Apoptosis, the original regulated cell death programme, is an established controller of cell populations in all tissues. The hub of the apoptosis machinery, the caspase cascade, mediates the cleavage of hundreds of protein substrates to ultimately effect disabling and dismantling of cells, deleting them in a cell-autonomous fashion that is well understood. Of lesser renown are the intercellular communicative properties of apoptotic cells which, as burgeoning evidence indicates, compound the signalling processes that culminate in the famously speedy, and most commonly non-phlogistic, phagocytic clearance of dying/dead cells and their fragments (efferocytosis) by macrophages and other efferocytes. Here, we discuss more broadly the curious variety of instructive intercellular signals that apoptotic cells can deploy in order to reprogramme their living neighbours, be they close or distant, efferocytes or not. We review the literature, and we consider conceptually the potential of such non-cell-autonomous, apoptotic cell-dependent communication (ACDC) mechanisms to profoundly impact cell populations in health and disease. We illustrate our discourse firstly with the biology of lymphoid germinal centres (GC) and their constituent efferocytic, 'tingible body' macrophages, and secondly with the pathology of malignancies in which counterpart efferocytic, 'starry sky' macrophages feature prominently. In conclusion, we suggest several key avenues in ACDC research.

Introduction: Erythroblastic island (EBI) macrophages play an essential role in the production and maturation of the vast numbers of red blood cells (RBCs) that are produced throughout life. Their location within the bone marrow makes it difficult to study the cellular and molecular interactions associated with their action so we have used an in vitro model of the EBI niche using macrophages derived from human induced pluripotent stem cells (hiPSCs). We previously demonstrated that the activation of the transcription factor KLF1 enhanced the activity of hiPSC-derived EBI macrophages.Methods: To elucidate the mechanisms associated with EBI-like activity we carried out a quantitative proteomic analysis and assessed the role of extracellular vesicles using Nanosight Tracking analyses and media filtration.Results and Discussion: Gene ontology analysis showed that many of the proteins upregulated by KLF1 were protein-binding factors, some of which were associated with the cell membrane or extracellular vesicles We demonstrated that filtration of macrophage-conditioned media resulted in a reduction in the supportive effects on erythroid cell viability and maturation implying a role for extracellular vesicles but this was not KLF1 dependent. Pathway analyses of the proteomic data revealed that proteins upregulated by KLF1 were associated with the citric acid cycle, pyruvate metabolism and ATP synthesis indicating that KLF1-activated macrophages had a metabolic profile comparable to a pro-reparative phenotype. This study has generated a proteomic dataset that could provide new insights into the role of macrophages within the EBI niche and has indicated a potential role for extracellular vesicles in the differentiation and maturation of RBCs in vitro. Further research will aid in the production of RBCs in vitro for use in disease modelling and cell therapy.

Extracellular vesicles (EVs) are lipid bilayer‐enclosed subcellular bodies produced by most, if not all cells. Research over the last two decades has recognised the importance of EVs in intercellular communication and horizontal transfer of biological material. EVs range in diameter from tens of nanometres up to several micrometres and are able to transfer a spectrum of biologically active cargoes – from whole organelles, through macromolecules including nucleic acids and proteins, to metabolites and small molecules – from their cells of origin to recipient cells, which may consequently become physiologically or pathologically altered. Based on their modes of biogenesis, the most renowned EV classes are (1) microvesicles, (2) exosomes (both produced by healthy cells), and (3) EVs from cells undergoing regulated death by apoptosis (ApoEVs). Microvesicles bud directly from the plasma membrane, while exosomes are derived from endosomal compartments. Current knowledge of the formation and functional properties of ApoEVs lags behind that of microvesicles and exosomes, but burgeoning evidence indicates that ApoEVs carry manifold cargoes, including mitochondria, ribosomes, DNA, RNAs, and proteins, and perform diverse functions in health and disease. Here we review this evidence, which demonstrates substantial diversity in the luminal and surface membrane cargoes of ApoEVs, permitted by their very broad size range (from around 50 nm to >5 μm; the larger often termed apoptotic bodies), strongly suggests their origins through both microvesicle‐ and exosome‐like biogenesis pathways, and indicates routes through which they interact with recipient cells. We discuss the capacity of ApoEVs to recycle cargoes and modulate inflammatory, immunological, and cell fate programmes in normal physiology and in pathological scenarios such as cancer and atherosclerosis. Finally, we provide a perspective on clinical applications of ApoEVs in diagnostics and therapeutics. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Objective To review the role of extracellular vesicles (EVs) released from the male reproductive tract and their impact on developing sperm. We discuss how sperm exiting the seminiferous tubules, although developmentally mature, require further modification. Acquisition of various functions including increased motility, transfer of cargoes and ability to undertake the acrosome reaction is mediated through the interaction between sperm and EVs. Methods A review of the literature identified that EVs are released from different portions of the male reproductive tract, notably the epididymis and prostate. These EVs interact with sperm as they pass from the seminiferous tubules to the epididymis and vas deferens prior to ejaculation. Results EVs are small lipid-bound particles carrying bespoke RNA, protein and lipid cargoes. These cargoes are loaded based on the state of the parent cell and are used to communicate with recipient cells. In sperm, these cargoes are essential for post-testicular modification. Conclusions Interactions between developing sperm and EVs are important for the subsequent function of sperm. Prior to ejaculation, these interactions confer important changes for the post-testicular modification and development of sperm. Little is known about the interaction between EVs from the testes and the spermatogonial stem cell niche or developing sperm within the seminiferous tubules. However, the numerous roles of EVs in the post-testicular modification of sperm have led many to suspect that they may also play important roles in developing sperm within the testes. Lay summary Sperm are crucial for successful fertility. In order to do this, they must be able to swim a large distance to meet the egg in the female reproductive tract and fertilise it. Once released from the testes, sperm may appear to be fully developed, but this is not the case. Several important modifications are required in order for them to swim and fertilise an egg. These modifications are carried out by sending sperm small packages from other cells which contain messages and cargo. We discuss the release of these small packages along with different parts of the male reproductive tract and how they change the way sperm behave and function. This article reviews the literature and known functions of these packages called extracellular vesicles, which are released by the male reproductive tract and modify sperm, transforming their function, before they are ejaculated.

Extracellular vesicles (EVs) are small lipid bound structures released from cells containing bioactive cargoes. Both the type of cargo and amount loaded varies compared to that of the parent cell. The characterisation of EVs in cancers of the male urogenital tract has identified several cargoes with promising diagnostic and disease monitoring potential. EVs released by cancers of the male urogenital tract promote cell-to-cell communication, migration, cancer progression and manipulate the immune system promoting metastasis by evading the immune response. Their use as diagnostic biomarkers represents a new area of screening and disease detection, potentially reducing the need for invasive biopsies. Many validated EV cargoes have been found to have superior sensitivity and specificity than current diagnostic tools currently in use. The use of EVs to improve disease monitoring and develop novel therapeutics will enable clinicians to individualise patient management in the exciting era of personalised medicine.

The term apoptosis describes a series of gross cellular changes characteristic of an active or programmed form of cell death that is now accepted as a physiological, as opposed to accidental, mode of cell deletion. Active cell death represents a normal control mechanism as profoundly important to multicellular organisms as cell proliferation or differentiation. In mammals, the significance of physiological cell death is illustrated by the removal of cells through activation of their apoptotic mechanism during tissue modeling and organogenesis in embryonic development, and in the control of homeostasis in a diversity of tissue types [1]. There is much evidence to support the view that programmed cell death is a cell-autonomous process [2]. It has also been suggested that, with few exceptions, all cells from all lineages are programmed to undergo active self-destruction unless they receive survival signals to suppress that program [3].

Extracellular vesicles (EVs) have numerous potential applications in the field of healthcare and diagnostics, and research into their biological functions is rapidly increasing. Mainly because of their small size and heterogeneity, there are significant challenges associated with their analysis and despite overt evidence of the potential of EVs in clinical diagnostic practice, guidelines for analytical procedures have not yet been properly established. Here, we present an overview of the main methods for studying the properties of EVs based on the principles of fluorescence. Setting aside the isolation, purification and physicochemical characterization strategies which answer questions about the size, surface charge and stability of EVs (reviewed elsewhere), we focus on available optical tools that enable the direct analysis of phenotype and mechanisms of interaction with tissues. In brief, the topics on which we elaborate range from the most popular approaches such as nanoparticle tracking analysis and flow cytometry, to less commonly used techniques such as fluorescence depolarization and microarrays as well as emerging areas such as fast fluorescence lifetime imaging microscopy (FLIM). We highlight that understanding the strengths and limitations of each method is essential for choosing the most appropriate combination of analytical tools. Finally, future directions of this rapidly developing area of medical diagnostics are discussed.

Apoptosis is the process by which single cells die in the midst of living tissues. It is responsible for most—perhaps all—of the cell-death events that occur during the formation of the early embryo and the sculpting and moulding of organs. Apoptotic cell death continues to play a critical role in the maintenance of cell numbers in those tissues in which cell turnover persists into adult life, such as the epithelium of the gastrointestinal tract, the bone marrow, and lymphoid system including both B- and T-cell lineages. Apoptosis is the usual mode of death in the targets of natural killer (NK) cells and cytotoxic T-cells, and in involution and atrophy induced by hormonal and other stimuli. It also appears in the reaction of many tissues to injury, including mild degrees of ischaemia, exposure to ionizing and ultraviolet radiation, or treatment with cancer chemotherapeutic drugs. Excessive or too little apoptosis play a significant part in the pathogenesis of autoimmunity, infectious disease, AIDS, stroke, myocardial disease, and cancer. When cancers regress, apoptosis is part of the mechanism involved. Here the cellular processes and molecular mechanisms of apoptosis are set out, together with a conspectus of its involvement in many diseases....

The disposal of apoptotic bodies by professional phagocytes is crucial to effective inflammation resolution. Our ability to improve the disposal of apoptotic bodies by professional phagocytes is impaired by a limited understanding of the molecular mechanisms that regulate the engulfment and digestion of the efferocytic cargo. Macrophages are professional phagocytes necessary for liver inflammation, fibrosis, and resolution, switching their phenotype from proinflammatory to restorative. Using sterile liver injury models, we show that the STAT3-IL-10-IL-6 axis is a positive regulator of macrophage efferocytosis, survival, and phenotypic conversion, directly linking debris engulfment to tissue repair.

The cell-death programme, apoptosis, is well established as a tumour suppressor mechanism. Paradoxically, high levels of apoptosis in tumours are closely coupled with poor prognosis. Indeed, where it has been studied, cell loss is a striking feature of high-grade cancers, illustrating the importance of considering malignant disease as an imbalance between cell gain and cell loss that favours cell gain rather than as a unidirectional disorder of cell gain alone. In addition to orchestrating cell loss, apoptosis can signal regenerative responses-for example compensatory proliferation-in neighbouring cells. Accumulating evidence suggests that normal tissue repair and regenerative processes are hijacked in the malignant tissue microenvironment such that cancer may be likened to a 'wound that fails to stop repairing'. We have proposed that a critical requirement for the successful growth, progression and re-growth of malignant tumours is a complex milieu, conceptually termed the 'onco-regenerative niche', which is composed, in addition to transformed neoplastic cells, of a network of normal cells and factors activated as if in tissue repair and regeneration. Our work is based around the hypothesis that tumour cell apoptosis, macrophage activation and endothelial activation are key, interlinked elements of the onco-regenerative niche and that apoptotic tumour cell-derived extracellular vesicles provide critical intercellular communication vehicles of the niche. In aggressive B-cell lymphoma, tumour cell apoptosis promotes both angiogenesis and the accumulation of pro-tumour macrophages in the lymphoma microenvironment. Furthermore, apoptotic lymphoma-derived extracellular vesicles have potent pro-tumour potential. These findings have important implications for the roles of apoptosis in regulation of malignant diseases and for the efficacy of apoptosis-inducing anti-cancer therapies.This article is part of the discussion meeting issue 'Extracellular vesicles and the tumour microenvironment'.

This book discusses properties of apoptosis and other cell death modalities in cancer pathogenesis and treatment. Its nine chapters discuss modulation of anti-tumor inflammatory and immune responses,

Although apoptosis is well recognized as a cell death program with clear anticancer roles, accumulating evidence linking apoptosis with tissue repair and regeneration indicates that its relationship with malignant disease is more complex than previously thought. Here we review how the responses of neighboring cells in the microenvironment of apoptotic tumor cells may contribute to the cell birth/cell death disequilibrium that provides the basis for cancerous tissue emergence and growth. We describe the bioactive properties of apoptotic cells and consider, in particular, how apoptosis of tumor cells can engender a range of responses including pro-oncogenic signals having proliferative, angiogenic, reparatory, and immunosuppressive features. Drawing on the parallels between wound healing, tissue regeneration and cancer, we propose the concept of the "onco-regenerative niche," a cell death-driven generic network of tissue repair and regenerative mechanisms that are hijacked in cancer. Finally, we consider how the responses to cell death in tumors can be targeted to provide more effective and long-lasting therapies.

Christopher D. Gregory and Zsuzsa SzondyZsolt Sarang, Gergely Joos, Eva Garabuczi, Ralph Ruhl,http://www.jimmunol.org/content/192/12/5730doi: 10.4049/jimmunol.1400284May 2014;J Immunol€2014; 192:5730-5738; Prepublished online 21Referenceshttp://www.jimmunol.org/content/192/12/5730.full#ref-list-1This article cites 53 articles, 20 of which you can access for free at: Subscriptionshttp://jimmunol.org/subscriptionsInformation about subscribing to The Journal of Immunology is online at: Permissionshttp://www.aai.org/ji/copyright.htmlSubmit copyright permission requests at: Email Alertshttp://jimmunol.org/cgi/alerts/etocReceive free email-alerts when new articles cite this article. Sign up at:

Induction of apoptosis is one of the main defenses of the body against cells that have acquired malicious mutations. It may seem counter-intuitive then, that massive cell death is observed in many malignant tumors (1, 2). Despite high rates of apoptosis, these tumors continue to grow rapidly. Thus, tumor cell growth must outbalance tumor cell death. Intuitively presumed only to inhibit tumor growth, apoptotic cells may actually promote net tumor growth (3, 4). As long ago as 1956, Revesz showed that cell death can enhance tumor growth (5). Moreover, new studies in progress in our laboratory show that apoptosis in tumor cells promotes growth rates in aggressive B-cell lymphoma. Cells undergoing apoptosis are difficult to observe in vivo, as they are rapidly cleared by phagocytosis, most obviously by macrophages. Accumulation of macrophages, sometimes engorged with apoptotic cells, is observed in many malignant tumors and is generally associated with poor prognosis (6, 7). Inflammatory cells, in particular macrophages, are key elements of the tumor environment, providing support for the continually expanding “rogue” tissue. The tumor microenvironment resembles that of a wound that fails to heal (8), where macrophages not only clear and repair, but also promote tissue regeneration and support. Tumor-associated macrophages (TAM) display a phenotype that is reminiscent of wound-healing macrophages. They have been shown to promote angiogenesis, tissue remodeling, and anti-inflammatory responses, which results in the support of tumor cell growth and metastasis (9–12). Apoptosis of tumor cells in a growing malignant tissue may therefore be rationalized as a “sinister sacrifice” of some cancer cells that ultimately facilitates cancer progression. We hypothesize that apoptotic cells play a key role in driving oncogenesis, both through the release of soluble and microvesicle-associated signaling factors, as well as through direct interaction with phagocytes. Here we postulate lactoferrin (Lf) as an important signaling factor maintaining an anti-inflammatory tumor microenvironment, and stress the importance of apoptotic cell engulfment by macrophages for driving a pro-tumor phenotype in TAM (Figure ​(Figure11). Figure 1 Tumor cell apoptosis potentially activates multiple oncogenic pathways, promoting tumor cell growth and survival, angiogenesis, remodeling, and metastasis, while inhibiting anti-tumor immune responses. We propose that TAM interacting with apoptotic tumor ...

It is well known that an important property of apoptosis is the prevention of cancer. However, it is becoming increasingly clear that apoptotic cells have the capacity to activate reparative and regenerative responses, which have the potential to make significant impact on pathological processes underlying autoimmune disease and cancer. Those properties of apoptotic cells that influence their immediate microenvironment are reviewed here in the context of cancer progression and treatment.

Embryonic stem cells provide a potentially convenient source of macrophages in the laboratory. Given the propensity of macrophages for plasticity in phenotype and function, standardised culture and differentiation protocols are required to ensure consistency in population output and activity in functional assays. Here we detail the development of an optimised culture protocol for the production of murine embryonic stem cell-derived macrophages (ESDM). This protocol provides improved yields of ESDM and we demonstrate that the cells are suitable for application to the study of macrophage responses to apoptotic cells. ESDM so produced were of higher purity than commonly used primary macrophage preparations and were functional in chemotaxis assays and in phagocytosis of apoptotic cells. Maturation of ESDM was found to be associated with reduced capacity for directed migration and increased capacity for phagocytic clearance of apoptotic cells. These results show ESDM to be functionally active in sequential phases of interaction with apoptotic cells and establish these macrophage populations as useful models for further study of molecular mechanisms underlying the recognition and removal of apoptotic cells.

Here we consider the impact of the physiological cell-death programme on normal tissue homeostasis and on disease pathogenesis, with particular reference to evolution and progression of neoplasia. We seek to describe the direct contributions played by apoptosis in creating the microenvironments of normal and malignant tissues and to discuss the molecular mechanisms underlying the elements of the '3Rs' that define the meaning of apoptosis: recognition, response, and removal. Apoptotic cells elicit responses in other cell types-both phagocytic and non-phagocytic-through short- and long-range signalling modes that range from direct contact to intercellular communication via membrane-bound microparticles. Such cellular responses include migration, proliferation, and differentiation, as well as production of immunomodulatory and anti-inflammatory mediators together with, in the case of phagocytes, engulfment, and breakdown of apoptotic cells. In normal tissues, the removal of apoptotic cells is rapid and typically non-phlogistic. We discuss the importance of this clearance process in tissue homeostasis and the consequences of its failure in disease pathogenesis. Using the typical cell culture environment in vitro as an illustrative example in which apoptosis occurs commonly in the absence of the removal mechanisms, we also discuss the inhibitory effects of persistent apoptotic cells on their otherwise viable neighbours. Since apoptosis is a common and sustained event in high-grade malignancies, we hypothesize on its purposeful role in conditioning the tumour microenvironment. We propose that apoptosis subserves several pro-tumour functions-trophic, anti-inflammatory, and immunomodulatory-and we identify strategies targeting host responses to apoptotic cells as promising modes of future therapies that could be applied to multiple cancer types in which tumour-cell apoptosis is active. Copyright. (C) 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.