Engulfment Of Apoptotic Cells Research Articles

CED-6/GULP and Components of the Clathrin-mediated Endocytosis Machinery Act Redundantly to Correctly Display CED-1 on the Cell Membrane in C. elegans.

CED-1 is a transmembrane receptor involved in the recognition of "eat-me" signals displayed on the surface of apoptotic cells and thus central for the subsequent engulfment of the cell corpse in C. elegans. The roles of CED-1 in engulfment are well established, as are its downstream effectors. The latter includes the adapter protein CED-6/GULP and the ABC family homolog CED-7. However, how CED-1 is maintained on the plasma membrane in the absence of engulfment is currently unknown. Here, we show that CED-6 and CED-7 have a novel role in maintaining CED-1 correctly on the plasma membrane. We propose that the underlying mechanism is via endocytosis as CED-6 and CED-7 act redundantly with clathrin and its adaptor, the AP2 complex, in ensuring correct CED-1 localization. In conclusion, CED-6 and CED-7 impact other cellular processes than engulfment of apoptotic cells.

Novel Role of Endothelial CD47 in the Regulation of Pathogenesis of Coronary Artery Disease

Background: The anti-phagocytic molecule CD47 is upregulated in atherosclerotic plaques of patients with CVD. However, the role of endothelial cell-specific expression of CD47 in the pathogenesis and progression of atherosclerosis has not yet been fully defined. Kojima et.al. described atheroprotective effect of anti-CD47 antibody by enhancing efferocytosis in atherosclerosis. In this study, we determined whether endothelial cell-specific CD47 regulates efferocytosis in atherosclerosis. Methods: Endothelial cell-specific, tamoxifen-inducible CD47knockout (CD47iECKO) mice were injected with a single dose of AAV-mutant PCSK9 via the tail vein. Susceptibility to atherosclerosis was evaluated in cross sections of the aortic sinus in mice fed Western Diet for 12 weeks. Using single-cell RNA sequencing and in vitro efferocytosis assays, we investigated the role of CD47 in regulating efferocytosis in an atherosclerotic mouse model. Results: Endothelial cell-specific deletion of CD47 led to a significant plaque reduction in the aortic sinus of athero-CD47iECKO mice. Cluster analysis and gene expression profiles of 20,000 aortic endothelial cells from athero-control and athero-CD47iECKO mice identified 10 major cell types from the integrated data: endothelial cells, vascular smooth muscle cells, fibroblasts, immune cells (B cells, T cells, macrophage), erythrocytes, oligodendrocytes cells, Schwann cells, and pericytes. After Western Diet intake, in the CD47iECKO group, a subpopulation of endothelial cells as well as fibroblasts, T-cells, B-cells, resident aortic macrophages, erythrocytes, oligodendrocytes, and pericytes were increased, while vascular smooth muscle cell numbers decreased. Using an in vitro efferocytosis assay on isolated primary endothelial cells from control and CD47iECKO mice, we observed CD47 deletion induced engulfment of apoptotic Jurkat cells, which had been exposed to BAY11-7082 (NF-κB inhibitor) to stimulate the apoptotic attributes. Quantitative PCR analyses showed that CD47 deletion increases the expression of efferocytosis receptor FasL, CX3CL1, Gpr132, MerTK, Scarb1, Sirpa, TSP1, and HMGB1. Conclusion: Our study indicates that genetic deletion of CD47 in endothelial cells attenuates atheroma progression by enhancing efferocytosis. This study also reveals phenotypic heterogenicity of endothelial cells at the single cell level during atherogenesis and provides a deeper understanding of endothelial cell biology in atherosclerosis that may facilitate the development of novel therapeutic interventions for this cardiovascular disease. NIH, AHA. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

EpHero – a tandem-fluorescent probe to track the fate of apoptotic cells during efferocytosis

The efficient removal of apoptotic cells via efferocytosis is critical for maintaining optimal tissue function. This involves the binding and engulfment of apoptotic cells by phagocytes and the subsequent maturation of the phagosome, culminating in lysosomal fusion and cargo destruction. However, current approaches to measure efferocytosis rely on labelling apoptotic targets with fluorescent dyes, which do not sufficiently distinguish between changes to the engulfment and acidification of apoptotic material. To address this limitation, we have developed a genetically coded ratiometric probe epHero which when expressed in the cytoplasm of target cells, bypasses the need for additional labelling steps. We demonstrate that epHero is a pH-sensitive reporter for efferocytosis and can be used to simultaneously track changes to apoptotic cell uptake and acidification, both in vitro and in mice. As proof-of-principle, we modify extracellular nutrition to show how epHero can distinguish between changes to cargo engulfment and acidification. Thus, tracking efferocytosis with epHero is a simple, cost-effective improvement on conventional techniques.

Abstract 6535: Targeting estrogen receptor signaling in tumor associated myeloid cells to enhance anti-tumor immunity

Abstract Immune checkpoint inhibitors (ICB) have significantly improved outcomes across the spectrum of cancers, especially in patients with metastatic melanoma or non-small cell lung cancer (NSCLC). Despite encouraging efficacy in some patients, the development of primary and acquired resistance and severe toxicity associated with these treatment regimens, together with the overall low response rates, have limited their widespread use. Mining publicly available transcriptomic datasets, we have demonstrated that the response to ICB in patients with melanoma is influenced by the functionality of tumor associated macrophages (TAMs). Thus, it is of significance that we have demonstrated that 17-β estradiol (E2), working through myeloid cell intrinsic estrogen receptor alpha (ERα), promotes tumor growth in preclinical mouse models by polarizing tumor associated macrophages towards an immune-suppressive state in a manner that suppresses CD8+T cell functionality and limits ICB efficacy independent of gender. Transcriptomic analysis of tumor infiltrating myeloid cells isolated from E2-treated syngeneic tumors revealed that the type I interferon (IFN) signaling pathway was suppressed and that this activity could be reversed by the addition of fulvestrant a Selective Estrogen Receptor Downregulator. The importance of type I IFN signaling in E2-regulated tumor growth was confirmed in syngeneic mouse tumor models (B16F10-melanoma and LLC1-NSCLC cells) by showing that inhibiting type I interferon receptor (IFNAR) activity using blocking antibodies reversed the protective effects of E2 deprivation (ovariectomy) on tumor growth. Further, using in vitro cultures of macrophages from mouse bone marrow or peripheral blood mononuclear cells from deidentified human donors, we demonstrated that E2-dependent inhibition of type I IFN signaling could be attributed to increased efferocytosis by myeloid cells. These findings were recapitulated in vivo using an IFN reporter mouse (Mx1-egfp) implanted with labelled tumor cells (mCherry-Spectrin labelled LLC1), where we demonstrated that E2 mediated-suppression of myeloid cell intrinsic interferon signaling within the tumor microenvironment is contingent upon their engulfment of apoptotic cancer cells. Mechanistically, this enhanced efferocytosis was attributed to increased expression of the fractalkine receptor (CX3CR1) in E2-treated myeloid cells- as either genetic or pharmacological depletion of CX3CR1 in mouse reversed E2-mediated tumor growth (B16F10 and LLC1), myeloid cell polarization (from immunosuppressive to proinflammatory), myeloid cell intrinsic type I IFN signaling and reinvigorated CD8+T cell function. Taken together, our results highlight the potential clinical utility of targeting the E2/ER-CX3CR1 pathway as a means to enhance response to ICBs. Citation Format: Binita Chakraborty, Michael Brown, Prabuddha Chakraborty, Aditi Goyal, Ching Yi Chang, Donald McDonnell. Targeting estrogen receptor signaling in tumor associated myeloid cells to enhance anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6535.

Abstract 4291: Bai1 inhibition promotes ferroptosis of breast cancer cells to prevent tumor growth in human xenograft tumor models

Abstract The adhesion G-protein coupled- receptor 1 ADGRB1/Bai1 was identified as a phosphatidylserine receptor that cooperates with ELMO/Dock180/Rac to promote maximal engulfment of apoptotic cells. This function of Bai1 was also shown to promote myoblasts fusion. Cell-cell fusion has been demonstrated to occur in normal physiological functions as well as in diseases. Studies have shown in cancer that cell-cell fusion increases tumor heterogeneity, drug resistance, and metastasis as hybrids acquire new properties that allow them to migrate and proliferate at new sites. Activation of ELMO-Dock signaling pathway has been associated with breast cancer metastasis. Moreover, ELMO1 has been identified as a modifier of breast cancer risk for BRCA mutation carriers. Rac1 was also shown to be overexpressed in proliferative breast disease, preinvasive and invasive breast carcinoma as well as lymph node metastases. Our recent studies showed that breast cancer cell fusion with bone marrow-derived mesenchymal stem cells was modulated by hypoxic conditions and promoted by apoptosis. Our hypothesis is that hypoxia stress induced apoptosis activates the phosphatidylserine receptor Bai1, and it signals through the ELMO/DOCK180/Rac to promote breast cancer cell fusion-driven tumor heterogeneity and metastasis. In this study, we determined the ability of Bai1 depleted breast cancer cells to induce tumor growth in immunocompromised mice. We used Bai1 sgRNA to inhibit Bai1 expression in the breast cancer cell MDA-MB-231. These cells (1x 106) together with bone marrow-derived mesenchymal stem cells (MSCs) (1x 106) were injected into the fat pad of the breast of 15 NOD SCID female mice. Control mice (15) received the same cells with no inhibition of Bai1. The mice were monitored for eight weeks. We found that Bai1 inhibition significantly reduced tumor growth in these mice in the course of time (P<0.05). The average tumor weight was also significantly reduced when Bai1 was inhibited (P<0.001). RNA sequencing of tumors generated from these mice revealed a significant increase in the expression of genes driving ferroptosis and a reduction in critical oncogenes in the experiment group compared to the control group. These findings will be helpful in the development of new classes of drugs effective in breast cancer treatment. Grant Support The NIH/NIMHD grant 5U54MD015929-03, the Society for Investigative Dermatology (SID) Freinkel Diversity Fellowship, and NSF Award Notice for Award ID 2142465 (awarded to FNK). Citation Format: Felicite Kamdem Noubissi, Oluwatowin Odubanjo, Hung-Chung Huang, Brenda Ogle, Paul Tchounwou. Bai1 inhibition promotes ferroptosis of breast cancer cells to prevent tumor growth in human xenograft tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4291.

Tim4 deficiency reduces CD301b+ macrophage and aggravates periodontitis bone loss

Periodontitis is a common chronic inflammatory disease that causes the periodontal bone destruction and may ultimately result in tooth loss. With the progression of periodontitis, the osteoimmunology microenvironment in periodontitis is damaged and leads to the formation of pathological alveolar bone resorption. CD301b+ macrophages are specific to the osteoimmunology microenvironment, and are emerging as vital booster for conducting bone regeneration. However, the key upstream targets of CD301b+ macrophages and their potential mechanism in periodontitis remain elusive. In this study, we concentrated on the role of Tim4, a latent upstream regulator of CD301b+ macrophages. We first demonstrated that the transcription level of Timd4 (gene name of Tim4) in CD301b+ macrophages was significantly upregulated compared to CD301b− macrophages via high-throughput RNA sequencing. Moreover, several Tim4-related functions such as apoptotic cell clearance, phagocytosis and engulfment were positively regulated by CD301b+ macrophages. The single-cell RNA sequencing analysis subsequently discovered that Cd301b and Timd4 were specifically co-expressed in macrophages. The following flow cytometric analysis indicated that Tim4 positive expression rates in total macrophages shared highly synchronized dynamic changes with the proportions of CD301b+ macrophages as periodontitis progressed. Furthermore, the deficiency of Tim4 in mice decreased CD301b+ macrophages and eventually magnified alveolar bone resorption in periodontitis. Additionally, Tim4 controlled the p38 MAPK signaling pathway to ultimately mediate CD301b+ macrophages phenotype. In a word, Tim4 might regulate CD301b+ macrophages through p38 MAPK signaling pathway in periodontitis, which provided new insights into periodontitis immunoregulation as well as help to develop innovative therapeutic targets and treatment strategies for periodontitis.

Efferocytosis reprograms the tumor microenvironment to promote pancreatic cancer liver metastasis.

Pancreatic ductal adenocarcinoma is a highly metastatic disease and macrophages support liver metastases. Efferocytosis, or engulfment of apoptotic cells by macrophages, is an essential process in tissue homeostasis and wound healing, but its role in metastasis is less well understood. Here, we found that the colonization of the hepatic metastatic site is accompanied by low-grade tissue injury and that efferocytosis-mediated clearance of parenchymal dead cells promotes macrophage reprogramming and liver metastasis. Mechanistically, progranulin expression in macrophages is necessary for efficient efferocytosis by controlling lysosomal acidification via cystic fibrosis transmembrane conductance regulator and the degradation of lysosomal cargo, resulting in LXRα/RXRα-mediated macrophage conversion and upregulation of arginase 1. Pharmacological blockade of efferocytosis or macrophage-specific genetic depletion of progranulin impairs macrophage conversion, improves CD8+ T cell functions, and reduces liver metastasis. Our findings reveal how hard-wired functions of macrophages in tissue repair contribute to liver metastasis and identify potential targets for prevention of pancreatic ductal adenocarcinoma liver metastasis.

Redistribution of the glycocalyx exposes phagocytic determinants on apoptotic cells

Phagocytes remove dead and dying cells by engaging "eat-me" ligands such as phosphatidylserine (PtdSer) on the surface of apoptotic targets. However, PtdSer is obscured by the bulky exofacial glycocalyx, which also exposes ligands that activate "don't-eat-me" receptors such as Siglecs. Clearly, unshielding the juxtamembrane "eat-me" ligands is required for the successful engulfment of apoptotic cells, but the mechanisms underlying this process have not been described. Using human and murine cells, we find that apoptosis-induced retraction and weakening of the cytoskeleton that anchors transmembrane proteins cause an inhomogeneous redistribution of the glycocalyx: actin-depleted blebs emerge, lacking the glycocalyx, while the rest of the apoptotic cell body retains sufficient actin to tether the glycocalyx in place. Thus, apoptotic blebs can be engaged by phagocytes and are targeted for engulfment. Therefore, in cells with an elaborate glycocalyx, such as mucinous cancer cells, this "don't-come-close-to-me" barrier must be removed to enable clearance by phagocytosis.

Potent CTLs can be induced against tumor cells in an environment of lower levels of systemic MFG-E8.

The direction and magnitude of immune responses are critically affected when dead cells are disposed of. Milk fat globule-epidermal growth factor-factor 8 (MFG-E8) promotes the engulfment of apoptotic normal and cancerous cells without inducing inflammation. We have previously reported that a certain proportion of the cancer cells express abundant MFG-E8, and that such expression is associated with the shorter survival of patients with esophageal cancer who had received chemotherapy before surgery. However, the influence of tumor-derived and systemically existing MFG-E8 on antitumor immune responses has not yet been fully investigated. Herein, we showed that CTL-dependent antitumor immune responses were observed in mice with no or decreased levels of systemic MFG-E8, and that such responses were enhanced further with the administration of anti-PD-1 antibody. In mice with decreased levels of systemic MFG-E8, the dominance of regulatory T cells in tumor-infiltrating lymphocytes was inverted to CD8+ T cell dominance. MFG-E8 expression by tumor cells appears to affect antitumor immune responses only when the level of systemic MFG-E8 is lower than the physiological status. We have also demonstrated in the clinical setting that lower levels of plasma MFG-E8, but not MFG-E8 expression in tumor cells, before the treatment was associated with objective responses to anti-PD-1 therapy in patients with non-small cell lung cancer. These results suggest that systemic MFG-E8 plays a critical role during the immunological initiation process of antigen-presenting cells to increase tumor-specific CTLs. Regulation of the systemic level of MFG-E8 might induce efficient antitumor immune responses and enhance the potency of anti-PD-1 therapy.

Potential roles for efferocytosis in glioblastoma immune evasion.

Glioblastoma is an aggressive and incurable brain cancer. This cancer establishes both local and systemic immunosuppression that creates a major obstacle to effective immunotherapies. Many studies point to tumor-resident myeloid cells (primarily microglia and macrophages) as key mediators of this immunosuppression. Myeloid cells exhibit a high level of plasticity with respect to their phenotype and are capable of both stimulating and repressing immune responses. How glioblastomas recruit myeloid cells and exploit them to avoid the immune system is an active area of research. Macrophages can acquire an immunosuppressive phenotype as a consequence of exposure to cytokines such as TGFB1 or IL4; in addition, macrophages can acquire an immunosuppressive phenotype as a consequence of the engulfment of apoptotic cells, a process referred to as efferocytosis. There is substantial evidence that glioblastoma cells are able to secrete cytokines and other factors that induce an immunosuppressive phenotype in macrophages and microglia. However, less is known about the contribution of efferocytosis to immunosuppression in glioblastoma. Here I review the literature in this area and discuss the potential of efferocytosis inhibition to improve glioblastoma response to immunotherapy.

Radio-chemotherapy of glioblastoma cells promotes phagocytosis by macrophages in vitro

Background and PurposeImmunotherapy is actively explored in glioblastoma (GBM) to improve patient prognosis. Tumor-associated macrophages (TAMs) are abundant in GBM and harnessing their function for anti-tumor immunity is of interest. They are plastic cells that are influenced by the tumor microenvironment, by radio-chemotherapy and by their own phagocytic activity. Indeed, the engulfment of necrotic cells promotes pro-inflammatory (and anti-tumoral) functions while the engulfment of apoptotic cells promotes anti-inflammatory (and pro-tumoral) functions through efferocytosis. Materials and MethodsTo model the effect of radio-chemotherapy on the GBM microenvironment, we exposed human macrophages to supernatant of treated GBM cells in vitro. Macrophages were derived from human monocytes and GBM cells from patient-resected tumors. GBM cells were exposed to therapeutically relevant doses of irradiation and chemotherapy. Apoptosis and phagocytic activity were assessed by flow cytometry. ResultsThe phagocytic activity of macrophages was increased, and it was correlated with the proportion of apoptotic GBM cells producing the supernatant. Whether uptake of apoptotic tumor cells could occur would depend upon the expression of efferocytosis-associated receptors. Indeed, we showed that efferocytosis-associated receptors, such as AXL, were upregulated. Conclusions and PerspectivesWe showed that macrophage phagocytic activity increased when exposed to supernatant from GBM cells treated by radio-chemotherapy. However, as efferocytosis-associated receptors were up-regulated, this effect could be deleterious for the anti-GBM immune response. We speculate that by inducing GBM cell apoptosis in parallel to an increase in efferocytosis receptor expression, the impact of radio-chemotherapy on phagocytic activity could promote anti-inflammatory and pro-tumoral TAM functions.

Studying Efferocytosis Dynamics in Tissue-Resident Macrophages Ex Vivo.

In vitro cocultures of macrophages and apoptotic cells (ACs) provide a practical and useful tool to study efferocytosis. Here, we describe a method for automated quantification and imaging of recognition and engulfment of apoptotic cells by primary macrophages using imaging flow cytometry (IFC). IFC-based analysis allows us to successfully quantify efferocytosis, clearly distinguishing phagocytic from nonphagocytic macrophages and, more importantly, from those in recognition stage, which is not achievable by standard flow cytometrical analysis. To this end, we established a universally employable analysis pipeline to address efferocytosis that can be easily adapted to any macrophage population from samples of different origins.

LXR signaling controls homeostatic dendritic cell maturation.

Dendritic cells (DCs) mature in an immunogenic or tolerogenic manner depending on the context in which an antigen is perceived, preserving the balance between immunity and tolerance. Whereas the pathways driving immunogenic maturation in response to infectious insults are well-characterized, the signals that drive tolerogenic maturation during homeostasis are still poorly understood. We found that the engulfment of apoptotic cells triggered homeostatic maturation of type 1 conventional DCs (cDC1s) within the spleen. This maturation process could be mimicked by engulfment of empty, nonadjuvanted lipid nanoparticles (LNPs), was marked by intracellular accumulation of cholesterol, and was highly specific to cDC1s. Engulfment of either apoptotic cells or cholesterol-rich LNPs led to the activation of the liver X receptor (LXR) pathway, which promotes the efflux of cellular cholesterol, and repressed genes associated with immunogenic maturation. In contrast, simultaneous engagement of TLR3 to mimic viral infection via administration of poly(I:C)-adjuvanted LNPs repressed the LXR pathway, thus delaying cellular cholesterol efflux and inducing genes that promote T cell-mediated immunity. These data demonstrate that conserved cellular cholesterol efflux pathways are differentially regulated in tolerogenic versus immunogenic cDC1s and suggest that administration of nonadjuvanted cholesterol-rich LNPs may be an approach for inducing tolerogenic DC maturation.

Abstract 338: Novel Role Of Endothelial Cd47 In The Regulation Of Pathogenesis Of Atherosclerosis

Background: The anti-phagocytic molecule CD47 is upregulated in atherosclerotic plaques of patients with cerebrovascular disease. However, the role of endothelial cell-specific CD47 in the pathogenesis of atherosclerosis has not yet been defined. Kojima et.al . described atheroprotective effect of anti-CD47 antibody by enhancing efferocytosis in atherosclerosis. In this study, we determined whether endothelialCD47 regulates efferocytosis in atherosclerosis. Methods: Susceptibility to atherosclerosis was evaluated in cross sections of the aortic sinus in atherosclerotic mice. Using single-cell RNA sequencing and efferocytosis assays, we investigated the role of CD47 in regulating efferocytosis in atherosclerotic mouse model. Results: Endothelial cell-specific deletion of CD47 led to significant plaque reduction in aortic sinus of athero-CD47iECKO mice. Cluster analysis and gene expression profiles of 20,000 aortic endothelial cells from atherosclerotic mice identified 10 major cell types: endothelial cells, VSMCs, fibroblasts, B cells, T cells, macrophage, erythrocytes, oligodendrocytes cells, Schwann cells, and pericytes. In athero-CD47iECKO group, subpopulation of endothelial cells as well as fibroblasts, T-cells, B-cells, macrophages, erythrocytes, oligodendrocytes, and pericytes were increased, while vascular smooth muscle cell numbers decreased. Using efferocytosis assay on isolated primary endothelial cells from control and CD47iECKO mice, we observed CD47 deletion induced engulfment of apoptotic Jurkat cells, which had been exposed to NF-κB inhibitor, BAY11-7082 to stimulate apoptotic attributes. Quantitative PCR analyses showed that CD47 deletion increases the expression of efferocytosis receptor FasL, CX3CL1, Gpr132, MerTK, Scarb1, Sirpa, TSP1, and HMGB1. Conclusion: Our study indicates that genetic deletion of CD47 in endothelial cells attenuates atheroma progression by enhancing efferocytosis. This study also reveals phenotypic heterogenicity of endothelial cells at the single-cell level during atherogenesis and provides deeper understanding of endothelial cell biology in atherosclerosis that may facilitate the development of novel therapeutic interventions for cardiovascular disease.

Protective effects of an electrophilic metabolite of docosahexaenoic acid on UVB-induced oxidative cell death, dermatitis, and carcinogenesis

Docosahexaenoic acid (DHA), a representative omega-3 (ω-3) polyunsaturated fatty acids, undergoes metabolism to produce biologically active electrophilic species. 17-Oxo-DHA is one such reactive metabolite generated from DHA by cyclooxygenase-2 and dehydrogenase in activated macrophages. The present study was aimed to investigate the effects of 17-oxo-DHA on ultraviolet B (UVB)-induced oxidative stress, inflammation, and carcinogenesis in mouse skin. UVB-induced epidermal cell death was ameliorated by topically applied 17-oxo-DHA. Topical application of 17-oxo-DHA onto hairless mouse skin inhibited UVB-induced phosphorylation of the proinflammatory transcription factor, STAT3 on tyrosine 705 (Tyr705). The 17-oxo-DHA treatment also reduced the levels of oxidative stress markers, 4-hydroxynonenal-modified protein, malondialdehyde, and 8-oxo-2′-deoxyguanosine. The protective effects of 17-oxo-DHA against oxidative damage in UVB-irradiated mouse skin were associated with activation of Nrf2. 17-Oxo-DHA enhanced the engulfment of apoptotic JB6 cells by macrophages, which was related to the increased expression of the scavenger receptor CD36. The 17-oxo-DHA-mediated potentiation of efferocytic activity of macrophages was attenuated by the pharmacologic inhibition or knockout of Nrf2. The pretreatment with 17-oxo-DHA reduced the UVB-induced skin carcinogenesis and tumor angiogenesis. It was also confirmed that 17-oxo-DHA treatment significantly inhibited the phosphorylation of the Tyr705 residue of STAT3 and decreased the expression of its target proteins in cutaneous papilloma. In conclusion, 17-oxo-DHA protects against UVB-induced oxidative cell death, dermatitis, and carcinogenesis. These effects were associated with inhibition of STAT3-mediated proinflammatory signaling and also activation of Nrf2 with subsequent upregulation of antioxidant and anti-inflammatory gene expression.

Gulp1 deficiency augments bone mass in malemice by affecting osteoclasts due to elevated 17β-estradiol levels.

The engulfment adaptor phosphotyrosine-binding domain containing 1 (GULP1) is an adaptor protein involved in the engulfment of apoptotic cells via phagocytosis. Gulp1 was first found to promote the phagocytosis of apoptotic cells by macrophages, and its role in various tissues, including neurons and ovaries, has been well studied. However, the expression and function of GULP1 in bone tissue are poorly understood. Consequently, to determine whether GULP1 plays a role in the regulation of bone remodeling in vitro and in vivo, we generated Gulp1 knockout (KO) mice. Gulp1 was expressed in bone tissue, mainly in osteoblasts, while its expression is very low in osteoclasts. Microcomputed tomography and histomorphometry analysis in 8-week-old male Gulp1 KO mice revealed a high bone mass in comparison with male wild-type (WT) mice. This was a result of decreased osteoclast differentiation and function in vivo and in vitro as confirmed by a reduced actin ring and microtubule formation in osteoclasts. Gas chromatography-mass spectrometry analysis further showed that both 17β-estradiol (E2) and 2-hydroxyestradiol levels, and the E2/testosterone metabolic ratio, reflecting aromatase activity, were also higher in the bone marrow of male Gulp1 KO mice than in male WT mice. Consistent with mass spectrometry analysis, aromatase enzymatic activity was significantly higher in the bone marrow of male Gulp1 KO mice. Altogether, our results suggest that GULP1 deficiency decreases the differentiation and function of osteoclasts themselves and increases sex steroid hormone-mediated inhibition of osteoclast differentiation and function, rather than affecting osteoblasts, resulting in a high bone mass in male mice. To the best of our knowledge, this is the first study to explore the direct and indirect roles of GULP1 in bone remodeling, providing new insights into its regulation.

Tim4, a macrophage receptor for apoptotic cells, binds polystyrene microplastics via aromatic-aromatic interactions

Understanding the interface between microplastics and biological systems will provide new insights into the impacts of microplastics on living organisms. When microplastics enter the body, they are engulfed preferentially by phagocytes such as macrophages. However, it is not fully understood how phagocytes recognize microplastics and how microplastics impact phagocyte functions. In this study, we demonstrate that T cell immunoglobulin mucin 4 (Tim4), a macrophage receptor for phosphatidylserine (PtdSer) on apoptotic cells, binds polystyrene (PS) microparticles as well as multi-walled carbon nanotubes (MWCNTs) through the extracellular aromatic cluster, revealing a novel interface between microplastics and biological systems via aromatic-aromatic interactions. Genetic deletion of Tim4 demonstrated that Tim4 is involved in macrophage engulfment of PS microplastics as well as of MWCNTs. While Tim4-mediated engulfment of MWCNTs causes NLRP3-dependent IL-1β secretion, that of PS microparticles does not. PS microparticles neither induce TNF-α, reactive oxygen species, nor nitric oxide production. These data indicate that PS microparticles are not inflammatory. The PtdSer-binding site of Tim4 contains an aromatic cluster that binds PS, and Tim4-mediated macrophage engulfment of apoptotic cells, a process called efferocytosis, was competitively blocked by PS microparticles. These data suggest that PS microplastics do not directly cause acute inflammation but perturb efferocytosis, raising concerns that chronic exposure to large amounts of PS microplastics may cause chronic inflammation leading to autoimmune diseases.

HIV vaccine candidate efficacy in female macaques mediated by cAMP-dependent efferocytosis and V2-specific ADCC

The development of an effective vaccine to protect against HIV acquisition will be greatly bolstered by in-depth understanding of the innate and adaptive responses to vaccination. We report here that the efficacy of DNA/ALVAC/gp120/alum vaccines, based on V2-specific antibodies mediating apoptosis of infected cells (V2-ADCC), is complemented by efferocytosis, a cyclic AMP (cAMP)-dependent antiphlogistic engulfment of apoptotic cells by CD14+ monocytes. Central to vaccine efficacy is the engagement of the CCL2/CCR2 axis and tolerogenic dendritic cells producing IL-10 (DC-10). Epigenetic reprogramming in CD14+ cells of the cyclic AMP/CREB pathway and increased systemic levels of miRNA-139-5p, a negative regulator of expression of the cAMP-specific phosphodiesterase PDE4D, correlated with vaccine efficacy. These data posit that efferocytosis, through the prompt and effective removal of apoptotic infected cells, contributes to vaccine efficacy by decreasing inflammation and maintaining tissue homeostasis.

Analysis of Efferocytic Receptor Dynamics and Synapse Formation in a Frustrated Efferocytosis Model.

Efferocytes express multiple receptors that mediate the recognition and engulfment of apoptotic cells through a process known as efferocytosis. Ligation of these receptors induces the formation of a structured efferocytic synapse that mediates the engulfment of the apoptotic cell by the efferocyte. The lateral diffusion of these receptors allows for clustering-mediated receptor activation and is central for the formation of the efferocytic synapse. This chapter describes a single particle tracking protocol to analyze the diffusion of efferocytic receptors within a frustrated efferocytosis model. This enables high-resolution tracking of efferocytic receptors throughout synapse formation, allowing the user to simultaneously quantify synapse formation and the dynamics of receptor diffusion as the efferocytic synapse evolves.

Apoptosis and Phagocytosis as Antiviral Mechanisms.

Viruses are infectious entities that make use of the replication machinery of their hosts to produce more progenies, causing disease and sometimes death. To counter viral infection, metazoan hosts are equipped with various defense mechanisms, from the rapid-evoking innate immune responses to the most advanced adaptive immune responses. Previous research demonstrated that cells in fruit flies and mice infected with Drosophila C virus and influenza, respectively, undergo apoptosis, which triggers the engulfment of apoptotic virus-infected cells by phagocytes. This process involves the recognition of eat-me signals on the surface of virus-infected cells by receptors of specialized phagocytes, such as macrophages and neutrophils in mice and hemocytes in fruit flies, to facilitate the phagocytic elimination of virus-infected cells. Inhibition of phagocytosis led to severe pathologies and death in both species, indicating that apoptosis-dependent phagocytosis of virus-infected cells is a conserved antiviral mechanism in multicellular organisms. Indeed, our understanding of the mechanisms underlying apoptosis-dependent phagocytosis of virus-infected cells has shed a new perspective on how hosts defend themselves against viral infection. This chapter explores the mechanisms of this process and its potential for developing new treatments for viral diseases.