Engulfment Of Cells Research Articles

Monitoring the Recruitment and Fusion of Autophagosomes to Phagosomes During the Clearance of Apoptotic Cells in the Nematode Caenorhabditis elegans.

During an animal's development, a large number of cells undergo apoptosis, a suicidal form of death. These cells are promptly phagocytosed by other cells and degraded inside phagosomes. The recognition, engulfment, and degradation of apoptotic cells is an evolutionarily conserved process occurring in all metazoans. Recently, we discovered a novel event in the nematode Caenorhabditis elegans: the double-membrane autophagosomes are recruited to the surface of phagosomes; subsequently, the outer membrane of an autophagosome fuses with the phagosomal membrane, allowing the inner vesicle to enter the phagosomal lumen and accumulate there over time. This event facilitates the degradation of the apoptotic cell inside the phagosome. During this study, we developed a real-time imaging protocol monitoring the recruitment and fusion of autophagosomes to phagosomes over two hours during embryonic development. This protocol uses a deconvolution-based microscopic imaging system with an optimized setting to minimize photodamage of the embryo during the recording period for high-resolution images. Furthermore, acid-resistant fluorescent reporters are chosen to label autophagosomes, allowing the inner vesicles of an autophagosome to remain visible after entering the acidic phagosomal lumen. The methods described here, which enable high sensitivity, quantitative measurement of each step of the dynamic incorporation in developing embryos, are novel since the incorporation of autophagosomes to phagosomes has not been reported previously. In addition to studying the degradation of apoptotic cells, this protocol can be applied to study the degradation of non-apoptotic cell cargos inside phagosomes, as well as the fusion between other types of intracellular organelles in living C. elegans embryos. Furthermore, its principle of detecting the membrane fusion event can be adapted to study the relationship between autophagosomes and phagosomes or other intracellular organelles in any biological system in which real-time imaging can be conducted. This protocol was validated in: eLife (2022), DOI: 10.7554/eLife.72466.

Efferocytosis during Skeletal Muscle Regeneration.

Efferocytosis, i.e., engulfment of dead cells by macrophages, is a crucial step during tissue repair after an injury. Efferocytosis delineates the transition from the pro-inflammatory phase of the inflammatory response to the recovery phase that ensures tissue reconstruction. We present here the role of efferocytosis during skeletal muscle regeneration, which is a paradigm of sterile tissue injury followed by a complete regeneration. We present the molecular mechanisms that have been described to control this process, and particularly the metabolic control of efferocytosis during skeletal muscle regeneration.

Haemonchus contortus Transthyretin-Like Protein TTR-31 Plays Roles in Post-Embryonic Larval Development and Potentially Apoptosis of Germ Cells.

Transthyretin (TTR)-like proteins play multi-function roles in nematode and are important component of excretory/secretory product in Haemonchus contortus. In this study, we functionally characterised a secretory transthyretin-like protein in the barber’s pole worm H. contortus. A full-length of transthyretin-like protein-coding gene (Hc-ttr-31) was identified in this parasitic nematode, representing a counterpart of Ce-ttr-31 in Caenorhabditis elegans. High transcriptional levels of Hc-ttr-31 were detected in the egg and early larval stages of H. contortus, with the lowest level measured in the adult stage, indicating a decreased transcriptional pattern of this gene during nematode development. Localisation analysis indicated a secretion of TTR-31 from the intestine to the gonad, suggesting additional roles of Hc-ttr-31 in nematode reproduction. Expression of Hc-ttr-31 and Ce-ttr-31 in C. elegans did not show marked influence on the nematode development and reproduction, whereas Hc-ttr-31 RNA interference-mediated gene knockdown of Ce-ttr-31 shortened the lifespan, decreased the brood size, slowed the pumping rate and inhibited the growth of treated worms. Particularly, gene knockdown of Hc-ttr-31 in C. elegans was linked to activated apoptosis signalling pathway, increased general reactive oxygen species (ROS) level, apoptotic germ cells and facultative vivipary phenotype, as well as suppressed germ cell removal signalling pathways. Taken together, Hc-ttr-31 appears to play roles in regulating post-embryonic larval development, and potentially in protecting gonad from oxidative stress and mediating engulfment of apoptotic germ cells. A better knowledge of these aspects should contribute to a better understanding of the developmental biology of H. contortus and a discovery of potential targets against this and related parasitic worms.

PLGA+Fe₃O₄+PFP Nanoparticles Drug-Delivery Demonstrates Potential Anti-Tumor Effects on Tumor Cells.

BackgroundNanoparticles are proven as a potential tool for treating various disorders. However, efficient nanoparticle delivery of anti-tumor drugs is urgently needed for tumor treatment. This study aimed to generate a drug-delivery nanoparticle with higher efficacy and safety.Material/MethodsWe developed a poly-(lactide-co-glycolide) (PLGA) nanoparticle (FLGA-Fe3O4+PFP) embedded with super-paramagnetic iron oxide (Fe3O4) and perfluoropentane (PFP). Characteristics of FLGA-Fe3O4+PFP nanoparticles were observed using optical microscopy, scanning electron microscopy, and transmission electron microscopy. HNE1 and HepG2 cells were cultured and used for experiments. MTT was used to evaluate cytotoxic effects of FLGA-Fe3O4+PFP nanoparticles on HNE1 and HepG2 cells. Cell engulfment capacity was examined and a cell targeting experiment was conducted to evaluate invasive capability and binding efficiency of PLGA+Fe3O4+PFP nanoparticles, respectively. Biological toxicity of PLGA+Fe3O4+PFP nanoparticles in rats was evaluated by determining CK, LDH, creatinine, and UA levels, and ALT and AST activities.ResultsPLGA+Fe3O4+PFP nanoparticles demonstrated well-defined spherical and dispersed morphology with smooth surfaces. There were scattered black spots on shells of PLGA+Fe3O4+PFP nanoparticles. PLGA+Fe3O4+PFP nanoparticles did not trigger obvious effects on cell viability of HNE1 and HepG2 cells. HNE1 and HepG2 cells demonstrated higher engulfment capacity for PLGA+ Fe3O4+PFP nanoparticles. PLGA+Fe3O4+PFP nanoparticles demonstrated higher targeting CDDP delivery efficacy and promoted binding efficiency of targeting CDDP with cells. PLGA+Fe3O4+PFP nanoparticles demonstrated no obvious toxic effects on heart, kidney, liver (without effects on CK, LDH, creatinine, UA levels, and ALT and AST activities).ConclusionsPLGA+Fe3O4+PFP nanoparticles were safe, with higher invasive ability and binding efficiency of targeting CDDP with tumor cells. Therefore, PLGA+Fe3O4+PFP nanoparticles demonstrated potential anti-tumor effects after transplantation.

Bacterial minicells to the rescue: cyto-Immunotherapy for the treatment of late stage cancers with minimal to no toxicity.

Bacterial minicells to the rescue: cyto-Immunotherapy for the treatment of late stage cancers with minimal to no toxicity.

In situ and transcriptomic identification of microglia in synapse-rich regions of the developing zebrafish brain

Microglia are brain resident macrophages that play vital roles in central nervous system (CNS) development, homeostasis, and pathology. Microglia both remodel synapses and engulf apoptotic cell corpses during development, but whether unique molecular programs regulate these distinct phagocytic functions is unknown. Here we identify a molecularly distinct microglial subset in the synapse rich regions of the zebrafish (Danio rerio) brain. We found that ramified microglia increased in synaptic regions of the midbrain and hindbrain between 7 and 28 days post fertilization. In contrast, microglia in the optic tectum were ameboid and clustered around neurogenic zones. Using single-cell mRNA sequencing combined with metadata from regional bulk sequencing, we identified synaptic-region associated microglia (SAMs) that were highly enriched in the hindbrain and expressed multiple candidate synapse modulating genes, including genes in the complement pathway. In contrast, neurogenic associated microglia (NAMs) were enriched in the optic tectum, had active cathepsin activity, and preferentially engulfed neuronal corpses. These data reveal that molecularly distinct phagocytic programs mediate synaptic remodeling and cell engulfment, and establish the zebrafish hindbrain as a model for investigating microglial-synapse interactions.

Novel Mechanisms of Exosome-Mediated Phagocytosis of Dead Cells in Injured Heart.

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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.

Dynamics of Cell Membrane and Cell Wall Development during Generative Cell Engulfment by the Pollen Tube Cell in <i>Liriope muscari</i>

Dynamics of Cell Membrane and Cell Wall Development during Generative Cell Engulfment by the Pollen Tube Cell in <i>Liriope muscari</i>

Amino acids and mechanistic target of rapamycin regulate the fate of live engulfed cells.

Metabolic stress contributes to the regulation of cell death in normal and diseased tissues. While different forms of cell death are known to be regulated by metabolic stress, how the cell engulfment and killing mechanism entosis is regulated is not well understood. Here we find that the death of entotic cells is regulated by the presence of amino acids and activity of the mechanistic target of rapamycin (mTOR). Amino acid withdrawal or mTOR inhibition induces apoptosis of engulfed cells and blocks entotic cell death that is associated with the lipidation of the autophagy protein microtubule‐associated protein light chain 3 (LC3) to entotic vacuoles. Two other live cell engulfment programs, homotypic cell cannibalism (HoCC) and anti‐CD47 antibody‐mediated phagocytosis, known as phagoptosis, also undergo a similar vacuole maturation sequence involving LC3 lipidation and lysosome fusion, but only HoCC involves mTOR‐dependent regulation of vacuole maturation and engulfed cell death similar to entosis. We further find that the regulation of cell death by mTOR is independent of autophagy activation and instead involves the 4E‐BP1/2 proteins that are known regulators of mRNA translation. Depletion of 4E‐BP1/2 proteins can restore the mTOR‐regulated changes of entotic death and apoptosis rates of engulfed cells. These results identify amino acid signaling and the mTOR‐4E‐BP1/2 pathway as an upstream regulation mechanism for the fate of live engulfed cells formed by entosis and HoCC.

The Multiple Facets of Iron Recycling.

The production of around 2.5 million red blood cells (RBCs) per second in erythropoiesis is one of the most intense activities in the body. It continuously consumes large amounts of iron, approximately 80% of which is recycled from aged erythrocytes. Therefore, similar to the “making”, the “breaking” of red blood cells is also very rapid and represents one of the key processes in mammalian physiology. Under steady-state conditions, this important task is accomplished by specialized macrophages, mostly liver Kupffer cells (KCs) and splenic red pulp macrophages (RPMs). It relies to a large extent on the engulfment of red blood cells via so-called erythrophagocytosis. Surprisingly, we still understand little about the mechanistic details of the removal and processing of red blood cells by these specialized macrophages. We have only started to uncover the signaling pathways that imprint their identity, control their functions and enable their plasticity. Recent findings also identify other myeloid cell types capable of red blood cell removal and establish reciprocal cross-talk between the intensity of erythrophagocytosis and other cellular activities. Here, we aimed to review the multiple and emerging facets of iron recycling to illustrate how this exciting field of study is currently expanding.

Cell-in-cell phenomenon: leukocyte engulfment by non-tumorigenic cells and cancer cell lines

BackgroundResearch on cell-in-cell (CIC) phenomena, including entosis, emperipolesis and cannibalism, and their biological implications has increased in recent years. Homotypic and heterotypic engulfment of various target cells by numerous types of host cells has been studied in vitro and in tissue sections. This work has identified proteins involved in the mechanism and uncovered evidence for CIC as a potential histopathologic predictive and prognostic marker in cancer. Our experimental study focused on non-professional phagocytosis of leukocytes.ResultsWe studied the engulfment of peripheral blood mononuclear cells isolated from healthy donors by counting CIC structures. Two non-tumorigenic cell lines (BEAS-2B, SBLF-9) and two tumour cell lines (BxPC3, ICNI) served as host cells. Immune cells were live-stained and either directly co-incubated or treated with irradiation or with conventional or microwave hyperthermia. Prior to co-incubation, we determined leukocyte viability for each batch via Annexin V-FITC/propidium iodide staining.All host cells engulfed their targets, with uptake rates ranging from 1.0% ± 0.5% in BxPC3 to 8.1% ± 5.0% in BEAS-2B. Engulfment rates of the cancer cell lines BxPC3 and ICNI (1.6% ± 0.2%) were similar to those of the primary fibroblasts SBLF-9 (1.4% ± 0.2%). We found a significant negative correlation between leukocyte viability and cell-in-cell formation rates. The engulfment rate rose when we increased the dose of radiotherapy and prolonged the impact time. Further, microwave hyperthermia induced higher leukocyte uptake than conventional hyperthermia.Using fluorescent immunocytochemistry to descriptively study the proteins involved, we detected ring-like formations of diverse proteins around the leukocytes, consisting, among others, of α-tubulin, integrin, myosin, F-actin, and vinculin. These results suggest the involvement of actomyosin contraction, cell-cell adhesion, and the α-tubulin cytoskeleton in the engulfment process.ConclusionsBoth non-tumorigenic and cancer cells can form heterotypic CIC structures by engulfing leukocytes. Decreased viability and changes caused by microwave and X-ray irradiation trigger non-professional phagocytosis.

Macrophage phagocytosis after spinal cord injury: when friends become foes.

After spinal cord injury, macrophages can exert either beneficial or detrimental effects depending on their phenotype. Aside from their critical role in inflammatory responses, macrophages are also specialized in the recognition, engulfment, and degradation of pathogens, apoptotic cells, and tissue debris. They promote remyelination and axonal regeneration by removing inhibitory myelin components and cellular debris. However, excessive intracellular presence of lipids and dysregulated intracellular lipid homeostasis result in the formation of foamy macrophages. These develop a pro-inflammatory phenotype that may contribute to further neurological decline. Additionally, myelin-activated macrophages play a crucial role in axonal dieback and retraction. Here, we review the opposing functional consequences of phagocytosis by macrophages in spinal cord injury, including remyelination and regeneration versus demyelination, degeneration, and axonal dieback. Furthermore, we discuss how targeting the phagocytic ability of macrophages may have therapeutic potential for the treatment of spinal cord injury.

Abstract 2752: LC3-associated phagocytosis in bone marrow macrophages suppresses AML progression through TIM-4 mediated STING activation

Abstract Acute myeloid leukemia (AML) is a tumor dependent on its interactions within the bone marrow (BM) microenvironment. LC3-associated phagocytosis (LAP) maintains tissue homeostasis by regulating immune responses, including in tumor immunity. Here we investigate the function of LAP in the AML BM microenvironment. We used two syngeneic leukemia models (HOXA9/Meis1 and MN1)to investigate the role of LAP on AML proliferation. AML cells were injected into LAP deficient (LAP-/-; ATG16E230-/-) and wild-type (WT) mice. LAP-/- mice had increased AML engraftment in the BM compared to WT mice, as well as reduced animal survival. Flow cytometry (Annexin+) of the BM microenvironment showed an increase in apoptosis in the BM compartment of mice engrafted with AML. This was further increased in LAP-/- mice with AML. The number of BM macrophages (MØ) (CD45+, GR1-, F4/80+ CD115INT) did not differ between the WT and LAP-/- mice. Next, we quantified MØ numbers in the BM of WT and LAP-/- mice with AML. We found increased numbers of tumor associated CD206+ BM MØ in LAP-/- mice with AML compared to WT animals with AML. Gene expression analysis showed up-regulation of type I interferons (IFNs) relating to the STING pathway in the WT engrafted mice. Inhibition of the STING pathway reversed the LAP dependent AML suppression of inflammatory cytokines, suggesting LAP processing of apoptotic cells is important for STING activation. AML has high mtDNA content compared to non-malignant cells, and as mtDNA can activate the STING pathway via cGAS we studied the effects of AML cells without mtDNA (AML ρ0) on STING pathway activation. Induction of apoptosis in AML ρ0 cells followed by co-culture with BM derived MØ (BMDM) for 24 hours did not activate the STING pathway in the MØ. In contrast, MØ co-culture with mitochondria containing apoptotic AML cells did activate the MØ STING pathway. STING pathway activation via type I IFNs induces recruitment of cytotoxic T cells, but no increase in CD8+ T cell numbers or activation (Granzyme-B and IFN-γ) was observed in the BM between LAP-/- and WT animals engrafted with AML. Type I IFNs produced by MØ have been shown to act in an autocrine manner, we therefore investigated MØ phagocytic capacity in AML. Ex-vivo analysis showed enhanced phagocytosis and LAP processing of Zymosan fluorescent beads and LC3 association in MØ from AML engrafted mice compared to controls. Engulfment of apoptotic cells by MØ requires recognition of phosphatidylserine by surface TIM-4 which mediates LAP. We found TIM-4 inhibition increased AML proliferation in vivo. Finally, ex vivo analysis of TIM-4 inhibited MØ confirmed reduced LAP. We report that BM MØ process apoptotic AML cells via LAP through TIM-4. Furthermore, AML apoptotic bodies containing mtDNA initiate MØ STING activation and inhibit AML proliferation. Citation Format: Jamie Aaron Moore, Jayna J. Mistry, Charlotte Hellmich, Aisha Jibril, Tom Wileman, Angela Collins, Kristian M. Bowles, Stuart A. Rushworth. LC3-associated phagocytosis in bone marrow macrophages suppresses AML progression through TIM-4 mediated STING activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2752.

VISTA facilitates phagocytic clearance of HIV infected CEM-SS T cells

Phagocytosis is a critical component of the innate immune response to viral infection, resulting in the clearance of infected cells while minimizing the exposure of uninfected cells. On the other hand, phagocytosis of HIV-infected T cells may cause phagocytes, such as macrophages and dendritic cells, to be infected, thus leading to HIV cell-to-cell transmission. V domain immunoglobulin suppressor of T cell activation (VISTA, gene Vsir, aliases Gi24, Dies-1, PD-1H, and DD1α) has been identified as an immune checkpoint molecule that possesses dual activities when expressed on APCs and T cells. Our study found that VISTA might play a significant role during the immune response to HIV infection via apoptosis upregulation and subsequent phagocytosis of infected CEM-SS T cells. HIV-induced apoptosis and monocytic cell engulfment were tested utilizing CEM-SS T cells as target cells and the monocytic cell line THP-1 as phagocytic cells. Cells were infected with a GFP-labeled HIV strain, NL4-3. HIV-infected CEM-SS T cells displayed greater apoptotic activity (approximately 18.0%) than mock-infected controls. Additionally, phagocytosis of HIV-infected CEM-SS T cells was increased approximately 4-fold. Expression of VISTA on infected CEM-SS T cells was detected in 16.7% of cells, which correlated with the increased phagocytosis observed. When an antagonistic antibody against VISTA was used, the number of phagocytosed cells was reduced by a factor of 2, which was replicated utilizing human stem cell-derived dendritic cells. Phagocytosis was also confirmed by the upregulation of IL-1β expression, which was 5-fold higher in infected cells than in control cells. We also found that VISTA overexpression on both phagocytes and HIV-infected CEM-SS T cells facilitated phagocytosis. Our study suggests that VISTA may act as a direct ligand in the phagocytosis of HIV-infected T cells.

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.

Apoptosis is not conserved in plants as revealed by critical examination of a model for plant apoptosis-like cell death

BackgroundAnimals and plants diverged over one billion years ago and evolved unique mechanisms for many cellular processes, including cell death. One of the most well-studied cell death programmes in animals, apoptosis, involves gradual cell dismantling and engulfment of cellular fragments, apoptotic bodies, through phagocytosis. However, rigid cell walls prevent plant cell fragmentation and thus apoptosis is not applicable for executing cell death in plants. Furthermore, plants are devoid of the key components of apoptotic machinery, including phagocytosis as well as caspases and Bcl-2 family proteins. Nevertheless, the concept of plant “apoptosis-like programmed cell death” (AL-PCD) is widespread. This is largely due to superficial morphological resemblances between plant cell death and apoptosis, and in particular between protoplast shrinkage in plant cells killed by various stimuli and animal cell volume decrease preceding fragmentation into apoptotic bodies.ResultsHere, we provide a comprehensive spatio-temporal analysis of cytological and biochemical events occurring in plant cells subjected to heat shock at 40–55 °C and 85 °C, the experimental conditions typically used to trigger AL-PCD and necrotic cell death, respectively. We show that cell death under both conditions was not accompanied by membrane blebbing or formation of apoptotic bodies, as would be expected during apoptosis. Instead, we observed instant and irreversible permeabilization of the plasma membrane and ATP depletion. These processes did not depend on mitochondrial functionality or the presence of Ca2+ and could not be prevented by an inhibitor of ferroptosis. We further reveal that the lack of protoplast shrinkage at 85 °C, the only striking morphological difference between cell deaths induced by 40–55 °C or 85 °C heat shock, is a consequence of the fixative effect of the high temperature on intracellular contents.ConclusionsWe conclude that heat shock-induced cell death is an energy-independent process best matching definition of necrosis. Although the initial steps of this necrotic cell death could be genetically regulated, classifying it as apoptosis or AL-PCD is a terminological misnomer. Our work supports the viewpoint that apoptosis is not conserved across animal and plant kingdoms and demonstrates the importance of focusing on plant-specific aspects of cell death pathways.

MER Tyrosine Kinase Mediates Efferocytosis Through a Novel β2 Integrin-Activating Signalling Pathway.

Abstract Efferocytosis – the phagocytic removal of apoptotic cells – is a central component of tissue homeostasis and contributes to inflammatory and autoimmune conditions including atherosclerosis and multiple sclerosis. MER tyrosine kinase (MERTK) is the predominant or sole efferocytic receptor in multiple tissues, and is the predominant efferocytic receptor on macrophages. MERTK is an opsonin-dependent receptor tyrosine kinase that triggers a poorly understood signaling pathway to mediate apoptotic cell engulfment. Using immunoprecipitation, mass spectrometry, and super-resolution microscopy, we have identified a pre-formed receptor complex on the macrophage plasma membrane comprised of ~100 nm clusters of MERTK, β2 integrins, and multiple signaling molecules including Src-family kinases, PI3-kinases, and the integrin regulatory proteins ILK and FAK. MERTK is unable to mediate efferocytosis in the absence of β2 integrins or their opsonins, while β2 integrins require activation via MERTK signaling to induce the engulfment of apoptotic cells. Using FRET microscopy, we determined that MERTK directly induces the conformational change of β2 integrins from the low to high-affinity form via a PI3-kinase, FAK and ILK-dependent signaling pathway. Activation of this pathway then drives the coalescence of the MERTK/β2 integrin clusters into a larger synapse through which efferocytosis occurs. The identification of the MERTK signaling pathway and the role of β2 integrins in this pathway provides new insights into the function of this critical homeostatic receptor and provides new insights into how MERTK mutations and signaling defects may contribute to inflammatory and autoimmune diseases.

Phagocytic clearance of apoptotic, necrotic, necroptotic and pyroptotic cells.

Although millions of cells in the human body will undergo programmed cell death each day, dying cells are rarely detected under homeostatic settings in vivo. The swift removal of dying cells is due to the rapid recruitment of phagocytes to the site of cell death which then recognise and engulf the dying cell. Apoptotic cell clearance — the engulfment of apoptotic cells by phagocytes — is a well-defined process governed by a series of molecular factors including ‘find-me’, ‘eat-me’, ‘don't eat-me’ and ‘good-bye’ signals. However, in recent years with the rapid expansion of the cell death field, the removal of other necrotic-like cell types has drawn much attention. Depending on the type of death, dying cells employ different mechanisms to facilitate engulfment and elicit varying functional impacts on the phagocyte, from wound healing responses to inflammatory cytokine secretion. Nevertheless, despite the mechanism of death, the clearance of dying cells is a fundamental process required to prevent the uncontrolled release of pro-inflammatory mediators and inflammatory disease. This mini-review summarises the current understandings of: (i) apoptotic, necrotic, necroptotic and pyroptotic cell clearance; (ii) the functional consequences of dying cell engulfment and; (iii) the outstanding questions in the field.

Enhancement of innate and adaptive anti-tumor immunity by serum obtained from vascular photodynamic therapy-cured BALB/c mouse.

Photodynamic therapy (PDT) is a clinically approved treatment for various types of cancer. Besides killing the tumor cells directly, PDT has also been reported to trigger anti-tumor immunity. In our previous study, BAM-SiPc-based PDT was shown to induce immunogenic cell death on CT26 murine colon tumor cells in vitro. Using the BALB/c mouse animal model and a vascular-PDT (VPDT) approach, it could also eradicate tumor in ∼ 70% of tumor-bearing mice and elicit an anti-tumor immune response. In the present study, the serum obtained from the VPDT-cured mice was studied and found to possess various immunomodulatory properties. In in vitro studies, it stimulated cytokine secretions of IL-6 and C-X-C motif chemokine ligands 1-3 in CT26 cells through the NF-κB and MAPK pathways. The complement protein C5a boosted in the serum was shown to be involved in the process. The serum also induced calreticulin exposure on CT26 cells and activated dendritic cells. It contained CT26-targeting antibodies which, through the Fc region, induced macrophage engulfment of the tumor cells. In in vivo studies, inoculation of the serum-treated CT26 cells to mice demonstrated a retarded tumor growth with leukocytes, particularly T cells, attracted to the tumor site. In addition, the VPDT-cured mice showed different degrees of resistance against challenge of other types of murine tumor cells, for example, the breast tumor 4T1 and EMT6 cells.