Non-autonomous cell death induced by the Draper phagocytosis receptor requires signaling through the JNK and SRC pathways.

The last step of cell death is cell clearance, a process critical for tissue homeostasis. For efficient cell clearance to occur, phagocytes and dead cells need to reciprocally signal to each other. One important phenomenon that is under-investigated, however, is that phagocytes not only engulf corpses but contribute to cell death progression. The aims of this study were to determine how the phagocytic receptor Draper non-autonomously induces cell death, using the Drosophila ovary as a model system. We found that Draper, expressed in epithelial follicle cells, requires its intracellular signaling domain to kill the adjacent nurse cell population. Kinases Src42A, Shark and JNK (Bsk) were required for Draper-induced nurse cell death. Signs of nurse cell death occurred prior to apparent engulfment and required the caspase Dcp-1, indicating that it uses a similar apoptotic pathway to starvation-induced cell death. These findings indicate that active signaling by Draper is required to kill nurse cells via the caspase Dcp-1, providing novel insights into mechanisms of phagoptosis driven by non-professional phagocytes.

Knockout of the P2Y6 Receptor Prevents Peri-Infarct Neuronal Loss after Transient, Focal Ischemia in Mouse Brain.

After stroke, there is a delayed neuronal loss in brain areas surrounding the infarct, which may in part be mediated by microglial phagocytosis of stressed neurons. Microglial phagocytosis of stressed or damaged neurons can be mediated by UDP released from stressed neurons activating the P2Y6 receptor on microglia, inducing microglial phagocytosis of such neurons. We show evidence here from a small trial that the knockout of the P2Y6 receptor, required for microglial phagocytosis of neurons, prevents the delayed neuronal loss after transient, focal brain ischemia induced by endothelin-1 injection in mice. Wild-type mice had neuronal loss and neuronal nuclear material within microglia in peri-infarct areas. P2Y6 receptor knockout mice had no significant neuronal loss in peri-infarct brain areas seven days after brain ischemia. Thus, delayed neuronal loss after stroke may in part be mediated by microglial phagocytosis of stressed neurons, and the P2Y6 receptor is a potential treatment target to prevent peri-infarct neuronal loss.

I'm Infected, Eat Me! Innate Immunity Mediated by Live, Infected Cells Signaling To Be Phagocytosed.

Innate immunity against pathogens is known to be mediated by barriers to pathogen invasion, activation of complement, recruitment of immune cells, immune cell phagocytosis of pathogens, death of infected cells, and activation of the adaptive immunity via antigen presentation. Here, we propose and review evidence for a novel mode of innate immunity whereby live, infected host cells induce phagocytes to phagocytose the infected cell, thereby potentially reducing infection. We discuss evidence that host cells, infected by virus, bacteria, or other intracellular pathogens (i) release nucleotides and chemokines as find-me signals, (ii) expose on their surface phosphatidylserine and calreticulin as eat-me signals, (iii) release and bind opsonins to induce phagocytosis, and (iv) downregulate don't-eat-me signals CD47, major histocompatibility complex class I (MHC1), and sialic acid. As long as the pathogens of the host cell are destroyed within the phagocyte, then infection can be curtailed; if antigens from the pathogens are cross-presented by the phagocyte, then an adaptive response would also be induced. Phagocytosis of live infected cells may thereby mediate innate immunity.

Inflammatory neuronal loss in the substantia nigra induced by systemic lipopolysaccharide is prevented by knockout of the P2Y6 receptor in mice.

Inflammation may contribute to multiple brain pathologies. One cause of inflammation is lipopolysaccharide/endotoxin (LPS), the levels of which are elevated in blood and/or brain during bacterial infections, gut dysfunction and neurodegenerative diseases, such as Parkinson's disease. How inflammation causes neuronal loss is unclear, but one potential mechanism is microglial phagocytosis of neurons, which is dependent on the microglial P2Y6 receptor. We investigated here whether the P2Y6 receptor was required for inflammatory neuronal loss. Intraperitoneal injection of LPS on 4 successive days resulted in specific loss of dopaminergic neurons (measured as cells staining with tyrosine hydroxylase or NeuN) in the substantia nigra of wild-type mice, but no neuronal loss in cortex or hippocampus. This supports the hypothesis that neuronal loss in Parkinson's disease may be driven by peripheral LPS. By contrast, there was no LPS-induced neuronal loss in P2Y6 receptor knockout mice. In vitro, LPS-induced microglial phagocytosis of cells was prevented by inhibition of the P2Y6 receptor, and LPS-induced neuronal loss was reduced in mixed glial-neuronal cultures from P2Y6 receptor knockout mice. This supports the hypothesis that microglial phagocytosis contributes to inflammatory neuronal loss, and can be prevented by blocking the P2Y6 receptor, suggesting that P2Y6 receptor antagonists might be used to prevent inflammatory neuronal loss in Parkinson's disease and other brain pathologies involving inflammatory neuronal loss.

Neuronal Loss after Stroke Due to Microglial Phagocytosis of Stressed Neurons.

After stroke, there is a rapid necrosis of all cells in the infarct, followed by a delayed loss of neurons both in brain areas surrounding the infarct, known as 'selective neuronal loss', and in brain areas remote from, but connected to, the infarct, known as 'secondary neurodegeneration'. Here we review evidence indicating that this delayed loss of neurons after stroke is mediated by the microglial phagocytosis of stressed neurons. After a stroke, neurons are stressed by ongoing ischemia, excitotoxicity and/or inflammation and are known to: (i) release "find-me" signals such as ATP, (ii) expose "eat-me" signals such as phosphatidylserine, and (iii) bind to opsonins, such as complement components C1q and C3b, inducing microglia to phagocytose such neurons. Blocking these factors on neurons, or their phagocytic receptors on microglia, can prevent delayed neuronal loss and behavioral deficits in rodent models of ischemic stroke. Phagocytic receptors on microglia may be attractive treatment targets to prevent delayed neuronal loss after stroke due to the microglial phagocytosis of stressed neurons.

Both the apoptotic suicide pathway and phagocytosis are required for a programmed cell death in Caenorhabditis elegans.

BACKGROUND: Programmed cell deaths in the nematode Caenorhabditis elegans are generally considered suicides. Dying cells are engulfed by neighboring cells in a process of phagocytosis. To better understand the interaction between the engulfment and death processes, we analyzed B.al/rapaav cell death, which has been previously described as engulfment-dependent and hence as a possible murder. RESULTS: We found that B.al/rapaav is resistant to caspase-pathway activation: the caspase-mediated suicide pathway initiates the cell-death process but is insufficient to cause B.al/rapaav death without the subsequent assistance of engulfment. When the engulfing cell P12.pa is absent, other typically non-phagocytic cells can display cryptic engulfment potential and facilitate this death. CONCLUSIONS: We term this death an "assisted suicide" and propose that assisted suicides likely occur in other organisms. The study of assisted suicides might provide insight into non-cell autonomous influences on cell death. Understanding the mechanism that causes B.al/rapaav to be resistant to activation of the caspase pathway might reveal the basis of differences in the sensitivity to apoptotic stimuli of tumor and normal cells, a key issue in the field of cancer therapeutics.

Phagocytosis executes delayed neuronal death after focal brain ischemia.

Delayed neuronal loss and brain atrophy after cerebral ischemia contribute to stroke and dementia pathology, but the mechanisms are poorly understood. Phagocytic removal of neurons is generally assumed to be beneficial and to occur only after neuronal death. However, we report herein that inhibition of phagocytosis can prevent delayed loss and death of functional neurons after transient brain ischemia. Two phagocytic proteins, Mer receptor tyrosine kinase (MerTK) and Milk fat globule EGF-like factor 8 (MFG-E8), were transiently up-regulated by macrophages/microglia after focal brain ischemia in vivo. Strikingly, deficiency in either protein completely prevented long-term functional motor deficits after cerebral ischemia and strongly reduced brain atrophy as a result of inhibiting phagocytosis of neurons. Correspondingly, in vitro glutamate-stressed neurons reversibly exposed the "eat-me" signal phosphatidylserine, leading to their phagocytosis by microglia; this neuronal loss was prevented in the absence of microglia and reduced if microglia were genetically deficient in MerTK or MFG-E8, both of which mediate phosphatidylserine-recognition. Thus, phagocytosis of viable neurons contributes to brain pathology and, surprisingly, blocking this process is strongly beneficial. Therefore, inhibition of specific phagocytic pathways may present therapeutic targets for preventing delayed neuronal loss after transient cerebral ischemia.

Inhibition of UDP/P2Y6 purinergic signaling prevents phagocytosis of viable neurons by activated microglia in vitro and in vivo.

Microglia activated through Toll-like receptor (TLR)-2 or -4 can cause neuronal death by phagocytosing otherwise-viable neurons-a form of cell death called "phagoptosis." UDP release from neurons has been shown to provoke microglial phagocytosis of neurons via microglial P2Y6 receptors, but whether inhibition of this process affects neuronal survival is unknown. We tested here whether inhibition of P2Y6 signaling could prevent neuronal death in inflammatory conditions, and whether UDP signaling can induce phagoptosis of stressed but viable neurons. We find that delayed neuronal loss and death in mixed neuronal/glial cultures induced by the TLR ligands lipopolysaccharide (LPS) or lipoteichoic acid was prevented by: apyrase (to degrade nucleotides), Reactive Blue 2 (to inhibit purinergic signaling), or MRS2578 (to specifically block P2Y6 receptors). In each case, inflammatory activation of microglia was not affected, and the rescued neurons remained viable for at least 7 days. Blocking P2Y6 receptors with MRS2578 also prevented phagoptosis of neurons induced by 250 nM amyloid beta 1-42, 5 µM peroxynitrite, or 50 µM 3-morpholinosydnonimine (which releases reactive oxygen and nitrogen species). Furthermore, the P2Y6 receptor agonist UDP by itself was sufficient to stimulate microglial phagocytosis and to induce rapid neuronal loss that was prevented by eliminating microglia or inhibiting phagocytosis. In vivo, injection of LPS into rat striatum induced microglial activation and delayed neuronal loss and blocking P2Y6 receptors with MRS2578 prevented this neuronal loss. Thus, blocking UDP/P2Y6 signaling is sufficient to prevent neuronal loss and death induced by a wide range of stimuli that activate microglial phagocytosis of neurons.

Integrin restriction by miR-34 protects germline progenitors from cell death during aging.

During aging, regenerative tissues must dynamically balance the two opposing processes of proliferation and cell death. While many microRNAs are differentially expressed during aging, their roles as dynamic regulators of tissue regeneration have yet to be described. We show that in the highly regenerative Drosophila testis, miR-34 levels are significantly elevated during aging. miR-34 modulates germ cell death and protects the progenitor germ cells from accelerated aging. However, miR-34 is not expressed in the progenitors themselves but rather in neighboring cyst cells that kill the progenitors. Transcriptomics followed by functional analysis revealed that during aging, miR-34 modifies integrin signaling by limiting the levels of the heterodimeric integrin receptor αPS2 and ßPS subunits. In addition, we found that in cyst cells, this heterodimer is essential for inducing phagoptosis and degradation of the progenitor germ cells. Together, these data suggest that the miR-34-integrin signaling axis acts as a sensor of progenitor germ cell death to extend progenitor functionality during aging.

Microglial Phagocytosis During Embryonic and Postnatal Development.

Microglia play decisive roles during the development of the central nervous system (CNS). Phagocytosis is one of the classical functions attributed to microglia, being involved in nearly all phases of the embryonic and postnatal development of the brain, such as rapid clearance of cell debris to avoid an inflammatory response, controlling the number of neuronal and glial cells or their precursors, contribution to axon guidance and to refinement of synaptic connections. To carry out all these tasks, microglial cells are equipped with a panoply of receptors, that convert microglia to the "professional phagocytes" of the nervous parenchyma. These receptors are modulated by spatiotemporal cues that adapt the properties of microglia to the needs of the developing CNS. Thus, in this chapter, we will discuss the role of microglial phagocytosis in all the aforementioned processes. First, we will explain the general phagocytic process, to describe afterward the performance of microglial cells in detail.

Live Imaging of Phagoptosis in ex vivo Drosophila Testis.

Phagoptosis is a prevalent type of programmed cell death (PCD) in adult tissues in which phagocytes non-autonomously eliminate viable cells. Therefore, phagoptosis can only be studied in the context of the entire tissue that includes both the phagocyte executors and the targeted cells doomed to die. Here, we describe an ex vivo live imaging protocol of Drosophila testis to study the dynamics of phagoptosis of germ cell progenitors that are spontaneously removed by neighboring cyst cells. Using this approach, we followed the pattern of exogenous fluorophores with endogenously expressed fluorescent proteins and revealed the sequence of events in germ cell phagoptosis. Although optimized for Drosophila testis, this easy-to-use protocol can be adapted to a wide variety of organisms, tissues, and probes, thus providing a reliable and simple means to study phagoptosis.

Murder on the Ovarian Express: A Tale of Non-Autonomous Cell Death in the Drosophila Ovary.

Throughout oogenesis, Drosophila egg chambers traverse the fine line between survival and death. After surviving the ten early and middle stages of oogenesis, egg chambers drastically change their size and structure to produce fully developed oocytes. The development of an oocyte comes at a cost, the price is the lives of the oocyte's 15 siblings, the nurse cells. These nurse cells do not die of their own accord. Their death is dependent upon their neighbors-the stretch follicle cells. Stretch follicle cells are nonprofessional phagocytes that spend the final stages of oogenesis surrounding the nurse cells and subsequently forcing the nurse cells to give up everything for the sake of the oocyte. In this review, we provide an overview of cell death in the ovary, with a focus on recent findings concerning this phagocyte-dependent non-autonomous cell death.

Classification of Cell-in-Cell Structures: Different Phenomena with Similar Appearance.

A phenomenon known for over 100 years named "cell-in-cell" (CIC) is now undergoing its renaissance, mostly due to modern cell visualization techniques. It is no longer an esoteric process studied by a few cell biologists, as there is increasing evidence that CICs may have prognostic and diagnostic value for cancer patients. There are many unresolved questions stemming from the difficulties in studying CICs and the limitations of current molecular techniques. CIC formation involves a dynamic interaction between an outer or engulfing cell and an inner or engulfed cell, which can be of the same (homotypic) or different kind (heterotypic). Either one of those cells appears to be able to initiate this process, which involves signaling through cell-cell adhesion, followed by cytoskeleton activation, leading to the deformation of the cellular membrane and movements of both cells that subsequently result in CICs. This review focuses on the distinction of five known forms of CIC (cell cannibalism, phagoptosis, enclysis, entosis, and emperipolesis), their unique features, characteristics, and underlying molecular mechanisms.

Fractalkine-Dependent Microglial Pruning of Viable Oligodendrocyte Progenitor Cells Regulates Myelination.

Oligodendrogenesis occurs during early postnatal development, coincident with neurogenesis and synaptogenesis, raising the possibility that microglia-dependent pruning mechanisms that modulate neurons regulate myelin sheath formation. Here we show a population of ameboid microglia migrating from the ventricular zone into the corpus callosum during early postnatal development, termed "the fountain of microglia," phagocytosing viable oligodendrocyte progenitor cells (OPCs) before onset of myelination. Fractalkine receptor-deficient mice exhibit a reduction in microglial engulfment of viable OPCs, increased numbers of oligodendrocytes, and reduced myelin thickness but no change in axon number. These data provide evidence that microglia phagocytose OPCs as a homeostatic mechanism for proper myelination. A hallmark of hypomyelinating developmental disorders such as periventricular leukomalacia and of adult demyelinating diseases such as multiple sclerosis is increased numbers of oligodendrocytes but failure to myelinate, suggesting that microglial pruning of OPCs may be impaired in pathological states and hinder myelination.

Lysosomal Machinery Drives Extracellular Acidification to Direct Non-apoptotic Cell Death.

Cell death is a fundamental aspect of development, homeostasis, and disease; yet, our understanding of non-apoptotic forms of cell death is limited. One such form is phagoptosis, in which one cell utilizes phagocytosis machinery to kill another cell that would otherwise continue living. We have previously identified a non-autonomous requirement of phagocytosis machinery for the developmental programmed cell death of germline nurse cells in the Drosophila ovary; however, the precise mechanism of death remained elusive. Here, we show that lysosomal machinery acting in epithelial follicle cells is used to non-autonomously induce the death of nearby germline cells. Stretch follicle cells recruit V-ATPases and chloride channels to their plasma membrane to extracellularly acidify the germline and release cathepsins that destroy the nurse cells. Our results reveal a role for lysosomal machinery acting at the plasma membrane to cause the death of neighboring cells, providing insight into mechanisms driving non-autonomous cell death.

Glial Phagocytic Receptors Promote Neuronal Loss in Adult Drosophila Brain.

Glial phagocytosis is critical for the development and maintenance of the CNS in vertebrates and flies and relies on the function of phagocytic receptors to remove apoptotic cells and debris. Glial phagocytic ability declines with age, which correlates with neuronal dysfunction, suggesting that increased glial phagocytosis may prevent neurodegeneration. Contradicting this hypothesis, we provide experimental evidence showing that an elevated expression of the phagocytic receptors Six-Microns-Under (SIMU) and Draper (Drpr) in adult Drosophila glia leads to a loss of both dopaminergic and GABAergic neurons, accompanied by motor dysfunction and a shortened lifespan. Importantly, this reduction in neuronal number is not linked to neuronal apoptosis, but rather to phosphatidylserine-mediated phagoptosis of live neurons by hyper-phagocytic glia. Altogether, our study reveals that the level of glial phagocytic receptors must be tightly regulated for proper brain function and that neurodegeneration occurs not only by defective, but also excessive glial cell function.

Anti-CD47 antibodies induce phagocytosis of live, malignant B cells by macrophages via the Fc domain, resulting in cell death by phagoptosis.

When expressed on the surface of cells, CD47 inhibits phagocytosis of these cells by phagocytes. Most human cancers overexpress CD47, and antibodies to CD47 have shown a remarkable ability to clear a range of cancers in animal models. However, the mechanism by which these antibodies cause cancer cell death is unclear. We find that CD47 is expressed on the surface of three B-cell lines from human malignancies: 697 (pre-B-ALL lymphoblasts), Ramos and DG-75 (both mature B-cells, Burkitt's lymphoma), and anti-CD47 antibodies greatly increase the phagocytosis of all three cell line by macrophages. In the presence of macrophages, the antibodies cause clearance of the lymphoblasts within hours, but in the absence of macrophages, the antibodies have no effect on lymphoblast viability. Macrophages engulf viable lymphoblasts containing mitochondria with a normal membrane potential, but following engulfment the mitochondrial membrane potential is lost indicating a loss of viability. Inhibition of phagocytosis protects lymphoblasts from death indicating that phagocytosis is required for anti-CD47 mediated cell death. Blocking either the antibody Fc domain or Fc receptors inhibits antibody-induced phagocytosis. Antibodies against cell surface markers CD10 or CD19 also induced Fc-domain-dependent phagocytosis, but at a lower level commensurate with expression. Thus, phagoptosis may contribute to the efficacy of a number of therapeutic antibodies used in cancer therapy, as well as potentially endogenous antibodies. We conclude that anti-CD47 antibodies induce phagocytosis by binding CD47 on lymphoblast and Fc receptors on macrophages, resulting in cell death by phagocytosis, i.e. phagoptosis.

The neuron-astrocyte-microglia triad in CA3 after chronic cerebral hypoperfusion in the rat: Protective effect of dipyridamole.

We investigated the quantitative and morphofunctional alterations of neuron-astrocyte-microglia triads in CA3 hippocampus, in comparison to CA1, after 2 Vessel Occlusion (2VO) and the protective effect of dipyridamole. We evaluated 3 experimental groups: sham-operated rats (sham, n=15), 2VO-operated rats treated with vehicle (2VO-vehicle, n=15), and 2VO-operated rats treated with dipyridamole from day 0 to day 7 (2VO-dipyridamole, n=15), 90days after 2VO. We analyzed Stratum Pyramidalis (SP), Stratum Lucidum (SL) and Stratum Radiatum (SR) of CA3. 1) ectopic neurons increased in SL and SR of 2VO-vehicle, and 2VO-dipyridamole rats; 2) apoptotic neurons increased in SP of 2VO-vehicle rats and dipyridamole reverted this effect; 3) astrocytes increased in SP, SL and SR of 2VO-vehicle and 2VO-dipyridamole rats; 4) TNF-α expression increased in astrocytes, blocked by dipyridamole, and in dendrites in SR of 2VO-vehicle rats; 5) total microglia increased in SL and SR of 2VO-vehicle and 2VO-dipyridamole rats; 6) triads increased in SR of 2VO-vehicle rats and dipyridamole reverted this effect. Microglia cooperated with astrocytes to phagocytosis of apoptotic neurons and debris, and engulfed ectopic non-fragmented neurons in SL of 2VO-vehicle and 2VO-dipyridamole rats, through a new mechanism called phagoptosis. CA3 showed a better adaptive capacity than CA1 to the ischemic insult, possibly due to the different behaviour of astrocytes and microglial cells. Dipyridamole had neuroprotective effects.

Stage 1 Registered Report: Effect of deficient phagocytosis on neuronal survival and neurological outcome after temporary middle cerebral artery occlusion (tMCAo).

Stroke is a major cause of death and disability worldwide. In addition to neuronal death resulting directly from energy depletion due to lack of blood supply, inflammation and microglial activation following ischemic brain injury has been increasingly recognized to be a key contributor to the pathophysiology of cerebrovascular disease. However, our understanding of the cross talk between the ischemic brain and the immune system is limited. Recently, we demonstrated that following focal ischemia, death of mature viable neurons can be executed through phagocytosis by microglial cells or recruited macrophages, i.e. through phagoptosis. It was shown that inhibition of phagocytic signaling pathways following endothelin-1 induced focal cerebral ischemia leads to increased neuronal survival and neurological recovery. This suggests that inhibition of specific phagocytic pathways may prevent neuronal death during cerebral ischemia. To further explore this potential therapeutic target, we propose to assess the role of phagocytosis in an established model of temporary (45min) middle cerebral artery occlusion (tMCAo), and to evaluate neuronal survival and neurological recovery in mice with deficient phagocytosis. The primary outcome of this study will be forelimb function assessed with the staircase test. Secondary outcomes constitute Rotarod performance, stroke volume (quantified on MR imaging or brain sections, respectively), diffusion tensor imaging (DTI) connectome mapping, and histological analyses to measure neuronal and microglial densities, and phagocytic activity. Male mice aged 10-12 weeks will be used for experiments.

Alterations in the Interplay between Neurons, Astrocytes and Microglia in the Rat Dentate Gyrus in Experimental Models of Neurodegeneration.

The hippocampus is negatively affected by aging and neurodegenerative diseases leading to impaired learning and memory abilities. A diverse series of progressive modifications in the intercellular communication among neurons, astrocytes and microglia occur in the hippocampus during aging or inflammation. A detailed understanding of the neurobiological modifications that contribute to hippocampal dysfunction may reveal new targets for therapeutic intervention. The current study focussed on the interplay between neurons and astroglia in the Granule Layer (GL) and the Polymorphic Layer (PL) of the Dentate Gyrus (DG) of adult, aged and LPS-treated rats. In GL and PL of aged and LPS-treated rats, astrocytes were less numerous than in adult rats. In GL of LPS-treated rats, astrocytes acquired morphological features of reactive astrocytes, such as longer branches than was observed in adult rats. Total and activated microglia increased in the aged and LPS-treated rats, as compared to adult rats. In the GL of aged and LPS-treated rats many neurons were apoptotic. Neurons decreased significantly in GL and PL of aged but not in rats treated with LPS. In PL of aged and LPS-treated rats many damaged neurons were embraced by microglia cells and were infiltrated by branches of astrocyte, which appeared to be bisecting the cell body, forming triads. Reactive microglia had a scavenging activity of dying neurons, as shown by the presence of neuronal debris within their cytoplasm. The levels of the chemokine fractalkine (CX3CL1) increased in hippocampal homogenates of aged rats and rats treated with LPS, and CX3CL1 immunoreactivity colocalized with activated microglia cells. Here we demonstrated that in the DG of aged and LPS-treated rats, astrocytes and microglia cooperate and participate in phagocytosis/phagoptosis of apoptotic granular neurons. The differential expression/activation of astroglia and the alteration of their intercommunication may be responsible for the different susceptibility of the DG in comparison to the CA1 and CA3 hippocampal areas to neurodegeneration during aging and inflammation.