Kodi S. Ravichandran

Nucleotides released by apoptotic cells act as a find-me signal to promote phagocytic clearance

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 P2Y2 as a critical sensor of nucleotides released by apoptotic cells using RNA interference-mediated depletion studies in monocytes, and macrophages from P2Y2-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 P2Y2-/- (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 P2Y2-/- mice). These results identify nucleotides as a critical find-me cue released by apoptotic cells to promote P2Y2-dependent recruitment of phagocytes, and provide evidence for a clear relationship between a find-me signal and efficient corpse clearance in vivo.

Pannexin 1 channels mediate /`find-me/' signal release and membrane permeability during apoptosis

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.

BAI1 is an engulfment receptor for apoptotic cells upstream of the ELMO/Dock180/Rac module

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.

Apoptotic cell clearance: basic biology and therapeutic potential

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.

Clearance of apoptotic cells: implications in health and disease

Recent advances in defining the molecular signaling pathways that regulate the phagocytosis of apoptotic cells have improved our understanding of this complex and evolutionarily conserved process. Studies in mice and humans suggest that the prompt removal of dying cells is crucial for immune tolerance and tissue homeostasis. Failed or defective clearance has emerged as an important contributing factor to a range of disease processes. This review addresses how specific molecular alterations of engulfment pathways are linked to pathogenic states. A better understanding of the apoptotic cell clearance process in healthy and diseased states could offer new therapeutic strategies.

Identification of a Novel Macrophage Phenotype That Develops in Response to Atherogenic Phospholipids via Nrf2

Rationale: Macrophages change their phenotype and biological functions depending on the microenvironment. In atherosclerosis, oxidative tissue damage accompanies chronic inflammation; however, macrophage phenotypic changes in response to oxidatively modified molecules are not known. Objective: To examine macrophage phenotypic changes in response to oxidized phospholipids that are present in atherosclerotic lesions. Methods and Results: We show that oxidized phospholipid-treated murine macrophages develop into a novel phenotype (Mox) that is strikingly different from the conventional M1 and M2 macrophage phenotypes. Compared to M1 and M2, Mox macrophages show a different gene expression pattern, as well as decreased phagocytotic and chemotactic capacity. Treatment with oxidized phospholipids induces both M1 and M2 macrophages to switch to the Mox phenotype. Whole-genome expression array analysis and subsequent gene ontology clustering revealed that the Mox phenotype was characterized by abundant overrepresentation of Nrf2-mediated expression of redox-regulatory genes. In macrophages isolated from Nrf2−/− mice, oxidized phospholipid-induced gene expression and regulation of redox status were compromised. Moreover, we found that Mox macrophages comprise 30% of all macrophages in advanced atherosclerotic lesions of low-density lipoprotein receptor knockout (LDLR−/−) mice. Conclusions: Together, we identify Nrf2 as a key regulator in the formation of a novel macrophage phenotype (Mox) that develops in response to oxidative tissue damage. The unique biological properties of Mox macrophages suggest this phenotype may play an important role in atherosclerotic lesion development as well as in other settings of chronic inflammation.

Phagosome maturation: going through the acid test

Phagosome maturation is the process by which internalized particles (such as bacteria and apoptotic cells) are trafficked into a series of increasingly acidified membrane-bound structures, leading to particle degradation. The characterization of the phagosomal proteome and studies in model organisms and mammals have led to the identification of numerous candidate proteins that cooperate to control the maturation of phagosomes containing different particles. A subset of these candidate proteins makes up the first pathway to be identified for the maturation of apoptotic cell-containing phagosomes. This suggests that a machinery that is distinct from receptor-mediated endocytosis is used in phagosome maturation.

A pathway for phagosome maturation during engulfment of apoptotic cells

Removal of apoptotic cells is critical for the physiological well-being of the organism and defects in corpse removal have been linked to disease states. Genes regulating corpse recognition and internalization have been identified, but few molecules involved in the processing of internalized corpses are known. Through a combination of targeted and unbiased reverse genetic screens in Caenorhabditis elegans, and studies in mammalian cells, we have identified genes required for maturation of apoptotic-cell-containing phagosomes. We have further ordered these candidates, which include the GTPases RAB-5 and RAB-7 and the HOPS complex, into a coherent linear pathway for the maturation of apoptotic cells within phagosomes. In depth analysis of two additional candidate genes, the phosphatidylinositol 3 kinase (PI(3)K) vps-34 (A001762) and dyn-1/dynamin, showed an accumulation of internalized, but undegraded, corpses within abnormal Rab5-negative phagosomes. We ordered these candidates in our pathway, with DYN-1 functioning upstream of VPS-34 in the recruitment and/or retention of RAB-5 to the phagosome. Finally, we have also identified a previously undescribed biochemical complex containing Vps34, dynamin and Rab5GDP, thus providing a mechanism for Rab5 recruitment to the nascent phagosome.

Phagocytosis of apoptotic cells is regulated by a UNC-73/TRIO-MIG-2/RhoG signaling module and Armadillo repeats of CED-12/ELMO

BACKGROUND Phagocytosis of cells undergoing apoptosis is essential during development, cellular turnover, and wound healing. Failure to promptly clear apoptotic cells has been linked to autoimmune disorders. C. elegans CED-12 and mammalian ELMO are evolutionarily conserved scaffolding proteins that play a critical role in engulfment from worm to human. ELMO functions together with Dock180 (a guanine nucleotide exchange factor for Rac) to mediate Rac-dependent cytoskeletal reorganization during engulfment and cell migration. However, the components upstream of ELMO and Dock180 during engulfment remain elusive. RESULTS Here, we define a conserved signaling module involving the small GTPase RhoG and its exchange factor TRIO, which functions upstream of ELMO/Dock180/Rac during engulfment. Complementary studies in C. elegans show that MIG-2 (which we identify as the homolog of mammalian RhoG) and UNC-73 (the TRIO homolog) also regulate corpse clearance in vivo, upstream of CED-12. At the molecular level, we identify a novel set of evolutionarily conserved Armadillo (ARM) repeats within CED-12/ELMO that mediate an interaction with activated MIG-2/RhoG; this, in turn, promotes Dock180-mediated Rac activation and cytoskeletal reorganization. CONCLUSIONS The combination of in vitro and in vivo studies presented here identify two evolutionarily conserved players in engulfment, TRIO/UNC73 and RhoG/MIG-2, and the TRIO --> RhoG signaling module is linked by ELMO/CED-12 to Dock180-dependent Rac activation during engulfment. This work also identifies ARM repeats within CED-12/ELMO and their role in linking RhoG and Rac, two GTPases that function in tandem during engulfment.

Identification of two evolutionarily conserved genes regulating processing of engulfed apoptotic cells

Engulfment of apoptotic cells occurs throughout life in multicellular organisms. Impaired apoptotic cell clearance (due to defective recognition, internalization or degradation) results in autoimmune disease. One fundamental challenge in understanding how defects in corpse removal translate into diseased states is the identification of critical components orchestrating the different stages of engulfment. Here we use genetic, cell biological and molecular studies in Caenorhabditis elegans and mammalian cells to identify SAND-1 and its partner CCZ-1 as new factors in corpse removal. In worms deficient in either sand-1 or ccz-1, apoptotic cells are internalized and the phagosomes recruit the small GTPase RAB-5 but fail to progress to the subsequent RAB-7(+) stage. The mammalian orthologues of SAND-1, namely Mon1a and Mon1b, were similarly required for phagosome maturation. Mechanistically, Mon1 interacts with GTP-bound Rab5, identifying Mon1 as a previously unrecognized Rab5 effector. Moreover, a Mon1–Ccz1 complex (but not either protein alone) could bind Rab7 and could also influence Rab7 activation, suggesting Mon1–Ccz1 as an important link in progression from the Rab5-positive stage to the Rab7-positive stage of phagosome maturation. Taken together, these data identify SAND-1 (Mon1) and CCZ-1 (Ccz1) as critical and evolutionarily conserved components regulating the processing of ingested apoptotic cell corpses.