The Gasdermin‐D pore acts as a conduit for IL‐1β secretion in mice

The pro‐inflammatory cytokine IL‐1β is well known for its role in host defense and the initiation of potent inflammatory responses. It is processed from its inactive pro‐form by the inflammatory caspase‐1 into its mature bioactive form, which is then released from the cell via an unconventional secretion mechanism. Recently, gasdermin‐D has been identified as a new target of caspase‐1. After proteolytical cleavage of gasdermin‐D, the N‐terminal fragment induces pyroptosis, a lytic cell death, by forming large permeability pores in the plasma membrane. Here we show using the murine system that gasdermin‐D is required for IL‐1β secretion by macrophages, dendritic cells and partially in neutrophils, and that secretion is a cell‐lysis‐independent event. Liposome transport assays in vitro further demonstrate that gasdermin‐D pores are large enough to allow the direct release of IL‐1β. Moreover, IL‐18 and other small soluble cytosolic proteins can also be released in a lysis‐independent but gasdermin‐D‐dependent mode, suggesting that the gasdermin‐D pores allow passive the release of cytosolic proteins in a size‐dependent manner.     

Pyroptosis and the cellular consequences of gasdermin pores

The family of gasdermin proteins plays a key role in the host response against external and internal pathogenic signals by mediating the form of inflammatory regulated cell death known as pyroptosis. One of the most well-studied gasdermins within innate immunity is gasdermin D, which is cleaved, oligomerizes, and forms plasma membrane pores. Gasdermin D pores lead to a number of downstream cellular consequences including plasma membrane rupture, or cell lysis. In this review, we describe mechanisms of activation for each of the gasdermins, their cell type specificity and some disease associations. We then discuss downstream consequences of gasdermin pore formation, including cellular mechanisms of membrane repair. Finally, we present some important next steps to better understand pyroptosis and the cellular consequences of gasdermin pore formation.     

Molecular mechanisms and functions of pyroptosis,inflammatory caspases and inflammasomes in infectious diseases

Cell death is a fundamental biological phenomenon that is essential for the survival and development of an organism. Emerging evidence also indicates that cell death contributes to immune defense against infectious diseases. Pyroptosis is a form of inflammatory programmed cell death pathway activated by human and mouse caspase-1, human caspase-4 and caspase-5, or mouse caspase-11. These inflammatory caspases are used by the host to control bacterial, viral, fungal, or protozoan pathogens. Pyroptosis requires cleavage and activation of the pore-forming effector protein gasdermin D by inflammatory caspases. Physical rupture of the cell causes release of the pro-inflammatory cytokines IL-1β and IL-18, alarmins and endogenous danger-associated molecular patterns, signifying the inflammatory potential of pyroptosis. Here, we describe the central role of inflammatory caspases and pyroptosis in mediating immunity to infection and clearance of pathogens.     

Pyroptosis in cardiovascular diseases: Pumping gasdermin on the fire

Pyroptosis is a form of programmed cell death associated with activation of inflammasomes and inflammatory caspases, proteolytic cleavage of gasdermin proteins (forming pores in the plasma membrane), and selective release of proinflammatory mediators. Induction of pyroptosis results in amplification of inflammation, contributing to the pathogenesis of chronic cardiovascular diseases such as atherosclerosis and diabetic cardiomyopathy, and acute cardiovascular events, such as thrombosis and myocardial infarction. While engagement of pyroptosis during sepsis-induced cardiomyopathy and septic shock is expected and well documented, we are just beginning to understand pyroptosis involvement in the pathogenesis of cardiovascular diseases with less defined inflammatory components, such as atrial fibrillation. Due to the danger that pyroptosis represents to cells within the cardiovascular system and the whole organism, multiple levels of pyroptosis regulation have evolved. Those include regulation of inflammasome priming, post-translational modifications of gasdermins, and cellular mechanisms for pore removal. While pyroptosis in macrophages is well characterized as a dramatic pro-inflammatory process, pyroptosis in other cell types within the cardiovascular system displays variable pathways and consequences. Furthermore, different cells and organs engage in local and distant crosstalk and exchange of pyroptosis triggers (oxidized mitochondrial DNA), mediators (IL-1β, S100A8/A9) and antagonists (IL-9). Development of genetic tools, such as Gasdermin D knockout animals, and small molecule inhibitors of pyroptosis will not only help us fully understand the role of pyroptosis in cardiovascular diseases but may result in novel therapeutic approaches inhibiting inflammation and progression of chronic cardiovascular diseases to reduce morbidity and mortality from acute cardiovascular events.     

NLR-mediated control of inflammasome assembly in the host response against bacterial pathogens

The host response against diverse bacterial pathogens involves activation of specialized immune cells and elaboration of pro-inflammatory cytokines that help to coordinate appropriate host defense. Members of the interleukin-1 (IL-1) cytokine family, IL-1β and IL-18, are central players in this process. Extracellular release of the mature, active form of these cytokines requires their processing by the cysteine protease caspase-1, which therefore serves as a key regulator of the inflammatory response. In addition to its role in secretion of pro-inflammatory cytokines, caspase-1 is also required for a form of cell death, recently termed pyroptosis, that occurs in macrophages infected by certain bacterial pathogens. Caspase-1 itself is synthesized as a pro-enzyme, which must first be activated by autocatalytic cleavage. This activation requires recruitment of caspase-1 into multiprotein complexes known as inflammasomes. The Nod-like receptor (NLR) family of cytosolic proteins play an important role in detecting inflammatory stimuli and subsequently mediate inflammasome assembly. A common feature of NLR proteins that trigger inflammasome assembly in response to bacterial infection is that they appear to sense membrane perturbation or delivery of bacterial components into the cytosol through bacterial pore-forming toxins or bacterial secretion systems. This review will discuss the recent developments regarding caspase-1 activation in response to bacterial infection, cross-talk between caspase-1 and other pathways involved in regulating cell death, and recent findings that a number of bacterial pathogens possess mechanisms to inhibit caspase-1 activation.     

Lower expression of NINJ1 (Ninjurin 1), a mediator of plasma membrane rupture,is associated with advanced disease and worse prognosis in serous ovarian cancer

Gasdermin proteins (GSDMs) form pores in cell membranes upon various stimuli, leading to the release of certain proinflammatory molecules such as IL-1β and IL-18, and this ultimately results in pyroptotic cell death. NINJ1 (Ninjurin 1) has recently been identified as a cell membrane protein responsible for the final complete plasma membrane rupture following lytic cell death mechanisms including pyroptosis, causing the release of relatively larger molecules such as HMGB1 and LDH. In this study, we reported the presence of higher GSDMD and lower GSDME protein levels in ovarian tumors compared to surrounding non-malignant stroma in the tumor microenvironment. GSDME protein levels are also lower in the tumors of the omentum compared to adjacent stromal cells. We found that NINJ1 expression decreases from early to late stage in serous ovarian cancer, and the percentage of NINJ1 copy number loss events is the highest in ovarian cancer among other cancers. Moreover, we showed that low expression of NINJ1 is associated with shorter overall survival of patients with ovarian cancer. In support of the findings showing that low NINJ1 expression contributes to worse prognosis in this most lethal gynecological malignancy, NINJ1 expression was found to be lower in cisplatin-resistant ovarian cancer cells compared to cisplatin-sensitive counterparts in vitro. We suggest that the members of gasdermin family might have distinct functions in serous ovarian cancer, and low levels of NINJ1 might contribute, at least in part, to the progression and poorer prognosis of ovarian cancer. A complete picture of how pyroptosis and subsequent plasma membrane rupture are involved in ovarian cancer will be of high importance in order to identify actionable therapeutic vulnerabilities within this newly identified group of proteins.

     

Fusobacterium nucleatum infection activates the noncanonical inflammasome and exacerbates inflammatory response in DSS-induced colitis

Caspase activation results in pyroptosis, an inflammatory cell death that contributes to several inflammatory diseases by releasing inflammatory cytokines and cellular contents. Fusobacterium nucleatum is a periodontal pathogen frequently detected in human cancer and inflammatory bowel diseases. Studies have reported that F. nucleatum infection leads to NLRP3 activation and pyroptosis, but the precise activation process and disease association remain poorly understood. This study demonstrated that F. nucleatum infection exacerbates acute colitis in mice and activates pyroptosis through caspase-11-mediated gasdermin D cleavage in macrophages. Furthermore, F. nucleatum infection in colitis mice induces the enhancement of IL-1⍺ secretion from the colon, affecting weight loss and severe disease activities. Neutralization of IL-1⍺ protects F. nucleatum infected mice from severe colitis. Therefore, F. nucleatum infection facilitates inflammation in acute colitis with IL-1⍺ from colon tissue by activating noncanonical inflammasome through gasdermin D cleavage.     

Pyroptosis in neutrophils: Multimodal integration of inflammasome and regulated cell death signaling pathways

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.     

Sphingosine-1-phosphate differently regulates the cytokine production of IL-12, IL-23 and IL-27 in activated murine bone marrow derived dendritic cells

Sphingosine-1-phosphate (S1P) modulates many cell functions such as lymphocyte trafficking and signaling as well as keratinocyte proliferation. However, less is known about the specific effects of S1P on cytokine production, particularly on the interaction between dendritic cells (DCs) and keratinocytes, cell types which are crucial for the initiation and maintenance of chronic inflammatory skin diseases like atopic dermatitis or psoriasis. Especially the cytokines of the IL-12 family play a dominant role in many inflammatory diseases as they have a significant impact on T-helper cell function. In the present study we show that S1P decreased the production of the pro-inflammatory cytokines IL-12 and IL-23 in LPS-stimulated DCs via the common subunit p40 as well as in the crosstalk with activated keratinocytes. By using specific S1P receptor agonists (SEW2871, FTY720-P) and antagonist (JTE013) we identified an important role for S1P receptor 1 in the modulation of the cytokine profile. While diminishing IL-12 and IL-23 secretion, S1P enhanced IL-27 production in DCs. To elucidate the mechanism of the different impact on the IL-12 family cytokine production, we investigated the mitogen-activated protein kinase (MAPK) and phosphatidylinositide 3-kinase (PI3K) pathways in DCs. By using specific MAPK-Inhibitors (U0126, SB202190, SP600125) we demonstrated that ERK, p38 and JNK differently regulate each pathway of each cytokine. While p38 and JNK did not seem to play a role in the modulation properties of S1P on cytokine production, ERK is at least partially involved in the S1P mediated modulation of IL-12 and IL-27. The PI3K-Inhibitor abrogated the S1P-induced decrease of IL-12 and IL-23 secretion, while it had no influence on the S1P-induced increase of IL-27 production. These data implicate, that S1P has an anti-inflammatory impact on the production of IL-12 family cytokines, indicating therapeutic potential for S1P treatment of several inflammatory diseases like psoriasis.     

Pannexin-1 promotes NLRP3 activation during apoptosis but is dispensable for canonical or noncanonical inflammasome activation

Inflammasomes are multimeric protein complex that assemble in the cytosol upon microbial infection or cellular stress. Upon activation, inflammasomes drive the maturation of proinflammatory cytokines, IL-1β and IL-18, and also activate the pore-forming protein, gasdermin D to initiate a form of lytic cell death known as “pyroptosis”. Pannexin-1 is channel-forming glycoprotein that promotes membrane permeability and ATP release during apoptosis; and was implicated in canonical NLRP3 or noncanonical inflammasome activation. Here, by utilizing three different pannexin-1 channel inhibitors and two lines of Panx1^–/– macrophages, we provide genetic and pharmacological evidence that pannexin-1 is dispensable for canonical or noncanonical inflammasome activation. In contrast, we demonstrate that pannexin-1 cleavage and resulting channel activity during apoptosis promotes NLRP3 inflammasome activation.     

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.     

Regulation of immune response by P2X7 receptor

The P2X7 receptor is an ATP-gated cation channel that is widely expressed in cells of the immune system. Signal transduction is accompanied by fast influx of Ca2+ and Na+, and efflux of K+. This receptor differs from other members of the P2X family in its relatively low affinity for ATP, the presence of a long C-terminal region that contains several protein-protein interaction motifs, and the activation of two membrane conductance states following receptor ligation. In the immune system, this receptor has been implicated in the processing and release of cytokines such as IL-1 beta, and in the initiation of cell death via both apoptotic and necrotic pathways. As such, it has been proposed to function as a major regulator of inflammation. Consistent with this hypothesis, inactivation of this receptor in mice modulates disease pathogenesis in several animal models of inflammatory and autoimmune diseases. Loss-of-function polymorphisms have also been noted in the human population, and there is accumulating evidence that these polymorphisms are linked to certain diseases. In this article, we review the current status of research in this field, with particular emphasis on the signaling pathways activated by this receptor, the mechanisms involved in the initiation of cell death, and associations with disease states in mice and humans.     

Role of pyroptosis in inflammation and cancer

Pyroptosis is a form of programmed cell death mediated by gasdermin and is a product of continuous cell expansion until the cytomembrane ruptures, resulting in the release of cellular contents that can activate strong inflammatory and immune responses. Pyroptosis, an innate immune response, can be triggered by the activation of inflammasomes by various influencing factors. Activation of these inflammasomes can induce the maturation of caspase-1 or caspase-4/5/11, both of which cleave gasdermin D to release its N-terminal domain, which can bind membrane lipids and perforate the cell membrane. Here, we review the latest advancements in research on the mechanisms of pyroptosis, newly discovered influencing factors, antitumoral properties, and applications in various diseases. Moreover, this review also provides updates on potential targeted therapies for inflammation and cancers, methods for clinical prevention, and finally challenges and future directions in the field.     

Inflammasomes contributing to inflammation in arthritis

Inflammasomes are intracellular multiprotein signaling platforms that initiate inflammatory responses in response to pathogens and cellular damage. Active inflammasomes induce the enzymatic activity of caspase-1, resulting in the induction of inflammatory cell death, pyroptosis, and the maturation and secretion of inflammatory cytokines IL-1β and IL-18. Inflammasomes are activated in many inflammatory diseases, including autoinflammatory disorders and arthritis, and inflammasome-specific therapies are under development for the treatment of inflammatory conditions. In this review, we outline the different inflammasome platforms and recent findings contributing to our knowledge about inflammasome biology in health and disease. In particular, we discuss the role of the inflammasome in the pathogenesis of arthritic diseases, including rheumatoid arthritis, gout, ankylosing spondylitis, and juvenile idiopathic arthritis, and the potential of newly developed therapies that specifically target the inflammasome or its products for the treatment of inflammatory diseases.     

Role of interleukin-17F in chronic inflammatory and allergic lung disease

IL-17 family members belong to a distinct category of cytokines that coordinate local tissue inflammation by inducing the release of pro-inflammatory and neutrophil-mobilizing cytokines. The importance of the IL-17 family in inflammatory and autoimmune disease is becoming increasingly apparent. IL-17F is a recently discovered member of the IL-17 family that has a number of biological activities through induction of various cytokines, chemokines, and mediators. IL-17A, the founding member of the IL-17 family, and IL-17F are produced by several inflammatory cells, including activated T cells, in response to infectious and antigenic stimuli. Overexpression of IL-17A or IL-17F in the lungs results in induction of CXC chemokines and neutrophil recruitment. In a case-control study of 1125 unrelated Japanese subjects, a His161 to Arg161 (H161R) substitution in the third exon of the IL17F gene was shown to be associated with asthma and chronic obstructive pulmonary disease (COPD). Functionally, this variant failed to induce cytokines and chemokines, and interestingly, was able to antagonize the activity of wild-type IL-17F. These results provide an experimental basis for the observed genetic association with chronic inflammatory lung diseases, and also suggest the potential therapeutic utility of this antagonistic variant of IL-17F. Given that asthma and COPD are complex diseases involving a number of genetic and environmental factors, the genetic impact of IL-17F H161R with regard to the development of chronic airway inflammation likely varies among individuals with different genetic backgrounds and environmental exposures.     

Mycoplasma arginini TUH-14 membrane lipoproteins induce production of interleukin-1, interleukin-6, and tumor necrosis factor alpha by human monocytes.

Mycoplasma arginini TUH-14 partially purified membrane lipoproteins (TUH-14-pp) directly induce secretion of the cytokines involved in the inflammatory response, namely, interleukin 1 (IL-1), tumor necrosis factor alpha, and IL-6, by human monocytes cultured in the absence of serum. The biological activity of each cytokine correlates with its immunoreactivity. Upon stimulation with either TUH-14-pp or lipopolysaccharide, most tumor necrosis factor alpha and IL-6 is secreted in the extracellular compartment, whereas a significant amount of IL-1 remains cell associated. Finally, polymyxin B does not affect secretion of cytokines induced by TUH-14-pp, indicating that mycoplasma lipopolysaccharide does not account for their effects on monocytes. Altogether, our data show that direct interaction of mycoplasma membrane components with human blood monocytes induces secretion of high levels of cytokines known to trigger inflammatory responses. This new concept of membrane-bound active components of mycoplasma may explain its ability to efficiently initiate inflammatory reactions.     

An NLRP7-containing inflammasome mediates recognition of microbial lipopeptides in human macrophages

Cytosolic pathogen- and damage-associated molecular patterns are sensed by pattern recognition receptors, including members of the nucleotide-binding domain and leucine-rich repeat-containing gene family (NLR), which cause inflammasome assembly and caspase-1 activation to promote maturation and release of the inflammatory cytokines interleukin-1β (IL-1β) and IL-18 and induction of pyroptosis. However, the contribution of most of the NLRs to innate immunity, host defense, and inflammasome activation and their specific agonists are still unknown. Here we describe identification and characterization of an NLRP7 inflammasome in human macrophages, which is induced in response to microbial acylated lipopeptides. Activation of NLRP7 promoted ASC-dependent caspase-1 activation, IL-1β and IL-18 maturation, and restriction of intracellular bacterial replication, but not caspase-1-independent secretion of the proinflammatory cytokines IL-6 and tumor necrosis factor-α. Our study therefore increases our currently limited understanding of NLR activation, inflammasome assembly, and maturation of IL-1β and IL-18 in human macrophages.     

NLRP3 inflammasome activation and cell death

The NLRP3 inflammasome is a cytosolic multiprotein complex composed of the innate immune receptor protein NLRP3, adapter protein ASC, and inflammatory protease caspase-1 that responds to microbial infection, endogenous danger signals, and environmental stimuli. The assembled NLRP3 inflammasome can activate the protease caspase‐1 to induce gasdermin D-dependent pyroptosis and facilitate the release of IL-1β and IL-18, which contribute to innate immune defense and homeostatic maintenance. However, aberrant activation of the NLRP3 inflammasome is associated with the pathogenesis of various inflammatory diseases, such as diabetes, cancer, and Alzheimer’s disease. Recent studies have revealed that NLRP3 inflammasome activation contributes to not only pyroptosis but also other types of cell death, including apoptosis, necroptosis, and ferroptosis. In addition, various effectors of cell death have been reported to regulate NLRP3 inflammasome activation, suggesting that cell death is closely related to NLRP3 inflammasome activation. In this review, we summarize the inextricable link between NLRP3 inflammasome activation and cell death and discuss potential therapeutics that target cell death effectors in NLRP3 inflammasome-associated diseases.