David Brough

The NLRP3 inflammasome is released as a particulate danger signal that amplifies the inflammatory response

Assembly of the NLRP3 inflammasome activates caspase-1 and mediates the processing and release of the leaderless cytokine IL-1β and thereby serves a central role in the inflammatory response and in diverse human diseases. Here we found that upon activation of caspase-1, oligomeric NLRP3 inflammasome particles were released from macrophages. Recombinant oligomeric protein particles composed of the adaptor ASC or the p.D303N mutant form of NLRP3 associated with cryopyrin-associated periodic syndromes (CAPS) stimulated further activation of caspase-1 extracellularly, as well as intracellularly after phagocytosis by surrounding macrophages. We found oligomeric ASC particles in the serum of patients with active CAPS but not in that of patients with other inherited autoinflammatory diseases. Our findings support a model whereby the NLRP3 inflammasome, acting as an extracellular oligomeric complex, amplifies the inflammatory response.

Consensus guidelines for the detection of immunogenic cell death

Apoptotic cells have long been considered as intrinsically tolerogenic or unable to elicit immune responses specific for dead cell-associated antigens. However, multiple stimuli can trigger a functionally peculiar type of apoptotic demise that does not go unnoticed by the adaptive arm of the immune system, which we named “immunogenic cell death” (ICD). ICD is preceded or accompanied by the emission of a series of immunostimulatory damage-associated molecular patterns (DAMPs) in a precise spatiotemporal configuration. Several anticancer agents that have been successfully employed in the clinic for decades, including various chemotherapeutics and radiotherapy, can elicit ICD. Moreover, defects in the components that underlie the capacity of the immune system to perceive cell death as immunogenic negatively influence disease outcome among cancer patients treated with ICD inducers. Thus, ICD has profound clinical and therapeutic implications. Unfortunately, the gold-standard approach to detect ICD relies on vaccination experiments involving immunocompetent murine models and syngeneic cancer cells, an approach that is incompatible with large screening campaigns. Here, we outline strategies conceived to detect surrogate markers of ICD in vitro and to screen large chemical libraries for putative ICD inducers, based on a high-content, high-throughput platform that we recently developed. Such a platform allows for the detection of multiple DAMPs, like cell surface-exposed calreticulin, extracellular ATP and high mobility group box 1 (HMGB1), and/or the processes that underlie their emission, such as endoplasmic reticulum stress, autophagy and necrotic plasma membrane permeabilization. We surmise that this technology will facilitate the development of next-generation anticancer regimens, which kill malignant cells and simultaneously convert them into a cancer-specific therapeutic vaccine.

Understanding the mechanism of IL-1β secretion

The cytokine interleukin-1β (IL-1β) is a key mediator of the inflammatory response. Essential for the host-response and resistance to pathogens, it also exacerbates damage during chronic disease and acute tissue injury. It is not surprising therefore that there is a huge level of interest in how this protein is produced and exported from cells. However, the mechanism of IL-1β release has proven to be elusive. It does not follow the conventional ER-Golgi route of secretion. A literature full of disparate observations arising from numerous experimental systems, has contributed to a complicated mix of diverse proposals. Here we summarise these observations and propose that secretion of IL-1β occurs on a continuum, dependent upon stimulus strength and the extracellular IL-1β requirement.

Caspase-1-dependent processing of pro-interleukin-1beta is cytosolic and precedes cell death.

The pro-inflammatory cytokine interleukin-1β is a key mediator of inflammation and is implicated in the pathogenesis of diverse disease states. Despite its biological importance, the mechanisms of its processing to an active form and its trafficking to the extracellular compartment remain poorly understood. Interleukin-1β secretion is proposed to occur via several distinct mechanisms including microvesicle shedding and the regulated secretion of lysosomes. In this study, we report for the first time that caspase-1-dependent processing of pro-interleukin-1β can occur in the cytosol following activation of P2X7-receptor. We also provide evidence that the pathway of secretion in this model is independent of the lysosomal trafficking regulator, a protein involved in lysosome secretion. Although release of interleukin-1β occurred before the appearance of significant levels of lactate dehydrogenase in the supernatant, the cells ultimately died. It is clear that structural changes preceding cell death, occurring after caspase-1 activation, promote the cellular release of interleukin-1β. We investigated the involvement of lipid rafts in this process and discovered that depleting the plasma membrane of cholesterol did not adversely affect interleukin-1β secretion in response to ATP. We propose that, in macrophages, ATP-induced interleukin-1β processing occurs in the cytosol by a mechanism that resembles pyroptosis.

Extracellular ATP and P2X7 receptors in neurodegeneration

Neuronal injury and cell death in the central nervous system (CNS) are underlying features of neurodegenerative disorders. However, our understanding of the fundamental mechanisms involved is still limited. Inflammatory processes mediated by cytokines, and interleukin-1 (IL-1) in particular, play a significant role in neuronal death following pathological insults. Despite this growing area of research, very little is known about the factors regulating the expression, cleavage and release of interleukin-1 in the brain. Recent studies on immune cells demonstrate that extracellular ATP can act as a potent stimulus for the maturation and release of interleukin-1beta, via activation of P2X7 receptors. Stimulation of P2X7 receptors with ATP has dramatic cytotoxic properties and a wider role in neurodegenerative processes is possible. This review discusses the potential involvement of extracellular ATP and P2X7 receptors as regulators of interleukin-1-mediated neuropathologies and thus as a mediator of cell death following pathological insults.

Caspase-1: Is IL-1 just the tip of the ICEberg?

Caspase-1, formerly known as interleukin (IL)-1-converting enzyme is best established as the protease responsible for the processing of the key pro-inflammatory cytokine IL-1β from an inactive precursor to an active, secreted molecule. Thus, caspase-1 is regarded as a key mediator of inflammatory processes, and has become synonymous with inflammation. In addition to the processing of IL-1β, caspase-1 also executes a rapid programme of cell death, termed pyroptosis, in macrophages in response to intracellular bacteria. Pyroptosis is also regarded as a host response to remove the niche of the bacteria and to hasten their demise. These processes are generally accepted as the main roles of caspase-1. However, there is also a wealth of literature supporting a direct role for caspase-1 in non-infectious cell death processes. This is true in mammals, but also in non-mammalian vertebrates where caspase-1-dependent processing of IL-1β is absent because of the lack of appropriate caspase-1 cleavage sites. This literature is most prevalent in the brain where caspase-1 may directly regulate neuronal cell death in response to diverse insults. We attempt here to summarise the evidence for caspase-1 as a cell death enzyme and propose that, in addition to the processing of IL-1β, caspase-1 has an important and a conserved role as a cell death protease.

Purinergic (P2X7) Receptor Activation of Microglia Induces Cell Death via an Interleukin-1-Independent Mechanism

Activation of purinergic P2X7 receptors, principally by extracellular ATP, promotes the processing and release of the cytokine interleukin-1beta (IL-1beta) and induces cell death in activated microglia and macrophages. The objective of this study was to determine if IL-1beta release contributes directly to this cell death in microglia. Exposure of microglia to bacterial lipopolysaccharide (LPS) and ATP induced release of IL-1beta and IL-1alpha, as well as cell death. Neither cell death nor IL-1 release was observed in microglia lacking the P2X7 receptor. Microglia from mice lacking the IL-1beta gene demonstrated a profile of death identical to that of wild-type microglia in response to LPS and ATP. Thus, IL-1beta is not required for P2X7 receptor-stimulated microglial death.

Ca2+ Stores and Ca2+ Entry Differentially Contribute to the Release of IL-1β and IL-1α from Murine Macrophages

Interleukin-1 is a primary mediator of immune responses to injury and infection, but the mechanism of its cellular release is unknown. IL-1 exists as two agonist forms (IL-1α and IL-1β) present in the cytosol of activated monocytes/macrophages. IL-1β is synthesized as an inactive precursor that lacks a signal sequence, and its trafficking does not use the classical endoplasmic reticulum-Golgi route of secretion. Using primary cultured murine peritoneal macrophages, we demonstrate that P2X7 receptor activation causes release of IL-1β and IL-1α via a common pathway, dependent upon the release of Ca2+ from endoplasmic reticulum stores and caspase-1 activity. Increases in intracellular Ca2+ alone do not promote IL-1 secretion because a concomitant efflux of K+ through the plasmalemma is required. In addition, we demonstrate the existence of an alternative pathway for the secretion of IL-1α, independent of P2X7 receptor activation, but dependent upon Ca2+ influx. The identification of these mechanisms provides insight into the mechanism of IL-1 secretion, and may lead to the identification of targets for the therapeutic modulation of IL-1 action in inflammation.

Role of P2X7 Receptors in Ischemic and Excitotoxic Brain Injury In Vivo

Purinergic P2X7 receptors may affect neuronal cell death through their ability to regulate the processing and release of interleukin-1β (IL-1β), a key mediator in neurodegeneration. The authors tested the hypothesis that ATP, acting at P2X7 receptors, contributes to experimentally induced neuronal death in rodents in vivo. Deletion of P2X7 receptors (P2X7 knockout mice) did not affect cell death induced by temporary cerebral ischemia, which was reduced by treatment with IL-1 receptor antagonist (IL-1RA). Treatment of mice with P2X antagonists did not affect ischemic or excitotoxic cell death, suggesting that P2X7 receptors are not primary mediators of experimentally induced neuronal death.

Molecular and Translational Classifications of DAMPs in Immunogenic Cell Death

The immunogenicity of malignant cells has recently been acknowledged as a critical determinant of efficacy in cancer therapy. Thus, besides developing direct immunostimulatory regimens, including dendritic cell-based vaccines, checkpoint-blocking therapies, and adoptive T-cell transfer, researchers have started to focus on the overall immunobiology of neoplastic cells. It is now clear that cancer cells can succumb to some anticancer therapies by undergoing a peculiar form of cell death that is characterized by an increased immunogenic potential, owing to the emission of the so-called “damage-associated molecular patterns” (DAMPs). The emission of DAMPs and other immunostimulatory factors by cells succumbing to immunogenic cell death (ICD) favors the establishment of a productive interface with the immune system. This results in the elicitation of tumor-targeting immune responses associated with the elimination of residual, treatment-resistant cancer cells, as well as with the establishment of immunological memory. Although ICD has been characterized with increased precision since its discovery, several questions remain to be addressed. Here, we summarize and tabulate the main molecular, immunological, preclinical, and clinical aspects of ICD, in an attempt to capture the essence of this phenomenon, and identify future challenges for this rapidly expanding field of investigation.