Phagocytic clearance of dying cells and its implications

It is estimated that an average adult human turns over roughly 330 ± 20 billion cells every day as part of healthy living.^{1, 2} This translates to 0.4% of our body mass. Such a large number for cell turnover then begs the question—what are these cells and why? The reasons for this are multi-factorial. First, there are cells in the body that have a finite life span, such as neutrophils (~1 day) and erythrocytes (~120 days), and there are also other cell types such as many hematopoietic cells that have a life span of a few days to few weeks; these need to be removed after their useful life span and replaced by new cells. Second, there are many aspects of development where we generate excess cells, of which only a few are deemed fit to progress to full maturation, and the rest undergo death and need to be removed; examples of this include development of T cells in the thymus, B cells in the bone marrow, and also adult neurogenesis in the brain.¹ Third, there are also “damaged” cells that emerge daily in the body, such as due to light/UV damage, for example skin and photoreceptors of the eye.³ Thus, all these turnover events result in a large number of cells undergoing death essentially in all organs and tissues, albeit at different magnitude.² Although there are many different forms of death processes, the cells that are destined die via homeostatic turnover do so primarily via the process of caspase-dependent apoptosis.⁴

What happens to these dying cells? Despite the billions of dying cells per day, when one looks at tissues, it is hard to recognize dying cells, even in those with high cellular turnover. This is because the recognition and clearance of dying cells is remarkably efficient.^{5, 6} Just like there is a dedicated set of molecules and mechanisms to induce programmed cell death, we also possess a dedicated machinery to recognize and remove these dying cells.⁷ Such clearance under homeostasis conditions occurs quickly, efficiently, and from an immunological perspective, quietly.⁸ It is worth noting that just like the apoptotic cell death machinery, the clearance processes are also highly conserved evolutionarily, and studies from the nematode, flies, zebrafish, mice, and humans have established the conserved components of the clearance process.^{9, 10} This volume of Immunological Reviews focuses on different aspects of the cell clearance process and its implications to homeostasis and disease.

While there are different forms of phagocytosis, the recognition and clearance of apoptotic cells by phagocytes has been termed “efferocytosis,” a term originally coined by Dr. Peter Henson. (where “effero” means “carry to the grave”).¹¹ This should be distinguished from Fc receptor mediated phagocytosis or complement receptor-mediated phagocytosis, which involves opsonization of target cells by specific ligands and uptake via the respective receptors. A key distinction from the other forms of phagocytosis is that the apoptotic cell clearance does not induce an immune response; further efferocytosis is also actively anti-inflammatory.⁸ This makes sense when one considers the fact that if we were to induce an inflammatory response to billions of cells that we clear every day, we may all end up as walking bags of inflammation. However, the failure to clear the apoptotic cells promptly can induce secondary necrosis that can lead to inflammatory sequelae, as detailed by some of the chapters in this volume.

Works from a number of laboratories have now established that there are different steps to recognition and removal of dying cells.^{1, 12} The first step is the recognition of the dying cells. When phagocytes such as macrophages, dendritic cells, or healthy neighbors are in close proximity to the dying cell, specific molecules on the apoptotic cells are recognized by specific receptors on the engulfing cell leading to subsequent intracellular signaling, cytoskeletal reorganization, and corpse internalization.¹³ While there are many ligands exposed on cells undergoing apoptosis, the best detailed is the exposure of the phospholipid phosphatidylserine (PtdSer).^{14, 15} PtdSer is normally kept via an active and energy dependent process on the inner leaflet but gets exposed on the outer leaflet as part of the apoptotic process.^{15, 16} This PtdSer in turn, gets recognized by multiple PtdSer recognition receptors—either directly, or indirectly, via intermediary bridging molecules.^{16, 17} As part of the first chapter, Dr. Tal Brustyn-Cohen details one of the best described receptor families linked to clearance of apoptotic cells, namely the TAM receptors.¹⁸ These TAM receptors recognize PtdSer indirectly via Gas6 or Protein S that bind PtdSer, and this chapter details the role of TAM receptors in different contexts and highlights their role in other physiology.

One of the challenges in studying cell clearance in the mammals is that multiple homologues for engulfment receptors and signaling molecules, and the complexities that arise when individual knockouts do not often have a clear phenotype. Thus, defining the function of individual molecules, as well as visualizing cell death and clearance in vivo have been a challenge for the past couple of decades. Will Wood, Andrew Davidson, and colleagues detail the beautiful model systems for cell death and efferocytosis in the fruit fly Drosophila that have provided many new insights.¹⁹ They also detail new approaches that have been developed to visualize apoptosis and efferocytosis in vivo, plus the different molecular mechanisms of clearance employed by Drosophila macrophages to clear dying cells.

After a phagocyte engulfs an apoptotic cell, a second challenge ensues—that is, digesting the corpse. This is no small feat, as this involves digesting another cell that is often nearly the same size as the phagocyte itself. Further, many phagocytes engulf multiple apoptotic cells.²⁰ Mylvaganam and Freeman take a comprehensive approach to how a phagocyte resolves a phagolysosome as well as aspects of the membrane traffic, and the role of solute carriers in managing some of the contents of the corpse. They also put this in disease contexts with lysosomal storage disorders.²¹

Another inherent challenge that the phagocyte faces is how to handle all the excess biomass. To put it another way, when a phagocyte engulfs an apoptotic cell, it basically doubles its lipids, carbohydrates, and proteins, to name a few of the corpse contents. Further, phagocytes such as macrophages ingest multiple corpses in succession, leading to even greater challenge of dealing with all this excess metabolic overload.^{20, 22} Shilperoort, Tabas, and colleagues beautifully detail the many aspects of this macrophage immunometabolism.²³ They detail how amino acids such as arginine and methionine and their subsequent conversion within phagocytes impact continued uptake of additional corpses, macrophage responses, and in turn, disease processes. The authors also detail lactate regulation in macrophage responses, relevance to human disease such as atherosclerosis, and limitations to the current studies.

While phagocytes such as macrophages get a lot of attention as “professional phagocytes” capable of engulfing many corpses, there are also nonprofessional phagocytes. Although these phagocytes may do cell clearance with slower kinetics than macrophages, they play an important role in routine clearance of the many cells in the body. The retinal pigmented epithelial cells (RPE) of the retina provide a beautiful example, as they clear on a daily basis the “used” photoreceptors that are damaged from light during the day and they need to be removed to allow new photoreceptors to take their place.^{24, 25} Another beautiful aspect of the RPE is that they are postmitotic, and we are born and die with the same number of RPE. This means that the RPE cells do the clearance throughout the lifetime, in addition to their many nurse cell functions for the photoreceptors to maintain a healthy retina. Silvia Finnemann and colleagues detail the background on clearance by RPE cells, the receptors, and mechanisms of RPE-mediated clearance, as well as diseases that arise when this clearance is disturbed and lead to retinal inflammation.²⁶

Just like we do not fully appreciate the importance of garbage workers until they go on strike, the importance of the “cellular clearance crew” has gotten much better appreciation in the past two decades when failures in clearance, or complexities associated with cell death and cell clearance contribute to inflammatory diseases or links to cancer.⁸ This is detailed in four of the final chapters of this volume. First, Christopher Gregory details beautifully the complexity of cell death in the cancer context.²⁷ He details how apoptotic cells and their products (including extracellular vesicles and other factors released by the dying cells) regulate the tumor microenvironment; this includes how responses of the macrophages within solid tumors, either due to direct contact with the apoptotic cells or their released products, lead to reshaping the tumor microenvironment for tumor growth. This is followed by a detailed description by Wagoner, Michael Elliott, and colleagues on the antibody-mediated phagocytosis of tumor cells—which occurs when an antibody-bound tumor cell is recognized via Fc receptors, primarily by macrophages.²⁸ The authors also present challenges and modifications that occurs to the phagocytes as part of the FcR-mediated phagocytosis, and some approaches to overcome them.

It has become increasingly clear that many auto-inflammatory diseases such as atherosclerosis, arthritis, and certain forms of colitis have a component of defective or minimally the release of certain components from the late stage dying cells, that promote a pro-inflammatory milieu, and in turn, chronic inflammation.¹ Further, if some of the self-antigens are presented in this pro-inflammatory environment, this can evolve to autoimmunity. Schneider and Arandjelovic detail the inflammatory components of arthritis. Interestingly, some of the components of the engulfment machinery has additional roles, such as in neutrophil migration to the arthritic joints that in turn can also contribute to arthritis.²⁹ Lastly, Gabrielle Fredman and Sayeed Khan discuss the role of specialized pro-resolving mediators (SPMs) in clearance of dead cells.³⁰ They highlight the role of SPMs in facilitating clearance of not only apoptotic cells but also necroptotic cells, and further link these to non-resolving diseases such as atherosclerosis.

In sum, in the past couple of decades of investigations on how cells die, how they are removed, and the consequences of such regulated and efficient phagocytosis to homeostasis have exploded. This has led to a remarkable increase in knowledge on the molecules and mechanisms, and how defects in clearance contribute to disease states in specific tissue contexts. In this collection of reviews, the authors not only highlight contributions from their own laboratories, but they also put these discoveries in the larger context of what is known, the challenges, and how to go about addressing the next set of questions in the field. The research on cellular turnover, with a role in essentially every single tissue, is bound to continue, with modulating phagocytosis providing opportunities for treating multiple diseases.³¹

The author declares no competing interests.