Interleukin-1beta (IL-1beta) is an important pro-inflammatory cytokine that is secreted by unconventional means in a caspase-1-dependent manner. Using a one-step immunoprecipitation approach to isolate endogenous caspase-1 from the monocytic THP1 cell line, we identified previously undescribed binding partners using mass spectrometry. One of the proteins identified was Rab39a, a member of the Rab GTPase family, a group of proteins that have important roles in protein trafficking and secretion. We confirmed by co-immunoprecipitation that Rab39a binds caspase-1. Knock down of Rab39a with small interfering RNA resulted in diminished levels of secreted IL-1beta but had no effect on induction of pro-IL-1beta mRNA by lipopolysaccharide. Rab39a contains a highly conserved caspase-1 cleavage site and was cleaved in the presence of recombinant caspase-1 or lipopolysaccharide. Finally, overexpression of Rab39a results in an increase in IL-1beta secretion, and furthermore, overexpression of a Rab39a construct lacking the caspase-1 cleavage site leads to an additional increase in IL-1beta secretion. Altogether, our findings show that Rab39a interacts with caspase-1 and suggest that Rab39a functions as a trafficking adaptor linking caspase-1 to IL-1beta secretion.

Caspase-11, a member of the murine caspase family, has been shown to be an upstream activator of caspase-1 in regulating cytokine maturation. We demonstrate here that in addition to its defect in cytokine maturation, caspase-11-deficient mice have a reduced number of apoptotic cells and a defect in caspase-3 activation after middle cerebral artery occlusion (MCAO), a mouse model of stroke. Recombinant procaspase-11 can autoprocess itself in vitro. Purified active recombinant caspase-11 cleaves and activates procaspase-3 very efficiently. Using a positional scanning combinatorial library method, we found that the optimal cleavage site of caspase-11 was (I/L/V/P)EHD, similar to that of upstream caspases such as caspase-8 and -9. Our results suggest that caspase-11 is a critical initiator caspase responsible for the activation of caspase-3, as well as caspase-1 under certain pathological conditions.

Caspase-1, also known as interleukin-1β (IL-1β)-converting enzyme (ICE), regulates antimicrobial host defense, tissue repair, tumorigenesis, metabolism and membrane biogenesis. On activation within an inflammasome complex, caspase-1 induces pyroptosis and converts pro-IL-1β and pro-IL-18 into their biologically active forms. "ICE-/-" or "Casp1-/-" mice generated using 129 embryonic stem cells carry a 129-associated inactivating passenger mutation on the caspase-11 locus, essentially making them deficient in both caspase-1 and caspase-11. The overlapping and unique functions of caspase-1 and caspase-11 are difficult to unravel without additional genetic tools. Here, we generated caspase-1-deficient mouse (Casp1Null) on the C57BL/6 J background that expressed caspase-11. Casp1Null cells did not release IL-1β and IL-18 in response to NLRC4 activators Salmonella Typhimurium and flagellin, canonical or non-canonical NLRP3 activators LPS and ATP, Escherichia coli, Citrobacter rodentium and transfection of LPS, AIM2 activators Francisella novicida, mouse cytomegalovirus and DNA, and the infectious agents Listeria monocytogenes and Aspergillus fumigatus. We further demonstrated that caspase-1 and caspase-11 differentially contributed to the host defense against A. fumigatus infection and to endotoxemia.

Inflammasomes are cytosolic multiprotein complexes assembled by intracellular nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) and they initiate innate immune responses to invading pathogens and danger signals by activating caspase-1 (ref. 1). Caspase-1 activation leads to the maturation and release of the pro-inflammatory cytokines interleukin (IL)-1β and IL-18, as well as lytic inflammatory cell death known as pyroptosis. Recently, a new non-canonical inflammasome was described that activates caspase-11, a pro-inflammatory caspase required for lipopolysaccharide-induced lethality. This study also highlighted that previously generated caspase-1 knockout mice lack a functional allele of Casp11 (also known as Casp4), making them functionally Casp1 Casp11 double knockouts. Previous studies have shown that these mice are more susceptible to infections with microbial pathogens, including the bacterial pathogen Salmonella enterica serovar Typhimurium (S. typhimurium), but the individual contributions of caspase-1 and caspase-11 to this phenotype are not known. Here we show that non-canonical caspase-11 activation contributes to macrophage death during S. typhimurium infection. Toll-like receptor 4 (TLR4)-dependent and TIR-domain-containing adaptor-inducing interferon-β (TRIF)-dependent interferon-β production is crucial for caspase-11 activation in macrophages, but is only partially required for pro-caspase-11 expression, consistent with the existence of an interferon-inducible activator of caspase-11. Furthermore, Casp1(-/-) mice were significantly more susceptible to infection with S. typhimurium than mice lacking both pro-inflammatory caspases (Casp1(-/-) Casp11(-/-)). This phenotype was accompanied by higher bacterial counts, the formation of extracellular bacterial microcolonies in the infected tissue and a defect in neutrophil-mediated clearance. These results indicate that caspase-11-dependent cell death is detrimental to the host in the absence of caspase-1-mediated innate immunity, resulting in extracellular replication of a facultative intracellular bacterial pathogen.

Caspases are cysteine proteases with essential functions in the apoptotic pathway; their proteolytic activity toward various substrates is associated with the morphological changes of cells. Recent reports have described non-apoptotic functions of caspases, including autophagy. In this report, we searched for novel modifiers of the phenotype of Dcp-1 gain-of-function (GF) animals by screening promoter element- inserted Drosophila melanogaster lines (EP lines).

Alpha 1-antitrypsin (A1AT) is a 52 kDa serine protease inhibitor produced largely by hepatocytes but also by mononuclear phagocytes. A1AT chiefly inhibits neutrophil elastase and proteinase-3 but has also been reported to have immune modulatory functions including the ability to inhibit caspases. Its clinical availability for infusion suggests that A1AT therapy might modulate caspase related inflammation. Here we tested the ability of A1AT to modulate caspase-1 function in human mononuclear phagocytes.

The study has investigated the effect of isoflavone attenuates the caspase-1 and caspase-3 level in cell model of Parkinsonism. The subjects were PC12 cells. They were randomly divided into six groups: control, MPP(+) (250 μmol/L), isoflavone (10 μM), isoflavone (10 μM) + MPP(+) (250 μmol/L), Z-YVAD-CHO (10 nM) + MPP(+) group, and Z-DEVD-CHO (10 nM) + MPP(+) group. Cell viability was measured by MTT methods; the content of tyrosine hydroxylase was measured by immunocytochemistry method of avidinbiotin peroxidase complex; apoptosis ratio was measured by flow cytometry. The results showed that cell viability in the MPP(+) group was lower than in all other five groups. There was no difference in cell viability between isoflavone + MPP(+) and control group. Optical density of TH positive cells in isoflavone group was higher than in control, isoflavone + MPP(+), and MPP(+) only groups. The apoptosis ratio in the isoflavone + MPP(+) group and control group and the Z-YVAD-CHO + MPP(+) and Z-DEVD-CHO + MPP(+) group was similar, which was lower than in the MPP(+) group. The lowest apoptosis ratio was found in the isoflavone only group.

We investigated the role of the inflammasome effector caspases-1 and -11 during Salmonella enterica serovar Typhimurium infection of murine intestinal epithelial cells (IECs). Salmonella burdens were significantly greater in the intestines of caspase-1/11 deficient (Casp1/11-/-), Casp1-/- and Casp11-/- mice, as compared to wildtype mice. To determine if this reflected IEC-intrinsic inflammasomes, enteroid monolayers were derived and infected with Salmonella. Casp11-/- and wildtype monolayers responded similarly, whereas Casp1-/- and Casp1/11-/- monolayers carried significantly increased intracellular burdens, concomitant with marked decreases in IEC shedding and death. Pretreatment with IFN-γ to mimic inflammation increased caspase-11 levels and IEC death, and reduced Salmonella burdens in Casp1-/- monolayers, while high intracellular burdens and limited cell shedding persisted in Casp1/11-/- monolayers. Thus caspase-1 regulates inflammasome responses in IECs at baseline, while proinflammatory activation of IECs reveals a compensatory role for caspase-11. These results demonstrate the importance of IEC-intrinsic canonical and non-canonical inflammasomes in host defense against Salmonella.

Sensing bacterial products in the cytosol of mammalian cells by NOD-like receptors leads to the activation of caspase-1 inflammasomes, and the production of the pro-inflammatory cytokines interleukin (IL)-18 and IL-1β. In addition, mouse caspase-11 (represented in humans by its orthologs, caspase-4 and caspase-5) detects cytosolic bacterial LPS directly. Activation of caspase-1 and caspase-11 initiates pyroptotic host cell death that releases potentially harmful bacteria from the nutrient-rich host cell cytosol into the extracellular environment. Here we use single cell analysis and time-lapse microscopy to identify a subpopulation of host cells, in which growth of cytosolic Salmonella Typhimurium is inhibited independently or prior to the onset of cell death. The enzymatic activities of caspase-1 and caspase-11 are required for growth inhibition in different cell types. Our results reveal that these proteases have important functions beyond the direct induction of pyroptosis and proinflammatory cytokine secretion in the control of growth and elimination of cytosolic bacteria.

Millions of patients suffer from silicosis, but it remains an uncurable disease due to its unclear pathogenic mechanisms. Though the Nlrp3 inflammasome is involved in silicosis pathogenesis, inhibition of its classic downstream factors, Caspase-1 and Gsdmd, fails to block pyroptosis and cytokine release. To clarify the molecular mechanism of silicosis pathogenesis for new therapy, we examined samples from silicosis patients and genetic mouse models. We discovered an alternative pyroptotic pathway which requires cleavage of Gsdme by Caspases-3/8 in addition to Caspase-1/Gsdmd. Consistently, Gsdmd-/-Gsdme-/- mice showed markedly attenuated silicosis pathology, and Gsdmd-/-Gsdme-/- macrophages were resistant to silica-induced pyroptosis. Furthermore, we found that in addition to Caspase 1, Caspase-8 cleaved IL-1β in silicosis, explaining why Caspase-1-/- mice also suffered from silicosis. Finally, we found that inhibitors of Caspase-1, -3, -8 or an FDA approved drug, dimethyl fumarate, could dramatically alleviate silicosis pathology through blocking cleavage of Gsdmd and Gsdme. This study highlights that Caspase-1/Gsdmd and Caspase-3/8/Gsdme-dependent pyroptosis is essential for the development of silicosis, implicating new potential targets and drug for silicosis treatment.

Barrett's esophagus (BE) is an inflammatory condition and a neoplastic precursor to esophageal adenocarcinoma (EAC). Inflammasome signaling, which contributes to acute and chronic inflammation, results in caspase-1 activation leading to the secretion of IL-1β and IL-18, and inflammatory cell death (pyroptosis). This study aimed to characterize caspase-1 expression, and its functional importance, during disease progression to BE and EAC. Three models of disease progression (Normal-BE-EAC) were employed to profile caspase-1 expression: (1) a human esophageal cell line model; (2) a murine model of BE; and (3) resected tissue from BE-associated EAC patients. BE patient biopsies and murine BE organoids were cultured ex vivo in the presence of a caspase-1 inhibitor, to determine the importance of caspase-1 for inflammatory cytokine and chemokine secretion.Epithelial caspase-1 expression levels were significantly enhanced in BE (p < 0.01). In contrast, stromal caspase-1 levels correlated with histological inflammation scores during disease progression (p < 0.05). Elevated secretion of IL-1β from BE explanted tissue, compared to adjacent normal tissue (p < 0.01), confirmed enhanced activity of caspase-1 in BE tissue. Caspase-1 inhibition in LPS-stimulated murine BE organoids caused a significant reduction in IL-1β (p < 0.01) and CXCL1 (p < 0.05) secretion, confirming the importance of caspase-1 in the production of cytokines and chemokines associated with disease progression from BE to EAC. Targeting caspase-1 activity in BE patients should therefore be tested as a novel strategy to prevent inflammatory complications associated with disease progression.

Gasdermins oligomerize to form pores in the cell membrane, causing regulated lytic cell death called pyroptosis. Mammals encode five gasdermins that can trigger pyroptosis: GSDMA, B, C, D, and E. Caspase and granzyme proteases cleave the linker regions of and activate GSDMB, C, D, and E, but no endogenous activation pathways are yet known for GSDMA. Here, we perform a comprehensive evolutionary analysis of the gasdermin family. A gene duplication of GSDMA in the common ancestor of caecilian amphibians, reptiles and birds gave rise to GSDMA-D in mammals. Uniquely in our tree, amphibian, reptile and bird GSDMA group in a separate clade than mammal GSDMA. Remarkably, GSDMA in numerous bird species contain caspase-1 cleavage sites like YVAD or FASD in the linker. We show that GSDMA from birds, amphibians, and reptiles are all cleaved by caspase-1. Thus, GSDMA was originally cleaved by the host-encoded protease caspase-1. In mammals the caspase-1 cleavage site in GSDMA is disrupted; instead, a new protein, GSDMD, is the target of caspase-1. Mammal caspase-1 uses exosite interactions with the GSDMD C-terminal domain to confer the specificity of this interaction, whereas we show that bird caspase-1 uses a stereotypical tetrapeptide sequence to confer specificity for bird GSDMA. Our results reveal an evolutionarily stable association between caspase-1 and the gasdermin family, albeit a shifting one. Caspase-1 repeatedly changes its target gasdermin over evolutionary time at speciation junctures, initially cleaving GSDME in fish, then GSDMA in amphibians/reptiles/birds, and finally GSDMD in mammals.

Gasdermins oligomerize to form pores in the cell membrane, causing regulated lytic cell death called pyroptosis. Mammals encode five gasdermins that can trigger pyroptosis: GSDMA, B, C, D, and E. Caspase and granzyme proteases cleave the linker regions of and activate GSDMB, C, D, and E, but no endogenous activation pathways are yet known for GSDMA. Here, we perform a comprehensive evolutionary analysis of the gasdermin family. A gene duplication of GSDMA in the common ancestor of caecilian amphibians, reptiles, and birds gave rise to GSDMA-D in mammals. Uniquely in our tree, amphibian, reptile, and bird GSDMA group in a separate clade than mammal GSDMA. Remarkably, GSDMA in numerous bird species contain caspase-1 cleavage sites like YVAD or FASD in the linker. We show that GSDMA from birds, amphibians, and reptiles are all cleaved by caspase-1. Thus, GSDMA was originally cleaved by the host-encoded protease caspase-1. In mammals the caspase-1 cleavage site in GSDMA is disrupted; instead, a new protein, GSDMD, is the target of caspase-1. Mammal caspase-1 uses exosite interactions with the GSDMD C-terminal domain to confer the specificity of this interaction, whereas we show that bird caspase-1 uses a stereotypical tetrapeptide sequence to confer specificity for bird GSDMA. Our results reveal an evolutionarily stable association between caspase-1 and the gasdermin family, albeit a shifting one. Caspase-1 repeatedly changes its target gasdermin over evolutionary time at speciation junctures, initially cleaving GSDME in fish, then GSDMA in amphibians/reptiles/birds, and finally GSDMD in mammals.

Innate immune cells sense and respond to the cytoplasmic infection of bacterial pathogens through NLRP3, NLRC4 or AIM2 inflammasome depending on the unique molecular pattern of invading pathogens. The infection of flagellin- or type III secretion system (T3SS)-containing Gram-negative bacteria such as Salmonella enterica serovar Typhimurium (S. typhimurium) or Pseudomonas aeruginosa (P. aeruginosa) triggers NLRC4-dependent caspase-1 activation leading to the secretion of proinflammatory cytokines such as interleukin-1-beta (IL-1β) and IL-18. Previous studies have shown that apoptosis-associated speck-like protein containing a CARD (ASC) is also required for Salmonella-induced caspase-1 activation, but it is still unclear how ASC contributes to the activation of NLRC4 inflammasome in response to S. typhimurium infection. In this study, we demonstrate that S. typhimurium triggers the formation of ASC oligomer in a potassium depletion-independent manner as determined by in vitro crosslinking and in situ fluorescence imaging. Remarkably, inhibition of potassium efflux failed to block Salmonella-promoted caspase-1 activation and macrophage cell death. These results collectively suggest that ASC is substantially oligomerized to facilitate the activation of caspase-1 in response to S. typhimurium infection. Contrary to NLRP3 inflammasome, intracellular potassium depletion is not critical for NLRC4 inflammasome signaling by S. typhimurium.

HIV infection remains incurable to date and there are no compounds targeted at the viral release. We show here HIV viral release is not spontaneous, rather requires caspases activation and shedding of its adhesion receptor, CD62L. Blocking the caspases activation caused virion tethering by CD62L and the release of deficient viruses. Not only productive experimental HIV infections require caspases activation for viral release, HIV release from both viremic and aviremic patient-derived CD4 T cells also require caspase activation, suggesting HIV release from cellular viral reservoirs depends on apoptotic shedding of the adhesion receptor. Further transcriptomic analysis of HIV infected CD4 T cells showed a direct contribution of HIV accessory gene Nef to apoptotic caspases activation. Current HIV cure focuses on the elimination of latent cellular HIV reservoirs that are resistant to infection-induced cell death. This has led to therapeutic strategies to stimulate T cell apoptosis in a "kick and kill" approach. Our current work has shifted the paradigm on HIV-induced apoptosis and suggests such approach would risk to induce HIV release and thus be counter-productive. Instead, our study supports targeting of viral reservoir release by inhibiting of caspases activation.

The role of receptors for endogenous metabolic danger signals-associated molecular patterns has been characterized recently as bridging innate immune sensory systems for danger signals-associated molecular patterns to initiation of inflammation in bone marrow-derived cells, such as macrophages. However, it remains unknown whether endothelial cells (ECs), the cell type with the largest numbers and the first vessel cell type exposed to circulating danger signals-associated molecular patterns in the blood, can sense hyperlipidemia. This report determined whether caspase-1 plays a role in ECs in sensing hyperlipidemia and promoting EC activation.

Mononuclear phagocytes utilize caspase-1 activation as a means to respond to danger signals. Although caspase-1 was discovered using highly concentrated cell extracts that spontaneously activate caspase-1, it is now clear that in live cell models caspase-1 activation occurs in the process of its cellular release and is not an intracellular event. Therefore, we compared the characteristics of caspase-1 activation in the cell lysate model to that of caspase-1 that is released in response to exogenous inflammasome activation. Whereas both models generated active caspase-1, the cell-lysate induced caspase-1 required highly concentrated cell lysates and had a short half-life (~15 min) whereas, the activation induced released caspase-1 required 2-3 log fold fewer cells and was stable for greater than 12 h. Both forms were able to cleave proIL-1beta but unexpectedly, the released activity was unable to be immunodepleted by caspase-1 antibodies. Size exclusion chromatography identified two antigenic forms of p20 caspase-1 in the activation induced released caspase-1: one at the predicted size of tetrameric, p20/p10 caspase-1 and the other at >200 kDa. However, only the high molecular weight form had stable functional activity. These results suggest that released caspase-1 exists in a unique complex that is functionally stable and protected from immunodepletion whereas cell-extract generated active caspase-1 is rapidly inhibited in the cytosolic milieu.

Pyroptosis is a mechanism of inflammatory cell death mediated by the activation of the prolytic protein gasdermin D by caspase-1, caspase-4, and caspase-5 in human, and caspase-1 and caspase-11 in mouse. In addition, caspase-1 amplifies inflammation by proteolytic activation of cytokine interleukin-1β (IL-1β). Modern mammals of the order Carnivora lack the caspase-1 catalytic domain but express an unusual version of caspase-4 that can activate both gasdermin D and IL-1β. Seeking to understand the evolutionary origin of this caspase, we utilized the large amount of data available in public databases to perform ancestral sequence reconstruction of an inflammatory caspase of a Carnivora ancestor. We expressed the catalytic domain of this putative ancestor in Escherichia coli, purified it, and compared its substrate specificity on synthetic and protein substrates to extant caspases. We demonstrated that it activates gasdermin D but has reduced ability to activate IL-1β. Our reconstruction suggests that caspase-1 was lost in a Carnivora ancestor, perhaps upon a selective pressure for which the generation of biologically active IL-1β by caspase-1 was detrimental. We speculate that later, a Carnivora encountered selective pressures that required the production of IL-1β, and caspase-4 subsequently gained this activity. This hypothesis would explain why extant Carnivora possess an inflammatory caspase with caspase-1 catalytic function placed on a caspase-4 scaffold.

During herpes simplex virus 1 (HSV-1) infection, apoptosis is initiated by immediate early gene transcription and is later modulated by proteins synthesized in infected cells. We have previously shown that procaspase 3 levels are reduced during HSV-1 replication. We now demonstrate that a replication-defective HSV-1 recombinant virus which is incapable of packaging viral DNA into capsids activated caspase 3 but retained the ability to prevent the apoptotic process from killing the infected cells. This implies that HSV-1-dependent apoptosis is not merely a response to abortive infection. Maximum accumulation of the active form of caspase 3 accompanied complete HSV-1-dependent apoptosis. Additionally, caspase 7 was found to be activated during HSV-1-dependent apoptosis. Infected MCF-7 cells which ectopically express caspase 3 underwent more efficient apoptosis than their caspase 3-null parental counterparts, confirming that caspase 3 contributes to HSV-1-dependent apoptosis. However, caspase 3 reconstitution did not make the MCF-7 cells as sensitive as HEp-2 cells to HSV-1-dependent apoptosis, suggesting that other cellular factors may be involved in conferring resistance to this process. These results indicate that caspase 3 activation is a consequence of HSV-1 infection and have important implications in our understanding of the interactions of the virus with host cells.

Legionella pneumophila is a Gram-negative, flagellated bacterium that survives in phagocytes and causes Legionnaires' disease. Upon infection of mammalian macrophages, cytosolic flagellin triggers the activation of Naip/NLRC4 inflammasome, which culminates in pyroptosis and restriction of bacterial replication. Although NLRC4 and caspase-1 participate in the same inflammasome, Nlrc4-/- mice and their macrophages are more permissive to L. pneumophila replication compared with Casp1/11-/-. This feature supports the existence of a pathway that is NLRC4-dependent and caspase-1/11-independent. Here, we demonstrate that caspase-8 is recruited to the Naip5/NLRC4/ASC inflammasome in response to flagellin-positive bacteria. Accordingly, caspase-8 is activated in Casp1/11-/- macrophages in a process dependent on flagellin, Naip5, NLRC4 and ASC. Silencing caspase-8 in Casp1/11-/- cells culminated in macrophages that were as susceptible as Nlrc4-/- for the restriction of L. pneumophila replication. Accordingly, macrophages and mice deficient in Asc/Casp1/11-/- were more susceptible than Casp1/11-/- and as susceptible as Nlrc4-/- for the restriction of infection. Mechanistically, we found that caspase-8 activation triggers gasdermin-D-independent pore formation and cell death. Interestingly, caspase-8 is recruited to the Naip5/NLRC4/ASC inflammasome in wild-type macrophages, but it is only activated when caspase-1 or gasdermin-D is inhibited. Our data suggest that caspase-8 activation in the Naip5/NLRC4/ASC inflammasome enable induction of cell death when caspase-1 or gasdermin-D is suppressed.