signaling in BMM cultures - was SIRT1 dependent. Consistent with the latter, myeloid-specific Ikaros signaling augmented hepatic pyroptosis to aggravate pro-inflammatory responses in vivo by negatively regulating SIRT1 in an AMPK-dependent manner. Finally, myeloid-specific SIRT1 was required to suppress pyroptosis, pro-inflammatory phenotype, and ultimately mitigate hepatocellular injury in ischemia-stressed murine livers.ConclusionThese findings identify the Ikaros-SIRT1 axis as a novel mechanistic biomarker of pyroptosis and a putative checkpoint regulator of homeostasis in response to acute hepatic stress/injury in mouse and human livers.Lay summaryThis report describes how crosstalk between Ikaros and SIRT1, two major transcriptional regulators, influence acute hepatic inflammation in murine models of liver ischemia-reperfusion injury and liver transplant recipients. We show that the myeloid Ikaros-SIRT1 axis regulates inflammasome-pyroptotic cell death and hepatocellular damage in stressed livers. Thus, the Ikaros-SIRT1 axis may serve as a novel checkpoint regulator that is required for homeostasis in response to acute liver injury in mice and humans.

BACKGROUND: Hereditary alpha-tryptasemia (HαT) is characterized by elevated basal serum tryptase due to increased copies of the TPSAB1 gene. Individuals with HαT frequently present with multisystem complaints, including anaphylaxis and seemingly functional gastrointestinal (GI) symptoms. OBJECTIVE: We sought to determine the prevalence of HαT in an irritable bowel syndrome cohort and associated immunologic characteristics that may distinguish patients with HαT from patients without HαT. METHODS: Tryptase genotyping by droplet digital PCR, flow cytometry, cytometry by time-of-flight, immunohistochemistry, and other molecular biology techniques was used. RESULTS: HαT prevalence in a large irritable bowel syndrome cohort was 5% (N = 8/158). Immunophenotyping of HαT PBMCs (N ≥ 27) revealed increased total and class-switched memory B cells. In the small bowel, expansion of tissue mast cells with expression of CD203c, HLA-DR, and FcεRI, higher intestinal epithelial cell pyroptosis, and increased class-switched memory B cells were observed. IgG profiles in sera from individuals with HαT (N = 21) significantly differed from those in individuals with quiescent Crohn disease (N = 20) and non-HαT controls (N = 19), with increased antibodies directed against GI-associated proteins identified in individuals with HαT. CONCLUSIONS: Increased mast cell number and intestinal epithelial cell pyroptosis in the small intestine, and class-switched memory B cells in both the gut and peripheral blood associated with IgG reactive to GI-related proteins, distinguish HαT from functional GI disease. These innate and adaptive immunologic findings identified in association with HαT are suggestive of subclinical intestinal inflammation in symptomatic individuals.

Neutrophils are an important source of interleukin (IL)-1β and other cytokines because they are recruited to sites of infection and inflammation in high numbers. Although secretion of processed, bioactive IL-1β by neutrophils is dependent on NLRP3 and Gasdermin D (GSDMD), IL-1α secretion by neutrophils has not been reported. In this study, we demonstrate that neutrophils produce IL-1α following injection of Aspergillus fumigatus spores that express cell-surface β-glucan. Although IL-1α secretion by lipopolysaccharide (LPS)/ATP-activated macrophages and dendritic cells is GSDMD dependent, IL-1α secretion by β-glucan-stimulated neutrophils occurs independently of GSDMD. Instead, we found that bioactive IL-1α is in exosomes that were isolated from cell-free media of β-glucan-stimulated neutrophils. Further, the exosome inhibitor GW4869 significantly reduces IL-1α in extracellular vesicles (EVs) and total cell-free supernatant. Together, these findings identify neutrophils as a source of IL-1α and demonstrate a role for EVs, specifically exosomes, in neutrophil secretion of bioactive IL-1α.

Background & aimsIncreased hepatocyte death contributes to the pathology of acute and chronic liver diseases. However, the role of hepatocyte pyroptosis and extracellular inflammasome release in liver disease is unknown.MethodsWe used primary mouse and human hepatocytes, hepatocyte-specific leucine 351 to proline Nlrp3KICreA mice, and GsdmdKO mice to investigate pyroptotic cell death in hepatocytes and its impact on liver inflammation and damage. Extracellular NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasomes were isolated from mutant NLRP3-YFP HEK cells and internalisation was studied in LX2 and primary human hepatic stellate cells. We also examined a cohort of 154 adult patients with biopsy-proven non-alcoholic fatty liver disease (Sir Charles Gairdner Hospital, Nedlands, Western Australia).ResultsWe demonstrated that primary mouse and human hepatocytes can undergo pyroptosis upon NLRP3 inflammasome activation with subsequent release of NLRP3 inflammasome proteins that amplify and perpetuate inflammasome-driven fibrogenesis. Pyroptosis was inhibited by blocking caspase-1 and gasdermin D activation. The activated form of caspase-1 was detected in the livers and in serum from patients with non-alcoholic steatohepatitis and correlated with disease severity. Nlrp3KICreA mice showed spontaneous liver fibrosis under normal chow diet, and increased sensitivity to liver damage and inflammation after treatment with low dose lipopolysaccharide. Mechanistically, hepatic stellate cells engulfed extracellular NLRP3 inflammasome particles leading to increased IL-1β secretion and α-smooth muscle actin expression. This effect was abrogated when cells were pre-treated with the endocytosis inhibitor cytochalasin B.ConclusionsThese results identify hepatocyte pyroptosis and release of inflammasome components as a novel mechanism to propagate liver injury and liver fibrosis development.Lay summaryOur findings identify a novel mechanism of inflammation in the liver. Experiments in cell cultures, mice, and human samples show that a specific form of cell death, called pyroptosis, leads to the release of complex inflammatory particles, the NLRP3 inflammasome, from inside hepatocytes into the extracellular space. From there they are taken up by other cells and thereby mediate inflammatory and pro-fibrogenic stress signals. The discovery of this mechanism may lead to novel treatments for chronic liver diseases in the future.

Gasdermin-D (GSDMD) in inflammasome-activated macrophages is cleaved by caspase-1 to generate N-GSDMD fragments. N-GSDMD then oligomerizes in the plasma membrane (PM) to form pores that increase membrane permeability, leading to pyroptosis and IL-1β release. In contrast, we report that although N-GSDMD is required for IL-1β secretion in NLRP3-activated human and murine neutrophils, N-GSDMD does not localize to the PM or increase PM permeability or pyroptosis. Instead, biochemical and microscopy studies reveal that N-GSDMD in neutrophils predominantly associates with azurophilic granules and LC3+ autophagosomes. N-GSDMD trafficking to azurophilic granules causes leakage of neutrophil elastase into the cytosol, resulting in secondary cleavage of GSDMD to an alternatively cleaved N-GSDMD product. Genetic analyses using ATG7-deficient cells indicate that neutrophils secrete IL-1β via an autophagy-dependent mechanism. These findings reveal fundamental differences in GSDMD trafficking between neutrophils and macrophages that underlie neutrophil-specific functions during inflammasome activation.

Although pyroptosis is critical for macrophages against pathogen infection, its role and mechanism in cancer cells remains unclear. PD-L1 has been detected in the nucleus, with unknown function. Here we show that PD-L1 switches TNFα-induced apoptosis to pyroptosis in cancer cells, resulting in tumour necrosis. Under hypoxia, p-Stat3 physically interacts with PD-L1 and facilitates its nuclear translocation, enhancing the transcription of the gasdermin C (GSDMC) gene. GSDMC is specifically cleaved by caspase-8 with TNFα treatment, generating a GSDMC N-terminal domain that forms pores on the cell membrane and induces pyroptosis. Nuclear PD-L1, caspase-8 and GSDMC are required for macrophage-derived TNFα-induced tumour necrosis in vivo. Moreover, high expression of GSDMC correlates with poor survival. Antibiotic chemotherapy drugs induce pyroptosis in breast cancer. These findings identify a non-immune checkpoint function of PD-L1 and provide an unexpected concept that GSDMC/caspase-8 mediates a non-canonical pyroptosis pathway in cancer cells, causing tumour necrosis.

Programmed cell death (PCD) in filamentous fungi prevents cytoplasmic mixing following fusion between conspecific genetically distinct individuals (allorecognition) and serves as a defense mechanism against mycoparasitism, genome exploitation, and deleterious cytoplasmic elements (i.e., senescence plasmids). Recently, we identified regulatorof cell death-1 (rcd-1), a gene controlling PCD in germinated asexual spores in the filamentous fungus Neurospora crassarcd-1 alleles are highly polymorphic and fall into two haplogroups in N. crassa populations. Coexpression of alleles from the two haplogroups, rcd-1-1 and rcd-1-2, is necessary and sufficient to trigger a cell death reaction. Here, we investigated the molecular bases of rcd-1-dependent cell death. Based on in silico analyses, we found that RCD-1 is a remote homolog of the N-terminal pore-forming domain of gasdermin, the executioner protein of a highly inflammatory cell death reaction termed pyroptosis, which plays a key role in mammalian innate immunity. We show that RCD-1 localizes to the cell periphery and that cellular localization of RCD-1 was correlated with conserved positively charged residues on predicted amphipathic α-helices, as shown for murine gasdermin-D. Similar to gasdermin, RCD-1 binds acidic phospholipids in vitro, notably, cardiolipin and phosphatidylserine, and interacts with liposomes containing such lipids. The RCD-1 incompatibility system was reconstituted in human 293T cells, where coexpression of incompatible rcd-1-1/rcd-1-2 alleles triggered pyroptotic-like cell death. Oligomers of RCD-1 were associated with the cell death reaction, further supporting the evolutionary relationship between gasdermin and rcd-1 This report documents an ancient transkingdom relationship of cell death execution modules involved in organismal defense.

Cytolethal distending toxins (Cdt) are a family of toxins produced by several human pathogens which infect mucocutaneous tissue and induce inflammatory disease. We have previously demonstrated that the Aggregatibacter actinomycetemcomitans Cdt induces a pro-inflammatory response from human macrophages which involves activation of the NLRP3 inflammasome. We now demonstrate that in addition to activating caspase-1 (canonical inflammasome), Cdt treatment leads to caspase-4 activation and involvement of the noncanonical inflammasome. Cdt-treated cells exhibit pyroptosis characterised by cleavage of gasdermin-D (GSDMD), release of HMGB1 at 24 hr and LDH at 48 hr. Inhibition of either the canonical (caspase-1) or noncanonical (caspase-4) inflammasome blocks both Cdt-induced release of IL-1β and induction of pyroptosis. Analysis of upstream events indicates that Cdt induces Syk phosphorylation (activation); furthermore, blockade of Syk expression and inhibition of pSyk activity inhibit both Cdt-induced cytokine release and pyroptosis. Finally, we demonstrate that increases in pSyk are dependent upon Cdt-induced activation of GSK3β. These studies advance our understanding of Cdt function and provide new insight into the virulence potential of Cdt in mediating the pathogenesis of disease caused by Cdt-producing organisms such as A. actinomycetemcomitans.

The 2019-nCoV is officially called SARS-CoV-2 and the disease is named COVID-19. This viral epidemic in China has led to the deaths of over 1800 people, mostly elderly or those with an underlying chronic disease or immunosuppressed state. This is the third serious Coronavirus outbreak in less than 20 years, following SARS in 2002-2003 and MERS in 2012. While human strains of Coronavirus are associated with about 15% of cases of the common cold, the SARS-CoV-2 may present with varying degrees of severity, from flu-like symptoms to death. It is currently believed that this deadly Coronavirus strain originated from wild animals at the Huanan market in Wuhan, a city in Hubei province. Bats, snakes and pangolins have been cited as potential carriers based on the sequence homology of CoV isolated from these animals and the viral nucleic acids of the virus isolated from SARS-CoV-2 infected patients. Extreme quarantine measures, including sealing off large cities, closing borders and confining people to their homes, were instituted in January 2020 to prevent spread of the virus, but by that time much of the damage had been done, as human-human transmission became evident. While these quarantine measures are necessary and have prevented a historical disaster along the lines of the Spanish flu, earlier recognition and earlier implementation of quarantine measures may have been even more effective. Lessons learned from SARS resulted in faster determination of the nucleic acid sequence and a more robust quarantine strategy. However, it is clear that finding an effective antiviral and developing a vaccine are still significant challenges. The costs of the epidemic are not limited to medical aspects, as the virus has led to significant sociological, psychological and economic effects globally. Unfortunately, emergence of SARS-CoV-2 has led to numerous reports of Asians being subjected to racist behavior and hate crimes across the world.

Human immunodeficiency virus type 2 (HIV-2) infection results in a milder course of disease and slower progression to AIDS than does HIV-1. We hypothesized that this difference may be due to degradation of the sterile alpha motif and HD domain 1 (SAMHD1) host restriction factor by the HIV-2 Vpx gene product, thereby diminishing abortive infection and pyroptotic cell death within bystander CD4 T cells. We have compared CD4 T cell death in tonsil-derived human lymphoid aggregate cultures (HLACs) infected with wild-type HIV-2, HIV-2 ΔVpx, or HIV-1. In contrast to our hypothesis, HIV-2, HIV-2 ΔVpx, and HIV-1 induced similar levels of bystander CD4 T cell death. In all cases, cell death was blocked by AMD3100, a CXCR4 entry inhibitor, but not by raltegravir, an integrase, indicating that only early life cycle events were required. Cell death was also blocked by a caspase-1 inhibitor, a key enzyme promoting pyroptosis, but not by a caspase-3 inhibitor, an important enzyme in apoptosis. HIV-1-induced abortive infection and pyroptotic cell death were also not reduced by forced encapsidation of HIV-2 Vpx into HIV-1 virions. Together, these findings indicate that HIV-2 and HIV-1 support similar levels of CD4 T cell depletion in vitro despite HIV-2 Vpx-mediated degradation of the SAMHD1 transcription factor. The milder disease course observed with HIV-2 infection likely stems from factors other than abortive infection and caspase-1-dependent pyroptosis in bystander CD4 T cells.IMPORTANCE CD4 T cell depletion during HIV-1 infection involves the demise of bystander CD4 T cells due to abortive infection, viral DNA sensing, inflammasome assembly, and death by caspase-1-dependent pyroptosis. HIV-2 infection is associated with milder disease and lower rates of CD4 T cell loss. We hypothesized that HIV-2 infection produces lower levels of pyroptosis due to the action of its Vpx gene product. Vpx degrades the SAMHD1 restriction factor, potentially reducing abortive forms of infection. However, in tonsil cell cultures, HIV-2, HIV-2 ΔVpx, and HIV-1 induced indistinguishable levels of pyroptosis. Forced encapsidation of Vpx into HIV-1 virions also did not reduce pyroptosis. Thus, SAMHD1 does not appear to play a key role in the induction of bystander cell pyroptosis. Additionally, the milder clinical course of HIV-2-induced disease is apparently not explained by a decrease in this inflammatory form of programmed cell death.

Glioblastoma multiforme (GBM; grade IV astrocytoma) is the most prevalent and aggressive form of primary brain cancer. A subpopulation of multipotent cells termed GBM cancer stem cells (CSCs) play a critical role in tumor initiation, tumor maintenance, metastasis, drug resistance, and recurrence following surgery. Here we report the identification of a small molecule, termed RIPGBM, from a cell-based chemical screen that selectively induces apoptosis in multiple primary patient-derived GBM CSC cultures. The cell type-dependent selectivity of this compound appears to arise at least in part from redox-dependent formation of a proapoptotic derivative, termed cRIPGBM, in GBM CSCs. cRIPGBM induces caspase 1-dependent apoptosis by binding to receptor-interacting protein kinase 2 (RIPK2) and acting as a molecular switch, which reduces the formation of a prosurvival RIPK2/TAK1 complex and increases the formation of a proapoptotic RIPK2/caspase 1 complex. In an orthotopic intracranial GBM CSC tumor xenograft mouse model, RIPGBM was found to significantly suppress tumor formation in vivo. Our chemical genetics-based approach has identified a drug candidate and a potential drug target that provide an approach to the development of treatments for this devastating disease.

Sepsis claims an estimated 30 million episodes and 6 million deaths per year, and treatment options are rather limited. Human neutrophil peptides 1-3 (HNP1-3) are the most abundant neutrophil granule proteins but their neutrophil content varies because of unusually extensive gene copy number polymorphism. A genetic association study found that increased copy number of the HNP-encoding gene DEFA1/DEFA3 is a risk factor for organ dysfunction during sepsis development. However, direct experimental evidence demonstrating that these risk alleles are pathogenic for sepsis is lacking because the genes are present only in some primates and humans. Here, we generate DEFA1/DEFA3 transgenic mice with neutrophil-specific expression of the peptides. We show that mice with high copy number of DEFA1/DEFA3 genes have more severe sepsis-related vital organ damage and mortality than mice with low copy number of DEFA1/DEFA3 or wild-type mice, resulting from more severe endothelial barrier dysfunction and endothelial cell pyroptosis after sepsis challenge. Mechanistically, HNP-1 induces endothelial cell pyroptosis via P2X7 receptor-mediating canonical caspase-1 activation in a NLRP3 inflammasome-dependent manner. Based on these findings, we engineered a monoclonal antibody against HNP-1 to block the interaction with P2X7 and found that the blocking antibody protected mice carrying high copy number of DEFA1/DEFA3 from lethal sepsis. We thus demonstrate that DEFA1/DEFA3 copy number variation strongly modulates sepsis development in vivo and explore a paradigm for the precision treatment of sepsis tailored by individual genetic information.

Proper regulation of sterol biosynthesis is critical for eukaryotic cellular homeostasis. Cholesterol and isoprenoids serve key roles in eukaryotic cells by regulating membrane fluidity and correct localization of proteins. It is becoming increasingly appreciated that dysregulated sterol metabolism engages pathways that lead to inflammation. Of particular importance are inflammasomes, which are multiplatform protein complexes that activate caspase-1 in order to process the pro-inflammatory and pyrogenic cytokines IL-1β and IL-18. In this review, we highlight recent research that links altered sterol biosynthetic pathway activity to inflammasome activation. We discuss how clues from human genetics have led to new insights into how alterations in isoprenoid biosynthesis connect to inflammation. We also discuss new mechanisms that show how macrophage cholesterol buildup can lead to inflammasome activation.

Trichomonas vaginalis is a sexually transmitted, eukaryotic parasite that causes trichomoniasis, the most common nonviral, sexually transmitted disease in the USA and worldwide. Little is known about the molecular mechanisms involved in the host immune response to this widespread parasite. Here we report that T. vaginalis induces NLRP3 inflammasome activation in human macrophages, leading to caspase-1 activation and the processing of pro-IL-1β to the mature and bioactive form of the cytokine. Using inhibitor-based approaches, we show that NLRP3 activation by T. vaginalis involves host cell detection of extracellular ATP via P2X7 receptors and potassium efflux. In addition, our data reveal that T. vaginalis inflammasome activation induces macrophage inflammatory cell death by pyroptosis, known to occur via caspase-1 cleavage of the gasdermin D protein, which assembles to form pores in the host cell membrane. We found that T. vaginalis-induced cytolysis of macrophages is attenuated in gasdermin D knockout cells. Lastly, in a murine challenge model, we detected IL-1β production in vaginal fluids in response to T. vaginalis infection in vivo. Together, our findings mechanistically dissect how T. vaginalis contributes to the production of the proinflammatory IL-1β cytokine and uncover pyroptosis as a mechanism by which the parasite can trigger host macrophage cell death.

Natural killer (NK) cells are innate immune lymphocytes that recognize and destroy abnormal host cells, such as tumor cells or those infected by viral pathogens. To safely accomplish these functions, NK cells display activating receptors that detect stress molecules or viral ligands displayed at the cell surface, balanced by inhibitory receptors that bind to self-molecules. To date, such activating and inhibitory receptors on NK cells are not known to recognize bacterial determinants. Moreover, NK cell responses to direct interactions with extracellular bacteria are poorly explored. In this study, we observed the human neonatal pathogen group B Streptococcus (GBS) can directly engage human NK cells. The interaction was mediated through the B6N segment of streptococcal β-protein, binding to the inhibitory receptor Siglec-7 via its amino-terminal V-set domain. Unlike classical Siglec binding, the interaction is also independent of its sialic acid recognition property. In contrast to WT GBS, mutants lacking β-protein induced efficient pyroptosis of NK cells through the NLRP3 inflammasome, with production and secretion of the proinflammatory cytokine IL-1β and dissemination of the cytotoxic molecule granzyme B. We postulate that GBS evolved β-protein engagement of inhibitory human Siglec-7 to suppress the pyroptotic response of NK cells and thereby block recruitment of a broader innate immune response, i.e., by "silencing the sentinel."

Balancing cell death and survival is essential for normal development and homeostasis and for preventing diseases, especially cancer. Conventional cell death pathways include apoptosis, a form of programmed cell death controlled by a well-defined biochemical pathway, and necrosis, the lysis of acutely injured cells. New types of regulated cell death include necroptosis, pyroptosis, ferroptosis, phagoptosis, and entosis. Autophagy can promote survival or can cause death. Newly described processes of anastasis and resuscitation show that, remarkably, cells can recover from the brink of apoptosis or necroptosis. Important new work shows that epithelia achieve homeostasis by extruding excess cells, which then die by anoikis due to loss of survival signals. This mechanically regulated process both maintains barrier function as cells die and matches rates of proliferation and death. In this review, we describe these unconventional ways in which cells have evolved to die or survive, as well as the contributions that these processes make to homeostasis and cancer.

Alcoholic hepatitis (AH) continues to be a disease with high mortality and no efficacious medical treatment. Although severe AH is presented as acute on chronic liver failure, what underlies this transition from chronic alcoholic steatohepatitis (ASH) to AH is largely unknown. To address this question, unbiased RNA sequencing and proteomic analyses were performed on livers of the recently developed AH mouse model, which exhibits the shift to AH from chronic ASH upon weekly alcohol binge, and these results are compared to gene expression profiling data from AH patients. This cross-analysis has identified Casp11 (CASP4 in humans) as a commonly up-regulated gene known to be involved in the noncanonical inflammasome pathway. Immunoblotting confirms CASP11/4 activation in AH mice and patients, but not in chronic ASH mice and healthy human livers. Gasdermin-D (GSDMD), which induces pyroptosis (lytic cell death caused by bacterial infection) downstream of CASP11/4 activation, is also activated in AH livers in mice and patients. CASP11 deficiency reduces GSDMD activation, bacterial load in the liver, and severity of AH in the mouse model. Conversely, the deficiency of interleukin-18, the key antimicrobial cytokine, aggravates hepatic bacterial load, GSDMD activation, and AH. Furthermore, hepatocyte-specific expression of constitutively active GSDMD worsens hepatocellular lytic death and polymorphonuclear leukocyte inflammation.ConclusionThese results implicate pyroptosis induced by the CASP11/4-GSDMD pathway in the pathogenesis of AH. (Hepatology 2018;67:1737-1753).

The liver and the mononuclear phagocyte system are a frequent target for engineered nanomaterials, either as a result of particle uptake and spread from primary exposure sites or systemic administration of therapeutic and imaging nanoparticles. In this study, we performed a comparative analysis of the toxicological impact of 29 metal oxide nanoparticles (NPs), some commonly used in consumer products, in transformed or primary Kupffer cells (KCs) and hepatocytes. We not only observed differences between KCs and hepatocytes, but also differences in the toxicological profiles of transition-metal oxides (TMOs, e. g., Co3O4) versus rare-earth oxide (REO) NPs ( e. g., Gd2O3). While pro-oxidative TMOs induced the activation of caspases 3 and 7, resulting in apoptotic cell death in both cell types, REOs induced lysosomal damage, NLRP3 inflammasome activation, caspase 1 activation, and pyroptosis in KCs. Pyroptosis was accompanied by cell swelling, membrane blebbing, IL-1β release, and increased membrane permeability, which could be reversed by knockdown of the pore forming protein, gasdermin D. Though similar features were not seen in hepatocytes, the investigation of the cytotoxic effects of REO NPs could also be seen to affect macrophage cell lines such as J774A.1 and RAW 264.7 cells as well as bone marrow-derived macrophages. These phagocytic cell types also demonstrated features of pyroptosis and increased IL-1β production. Collectively, these findings demonstrate important mechanistic considerations that can be used for safety evaluation of metal oxides, including commercial products that are developed from these materials.

Group A Streptococcus (GAS) is among the top ten causes of infection-related mortality in humans. M protein is the most abundant GAS surface protein, and M1 serotype GAS strains are associated with invasive infections, including necrotizing fasciitis and toxic shock syndrome. Here, we report that released, soluble M1 protein triggers programmed cell death in macrophages (Mϕ). M1 served as a second signal for caspase-1-dependent NLRP3 inflammasome activation, inducing maturation and release of proinflammatory cytokine interleukin-1β (IL-1β) and macrophage pyroptosis. The structurally dynamic B-repeat domain of M1 was critical for inflammasome activation, which involved K+ efflux and M1 protein internalization by clathrin-mediated endocytosis. Mouse intraperitoneal challenge showed that soluble M1 was sufficient and specific for IL-1β activation, which may represent an early warning to activate host immunity against the pathogen. Conversely, in systemic infection, hyperinflammation associated with M1-mediated pyroptosis and IL-1β release could aggravate tissue injury.

Although the replicative life cycle of HIV within CD4 T cells is understood in molecular detail, less is known about how this human retrovirus promotes the loss of CD4 T lymphocytes. It is this cell death process that drives clinical progression to acquired immune deficiency syndrome (AIDS). Recent studies have highlighted how abortive infection of resting and thus nonpermissive CD4 T cells in lymphoid tissues triggers a lethal innate immune response against the incomplete DNA products generated by inefficient viral reverse transcription in these cells. Sensing of these DNA fragments results in pyroptosis, a highly inflammatory form of programmed cell death, that potentially further perpetuates chronic inflammation and immune activation. As discussed here, these studies cast CD4 T cell death during HIV infection in a different light. Further, they identify drug targets that may be exploited to both block CD4 T cell demise and the chronic inflammatory response generated during pyroptosis.

Progression to AIDS is driven by CD4 T cell depletion, mostly involving pyroptosis elicited by abortive HIV infection of CD4 T cells in lymphoid tissues. Inefficient reverse transcription in these cells leads to cytoplasmic accumulation of viral DNAs that are detected by the DNA sensor IFI16, resulting in inflammasome assembly, caspase-1 activation, and pyroptosis. Unexpectedly, we found that peripheral blood-derived CD4 T cells naturally resist pyroptosis. This resistance is partly due to their deeper resting state, resulting in fewer HIV-1 reverse transcripts and lower IFI16 expression. However, when co-cultured with lymphoid-derived cells, blood-derived CD4 T cells become sensitized to pyroptosis, likely recapitulating interactions occurring within lymphoid tissues. Sensitization correlates with higher levels of activated NF-κB, IFI16 expression, and reverse transcription. Blood-derived lymphocytes purified from co-cultures lose sensitivity to pyroptosis. These differences highlight how the lymphoid tissue microenvironment encountered by trafficking CD4 T lymphocytes dynamically shapes their biological response to HIV.

Muscle inflammation is often associated with its expansion. Bladder smooth muscle inflammation-induced cell death is accompanied by hyperplasia and hypertrophy as the primary cause for poor bladder function. In mice, DNA damage initiated by chemotherapeutic drug cyclophosphamide activated caspase 1 through the formation of the NLRP3 complex resulting in detrusor hyperplasia. A cyclophosphamide metabolite, acrolein, caused global DNA methylation and accumulation of DNA damage in a mouse model of bladder inflammation and in cultured bladder muscle cells. In correlation, global DNA methylation and NLRP3 expression was up-regulated in human chronic bladder inflammatory tissues. The epigenetic silencing of DNA damage repair gene, Ogg1, could be reversed by the use of demethylating agents. In mice, demethylating agents reversed cyclophosphamide-induced bladder inflammation and detrusor expansion. The transgenic knock-out of Ogg1 in as few as 10% of the detrusor cells tripled the proliferation of the remaining wild type counterparts in an in vitro co-culture titration experiment. Antagonizing IL-1β with Anakinra, a rheumatoid arthritis therapeutic, prevented detrusor proliferation in conditioned media experiments as well as in a mouse model of bladder inflammation. Radiation treatment validated the role of DNA damage in the NLRP3-associated caspase 1-mediated IL-1β secretory phenotype. A protein array analysis identified IGF1 to be downstream of IL-1β signaling. IL-1β-induced detrusor proliferation and hypertrophy could be reversed with the use of Anakinra as well as an IGF1 neutralizing antibody. IL-1β antagonists in current clinical practice can exploit the revealed mechanism for DNA damage-mediated muscular expansion.

Inflammasomes are intracellular sensors that couple detection of pathogens and cellular stress to activation of Caspase-1, and consequent IL-1β and IL-18 maturation and pyroptotic cell death. Here, we show that the absent in melanoma 2 (AIM2) and nucleotide-binding oligomerization domain-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasomes trigger Caspase-1-dependent mitochondrial damage. Caspase-1 activates multiple pathways to precipitate mitochondrial disassembly, resulting in mitochondrial reactive oxygen species (ROS) production, dissipation of mitochondrial membrane potential, mitochondrial permeabilization, and fragmentation of the mitochondrial network. Moreover, Caspase-1 inhibits mitophagy to amplify mitochondrial damage, mediated in part by cleavage of the key mitophagy regulator Parkin. In the absence of Parkin activity, increased mitochondrial damage augments pyroptosis, as indicated by enhanced plasma membrane permeabilization and release of danger-associated molecular patterns (DAMPs). Therefore, like other initiator caspases, Caspase-1 activation by inflammasomes results in mitochondrial damage.