Engulfment Receptors Research Articles

Experience-dependent glial pruning of synaptic glomeruli during the critical period

Critical periods are temporally-restricted, early-life windows when sensory experience remodels synaptic connectivity to optimize environmental input. In the Drosophila juvenile brain, critical period experience drives synapse elimination, which is transiently reversible. Within olfactory sensory neuron (OSN) classes synapsing onto single projection neurons extending to brain learning/memory centers, we find glia mediate experience-dependent pruning of OSN synaptic glomeruli downstream of critical period odorant exposure. We find glial projections infiltrate brain neuropil in response to critical period experience, and use Draper (MEGF10) engulfment receptors to prune synaptic glomeruli. Downstream, we find antagonistic Basket (JNK) and Puckered (DUSP) signaling is required for the experience-dependent translocation of activated Basket into glial nuclei. Dependent on this signaling, we find critical period experience drives expression of the F-actin linking signaling scaffold Cheerio (FLNA), which is absolutely essential for the synaptic glomeruli pruning. We find Cheerio mediates experience-dependent regulation of the glial F-actin cytoskeleton for critical period remodeling. These results define a sequential pathway for experience-dependent brain synaptic glomeruli pruning in a strictly-defined critical period; input experience drives neuropil infiltration of glial projections, Draper/MEGF10 receptors activate a Basket/JNK signaling cascade for transcriptional activation, and Cheerio/FLNA induction regulates the glial actin cytoskeleton to mediate targeted synapse phagocytosis.

Chimeric engulfment receptors: A new cell therapy approach for SIV and HIV infection

In this issue, authors Daniel Corey, Francoise Haeseleer, Joe Hou, and Lawrence Corey from CERo Therapeutics and the Fred Hutchinson Cancer Center describe a new approach to creating chimeric antigen receptors (CARs) in “Chimeric engulfment receptors (CERS): a new cell therapy approach for SIV- and HIV-infection”.1 Effective disease control with engineered T cells against targets other than hematologic malignancies, whether the application is for solid tumors or viral-specific CARs, remains elusive.

Novel engineered chimeric engulfment receptors trigger T cell effector functions against SIV-infected CD4+ T cells

Adoptive therapy with genetically engineered Tcells offers potential for infectious disease treatment in immunocompromised persons. HIV/simian immunodeficiency virus (SIV)-infected cells express phosphatidylserine (PS) early post infection. We tested whether chimeric engulfment receptor (CER) Tcells designed to recognize PS-expressing cells could eliminate SIV-infected cells. Lentiviral CER constructs composed of the extracellular domain of Tcell immunoglobulin and mucin domain containing 4 (TIM-4), the PS receptor, and engulfment signaling domains were transduced into primary rhesus macaque (RM) Tcells. We measured PS binding and Tcell engulfment of RM CD4+ Tcells infected with SIV expressing GFP and invitro, TIM-4 CER CD4+ Tcells effectively killed SIV-infected cells, which was dependent on TIM-4 binding to PS. Enhanced killing of SIV-infected CD4+ Tcells by CER and chimeric antigen receptor Tcell combinations was also observed. This installation of innate immune functions into Tcells presents an opportunity to enhance elimination of SIV-infected cells, and studies to evaluate their effect invivo are warranted.

Wun2-mediated integrin recycling promotes apoptotic cell clearance in Drosophila melanogaster.

Apoptotic cell (AC) clearance is a complex process in which phagocytes recognize, engulf, and digest ACs during organismal development and tissue homeostasis. Impaired efferocytosis results in developmental defects and autoimmune diseases. In the current study, we performed RNA-sequencing to systematically identify regulators involved in the phagocytosis of ACs by Drosophila melanogaster macrophage-like S2 cells, followed by targeted RNA interference screening. Wunen2 (Wun2), a homolog of mammalian lipid phosphate phosphatase (LPP), was deemed as required for efferocytosis both in vitro and in vivo. However, efferocytosis was independent of Wun2 phosphatase activity. Proteomic analysis further revealed that Rab11 and its effector Rip11 are interaction partners of Wun2. Therefore, Wun2 collaborates with Rip11 and Rab11 to mediate efficient recycling of the phagocytic receptor βν integrin subunit to the plasma membrane. The loss of Wun2 results in the routing of βv integrin subunit (Itgbn) into lysosomes, leading to its degradation. The deficiency of βv integrin subunit on the cell surface leads to aberrant and disorganized actin cytoskeleton, thereby influencing the formation of macrophage pseudopodia toward ACs and thus failure to engulf them. The findings of this study provide insights that clarify how phagocytes coordinate AC signals and adopt a precise mechanism for the maintenance of engulfment receptors at their cell membrane surface to regulate efferocytosis.

New-generation T cell immunotherapy at the intersection of adaptive and innate immunity

<div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://cdn.insights.bio/uploads/Daniel_Corey.png" data-image="1"></div><div class="author-det"> <b>Daniel Corey</b> is founder and chief executive officer of CERo Therapeutics. A board-certified hematologist, Dr. Corey led the foundational discovery research to identify and define the therapeutic utility of chimeric engulfment receptors and advance understanding of the basic biology of innate immune responses. Dr. Corey received his medical degree from the University of Washington Medical School and completed his residency in internal medicine at Duke. Dr. Corey completed a series of fellowships in cell biology, immunology and hematology at Duke University and Stanford University. </div></div></div><div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://cdn.insights.bio/uploads/Rossi_Conf.jpg" data-image="1"></div><div class="author-det"> <b>John Rossi</b> leads the Translational Medicine team at CERo. Prior to joining, Mr. Rossi was Senior Director of Translational Medicine and Head of Cell Therapy Clinical Pharmacology at Kite, a Gilead Company. While at Kite, Mr. Rossi led translational activities supporting the clinical development and global approvals of YESCARTA^®️ and TECARTUS^®️ CAR T cell therapies. Mr. Rossi’s team also supported work focusing on the mechanistic understanding of engineered cell therapy products under strategic collaborations with the NCI and numerous leading academic institutions. Before Kite, Mr. Rossi spent 13 years in oncology drug development at Amgen working with teams to identify novel biomarkers for hematologic and solid tumor drug development programs. </div></div></div><div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://cdn.insights.bio/uploads/Vezan copy.jpg" data-image="1"></div><div class="author-det"> <b>Remus Vezan</b> MD PhD, joined CERo in April 2021 from Kite, a Gilead Company, where he spent the last four years in cell therapy clinical development, most recently as Executive Director of Clinical Development. At Kite, Dr Vezan oversaw the clinical development of more than 20 CAR T-cell trials, as a single agent or in various combinations for the treatment of B-cell malignancies. Under his leadership, YESCARTA^®️ and TECARTUS^®️ were granted various global approvals. Before Kite, Dr Vezan served as Medical Director at Pharmacyclis, an Abbvie Company. Dr Vezan completed his medical training (MD and PhD) at the University of Medicine and Pharmacy Cluj, Romania and University of Bern, Switzerland. </div></div></div>

EphA2 Interacts with Tim-4 through Association between Its FN3 Domain and the IgV Domain of Tim-4.

Tim-4 promotes the engulfment of apoptotic cells or exogenous particles by securing them on phagocytes. It is unable to transduce signals by itself but helps other engulfment receptors sense and internalize them. However, the identity of the engulfment receptors collaborating with Tim-4 is still incompletely understood. In this study, we searched for a candidate transmembrane protein with a FN3 domain, important for interaction with Tim-4, in silico and investigated whether it indeed interacts with Tim-4 and is involved in Tim-4-mediated phagocytosis. We found that EphA2 containing a FN3 domain in the extracellular region interacted with Tim-4, which was mediated by the IgV domain of Tim-4 and the FN3 domain of EphA2. Nevertheless, we found that EphA2 expression failed to alter Tim-4-mediated phagocytosis of apoptotic cells or polystyrene beads. Taken together, our findings suggest that EphA2, a new Tim-4 interacting protein, may intervene in a Tim-4-mediated cellular event even if it is not phagocytosis of endogenous or exogenous particles and vice versa.

Modular Organization of Engulfment Receptors and Proximal Signaling Networks: Avenues to Reprogram Phagocytosis.

Transmembrane protein engulfment receptors expressed on the surface of phagocytes engage ligands on apoptotic cells and debris to initiate a sequence of events culminating in material internalization and immunologically beneficial outcomes. Engulfment receptors are modular, comprised of functionally independent extracellular ligation domains and cytosolic signaling motifs. Cognate kinases, adaptors, and phosphatases regulate engulfment by controlling the degree of receptor activation in phagocyte plasma membranes, thus acting as receptor-proximal signaling modules. Here, we review recent efforts to reprogram phagocytes using modular synthetic receptors composed of antibody-based extracellular domains fused to engulfment receptor signaling domains. To aid the development of new phagocyte reprogramming methods, we then define the kinases, adaptors, and phosphatases that regulate a conserved family of engulfment receptors. Finally, we discuss current challenges and opportunities for the field.

Role for phagocytosis in the prevention of neoplastic transformation in Drosophila.

Immunity is considered to be involved in the prevention of cancer. Although both humoral and cellular immune reactions may participate, underlying mechanisms have yet to be clarified. The present study was conducted to clarify this issue using a Drosophila model, in which neoplastic transformation was induced through the simultaneous inhibition of cell-cycle checkpoints and apoptosis. We first determined the location of hemocytes, blood cells of Drosophila playing a role of immune cells, in neoplasia-induced and normal larvae, but there was no significant difference between the two groups. When gene expression pattern in larval hemocytes was determined, the expression of immunity-related genes including those necessary for phagocytosis was reduced in the neoplasia model. We then asked the involvement of phagocytosis in the prevention of neoplasia examining animals where the expression of engulfment receptors instead of apoptosis was retarded. We found that the inhibition of engulfment receptor expression augmented the occurrence of neoplasia induced by a defect in cell-cycle checkpoints. This suggested a role for phagocytosis in the prevention of neoplastic transformation in Drosophila.

Transcription repressor-mediated control of engulfment receptor expression in Drosophila phagocytes

We previously reported that Drosophila phagocytes enhance their phagocytic activity after apoptotic cell engulfment accompanied by the activation of the transcription repressor Tailless and an increase in the levels of engulfment receptors. We herein investigated the underlying mechanisms. We found that Tailless phosphorylation levels decreased in Drosophila phagocytes following the stimulation with apoptotic cells. Anticipating the involvement of another transcription repressor, we examined the possible involvement of Krüppel, a bibliographically identified repressor whose expression is controlled by Tailless. The level of Krüppel in phagocytes decreased after the stimulation in a Tailless-dependent manner. The RNAi knockdown of Krüppel abrogated increases in the levels of engulfment receptors and phagocytic activity in stimulated phagocytes. The binding of Krüppel to the 5′-upstream regions of genes coding for engulfment receptors was demonstrated. These results suggest the following pathway: Tailless is activated by de-phosphorylation; Krüppel expression is inhibited by Tailless; the transcription of engulfment receptors-encoding genes is augmented due to a decrease of inhibition by Krüppel; and finally phagocytic activity is enhanced.

A scaffold for signaling of Tim-4-mediated efferocytosis is formed by fibronectin.

An essential step during clearance of apoptotic cells is the recognition of phosphatidylserine (PS) exposed on apoptotic cells by its receptors on phagocytes. Tim-4 directly binding to PS and functioning as a tethering receptor for phagocytosis of apoptotic cells has been extensively studied over the past decade. However, the molecular mechanisms by which Tim-4 collaborates with other engulfment receptors during efferocytosis remain elusive. By comparing efferocytosis induced by Tim-4 with that by Anxa5-GPI, an artificial tethering receptor, we found that Tim-4 possesses auxiliary machinery to induce a higher level of efferocytosis than Anxa5-GPI. To search for that, we performed a yeast two-hybrid screen and identified Fibronectin (Fn1) as a novel Tim-4-associating protein. Tim-4 directly associated with Fn1 and formed a complex with integrins via the association of Fn1. Through Tim-4-/- mice and cell-based assays, we found that modulation of the Fn1 level affected efferocytosis induced by Tim-4 and disruption of the interaction between Tim-4 and Fn1 abrogated Tim-4-mediated efferocytosis. In addition, Tim-4 depletion attenuated integrin signaling activation and perturbation of integrin signaling suppressed Tim-4-promoted efferocytosis. Taken together, the data suggest that Fn1 locates Tim-4 and integrins in close proximity by acting as a scaffold, resulting in synergistic cooperation of Tim-4 with integrins for efficient efferocytosis.

C. elegans Blastomeres Clear the Corpse of the Second Polar Body by LC3-Associated Phagocytosis

To understand how undifferentiated pluripotent cells cope with cell corpses, we examined the clearance of polar bodies born during female meiosis. We found that polar bodies lose membrane integrity and expose phosphatidylserine in Caenorhabditis elegans. Polar body signaling recruits engulfment receptors to the plasma membrane of embryonic blastomeres using the PI3K VPS-34, RAB-5 GTPase and the sorting nexin SNX-6. The second polar body is then phagocytosed using receptor-mediated engulfment pathways dependent on the Rac1 ortholog CED-10 but undergoes non-apoptotic programmed cell death independent of engulfment. RAB-7 GTPase is required for lysosome recruitment to the polar body phagosome, while LC3 lipidation is required for degradation of the corpse membrane after lysosome fusion. The polar body phagolysosome vesiculates in an mTOR- and ARL-8-dependent manner, which assists its timely degradation. Thus, we established a genetic model to study clearance by LC3-associated phagocytosis and reveal insights into the mechanisms of phagosome maturation and degradation.

Context-dependent compensation among phosphatidylserine-recognition receptors

Phagocytes express multiple phosphatidylserine (PtdSer) receptors that recognize apoptotic cells. It is unknown whether these receptors are interchangeable or if they play unique roles during cell clearance. Loss of the PtdSer receptor Mertk is associated with apoptotic corpse accumulation in the testes and degeneration of photoreceptors in the eye. Both phenotypes are linked to impaired phagocytosis by specialized phagocytes: Sertoli cells and the retinal pigmented epithelium (RPE). Here, we overexpressed the PtdSer receptor BAI1 in mice lacking MerTK (Mertk−/−Bai1Tg) to evaluate PtdSer receptor compensation in vivo. While Bai1 overexpression rescues clearance of apoptotic germ cells in the testes of Mertk−/− mice it fails to enhance RPE phagocytosis or prevent photoreceptor degeneration. To determine why MerTK is critical to RPE function, we examined visual cycle intermediates and performed unbiased RNAseq analysis of RPE from Mertk+/+ and Mertk−/− mice. Prior to the onset of photoreceptor degeneration, Mertk−/− mice had less accumulation of retinyl esters and dysregulation of a striking array of genes, including genes related to phagocytosis, metabolism, and retinal disease in humans. Collectively, these experiments establish that not all phagocytic receptors are functionally equal, and that compensation among specific engulfment receptors is context and tissue dependent.

Signaling pathway for phagocyte priming upon encounter with apoptotic cells

The phagocytic elimination of cells undergoing apoptosis is an evolutionarily conserved innate immune mechanism for eliminating unnecessary cells. Previous studies showed an increase in the level of engulfment receptors in phagocytes after the phagocytosis of apoptotic cells, which leads to the enhancement of their phagocytic activity. However, precise mechanisms underlying this phenomenon require further clarification. We found that the pre-incubation of a Drosophila phagocyte cell line with the fragments of apoptotic cells enhanced the subsequent phagocytosis of apoptotic cells, accompanied by an augmented expression of the engulfment receptors Draper and integrin αPS3. The DNA-binding activity of the transcription repressor Tailless was transiently raised in those phagocytes, depending on two partially overlapping signal-transduction pathways for the induction of phagocytosis as well as the occurrence of engulfment. The RNAi knockdown of tailless in phagocytes abrogated the enhancement of both phagocytosis and engulfment receptor expression. Furthermore, the hemocyte-specific RNAi of tailless reduced apoptotic cell clearance in Drosophila embryos. Taken together, we propose the following mechanism for the activation of Drosophila phagocytes after an encounter with apoptotic cells: two partially overlapping signal-transduction pathways for phagocytosis are initiated; transcription repressor Tailless is activated; expression of engulfment receptors is stimulated; and phagocytic activity is enhanced. This phenomenon most likely ensures the phagocytic elimination of apoptotic cells by stimulated phagocytes and is thus considered as a mechanism to prime phagocytes in innate immunity.

Mechanisms and Significance of Phagocytic Elimination of Cells Undergoing Apoptotic Death

Cells that have become unwanted by the body need to be selectively, rapidly, and safely removed. The removal of these cells is achieved by apoptosis-dependent phagocytosis: unwanted cells are induced to undergo apoptosis and given susceptibility to phagocytosis. Phagocytes recognize these cells using engulfment receptors that bind substances expressed on the surface of target cells during the apoptotic process. The phagocytic elimination of cells undergoing apoptosis is a mechanism that is conserved among multicellular organisms. Malfunctions in this process may lead to structural and functional defects in morphogenesis and tissue homeostasis. Therefore, molecules involved in this phenomenon may be targeted in medical treatments. The mechanisms responsible for the apoptosis-dependent phagocytosis of unwanted cells as well as its physiological and pathological consequences are described herein.

Abstract A165: Engineering approaches to uncover the mechanism of apoptotic cell clearance by a conserved signaling system

Abstract Introduction: Efficient apoptotic cell clearance after injury, chemotherapy, and other triggers of programmed cell death is required to avoid inflammation and further tissue damage. However the behaviors of engulfment signaling molecules and their contributions towards non-inflammatory resolution are poorly understood. In Drosophila glial cells the receptor Draper is proposed to drive engulfment of apoptotic neurons. An orthologous receptor, MEGF10, has been proposed to work in mammalian glia. Both Draper and the T cell receptor complex (TCR) are proposed to recruit downstream molecules to the plasma membrane but functional similarities and differences between T cell and engulfment signaling have not been described. We have reconstituted engulfment in order to uncover the mechanism of this important, conserved signaling system. Experimental systems: By expressing Draper in otherwise inept phagocytes we have dramatically increased the engulfment of apoptotic cells or physiological mimics that expose natural ligands. We have also generated an inducible ligand-receptor system that provides a platform to monitor rapid responses of Draper signaling that are otherwise hard to capture. We utilize a supported lipid bilayer system and total internal fluorescence (TIRF) imaging to monitor the localization of Draper and downstream signaling molecules at the plasma membrane. Results: Remarkable recent work in T cells has led to the development of chimeric antigen receptors (CARs) that link specific ligands to heterologous intracellular signaling pathways in order to kill tumor cells. We have taken a similar approach to engulfment signaling by engineering chimeric engulfment receptors. An inducible heterologous receptor-ligand pair is used in our chimeric engulfment receptor system. Like Draper, the engineered chimeric receptor recruits signaling machinery to the plasma membrane and drives engulfment. Our results indicate ligand flexibility in the system and that the mechanism of activation required for endogenous Draper can be mimicked by engineered chimeric receptors. Using our TIRF assay we have discovered that Draper forms dynamic clusters similar to TCR and that clustered receptors recruit cytosolic signaling proteins to the plasma membrane. Importantly, simultaneous imaging of engulfment receptors and filamentous-actin (F-actin) has revealed that engulfment receptors drive F-actin remodeling that is distinct from behavior seen during TCR signaling. Our TIRF work has revealed both similarities and significant differences between the cellular changes set into motion by Draper signaling as compared to T cell triggering. Conclusions: We have built reconstitution platforms to study engulfment signaling and are in a unique position to understand the essential factors and rate-limiting steps of clearance. We have discovered that engineered chimeric receptors drive efficient engulfment and that engulfment signaling is sparked by formation of dynamic clusters of signaling molecules. We are hopeful that the lessons we learn from our reconstitution systems can be applied to activate non-inflammatory clearance of apoptotic cells after injury or extensive cell death. Citation Format: Adam Williamson, Ron Vale. Engineering approaches to uncover the mechanism of apoptotic cell clearance by a conserved signaling system. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A165.

Protection of Insects against Viral Infection by Apoptosis-Dependent Phagocytosis.

We investigated whether phagocytosis participates in the protection of insects from viral infection using the natural host-virus interaction between Drosophila melanogaster and Drosophila C virus (DCV). Drosophila S2 cells were induced to undergo apoptotic cell death upon DCV infection. However, UV-inactivated virus was unable to cause apoptosis, indicating the need for productive infection for apoptosis induction. S2 cells became susceptible to phagocytosis by hemocyte-derived l(2)mbn cells after viral infection, and the presence of phagocytes in S2 cell cultures reduced viral proliferation. Phagocytosis depended, in part, on caspase activity in S2 cells, as well as the engulfment receptors Draper and integrin βν in phagocytes. To validate the in vivo situation, adult flies were abdominally infected with DCV, followed by the analysis of fly death and viral growth. DCV infection killed flies in a dose-responding manner, and the activation of effector caspases was evident, as revealed by the cleavage of a target protein ectopically expressed in flies. Furthermore, hemocytes isolated from infected flies contained DCV-infected cells, and preinjection of latex beads to inhibit the phagocytic activity of hemocytes accelerated fly death after viral infection. Likewise, viral virulence was exaggerated in flies lacking the engulfment receptors, and was accompanied by the augmented proliferation of virus. Finally, phagocytosis of DCV-infected cells in vitro was inhibited by phosphatidylserine-containing liposome, and virus-infected flies died early when a phosphatidylserine-binding protein was ectopically expressed. Collectively, our study demonstrates that the apoptosis-dependent, phosphatidylserine-mediated phagocytosis of virus-infected cells plays an important role in innate immune responses against viral infection in Drosophila.

Co-receptors are dispensable for tethering receptor-mediated phagocytosis of apoptotic cells.

During efferocytosis, phagocytic cells recognize dying cells by receptors binding to ligands specifically exposed on apoptotic cells. Multiple phagocytic receptors and some of their signaling pathways have been identified. However, the downstream pathways of tethering receptors that secure apoptotic cells remain elusive. It is generally assumed that tethering receptors induce signaling to mediate engulfment via interacting with co-receptors or other engulfment receptors located nearby. However, it is poorly understood whether co-receptors for tethering receptors exist during efferocytosis, and, if they do, whether they are indispensable for this process. Here, we address this issue using glycophosphatidylinositol (GPI)-anchored annexin A5 (Anxa5-GPI), an artificial tethering receptor without a putative co-receptor. Phagocytes expressing Anxa5-GPI exhibited enhanced binding of apoptotic cells, resulting in promoted ingestion of apoptotic cells in a phosphatidylserine-dependent manner. Anxa5-GPI-induced phagocytosis of apoptotic cells relied on the known cytoskeletal engulfment machinery but partially depended on the Elmo-Dock-Rac module or the integrin pathway. In addition, Anxa5-GPI-mediated efferocytosis provoked anti-inflammatory responses. Taken together, our work suggests that co-receptors are dispensable for tethering receptor-induced efferocytosis and that tethering receptors mediate the engulfment of apoptotic cells through multiple engulfment signaling pathways.

Cyclin-dependent kinase inhibitor 2B regulates efferocytosis and atherosclerosis

Genetic variation at the chromosome 9p21 risk locus promotes cardiovascular disease; however, it is unclear how or which proteins encoded at this locus contribute to disease. We have previously demonstrated that loss of one candidate gene at this locus, cyclin-dependent kinase inhibitor 2B (Cdkn2b), in mice promotes vascular SMC apoptosis and aneurysm progression. Here, we investigated the role of Cdnk2b in atherogenesis and found that in a mouse model of atherosclerosis, deletion of Cdnk2b promoted advanced development of atherosclerotic plaques composed of large necrotic cores. Furthermore, human carriers of the 9p21 risk allele had reduced expression of CDKN2B in atherosclerotic plaques, which was associated with impaired expression of calreticulin, a ligand required for activation of engulfment receptors on phagocytic cells. As a result of decreased calreticulin, CDKN2B-deficient apoptotic bodies were resistant to efferocytosis and not efficiently cleared by neighboring macrophages. These uncleared SMCs elicited a series of proatherogenic juxtacrine responses associated with increased foam cell formation and inflammatory cytokine elaboration. The addition of exogenous calreticulin reversed defects associated with loss of Cdkn2b and normalized engulfment of Cdkn2b-deficient cells. Together, these data suggest that loss of CDKN2B promotes atherosclerosis by increasing the size and complexity of the lipid-laden necrotic core through impaired efferocytosis.

Nr4a1 regulates thymic resident macrophage development and function (P4468)

Abstract Mononuclear phagocytes, including monocytes, macrophages, and dendritic cells, function in tissue homeostasis, and also regulate immune responses. Tissue resident macrophages in a variety of tissues arise from the yolk sac, and are generated independently of hematopoietic stem cells. However, the origin of thymic macrophages remains poorly understood. Nr4a1 is an orphan nuclear receptor that limits pro-inflammatory cytokine production in macrophages, and functions as the master regulator of Ly6C- patrolling monocyte development. Here, we report a significant defect in the development of thymic resident macrophages in Nr4a1-deficient animals. Thymic macrophage levels were reduced over two-fold compared with wild-type controls. However, Nr4a1 does not globally regulate the development of tissue macrophages, as macrophages resident in the spleen, lung, brain, pancreas, and peritoneum from Nr4a1-deficient mice develop normally. Moreover, Nr4a1-deficient thymic macrophages fail to clear apoptotic thymocytes in vivo, and express reduced levels of genes important for apoptotic cell engulfment, including engulfment receptors Mertk, and Axl. Defective thymic macrophage development likely impairs proper T cell selection, thus altering the immune system as a whole. Together, these data show that Nr4a1 regulates the development and functional capacity of tissue resident macrophages in the thymus, and provide a novel mechanism for maintenance of thymic homeostasis.

Cross Talk between Engulfment Receptors Stabilin-2 and Integrin αvβ5 Orchestrates Engulfment of Phosphatidylserine-Exposed Erythrocytes

Efficient cell corpse clearance is critical for health in organisms. Apoptotic cells displaying phosphatidylserine (PS) are recognized by engulfment receptors and ingested through two conserved pathways. In one pathway, engulfment receptor brain-specific angiogenesis inhibitor 1 (BAI-1) or integrin functions upstream of ELMO/DOCK180 and activate the small GTPase Rac1. In the other pathway, engulfment receptor CED-1 or stabilin-2 acts in concert with the adaptor protein GULP to activate Rac1. Stabilin-2, a PS receptor, facilitates phagocytosis of apoptotic cells and mediates the production of anti-inflammatory cytokines. Here, we propose that the stabilin-2 extracellular domain consisting of integrin-binding fasciclin 1 (FAS1) domains coordinates the activities of the two phagocytic pathways via direct interactions with integrin. Interactions between stabilin-2 and integrin were determined using biochemical assays, including coimmunoprecipitation and fluorescence resonance energy transfer (FRET). These interactions appear to have functional relevance, since knockdown of endogenous αvβ5 expression or treatment with a function-blocking αvβ5 antibody significantly decreased stabilin-2-mediated phagocytosis in the absence of soluble factors. Our data collectively suggest that the engulfment receptors of the two phagocytic pathways communicate with each other to orchestrate engulfment of damaged erythrocytes. Coordinated phagocytic signaling would be advantageous for physiological and pathological circumstances that require rapid clearance of abnormal (apoptotic or aged) cells.