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Macrophage antibody (Ab)-dependent cellular phagocytosis (ADCP) is a major cytotoxic mechanism for both therapeutic unconjugated monoclonal Abs (mAbs) such as rituximab and Ab-induced hemolytic anemia and immune thrombocytopenia. Here, we studied the mechanisms controlling the rate and capacity of macrophages to carry out ADCP in settings of high target/effector cell ratios, such as those seen in patients with circulating tumor burden in leukemic phase disease. Using quantitative live-cell imaging of primary human and mouse macrophages, we found that, upon initial challenge with mAb-opsonized lymphocytes, macrophages underwent a brief burst (<1 hour) of rapid phagocytosis, which was then invariably followed by a sharp reduction in phagocytic activity that could persist for days. This previously unknown refractory period of ADCP, or hypophagia, was observed in all macrophage, mAb, and target cell conditions tested in vitro and was also seen in vivo in Kupffer cells from mice induced to undergo successive rounds of αCD20 mAb-dependent clearance of circulating B cells. Importantly, hypophagia had no effect on Ab-independent phagocytosis and did not alter macrophage viability. In mechanistic studies, we found that the rapid loss of activating Fc receptors from the surface and their subsequent proteolytic degradation were the primary mechanisms responsible for the loss of ADCP activity in hypophagia. These data suggest hypophagia is a critical limiting step in macrophage-mediated clearance of cells via ADCP, and understanding such limitations to innate immune system cytotoxic capacity will aid in the development of mAb regimens that could optimize ADCP and improve patient outcome., (© 2020 by The American Society of Hematology.)

Phagocytosis is a dynamic process central to immunity and tissue homeostasis. Current methods for quantification of phagocytosis largely rely on indirect or static measurements, such as target clearance or dye uptake, and thus provide limited information about engulfment rates or target processing. Improved kinetic measurements of phagocytosis could provide useful, basic insights in many areas. We present a live-cell, time-lapse and high-content microscopy imaging method based on the detection and quantification of fluorescent dye 'voids' within phagocytes that result from target internalization to quantify phagocytic events with high temporal resolution. Using this method, we measure target cell densities and antibody concentrations needed for optimal antibody-dependent cellular phagocytosis. We compare void formation and dye uptake methods for phagocytosis detection, and examine the connection between target cell engulfment and phagolysosomal processing. We demonstrate how this approach can be used to measure distinct forms of phagocytosis, and changes in macrophage morphology during phagocytosis related to both engulfment and target degradation. Our results provide a high-resolution method for quantifying phagocytosis that provides opportunities to better understand the cellular and molecular regulation of this fundamental biological process., Competing Interests: Competing interestsThe authors declare no conflict of interest. While we thank our funding sources for their support, none of these organizations, including Acerta Pharma LLC, contributed to the concept or design of this project, nor did they contribute to the data acquisition or data analysis presented here., (© 2020. Published by The Company of Biologists Ltd.)

Rapid and efficient removal of apoptotic cells by phagocytes is important during development, tissue homeostasis and in immune responses. Efficient clearance depends on the capacity of a single phagocyte to ingest multiple apoptotic cells successively, and to process the corpse-derived cellular material. However, the factors that influence continued clearance by phagocytes are not known. Here we show that the mitochondrial membrane potential of the phagocyte critically controls engulfment capacity, with lower potential enhancing engulfment and vice versa. The mitochondrial membrane protein Ucp2, which acts to lower the mitochondrial membrane potential, was upregulated in phagocytes engulfing apoptotic cells. Loss of Ucp2 reduced phagocytic capacity, whereas Ucp2 overexpression enhanced engulfment. Mutational and pharmacological studies indicated a direct role for Ucp2-mediated mitochondrial function in phagocytosis. Macrophages from Ucp2-deficient mice were impaired in phagocytosis in vitro, and Ucp2-deficient mice showed profound in vivo defects in clearing dying cells in the thymus and testes. Collectively, these data indicate that mitochondrial membrane potential and Ucp2 are key molecular determinants of apoptotic cell clearance. As Ucp2 is linked to metabolic diseases and atherosclerosis, this newly discovered role for Ucp2 in apoptotic cell clearance has implications for the complex aetiology and pathogenesis of these diseases.

Whereas thousands of new neurons are generated daily during adult life, only a fraction of them survive and become part of neural circuits; the rest die, and their corpses are presumably cleared by resident phagocytes. How the dying neurons are removed and how such clearance influences neurogenesis are not well understood. Here, we identify an unexpected phagocytic role for the doublecortin (DCX)-positive neuronal progenitor cells during adult neurogenesis. Our in vivo and ex vivo studies demonstrate that DCX(+) cells comprise a significant phagocytic population within the neurogenic zones. Intracellular engulfment protein ELMO1, which promotes Rac activation downstream of phagocytic receptors, was required for phagocytosis by DCX(+) cells. Disruption of engulfment in vivo genetically (in Elmo1-null mice) or pharmacologically (in wild-type mice) led to reduced uptake by DCX(+) cells, accumulation of apoptotic nuclei in the neurogenic niches and impaired neurogenesis. Collectively, these findings indicate a paradigm wherein DCX(+) neuronal precursors also serve as phagocytes, and that their phagocytic activity critically contributes to neurogenesis in the adult brain.

Phagocytic removal of apoptotic cells occurs efficiently in vivo such that even in tissues with significant apoptosis, very few apoptotic cells are detectable. This is thought to be due to the release of 'find-me' signals by apoptotic cells that recruit motile phagocytes such as monocytes, macrophages and dendritic cells, leading to the prompt clearance of the dying cells. However, the identity and in vivo relevance of such find-me signals are not well understood. Here, through several lines of evidence, we identify extracellular nucleotides as a critical apoptotic cell find-me signal. We demonstrate the caspase-dependent release of ATP and UTP (in equimolar quantities) during the early stages of apoptosis by primary thymocytes and cell lines. Purified nucleotides at these concentrations were sufficient to induce monocyte recruitment comparable to that of apoptotic cell supernatants. Enzymatic removal of ATP and UTP (by apyrase or the expression of ectopic CD39) abrogated the ability of apoptotic cell supernatants to recruit monocytes in vitro and in vivo. We then identified the ATP/UTP receptor P2Y(2) as a critical sensor of nucleotides released by apoptotic cells using RNA interference-mediated depletion studies in monocytes, and macrophages from P2Y(2)-null mice. The relevance of nucleotides in apoptotic cell clearance in vivo was revealed by two approaches. First, in a murine air-pouch model, apoptotic cell supernatants induced a threefold greater recruitment of monocytes and macrophages than supernatants from healthy cells did; this recruitment was abolished by depletion of nucleotides and was significantly decreased in P2Y(2)(-/-) (also known as P2ry2(-/-)) mice. Second, clearance of apoptotic thymocytes was significantly impaired by either depletion of nucleotides or interference with P2Y receptor function (by pharmacological inhibition or in P2Y(2)(-/-) mice). These results identify nucleotides as a critical find-me cue released by apoptotic cells to promote P2Y(2)-dependent recruitment of phagocytes, and provide evidence for a clear relationship between a find-me signal and efficient corpse clearance in vivo.

Engulfment of apoptotic cells by phagocytes is important throughout development and adult life. When phagocytes engulf apoptotic cells, they increase their cellular contents including cholesterol and phospholipids, but how the phagocytes respond to this increased load is poorly understood. Here, we identify one type of a phagocyte response, wherein the recognition of apoptotic cells triggers enhanced cholesterol efflux (to apolipoprotein A-I) from macrophages. Phosphatidylserine (PS) exposed on apoptotic cells was necessary and sufficient to stimulate the efflux response. A major mechanism for this enhanced efflux by macrophages was the upregulation of the mRNA and protein for ABCA1, a membrane transporter independently linked to cholesterol efflux as well as engulfment of apoptotic cells. This increase in phagocyte ABCA1 levels required the function of nuclear receptor LXRalpha/beta, a known regulator of cholesterol homeostasis in humans and mice. Taken together, these data reveal a "homeostatic program" initiated in phagocytes that include a proximal membrane signaling event initiated by PS recognition, a downstream signaling event acting through nuclear receptors, and an effector arm involving upregulation of ABCA1, in turn promoting reverse cholesterol transport from the phagocytes. These data also have implications for macrophage handling of contents derived from apoptotic versus necrotic cells in atherosclerotic lesions.

Since the pioneering work of Elie Metchnikoff and the discovery of cellular immunity, the phagocytic clearance of cellular debris has been considered an integral component of resolving inflammation and restoring function of damaged and infected tissues. We now know that the phagocytic clearance of dying cells (efferocytosis), particularly bymacrophages and other immune phagocytes, has profound consequences on innate and adaptive immune responses in inflamed tissues. These immunomodulatory effects result from an array of molecular signaling events between macrophages, dying cells, and other tissue-resident cells. In recent years, many of these molecular pathways have been identified and studied in the context of tissue inflammation, helping us better understand the relationship between efferocytosis and inflammation. We review specific types of efferocytosis-related signals that can impact macrophage immune responses and discuss their relevance to inflammation-related diseases. [ABSTRACT FROM AUTHOR]

The phagocytic clearance of dying cells in a tissue is a highly orchestrated series of intercellular events coordinated by a complex signaling network. Recent data from genetic, biochemical, and live-imaging approaches have greatly enhanced our understanding of the dynamics of cell clearance and how the process is orchestrated at the cellular and tissue levels. We discuss how networks regulating apoptotic cell clearance are integrated to enable a rapid, efficient, and high-capacity clearance system within tissues. [ABSTRACT FROM AUTHOR]

Apoptotic cells release 'find-me' signals at the earliest stages of death to recruit phagocytes. The nucleotides ATP and UTP represent one class of find-me signals, but their mechanism of release is not known. Here, we identify the plasma membrane channel pannexin 1 (PANX1) as a mediator of find-me signal/nucleotide release from apoptotic cells. Pharmacological inhibition and siRNA-mediated knockdown of PANX1 led to decreased nucleotide release and monocyte recruitment by apoptotic cells. Conversely, PANX1 overexpression enhanced nucleotide release from apoptotic cells and phagocyte recruitment. Patch-clamp recordings showed that PANX1 was basally inactive, and that induction of PANX1 currents occurred only during apoptosis. Mechanistically, PANX1 itself was a target of effector caspases (caspases 3 and 7), and a specific caspase-cleavage site within PANX1 was essential for PANX1 function during apoptosis. Expression of truncated PANX1 (at the putative caspase cleavage site) resulted in a constitutively open channel. PANX1 was also important for the 'selective' plasma membrane permeability of early apoptotic cells to specific dyes. Collectively, these data identify PANX1 as a plasma membrane channel mediating the regulated release of find-me signals and selective plasma membrane permeability during apoptosis, and a new mechanism of PANX1 activation by caspases. [ABSTRACT FROM AUTHOR]

Engulfment and subsequent degradation of apoptotic cells is an essential step that occurs throughout life in all multicellular organisms. ELMO/Dock180/Rac proteins are a conserved signalling module for promoting the internalization of apoptotic cell corpses; ELMO and Dock180 function together as a guanine nucleotide exchange factor (GEF) for the small GTPase Rac, and thereby regulate the phagocyte actin cytoskeleton during engulfment. However, the receptor(s) upstream of the ELMO/Dock180/Rac module are still unknown. Here we identify brain-specific angiogenesis inhibitor 1 (BAI1) as a receptor upstream of ELMO and as a receptor that can bind phosphatidylserine on apoptotic cells. BAI1 is a seven-transmembrane protein belonging to the adhesion-type G-protein-coupled receptor family, with an extended extracellular region and no known ligands. We show that BAI1 functions as an engulfment receptor in both the recognition and subsequent internalization of apoptotic cells. Through multiple lines of investigation, we identify phosphatidylserine, a key ‘eat-me’ signal exposed on apoptotic cells, as a ligand for BAI1. The thrombospondin type 1 repeats within the extracellular region of BAI1 mediate direct binding to phosphatidylserine. As with intracellular signalling, BAI1 forms a trimeric complex with ELMO and Dock180, and functional studies suggest that BAI1 cooperates with ELMO/Dock180/Rac to promote maximal engulfment of apoptotic cells. Last, decreased BAI1 expression or interference with BAI1 function inhibits the engulfment of apoptotic targets ex vivo and in vivo. Thus, BAI1 is a phosphatidylserine recognition receptor that can directly recruit a Rac–GEF complex to mediate the uptake of apoptotic cells. [ABSTRACT FROM AUTHOR]