Gasdermin D Deficiency Research Articles

Gasdermin D-mediated pyroptosis suppresses liver regeneration after 70% partial hepatectomy.

Pyroptosis is a kind of programmed cell death primarily mediated by gasdermin D (GSDMD) and shown to regulate multiple diseases. However, its contribution to liver regeneration, a fine‐tuned tissue repair process mediated primarily by hepatocytes after mass loss, remains unclear. Herein, we found that caspase‐11/GSDMD‐mediated pyroptosis was activated in regenerating liver after 70% partial hepatectomy. Impeding pyroptosis by deleting GSDMD significantly reduced liver injury and accelerated liver regeneration. Mechanistically, GSDMD deficiency up‐regulates the activation of hepatocyte growth factor/c‐Met and epidermal growth factor receptor mitogenic pathways at the initiation phase. Moreover, activin A and glypican 3 (GPC3), two terminators of liver regeneration, were inhibited when GSDMD was absent. In vitro study suggested the expressions of activin A and GPC3 were induced by interleukin (IL)–1β and IL‐18, whose maturations were regulated by GSDMD‐mediated pyroptosis. Similarly, pharmacologically inhibiting GSDMD recapitulates these phenomena. Conclusion: This study characterizes the role of GSDMD‐mediated pyroptosis in liver regeneration and lays the foundation for enhancing liver restoration by targeting GSDMD in liver patients with impaired regenerative capacity.

GSDMD deficiency drives immunopathology in cutaneous leishmaniasis

Abstract Cutaneous leishmaniasis is a neglected tropical disease that results in severe, chronic lesions that are frequently resistant to parasite-directed therapy. Studies in patients and murine models indicate that NLRP-3 dependent IL-1β release is associated with more severe disease and treatment failure. One mechanism leading to IL-1β release as well as pyroptosis is the formation of pores by gasdermin D (GSDMD). To determine if GSDMD might contribute to IL-1β release and subsequent increased disease, we infected GSDMD knockout mice and wild-type C57BL/6 (WT) mice with Leishmania major and followed the course of infection. Contrary to our expectations, GSDMD KO mice infected mice developed more severe lesions compared with WT mice, but without a change in parasite burden. Because inflammasome activation without pyroptosis has been reported to lead to prolonged IL-1β release from hyperactivated dendritic cells, we tested whether blocking IL-1β signaling would prevent severe pathology in GSDMD KO mice. However, GSDMD KO mice treated with anti-IL-1β developed pathology similar to the untreated GSDMD KO mice, indicating that IL-1β was not mediating the increased pathology. Therefore, we next compared the immune responses in WT and GSDMD KO mice. The only significant difference found was an increase in the production of IFN-g, and our working hypothesis is that excess IFN-g may contribute to the disease severity. Why the absence of GSDMD leads to increased disease remains unclear, but previously it was found that the loss of GSDMD can have consequences beyond IL-1β release and pyroptosis. Current studies are focused on defining the mechanism(s) leading to increased disease in GSDMD KO mice. Supported by grants from NIH (5R01AI125265-05, 1R01AI150606-01, T32AR7465).

Pyroptosis executor gasdermin D plays a key role in scleroderma and bleomycin-induced skin fibrosis

The NLRP3 inflammasome and IL-1β are essential for scleroderma pathogenesis. Nevertheless, the role of pyroptosis executor gasdermin D(GSDMD), which is a downstream molecule of NLRP3 and is required for IL-1β release in some situations, has not yet been well elucidated in scleroderma. Here, we found that GSDMD was significantly up-regulated and activated in the skin of scleroderma patients and bleomycin-induced mouse model. What’s more, the ablation of GSDMD ameliorates bleomycin-induced skin fibrosis according to HE staining, Masson staining and the detection of hydroxyproline contents. GSDMD deficiency also impaired macrophages infiltration and reduced inflammation response. Furthermore, the loss of GSDMD reduced Th17 differentiation in vivo and in vitro. Collectively, these findings provide the first demonstration that GSDMD related pyroptosis plays an important role in scleroderma pathogenesis.

GSDMD-Mediated Cardiomyocyte Pyroptosis Promotes Myocardial I/R Injury

Rationale: Pyroptosis is a morphologically and mechanistically distinct form of cell death and is characterized by gasdermin D (GSDMD) or gasdermin E (GSDME)-mediated necrosis with excessive inflammatory factor release. Cardiomyocyte necrosis and inflammation play key roles in the pathophysiology of myocardial ischemia/reperfusion (I/R) injury. However, whether cardiomyocytes undergo pyroptosis and the underlying mechanism in myocardial I/R injury remain unclear. Objective: We aimed to investigate the role of pyroptosis in myocardial I/R injury. Methods and Results: In vivo and in vitro experiments were used to investigate pyroptosis of cardiomyocyte and the associated mechanisms during I/R injury. Wild-type (WT), Myh6-Cre and cardiomyocyte-specific GSDMD-deficient (GSDMD-CKO) male mice were subjected to I/R. Human peripheral blood samples were collected from STEMI (acute ST-segment-elevation myocardial infarction) patients or control patients at 0, 1 and 24 h after PCI (percutaneous coronary intervention) in our department. The serum levels of GSDMD were measured by ELISA. H/R (hypoxia/reoxygenation) induced cardiomyocyte pyroptosis and the release of mature IL-18 but not IL-1β, which mechanistically resulted from GSDMD cleavage by caspase-11 in cardiomyocytes. Furthermore, GSDMD gene deletion blocked H/R-induced cardiomyocyte pyroptosis and IL-18 release. GSDMD and its pyroptosis-inducing N-terminal fragment (GSDMD-N) were upregulated in myocardial tissues after I/R injury. Immunofluorescence analysis showed that GSDMD was mainly localized in cardiomyocytes. GSDMD deficiency in cardiomyocytes significantly reduced the I/R-induced myocardial infarct size. Moreover, increased GSDMD serum levels were detected in patients exhibiting I/R injury 1 h after PCI for STEMI. Conclusions: Our results show that GSDMD-mediated cardiomyocyte pyroptosis is a key event during myocardial I/R injury and that the caspase-11/GSDMD pathway may be essential to this process. Additionally, GSDMD inhibition significantly reduces cardiomyocyte pyroptosis and I/R-induced myocardial injury.

GSDMD contributes to host defence against Staphylococcus aureus skin infection by suppressing the Cxcl1\u2013Cxcr2 axis

Gasdermin D (GSDMD), a member of the gasdermin protein family, is a caspase substrate, and its cleavage is required for pyroptosis and IL-1β secretion. To date, the role and regulatory mechanism of GSDMD during cutaneous microbial infection remain unclear. Here, we showed that GSDMD protected against Staphylococcus aureus skin infection by suppressing Cxcl1–Cxcr2 signalling. GSDMD deficiency resulted in larger abscesses, more bacterial colonization, exacerbated skin damage, and increased inflammatory cell infiltration. Although GSDMD deficiency resulted in defective IL-1β production, the critical role of IL-1β was counteracted by the fact that Caspase-1/11 deficiency also resulted in less IL-1β production but did not aggravate disease severity during S. aureus skin infection. Interestingly, GSDMD-deficient mice had increased Cxcl1 secretion accompanied by increased recruitment of neutrophils, whereas Caspase-1/11-deficient mice presented similar levels of Cxcl1 and neutrophils as wild-type mice. Moreover, the absence of GSDMD promoted Cxcl1 secretion in bone marrow-derived macrophages induced by live, dead, or different strains of S. aureus. Corresponding to higher transcription and secretion of Cxcl1, enhanced NF-κB activation was shown in vitro and in vivo in the absence of GSDMD. Importantly, inhibiting the Cxcl1–Cxcr2 axis with a Cxcr2 inhibitor or anti-Cxcl1 blocking antibody rescued host defence defects in the GSDMD-deficient mice. Hence, these results revealed an important role of GSDMD in suppressing the Cxcl1–Cxcr2 axis to facilitate pathogen control and prevent tissue damage during cutaneous S. aureus infection.

Host Gasdermin D restrains systemic endotoxemia by capturing Proteobacteria in the colon of high-fat diet-feeding mice

ABSTRACT Gasdermin D (GSDMD) functions as a key pyroptotic executor through its secreted N-terminal domain (GSDMD-N). However, the functional relevance and mechanistic basis of the precise roles of host colonic GSDMD in high-fat diet (HFD)-induced gut dysbiosis and systemic endotoxemia remain elusive. In this study, we demonstrate that HFD feeding triggers GSDMD-N secretion of both T-lymphocytes and enterocytes in mouse colons. GSDMD deficiency aggravates HFD-induced systemic endotoxemia, gut barrier impairment, and colonic inflammation. More importantly, active GSDMD-N kills the Proteobacteria phylum via directly interacting with Cardiolipin. Mechanistically, we identify that the Glu236 (a known residue for GSDMD protein cleavage) is a bona fide important site for the bacterial recognition of GSDMD. Collectively, our findings explain the mechanism by which colonic GSDMD-N maintains low levels of HFD-induced metabolic endotoxemia. A GSDMD-N mimetic containing an exposed Glu236 site could be an attractive strategy for the treatment of HFD-induced metabolic endotoxemia.

Pyroptosis executioner gasdermin D contributes to host defense and promotes Th 1 immune response during Neospora caninum infection

Neospora caninum (N. caninum) is an intracellular parasite and is the causative agent of neosporosis, which leads to reproductive failure in cattle. Pyroptosis is a recently discovered form of programmed cell death executed by gasdermin D (GSDMD). This cell death mechanism is an important host defense against intracellular pathogens. However, pyroptosis induced by N. caninum is poorly understood. The aim of this study was to explore the roles of GSDMD-mediated pyroptosis during N. caninum infection in vivo. N. caninum-infected wild type mice and GSDMD-deficient mice were used to evaluate host resistance and its ability to affect immune response against this parasite. The results showed that GSDMD deficiency significantly reduced survival and impaired the host’s abilities to clear parasite loads in tissues, monocytes/macrophages and neutrophils. Additionally, GSDMD was essential for circulating IL-18 and IFN-γ production induced by N. caninum infection, indicating that GSDMD can mediate the Th 1 immune response against N. caninum infection. Additional data revealed that treatment with exogenous recombinant IL-18 in N. caninum-infected Gsdmd−/− mice rescues the reduction of circulating IFN-γ production to help eliminate the parasite. Taken together, our data indicate that GSDMD-mediated pyroptosis plays a vital role in maintaining host resistance to N. caninum and is essential for clearing the parasite. This form of programmed cell death promotes the Th 1 immune response by controlling IL-18 release and is considered a host defense against N. caninum. This study expands our understanding of interactions between host immune response/defense and N. caninum infection.

Gasdermin D-independent release of interleukin-1β by living macrophages in response to mycoplasmal lipoproteins and lipopeptides.

Interleukin-1β (IL-1β) plays pivotal roles in controlling bacterial infections and is produced after the processing of pro-IL-1β by caspase-1, which is activated by the inflammasome. In addition, caspase-1 cleaves the cytosolic protein, gasdermin-D (GSDMD), whose N-terminal fragment subsequently forms a pore in the plasma membrane, leading to the pyroptic cell-death-mediated release of IL-1β. Living cells can also release IL-1β via GSDMD pores or other unconventional secretory pathways. However, the precise mechanisms are poorly defined. Here, we show that lipoproteins from Mycoplasma salivarium (MsLP) and Mycoplasma pneumoniae (MpLP) and an M.salivarium-derived lipopeptide (FSL-1), which are activators of the nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome, induce IL-1β release from mouse bone-marrow-derived macrophages (BMMs) without inducing cell death. The levels of IL-1β release induced by MsLP, MpLP and FSL-1 were more than 100 times lower than those induced by the canonical NLRP3 activator nigericin. The IL-1β release-inducing activities of MsLP, MpLP and FSL-1 were not attenuated in BMMs from GSDMD-deficient mice. Furthermore, both active caspase-1 and cleaved GSDMD were detected in response to transfection of FSL-1 into the cytosol of BMMs, but the release of IL-1β was unaffected by GSDMD deficiency. Meanwhile, punicalagin, a membrane-stabilizing agent, drastically down-regulated the release of IL-1β in response to FSL-1. These results suggest that mycoplasmal lipoprotein/lipopeptide-induced IL-1β release by living macrophages is not mediated via GSDMD but rather through changes in membrane permeability.

Epithelial-derived gasdermin D mediates nonlytic IL-1β release during experimental colitis.

Gasdermin D (GSDMD) induces pyroptosis via the pore-forming activity of its N-terminal domain, cleaved by activated caspases associated with the release of IL-1β. Here, we report a nonpyroptotic role of full-length GSDMD in guiding the release of IL-1β-containing small extracellular vesicles (sEVs) from intestinal epithelial cells (IECs). In response to caspase-8 inflammasome activation, GSDMD, chaperoned by Cdc37/Hsp90, recruits the E3 ligase, NEDD4, to catalyze polyubiquitination of pro-IL-1β, serving as a signal for cargo loading into secretory vesicles. GSDMD and IL-1β colocalize with the exosome markers CD63 and ALIX intracellularly, and GSDMD and NEDD4 are required for release of CD63+ sEVs containing IL-1β, GSDMD, NEDD4, and caspase-8. Importantly, increased expression of epithelial-derived GSDMD is observed both in patients with inflammatory bowel disease (IBD) and those with experimental colitis. While GSDMD-dependent release of IL-1β-containing sEVs is detected in cultured colonic explants from colitic mice, GSDMD deficiency substantially attenuates disease severity, implicating GSDMD-mediated release of IL-1β sEVs in the pathogenesis of intestinal inflammation, such as that observed in IBD.

Gasdermin D in macrophages restrains colitis by controlling cGAS-mediated inflammation.

The functional relevance and mechanistic basis of the effects of the pyroptosis executioner Gasdermin D (GSDMD) on colitis remain unclear. In this study, we observed that GSDMD protein was activated during intestinal inflammation in a model of chemically induced colitis. GSDMD deficiency exacerbated experimental colitis independent of changes in the microbiota and without affecting the production of antimicrobial peptides. GSDMD deficiency in macrophages, but not epithelial cells, was sufficient to drive this exacerbated experimental colitis. We further demonstrate that GSDMD functions in macrophages as a negative regulator to control cyclic GMP-AMP synthase (cGAS)-dependent inflammation, thereby protecting against colitis. Moreover, the administration of cGAS inhibitor can rescue the colitogenic phenotype in GSDMD-deficient mice. Collectively, these findings provide the first demonstration of GSDMD's role in controlling colitis and a detailed delineation of the underlying mechanism.

Inflammasome Activation Promotes Deep Vein Thrombosis through Pyroptosis

Venous thromboembolism (VTE), including pulmonary embolism (PE) and deep vein thrombosis (DVT), is one of the most common causes of cardiovascular death worldwide. Monocytic cells (including monocytes/macrophages) and their derived tissue factor have been reported to play important roles in the development of DVT. However, the mechanism by which monocytes contribute to the development DVT is not well elucidated. In this study, we reported a critical role of inflammasome activation and pyroptosis in the development of DVT. Using a flow restriction-induced mouse DVT model in the inferior vena cava, we show that deficiency of caspase-1 protected against flow restriction-induced DVT. Inflammasome activation leads to interleukin (IL)-1b and IL-18 maturation/release and pyroptosis. Recent studies show that caspases-1 cleaves Gasdermin D (GSDMD) and triggers pyroptosis-a form of programmed cell death with similar morphology to necrosis. We tested the hypothesis that GSDMD-dependent pyroptosis drives inflammasome-induced coagulation and DVT using Gsdmd-/- mice. Indeed, flow restriction-induced DVT was inhibited by GSDMD deficiency. After induction of DVT, fibrin was deposited in the vein as detected by Western blot with a monoclonal antibody that specifically recognizes mouse fibrin, which were inhibited in the caspase-1 deficient mice and GSDMD deficient mice. IL-1b was also increased in tissue of vein following induction of DVT, which was inhibited by caspase-1 or GSDMD deficiency. Following inflammasome activation, pyroptotic macrophages release tissue factor (TF), an essential initiator of coagulation cascades. Consistently, flow restriction-induced DVT was flow restriction-induced DVT inhibited in inducible TF deficient mice. Our data reveal a critical role of inflammasome activation and pyroptosis in the development of DVT. Disclosures No relevant conflicts of interest to declare.

Gasdermin D in peripheral myeloid cells drives neuroinflammation in experimental autoimmune encephalomyelitis.

The NLRP3 inflammasome is critical for EAE pathogenesis; however, the role of gasdermin D (GSDMD), a newly identified pyroptosis executioner downstream of NLRP3 inflammasome, in EAE has not been well defined. Here, we observed that the levels of GSDMD protein were greatly enhanced in the CNS of EAE mice, especially near the areas surrounding blood vessels. GSDMD was required for the pathogenesis of EAE, and GSDMD deficiency in peripheral myeloid cells impaired the infiltration of immune cells into the CNS, leading to the suppression of neuroinflammation and demyelination. Furthermore, the loss of GSDMD reduced the activation and differentiation of T cell in the secondary lymphoid organs and prevented T cell infiltration into CNS of EAE. The administration of inflammasome-related cytokines partially rescued the impairment of pathogenesis of EAE in GSDMD KO mice. Collectively, these findings provide the first demonstration of GSDMD in peripheral myeloid cells driving neuroinflammation during EAE pathogenesis.

Shiga Toxin/Lipopolysaccharide Activates Caspase-4 and Gasdermin D to Trigger Mitochondrial Reactive Oxygen Species Upstream of the NLRP3 Inflammasome

The non-canonical caspase-4 and canonical NLRP3 inflammasomes are both activated by intracellular lipopolysaccharide (LPS), but the crosstalk between these two pathways remains unclear. Shiga toxin 2 (Stx2)/LPS complex, from pathogenic enterohemorrhagic Escherichia coli, activates caspase-4, gasdermin D (GSDMD), and the NLRP3 inflammasome in human THP-1 macrophages, but not mouse macrophages that lack the Stx receptor CD77. Stx2/LPS-mediated IL-1β secretion and pyroptosis are dependent on mitochondrial reactive oxygen species (ROS) downstream of the non-canonical caspase-4 inflammasome and cleaved GSDMD, which is enriched at the mitochondria. Blockade of caspase-4 activation and ROS generation as well as GSDMD deficiency significantly reduces Stx2/LPS-induced IL-1β production and pyroptosis. The NLRP3 inflammasome plays a significant role in amplifying Stx2/LPS-induced GSDMD cleavage and pyroptosis, with significant reduction of these responses in NLRP3-deficientTHP-1 cells. Together, these data show that Stx2/LPS complex activates the non-canonical inflammasome and mitochondrial ROS upstream of the NLRP3 inflammasome to promote cytokine maturation and pyroptosis.

Gasdermin D Exerts Anti-Inflammatory Effects by Promoting Neutrophil Death

Gasdermin D (GSDMD) is considered a pro-inflammatory factor that mediates lytic pyroptotic cell death in macrophages to protect hosts from intracellular bacteria (He et al., 2015; Kayagaki et al., 2015; Shi et al., 2015a). However, GSDMD’s role in clearing extracellular pathogens has not been directly examined. Here we reveal that GSDMD deficiency unexpectedly and paradoxically augmented host responses to extracellular Escherichia coli, mainly by delaying neutrophil death, for the first time establishing GSDMD as a negative regulator of innate immunity. The levels of pro-inflammatory cytokines in the peritoneal cavity were significantly elevated in GSDMD-deficient mice, suggesting that the production of these cytokines was not mainly mediated by macrophage pyroptosis. This further confirmed that in E.coli-induced peritonitis GSDMD acted as an anti-inflammatory factor. In contrast to its activation in macrophages, in which activated inflammatory caspases cleave GSDMD to produce an N-terminal fragment (GSDMD-cNT) to trigger pyroptosis, GSDMD cleavage and activation in neutrophils was caspase independent and mediated by a neutrophil-specific serine protease, neutrophil elastase (ELANE), released from cytoplasmic granules into the cytosol during neutrophil death. ELANE-mediated GSDMD cleavage was upstream of the caspase cleavage site and produced a fully active ELANE-derived Nterminal fragment (GSDMD-eNT) that induced lytic cell death as efficiently as GSDMD-cNT. This is the first description of GSDMD’s role in neutrophil death and the negative regulation of neutrophil-mediated innate immunity. Thus, GSDMD is pleiotropic, exerting both pro- and anti-inflammatory effects that make it a unique target for novel anti-bacterial and anti-inflammatory therapies.