An essential role for calcium flux in phagocytes for apoptotic cell engulfment and the anti-inflammatory response.
ABSTRACT: Cells undergo programmed cell death/apoptosis throughout the lifespan of an organism. The subsequent immunologically silent removal of apoptotic cells plays a role in the maintenance of tolerance; defects in corpse clearance have been associated with autoimmune disease. A number of receptors and signaling molecules involved in this process have been identified, but intracellular signaling downstream of corpse recognition is only now being defined. Calcium plays a key role as a second messenger in many cell types, leading to the activation of downstream molecules and eventual transcription of effector genes; however, the role of calcium signaling during apoptotic cell removal is unclear. Here, using studies in cell lines and in the context of a whole organism, we show that apoptotic cell recognition induces both an acute and sustained calcium flux within phagocytes and that the genes required for calcium flux are essential for engulfment. Furthermore, we provide evidence that both the release of calcium from the endoplasmic reticulum and the entry of extracellular calcium through CRAC channels into the phagocytes are important during engulfment. Moreover, knockdown in Caenorhabditis elegans of stim-1 and jph-1, two genes linked to the entry of extracellular calcium into cells, led to increased persistence of apoptotic cells in the nematode. Loss of these genes seemed to affect early signaling events, leading to a decreased enrichment of actin adjacent to the apoptotic cell during corpse removal. We also show that calcium is crucial for the secretion of TGF-beta by the phagocytes during the engulfment of apoptotic cells. Taken together, these data point to an earlier unappreciated and evolutionarily conserved role for calcium flux at two distinguishable steps: the formation of the phagocytic cup and the internalization of the apoptotic cell, and the anti-inflammatory signaling induced in phagocytes by contact with apoptotic cells.
Project description:Clearance of apoptotic cells by engulfment plays an important role in the homeostasis and development of multicellular organisms. Despite the fact that the recognition of apoptotic cells by engulfment receptors is critical in inducing the engulfment process, the molecular mechanisms are still poorly understood. Here, we characterize a novel cell corpse engulfment pathway mediated by the integrin ? subunit PAT-2 in Caenorhabditis elegans and show that it specifically functions in muscle-mediated engulfment during embryogenesis. Inactivation of pat-2 results in a defect in apoptotic cell internalization. The PAT-2 extracellular region binds to the surface of apoptotic cells in vivo, and the intracellular region may mediate signaling for engulfment. We identify essential roles of small GTPase CDC-42 and its activator UIG-1, a guanine-nucleotide exchange factor, in PAT-2-mediated cell corpse removal. PAT-2 and CDC-42 both function in muscle cells for apoptotic cell removal and are co-localized in growing muscle pseudopods around apoptotic cells. Our data suggest that PAT-2 functions through UIG-1 for CDC-42 activation, which in turn leads to cytoskeletal rearrangement and apoptotic cell internalization by muscle cells. Moreover, in contrast to PAT-2, the other integrin ? subunit INA-1 and the engulfment receptor CED-1, which signal through the conserved signaling molecules CED-5 (DOCK180)/CED-12 (ELMO) or CED-6 (GULP) respectively, preferentially act in epithelial cells to mediate cell corpse removal during mid-embryogenesis. Our results show that different engulfing cells utilize distinct repertoires of receptors for engulfment at the whole organism level.
Project description: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.
Project description:During apoptosis, dying cells are swiftly removed by phagocytes. It is not fully understood how apoptotic cells are recognized by phagocytes. Here we report the identification and characterization of the Caenorhabditis elegans ttr-52 gene, which encodes a transthyretin-like protein and is required for efficient cell corpse engulfment. The TTR-52 protein is expressed in, and secreted from, C. elegans endoderm and clusters around apoptotic cells. Genetic analysis indicates that TTR-52 acts in the cell corpse engulfment pathway mediated by CED-1, CED-6 and CED-7 and affects clustering of the phagocyte receptor CED-1 around apoptotic cells. TTR-52 recognizes surface-exposed phosphatidylserine (PtdSer) in vivo and binds to both PtdSer and the extracellular domain of CED-1 in vitro. TTR-52 is therefore the first bridging molecule identified in C. elegans that mediates recognition of apoptotic cells by crosslinking the PtdSer 'eat me' signal with the phagocyte receptor CED-1.
Project description:The engulfment of apoptotic cells is essential for tissue homeostasis and recovering from damage. Engulfment is mediated by receptors that recognize ligands exposed on apoptotic cells such as phosphatidylserine (PS). In this study, we convert Drosophila melanogaster S2 cells into proficient phagocytes by transfecting the Draper engulfment receptor and replacing apoptotic cells with PS-coated beads. Similar to the T cell receptor (TCR), PS-ligated Draper forms dynamic microclusters that recruit cytosolic effector proteins and exclude a bulky transmembrane phosphatase, consistent with a kinetic segregation-based triggering mechanism. However, in contrast with the TCR, localized signaling at Draper microclusters results in time-dependent depletion of actin filaments, which facilitates engulfment. The Draper-PS extracellular module can be replaced with FRB and FKBP, respectively, resulting in a rapamycin-inducible engulfment system that can be programmed toward defined targets. Collectively, our results reveal mechanistic similarities and differences between the receptors involved in apoptotic corpse clearance and mammalian immunity and demonstrate that engulfment can be reprogrammed toward nonnative targets.
Project description:Phagocytes in different tissues recognize and remove apoptotic cells via the process of efferocytosis. Although it is well-established that efferocytosis elicits an anti-inflammatory response by phagocytes, the molecules and mechanisms that enforce this response in phagocytes are still being defined. In attempting to decipher gene programs induced after a phagocyte ingests a dying cell, we uncovered a chloride-sensing signaling pathway that controls both the ‘appetite’ of a phagocyte and how a phagocyte responds after corpse uptake. First, we noted that within phagocytes that have ingested a corpse, the solute carrier 12 (SLC12) family members SLC12A2 and SLC12A4 are actively modulated. Interfering with SLC12A2, either genetically or pharmacologically, led to significantly enhanced corpse uptake per phagocyte, while loss of SLC12A4 inhibited corpse uptake. Interestingly, when phagocytes with disrupted SLC12A2 engulfed apoptotic corpses, the typical homeostatic efferocytosis signature was perturbed, characterized by loss of the canonical anti-inflammatory program and replaced by pro-inflammatory and oxidative stress-associated gene programs. In further mechanistic studies, we observed efferocytosis was also regulated by the chloride-sensing pathway upstream of SLC12A2, including the kinases WNK1-OSR1-SPAK, and this involved chloride entry/exit across the plasma membrane of phagocytes during corpse engulfment. We also show that the ‘switch’ to pro-inflammatory sensing of apoptotic cells is specifically due to disruption of the chloride-sensing pathway and not due to corpse overload or poor degradation, and that the pro-inflammatory gene signature can be reversed using a chloride ionophore. Collectively, these data identify the WNK1-OSR1-SPAK-SLC12A2/SLC12A4 chloride-sensing pathway and chloride flux in phagocytes as key modifiers of how a phagocyte interprets an engulfed apoptotic corpse. Overall design: The first experiment consisted of four conditions: Phagocytes (LR73 cells) alone, treated with cytochalasin-D, co-cultured with apoptotic Jurkat lymphoma cells, or treated with cytochalasin-D and co-cultured with apoptotic Jurkat lymphoma cells. The second experiment consisted of two conditions, wildtype or Slc12a2-deficient LR73 phagocytes. Phagocytes were co-cultured with apoptotic Jurkat cellsfor 2h, then actively engulfing phagocytes (i.e. CypHer5E+) were FACS-sorted prior to RNA isolation. All conditions consisted of four biological replicates.
Project description:Billions of cells die in our bodies on a daily basis and are engulfed by phagocytes. Engulfment, or phagocytosis, can be broken down into five basic steps: attraction of the phagocyte, recognition of the dying cell, internalization, phagosome maturation, and acidification. In this study, we focus on the last two steps, which can collectively be considered corpse processing, in which the engulfed material is degraded. We use the Drosophila ovarian follicle cells as a model for engulfment of apoptotic cells by epithelial cells. We show that engulfed material is processed using the canonical corpse processing pathway involving the small GTPases Rab5 and Rab7. The phagocytic receptor Draper is present on the phagocytic cup and on nascent, phosphatidylinositol 3-phosphate (PI(3)P)- and Rab7-positive phagosomes, whereas integrins are maintained on the cell surface during engulfment. Due to the difference in subcellular localization, we investigated the role of Draper, integrins, and downstream signaling components in corpse processing. We found that some proteins were required for internalization only, while others had defects in corpse processing as well. This suggests that several of the core engulfment proteins are required for distinct steps of engulfment. We also performed double mutant analysis and found that combined loss of draper and ?PS3 still resulted in a small number of engulfed vesicles. Therefore, we investigated another known engulfment receptor, Crq. We found that loss of all three receptors did not inhibit engulfment any further, suggesting that Crq does not play a role in engulfment by the follicle cells. A more complete understanding of how the engulfment and corpse processing machinery interact may enable better understanding and treatment of diseases associated with defects in engulfment by epithelial cells.
Project description:The rapid clearance of dying cells is important for the well-being of multicellular organisms. In C. elegans, cell corpse removal is mainly mediated by three parallel engulfment signaling cascades. These pathways include two small GTPases, MIG-2/RhoG and CED-10/Rac1. Here we present the identification and characterization of CDC-42 as a third GTPase involved in the regulation of cell corpse clearance. Genetic analyses performed by both loss of cdc-42 function and cdc-42 overexpression place cdc-42 in parallel to the ced-2/5/12 signaling module, in parallel to or upstream of the ced-10 module, and downstream of the ced-1/6/7 module. CDC-42 accumulates in engulfing cells at membranes surrounding apoptotic corpses. The formation of such halos depends on the integrins PAT-2/PAT-3, UNC-112 and the GEF protein UIG-1, but not on the canonical ced-1/6/7 or ced-2/5/12 signaling modules. Together, our results suggest that the small GTPase CDC-42 regulates apoptotic cell engulfment possibly upstream of the canonical Rac GTPase CED-10, by polarizing the engulfing cell toward the apoptotic corpse in response to integrin signaling and ced-1/6/7 signaling in C. elegans.
Project description:Apoptotic cell clearance (efferocytosis) elicits an anti-inflammatory response by phagocytes, but the mechanisms that underlie this response are still being defined. Here, we uncover a chloride-sensing signalling pathway that controls both the phagocyte 'appetite' and its anti-inflammatory response. Efferocytosis transcriptionally altered the genes that encode the solute carrier (SLC) proteins SLC12A2 and SLC12A4. Interfering with SLC12A2 expression or function resulted in a significant increase in apoptotic corpse uptake per phagocyte, whereas the loss of SLC12A4 inhibited corpse uptake. In SLC12A2-deficient phagocytes, the canonical anti-inflammatory program was replaced by pro-inflammatory and oxidative-stress-associated gene programs. This 'switch' to pro-inflammatory sensing of apoptotic cells resulted from the disruption of the chloride-sensing pathway (and not due to corpse overload or poor degradation), including the chloride-sensing kinases WNK1, OSR1 and SPAK-which function upstream of SLC12A2-had a similar effect on efferocytosis. Collectively, the WNK1-OSR1-SPAK-SLC12A2/SLC12A4 chloride-sensing pathway and chloride flux in phagocytes are key modifiers of the manner in which phagocytes interpret the engulfed apoptotic corpse.
Project description:Phagocytosis of apoptotic cell corpses is a conserved and well-regulated process and is required to maintain tissue homeostasis within an organism. Evidence suggests that apoptotic cell engulfment by macrophages is dependent upon the externalization of phosphatidylserine (PS) on the plasma membrane of the dying cell. Furthermore, oxidation of PS and other phospholipids may serve to facilitate cell corpse removal. However, our understanding of how these various lipid "eat-me" signals are recognized by macrophages has been limited. Using a combination of cellular and animal models, along with an array of biophysical methods, Hazen and his associates (Greenberg et al., J. Exp. Med., 2006, 203, 2613-2625;Li et al., Biochemistry, 2007, 46, 5009-5017) have now identified the scavenger receptor CD36 as a putative receptor for oxidized PS on apoptotic cells; moreover, they have deduced the conformation of the oxidized lipid ligand that is recognized by this receptor, thus providing structural insight into how phagocytes recognize senescent or apoptotic cells.
Project description:Clathrin and the multi-subunit adaptor protein complex AP2 are central players in clathrin-mediated endocytosis by which the cell selectively internalizes surface materials. Here, we report the essential role of clathrin and AP2 in phagocytosis of apoptotic cells. In Caenorhabditis elegans, depletion of the clathrin heavy chain CHC-1 and individual components of AP2 led to a significant accumulation of germ cell corpses, which resulted from defects in both cell corpse engulfment and phagosome maturation required for corpse removal. CHC-1 and AP2 components associate with phagosomes in an inter-dependent manner. Importantly, we found that the phagocytic receptor CED-1 interacts with the ? subunit of AP2, while the CED-6/Gulp adaptor forms a complex with both CHC-1 and the AP2 complex, which likely mediates the rearrangement of the actin cytoskeleton required for cell corpse engulfment triggered by the CED-1 signaling pathway. In addition, CHC-1 and AP2 promote the phagosomal association of LST-4/Snx9/18/33 and DYN-1/dynamin by forming a complex with them, thereby facilitating the maturation of phagosomes necessary for corpse degradation. These findings reveal a non-classical role of clathrin and AP2 and establish them as indispensable regulators in phagocytic receptor-mediated apoptotic cell clearance.