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Heparanase is a β-D-endoglucuronidase that cleaves heparan sulfate (HS), facilitating degradation of the extracellular matrix (ECM) and the release of HS-bound biomolecules including cytokines. The remodeling of the ECM by heparanase is important for various physiological and pathological processes, including inflammation, wound healing, tumour angiogenesis and metastasis. Although heparanase has been proposed to facilitate leukocyte migration through degradation of the ECM, its role in inflammation by regulating the expression and release of cytokines has not been fully defined. In this study, the role of heparanase in regulating the expression and release of cytokines from human and murine immune cells was examined. Human peripheral blood mononuclear cells treated ex vivo with heparanase resulted in the release of a range of pro-inflammatory cytokines including IL-1β, IL-6, IL-8, IL-10 and TNF. In addition, mouse splenocytes treated ex vivo with heparanase resulted in the release of IL-6, MCP-1 and TNF. A similar pattern of cytokine release was also observed when cells were treated with soluble HS. Furthermore, heparanase-induced cytokine release was abolished by enzymatic-inhibitors of heparanase, suggesting this process is mediated via the enzymatic release of cell surface HS fragments. As soluble HS can signal through the Toll-like receptor (TLR) pathway, heparanase may promote the upregulation of cytokines through the generation of heparanase-cleaved fragments of HS. In support of this hypothesis, mouse spleen cells lacking the key TLR adaptor molecule MyD88 demonstrated an abolition of cytokine release after heparanase stimulation. Furthermore, TLR4-deficient spleen cells showed reduced cytokine release in response to heparanase treatment, suggesting that TLR4 is involved in this response. Consistent with these observations, the pathway involved in cytokine upregulation was identified as being NF-κB-dependent. These data identify a new mechanism for heparanase in promoting the release of pro-inflammatory cytokines that is likely to be important in regulating cell migration and inflammation.

Plasma membrane pannexin 1 channels (PANX1) release nucleotide find-me signals from apoptotic cells to attract phagocytes. Here we show that the quinolone antibiotic trovafloxacin is a novel PANX1 inhibitor, by using a small-molecule screen. Although quinolones are widely used to treat bacterial infections, some quinolones have unexplained side effects, including deaths among children. PANX1 is a direct target of trovafloxacin at drug concentrations seen in human plasma, and its inhibition led to dysregulated fragmentation of apoptotic cells. Genetic loss of PANX1 phenocopied trovafloxacin effects, revealing a non-redundant role for pannexin channels in regulating cellular disassembly during apoptosis. Increase in drug-resistant bacteria worldwide and the dearth of new antibiotics is a major human health challenge. Comparing different quinolone antibiotics suggests that certain structural features may contribute to PANX1 blockade. These data identify a novel linkage between an antibiotic, pannexin channels and cellular integrity, and suggest that re-engineering certain quinolones might help develop newer antibacterials. The pannexin 1 channel on the plasma membrane of apoptotic cells mediates the release of find-me molecular signals to attract phagocytic cells for clearance of the apoptotic cells; here the quinolone antibiotic trovafloxacin is identified as a direct inhibitor of pannexin 1, which results in dysregulated fragmentation of apoptotic cells and may partly explain quinolone toxicity. Dead-cell disposal mechanism clarified The pannexin 1 channel on the plasma membrane of apoptotic cells mediates the release of find-me molecular signals that attract phagocytic cells tasked to clear away the dead cells. In an unbiased screen of small molecules, Kodi Ravichandran and colleagues identify the quinolone antibiotic trovafloxacin as a direct inhibitor of the pannexin 1 channel activity, which results in dysregulated fragmentation of apoptotic cells. This work establishes an essential role for pannexin channels in orderly disassembly of apoptotic cells, and could also re-invigorate interest in quinolone antibiotics without cross-reactivity with the pannexin 1 channel, which may explain the particular toxicity that emerged during clinical trials of trovafloxacin.

The many functions of extracellular vesicles (EVs) like exosomes and microvesicles released from healthy cells have been well characterized, particularly in relation to their roles in immune modulation. Apoptotic bodies, a major class of EV released as a product of apoptotic cell disassembly, and other types of EVs released from dying cells are also becoming recognized as key players in this emerging field. There is now increasing evidence to suggest that EVs produced during apoptosis have important immune regulatory roles, a concept relevant across different disease settings including autoimmunity, cancer, and infection. Therefore, this review focuses on how the formation of EVs during apoptosis could be a key mechanism of immune modulation by dying cells.

Phosphorylated phosphatidylinositol lipids, or phosphoinositides, critically regulate diverse cellular processes, including signalling transduction, cytoskeletal reorganisation, membrane dynamics and cellular trafficking. However, phosphoinositides have been inadequately investigated in the context of cell death, where they are mainly regarded as signalling secondary messengers. However, recent studies have begun to highlight the importance of phosphoinositides in facilitating cell death execution. Here, we cover the latest phosphoinositide research with a particular focus on phosphoinositides in the mechanisms of cell death. This progress article also raises key questions regarding the poorly defined role of phosphoinositides, particularly during membrane-associated events in cell death such as apoptosis and secondary necrosis. The review then further discusses important future directions for the phosphoinositide field, including therapeutically targeting phosphoinositides to modulate cell death.

Mucosa-associated invariant T (MAIT) cells are abundant antimicrobial T cells in humans and recognize antigens derived from the microbial riboflavin biosynthetic pathway presented by the MHC-Ib-related protein (MR1). However, the mechanisms responsible for MAIT cell antimicrobial activity are not fully understood, and the efficacy of these mechanisms against antibiotic resistant bacteria has not been explored. Here, we show that MAIT cells mediate MR1-restricted antimicrobial activity against Escherichia coli clinical strains in a manner dependent on the activity of cytolytic proteins but independent of production of pro-inflammatory cytokines or induction of apoptosis in infected cells. The combined action of the pore-forming antimicrobial protein granulysin and the serine protease granzyme B released in response to T cell receptor (TCR)-mediated recognition of MR1-presented antigen is essential to mediate control against both cell-associated and free-living, extracellular forms of E. coli. Furthermore, MAIT cell-mediated bacterial control extends to multidrug-resistant E. coli primary clinical isolates additionally resistant to carbapenems, a class of last resort antibiotics. Notably, high levels of granulysin and granzyme B in the MAIT cell secretomes directly damage bacterial cells by increasing their permeability, rendering initially resistant E. coli susceptible to the bactericidal activity of carbapenems. These findings define the role of cytolytic effector proteins in MAIT cell-mediated antimicrobial activity and indicate that granulysin and granzyme B synergize to restore carbapenem bactericidal activity and overcome carbapenem resistance in E. coli.

Crocodilians are an order of ancient reptiles that thrive in pathogen-rich environments. The ability to inhabit these harsh environments is indicative of a resilient innate immune system. Defensins, a family of cysteine-rich cationic host defence peptides, are a major component of the innate immune systems of all plant and animal species, however crocodilian defensins are poorly characterised. We now show that the saltwater crocodile defensin CpoBD13 harbors potent antifungal activity that is mediated by a pH-dependent membrane-targeting action. CpoBD13 binds the phospholipid phosphatidic acid (PA) to form a large helical oligomeric complex, with specific histidine residues mediating PA binding. The utilisation of histidine residues for PA engagement allows CpoBD13 to exhibit differential activity at a range of environmental pH values, where CpoBD13 is optimally active in an acidic environment. Defensins are a class of host defence peptides that contribute to the immune system of eukaryotes. Here, the authors report the structure of saltwater crocodile defensin CpoBD13 and the mechanism of pH-dependent antifungal activity.

To maintain physiological homeostasis, cell turnover occurs every day in the body via a form of programmed cell death called apoptosis. During apoptosis, cells undergo distinct morphological changes culminating in the disassembly of the dying cell into smaller fragments known as apoptotic bodies (ApoBDs). Dysregulation of apoptosis is associated with diseases including infection, cancer and atherosclerosis. Although the development of atherosclerosis is largely attributed to the accumulation of lipids and inflammatory debris in vessel walls, it is also associated with apoptosis of macrophages, smooth muscle cells (SMCs) and endothelial cells. During cellular activation and apoptosis, endothelial cells can release several types of membrane-bound extracellular vesicles (EVs) including exosomes, microvesicles (MVs)/microparticles and ApoBDs. Emerging evidence in the field suggests that these endothelial cell-derived EVs (EndoEVs) can contribute to intercellular communication during the development of atherosclerosis via the transfer of cellular contents such as protein and microRNA, which may prevent or promote disease progression depending on the context. This review provides an up-to-date overview of the known causes and consequences of endothelial cell death during atherosclerosis along with highlighting current methodological approaches to studying EndoEVs and the potential roles of EndoEVs in atherosclerosis development.

Apoptotic cells communicate to phagocytic cells through releasing soluble factors and apoptotic cell-derived extracellular vesicles. However, whether there are additional factors that remain attached at the site of cell death to signal to phagocytic cells is currently unknown. Here we show that apoptotic cell retraction generates a membrane-encased, F-actin-rich ‘footprint’ tightly anchored to the substrate that marks the site of cell death, coined ‘the FOotprint Of Death’ or FOOD. Formation of FOOD is observed frequently across many different cell types, apoptotic stimuli and surface composition. Mechanistically, FOOD formation is regulated by the protein kinase ROCK1. 3D time-lapse microscopy studies revealed that FOOD vesicularises into distinct large extracellular vesicles. These extracellular vesicles expose the ‘eat-me’ signal phosphatidylserine and can function to ‘flag’ the site of cell death to neighbouring phagocytes for efferocytosis. Under a viral infection setting, FOOD can harbour viral proteins and virions, and propagate infection to healthy cells. Together, this study has revealed another route of apoptotic cell-phagocyte communication. Apoptotic cells often release extracellular vesicles that aid in their clearance and provide molecular information to cellular neighbours. Here, the authors show that some adherent apoptotic cells also create vesicles that remain attached at the site of death.

Key Points Apoptotic cells are rapidly removed by tissue-resident professional phagocytes and/or by neighbouring non-professional phagocytes under homeostatic conditions. The prompt clearance of apoptotic cells involves molecular steps that include the recruitment of phagocytes towards apoptotic cells through 'find-me' signals and the recognition of 'eat-me' signals on apoptotic cells that trigger engulfment. Apoptotic cell clearance under physiological conditions is generally anti-inflammatory and immunologically silent. Defects in apoptotic cell removal are associated with the initiation and progression of a number of pathological conditions, including inflammation and autoimmunity. The process of apoptotic cell clearance can be manipulated potentially by pharmacological means to treat a variety of human diseases. The removal of apoptotic cells by phagocytes is essential for the maintenance of tissue homeostasis and is usually immunologically silent. However, dysregulation of this process is associated with numerous inflammatory and autoimmune diseases, and thus the therapeutic manipulation of apoptotic cell clearance by phagocytes may be a means to treat these diseases. The prompt removal of apoptotic cells by phagocytes is important for maintaining tissue homeostasis. The molecular and cellular events that underpin apoptotic cell recognition and uptake, and the subsequent biological responses, are increasingly better defined. The detection and disposal of apoptotic cells generally promote an anti-inflammatory response at the tissue level, as well as immunological tolerance. Consequently, defects in apoptotic cell clearance have been linked with various inflammatory diseases and autoimmunity. Conversely, under certain conditions, such as the killing of tumour cells by specific cell-death inducers, the recognition of apoptotic tumour cells can promote an immunogenic response and antitumour immunity. Here, we review the current understanding of the complex process of apoptotic cell clearance in physiology and pathology, and discuss how this knowledge could be harnessed for new therapeutic strategies.