Heparanase promotes the onset and progression of atherosclerosis in apolipoprotein E gene knockout mice.

BACKGROUND AND AIMS: Atherosclerosis is the primary underlying cause of myocardial infarction and stroke, which are the major causes of death globally. Heparanase (Hpse) is a pro-inflammatory extracellular matrix degrading enzyme that has been implicated in atherogenesis. However, to date the precise roles of Hpse in atherosclerosis and its mechanisms of action are not well defined. This study aims to provide new insights into the contribution of Hpse in different stages of atherosclerosis in vivo. METHODS: We generated Hpse gene-deficient mice on the atherosclerosis-prone apolipoprotein E gene knockout (ApoE-/-) background to investigate the impact of Hpse gene deficiency on the initiation and progression of atherosclerosis after 6 and 14 weeks high-fat diet feeding, respectively. Atherosclerotic lesion development, blood serum profiles, lesion composition and aortic immune cell populations were evaluated. RESULTS: Hpse-deficient mice exhibited significantly reduced atherosclerotic lesion burden in the aortic sinus and aorta at both time-points, independent of changes in plasma cholesterol levels. A significant reduction in the necrotic core size and an increase in smooth muscle cell content were also observed in advanced atherosclerotic plaques of Hpse-deficient mice. Additionally, Hpse deficiency reduced circulating and aortic levels of VCAM-1 at the initiation and progression stages of disease and circulating MCP-1 levels in the initiation but not progression stage. Moreover, the aortic levels of total leukocytes and dendritic cells in Hpse-deficient ApoE-/- mice were significantly decreased compared to control ApoE-/-mice at both disease stages. CONCLUSIONS: This study identifies Hpse as a key pro-inflammatory enzyme driving the initiation and progression of atherosclerosis and highlighting the potential of Hpse inhibitors as novel anti-inflammatory treatments for cardiovascular disease.

Crocodile defensin (CpoBD13) antifungal activity via pH-dependent phospholipid targeting and membrane disruption.

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.

Translating extracellular vesicle packaging into therapeutic applications.

Extracellular vesicles (EVs) are membrane-bound particles released by cells in various (patho)physiological conditions. EVs can transfer effector molecules and elicit potent responses in recipient cells, making them attractive therapeutic agents and drug delivery platforms. In contrast to their tremendous potential, only a few EV-based therapies and drug delivery have been approved for clinical use, which is largely attributed to limited therapeutic loading technologies and efficiency. As EV cargo has major influence on their functionality, understanding and translating the biology underlying the packaging and transferring of biomolecule cargos (e.g. miRNAs, pathogen antigens, small molecule drugs) into EVs is key in harnessing their therapeutic potential. In this review, through recent insights into EVs' content packaging, we discuss different mechanisms utilized by EVs during cargo packaging, and how one might therapeutically exploit this process. Apart from the well-characterized EVs like exosomes and microvesicles, we also cover the less-studied and other EV subtypes like apoptotic bodies, large oncosomes, bacterial outer membrane vesicles, and migrasomes to highlight therapeutically-diverse opportunities of EV armoury.

Human ß-Defensin 2 (HBD-2) Displays Oncolytic Activity but Does Not Affect Tumour Cell Migration.

Defensins form an integral part of the cationic host defence peptide (HDP) family, a key component of innate immunity. Apart from their antimicrobial and immunomodulatory activities, many HDPs exert multifaceted effects on tumour cells, notably direct oncolysis and/or inhibition of tumour cell migration. Therefore, HDPs have been explored as promising anticancer therapeutics. Human ß-defensin 2 (HBD-2) represents a prominent member of human HDPs, being well-characterised for its potent pathogen-killing, wound-healing, cytokine-inducing and leukocyte-chemoattracting functions. However, its anticancer effects remain largely unknown. Recently, we demonstrated that HBD-2 binds strongly to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), a key mediator of defensin-induced cell death and an instructional messenger during cell migration. Hence, in this study, we sought to investigate the lytic and anti-migratory effects of HBD-2 on tumour cells. Using various cell biological assays and confocal microscopy, we showed that HBD-2 killed tumour cells via acute lytic cell death rather than apoptosis. In addition, our data suggested that, despite the reported PI(4,5)P2 interaction, HBD-2 does not affect cytoskeletal-dependent tumour cell migration. Together, our findings provide further insights into defensin biology and informs future defensin-based drug development.

Defensin-lipid interactions in membrane targeting: mechanisms of action and opportunities for the development of antimicrobial and anticancer therapeutics.

Defensins are a class of host defence peptides (HDPs) that often harbour antimicrobial and anticancer activities, making them attractive candidates as novel therapeutics. In comparison with current antimicrobial and cancer treatments, defensins uniquely target specific membrane lipids via mechanisms distinct from other HDPs. Therefore, defensins could be potentially developed as therapeutics with increased selectivity and reduced susceptibility to the resistance mechanisms of tumour cells and infectious pathogens. In this review, we highlight recent advances in defensin research with a particular focus on membrane lipid-targeting in cancer and infection settings. In doing so, we discuss strategies to harness lipid-binding defensins for anticancer and anti-infective therapies.

Staphylococcus aureus membrane vesicles contain immunostimulatory DNA, RNA and peptidoglycan that activate innate immune receptors and induce autophagy.

Gram-positive bacteria ubiquitously produce membrane vesicles (MVs), and although they contribute to biological functions, our knowledge regarding their composition and immunogenicity remains limited. Here we examine the morphology, contents and immunostimulatory functions of MVs produced by three Staphylococcus aureus strains; a methicillin resistant clinical isolate, a methicillin sensitive clinical isolate and a laboratory-adapted strain. We observed differences in the number and morphology of MVs produced by each strain and showed that they contain microbe-associated molecular patterns (MAMPs) including protein, nucleic acids and peptidoglycan. Analysis of MV-derived RNA indicated the presence of small RNA (sRNA). Furthermore, we detected variability in the amount and composition of protein, nucleic acid and peptidoglycan cargo carried by MVs from each S. aureus strain. S. aureus MVs activated Toll-like receptor (TLR) 2, 7, 8, 9 and nucleotide-binding oligomerization domain containing protein 2 (NOD2) signalling and promoted cytokine and chemokine release by epithelial cells, thus identifying that MV-associated MAMPs including DNA, RNA and peptidoglycan are detected by pattern recognition receptors (PRRs). Moreover, S. aureus MVs induced the formation of and colocalized with autophagosomes in epithelial cells, while inhibition of lysosomal acidification using bafilomycin A1 resulted in accumulation of autophagosomal puncta that colocalized with MVs, revealing the ability of the host to degrade MVs via autophagy. This study reveals the ability of DNA, RNA and peptidoglycan associated with MVs to activate PRRs in host epithelial cells, and their intracellular degradation via autophagy. These findings advance our understanding of the immunostimulatory roles of Gram-positive bacterial MVs in mediating pathogenesis, and their intracellular fate within the host.

Pannexin-1 channel regulates nuclear content packaging into apoptotic bodies and their size.

Apoptotic bodies (ApoBDs), which are large extracellular vesicles exclusively released by apoptotic cells, possess therapeutically exploitable properties including biomolecule loadability and transferability. However, current limited understanding of ApoBD biology has hindered its exploration for clinical use. Particularly, as ApoBD-accompanying cargoes (e.g., nucleic acids and proteins) have major influence on their functionality, further insights into the mechanism of biomolecule sorting into ApoBDs are critical to unleash their therapeutic potential. Previous studies suggested pannexin 1 (PANX1) channel, a negative regulator of ApoBD biogenesis, can modify synaptic vesicle contents. We also reported that trovafloxacin (a PANX1 inhibitor) increases proportion of ApoBDs containing DNA. Therefore, we sought to define the role of PANX1 in regulating the sorting of nuclear content into ApoBDs. Here, using flow cytometry and label-free quantitative proteomic analyses, we showed that targeting PANX1 activity during apoptosis, via either pharmacological inhibition or genetic disruption, resulted in enrichment of both DNA and nuclear proteins in ApoBDs that were unexpectedly smaller in size. Our data suggest that PANX1, besides being a key regulator of ApoBD formation, also functions as a negative regulator of nuclear content packaging and modulator of ApoBD size. Together, our findings provide further insights into ApoBD biology and form a novel conceptual framework for ApoBD-based therapies through pharmacologically manipulating ApoBD contents.

TREML4 receptor regulates inflammation and innate immune cell death during polymicrobial sepsis.

Sepsis is a biphasic disease characterized by an acute inflammatory response, followed by a prolonged immunosuppressive phase. Therapies aimed at controlling inflammation help to reduce the time patients with sepsis spend in intensive care units, but they do not lead to a reduction in overall mortality. Recently, the focus has been on addressing the immunosuppressive phase, often caused by apoptosis of immune cells. However, molecular triggers of these events are not yet known. Using whole-genome CRISPR screening in mice, we identified a triggering receptor expressed on myeloid cells (TREM) family receptor, TREML4, as a key regulator of inflammation and immune cell death in sepsis. Genetic ablation of Treml4 in mice demonstrated that TREML4 regulates calcium homeostasis, the inflammatory cytokine response, myeloperoxidase activation, the endoplasmic reticulum stress response and apoptotic cell death in innate immune cells, leading to an overall increase in survival rate, both during the acute and chronic phases of polymicrobial sepsis.

Unleashing the therapeutic potential of apoptotic bodies.

Extracellular vesicles (EVs), membrane-bound vesicles that are naturally released by cells, have emerged as new therapeutic opportunities. EVs, particularly exosomes and microvesicles, can transfer effector molecules and elicit potent responses in recipient cells, making them attractive therapeutic targets and drug delivery platforms. Furthermore, containing predictive biomarkers and often being dysregulated in various disease settings, these EVs are being exploited for diagnostic purposes. In contrast, the therapeutic application of apoptotic bodies (ApoBDs), a distinct type of EVs released by cells undergoing a form of programmed cell death called apoptosis, has been largely unexplored. Recent studies have shed light on ApoBD biogenesis and functions, promisingly implicating their therapeutic potential. In this review, we discuss many strategies to develop ApoBD-based therapies as well as highlight their advantages and challenges, thereby positioning ApoBD for potential EV-based therapy.

Human MAIT cell cytolytic effector proteins synergize to overcome carbapenem resistance in Escherichia coli.

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.

Monocyte apoptotic bodies are vehicles for influenza A virus propagation.

The disassembly of apoptotic cells into small membrane-bound vesicles termed apoptotic bodies (ApoBDs) is a hallmark of apoptosis; however, the functional significance of this process is not well defined. We recently discovered a new membrane protrusion (termed beaded apoptopodia) generated by apoptotic monocytes which fragments to release an abundance of ApoBDs. To investigate the function of apoptotic monocyte disassembly, we used influenza A virus (IAV) infection as a proof-of-concept model, as IAV commonly infects monocytes in physiological settings. We show that ApoBDs generated from IAV-infected monocytes contained IAV mRNA, protein and virions and consequently, could facilitate viral propagation in vitro and in vivo, and induce a robust antiviral immune response. We also identified an antipsychotic, Haloperidol, as an unexpected inhibitor of monocyte cell disassembly which could impair ApoBD-mediated viral propagation under in vitro conditions. Together, this study reveals a previously unrecognised function of apoptotic monocyte disassembly in the pathogenesis of IAV infections.

ROCK1 but not LIMK1 or PAK2 is a key regulator of apoptotic membrane blebbing and cell disassembly.

Many cell types are known to undergo a series of morphological changes during the progression of apoptosis, leading to their disassembly into smaller membrane-bound vesicles known as apoptotic bodies (ApoBDs). In particular, the formation of circular bulges called membrane blebs on the surface of apoptotic cells is a key morphological step required for a number of cell types to generate ApoBDs. Although apoptotic membrane blebbing is thought to be regulated by kinases including ROCK1, PAK2 and LIMK1, it is unclear whether these kinases exhibit overlapping roles in the disassembly of apoptotic cells. Utilising both pharmacological and CRISPR/Cas9 gene editing based approaches, we identified ROCK1 but not PAK2 or LIMK1 as a key non-redundant positive regulator of apoptotic membrane blebbing as well as ApoBD formation. Functionally, we have established an experimental system to either inhibit or enhance ApoBD formation and demonstrated the importance of apoptotic cell disassembly in the efficient uptake of apoptotic materials by various phagocytes. Unexpectedly, we also noted that ROCK1 could play a role in regulating the onset of secondary necrosis. Together, these data shed light on both the mechanism and function of cell disassembly during apoptosis.

Combating Human Pathogens and Cancer by Targeting Phosphoinositides and Their Metabolism.

Pathogens and tumor cells have adopted various adept strategies to evade immunosurveillance and promote their growth and survival. There has been substantial evidence demonstrating phosphoinositide lipids and their modifying enzymes as essential host targets that are often hijacked by pathogens and tumor cells. The common dependence of pathogen virulence and tumor progression on phosphoinositides presents an exciting disease-combating potential, particularly combinatorial therapeutics. While traditional approaches to pharmacologically inhibit phosphoinositide-metabolizing enzymes has shown some promise, the direct targeting of phosphoinositides has recently emerged as a novel therapeutic strategy. Our review provides a current picture of the role of phosphoinositides during pathogen virulence and tumorigenesis as well as a thorough discussion on promises, challenges, and new perspectives of phosphoinositide-targeting drug development.

Plexin B2 Is a Regulator of Monocyte Apoptotic Cell Disassembly.

Billions of cells undergo apoptosis daily and often fragment into small, membrane-bound extracellular vesicles termed apoptotic bodies (ApoBDs). We demonstrate that apoptotic monocytes undergo a highly coordinated disassembly process and form long, beaded protrusions (coined as beaded apoptopodia), which fragment to release ApoBDs. Here, we find that the protein plexin B2 (PlexB2), a transmembrane receptor that regulates axonal guidance in neurons, is enriched in the ApoBDs of THP1 monocytes and is a caspase 3/7 substrate. To determine whether PlexB2 is involved in the disassembly of apoptotic monocytes, we generate PlexB2-deficient THP1 monocytes and demonstrate that lack of PlexB2 impairs the formation of beaded apoptopodia and ApoBDs. Consequently, the loss of PlexB2 in apoptotic THP1 monocytes impairs their uptake by both professional and non-professional phagocytes. Altogether, these data identify PlexB2 as a positive regulator of apoptotic monocyte disassembly and demonstrate the importance of this process in apoptotic cell clearance.

Correction to: Defining the role of cytoskeletal components in the formation of apoptopodia and apoptotic bodies during apoptosis.

The original version of the article unfortunately contained a typo in the fourth author name. The author name was incorrectly listed as Rochelle Tixeria. The correct name should be Rochelle Tixeira. The original article has been corrected.

Defining the role of cytoskeletal components in the formation of apoptopodia and apoptotic bodies during apoptosis.

During apoptosis, dying cells undergo dynamic morphological changes that ultimately lead to their disassembly into fragments called apoptotic bodies (ApoBDs). Reorganisation of the cytoskeletal structures is key in driving various apoptotic morphologies, including the loss of cell adhesion and membrane bleb formation. However, whether cytoskeletal components are also involved in morphological changes that occur later during apoptosis, such as the recently described generation of thin apoptotic membrane protrusions called apoptopodia and subsequent ApoBD formation, is not well defined. Through monitoring the progression of apoptosis by confocal microscopy, specifically focusing on the apoptopodia formation step, we characterised the presence of F-actin and microtubules in a subset of apoptopodia generated by T cells and monocytes. Interestingly, targeting actin polymerisation and microtubule assembly pharmacologically had no major effect on apoptopodia formation. These data demonstrate apoptopodia as a novel type of membrane protrusion that could be formed in the absence of actin polymerisation and microtubule assembly.

Moving beyond size and phosphatidylserine exposure: evidence for a diversity of apoptotic cell-derived extracellular vesicles in vitro.

Apoptosis is a form of programmed cell death that occurs throughout life as part of normal development as well as pathologic processes including chronic inflammation and infection. Although the death of a cell is often considered as the only biological outcome of a cell committed to apoptosis, it is becoming increasingly clear that the dying cell can actively communicate with other cells via soluble factors as well as membrane-bound extracellular vesicles (EVs) to regulate processes including cell clearance, immunity and tissue repair. Compared to EVs generated from viable cells such as exosomes and microvesicles, apoptotic cell-derived EVs (ApoEVs) are less well defined and the basic criteria for ApoEV characterization have not been established in the field. In this study, we will examine the current understanding of ApoEVs, in particular, the ApoEV subtype called apoptotic bodies (ApoBDs). We described that a subset of ApoBDs can be larger than 5 µm and smaller than 1 µm based on flow cytometry and live time-lapse microscopy analysis, respectively. We also described that a subset of ApoBDs can expose a relatively low level of phosphatidylserine on its surface based on annexin A5 staining. Furthermore, we characterized the presence of caspase-cleaved proteins (in particular plasma membrane-associated or cytoplasmic proteins) in samples enriched in ApoBDs. Lastly, using a combination of biochemical-, live imaging- and flow cytometry-based approaches, we characterized the progressive lysis of ApoBDs. Taken together, these results extended our understanding of ApoBDs.

Analysis of extracellular vesicles generated from monocytes under conditions of lytic cell death.

Extracellular vesicles (EVs) are an important class of membrane-bound structures that have been widely investigated for their roles in intercellular communication in the contexts of tumor progression, vascular function, immunity and regenerative medicine. Much of the current knowledge on the functions of EVs pertains to those derived from viable cells (e.g. exosomes and microvesicles) or apoptotic cells (e.g. apoptotic bodies) whilst the generation of EVs from dying cells under non-apoptotic conditions remains poorly characterized. Herein, the release of EVs from THP-1 monocytes under conditions of primary necrosis, secondary necrosis and pyroptosis, was investigated. A comprehensive analysis of THP-1-derived EVs revealed that cells undergoing lytic forms of cell death generated a high number of EVs compared with viable or apoptotic cells in vitro. Differential centrifugation via 16,000 g and 100,000 g revealed that dying THP-1 cells release both medium and small EVs, respectively, consistent with the known characteristics of microvesicles and/or exosomes. In addition, large EVs isolated via 2000 g centrifugation were also present in all samples. These findings suggest that lytic cell death under both sterile and non-sterile inflammatory conditions induces monocytes to generate EVs, which could potentially act as mediators of cell-to-cell communication.