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Resolution of acute inflammation is an active process orchestrated by endogenous mediators and mechanisms pivotal in host defense and homeostasis. The macrophage mediator in resolving inflammation, maresin 1 (MaR1), is a potent immunoresolvent, stimulating resolution of acute inflammation and organ protection. Using an unbiased screening of greater than 200 GPCRs, we identified MaR1 as a stereoselective activator for human leucine-rich repeat containing G protein–coupled receptor 6 (LGR6), expressed in phagocytes. MaR1 specificity for recombinant human LGR6 activation was established using reporter cells expressing LGR6 and functional impedance sensing. MaR1-specific binding to LGR6 was confirmed using (3)H-labeled MaR1. With human and mouse phagocytes, MaR1 (0.01–10 nM) enhanced phagocytosis, efferocytosis, and phosphorylation of a panel of proteins including the ERK and cAMP response element-binding protein. These MaR1 actions were significantly amplified with LGR6 overexpression and diminished by gene silencing in phagocytes. Thus, we provide evidence for MaR1 as an endogenous activator of human LGR6 and a novel role of LGR6 in stimulating MaR1’s key proresolving functions of phagocytes.

Background/aims Heme oxygenase-1 (HO-1) plays a central role in cellular defense against inflammatory insults, and its induction in macrophages potentiates their efferocytic activity. In this study, we explored the potential role of macrophage HO-1 in the resolution of experimentally induced colitis. Methods To induce colitis, male C57BL/6 mice were treated with 2% dextran sulfate sodium (DSS) in the drinking water for 7 days. To investigate efferocytosis, apoptotic colon epithelial CCD 841 CoN cells were coincubated with bone marrow-derived macrophages (BMDMs). Results Administration of the HO-1 inhibitor zinc protoporphyrin IX (ZnPP) blunted the resolution of DSS-induced intestinal inflammation and expression of the proresolving M2 macrophage marker CD206. BMDMs treated with apoptotic colonic epithelial cells showed significantly elevated expression of HO-1 and its regulator Nrf2. Under the same experimental conditions, the proportion of CD206-expressing macrophages was also enhanced. ZnPP treatment abrogated the upregulation of CD206 expression in BMDMs engulfing apoptotic colonic epithelial cells. This result was verified with BMDMs isolated from HO-1-knockout mice. BMDMs, when stimulated with lipopolysaccharide, exhibited increased expression of CD86, a marker of M1 macrophages. Coculture of lipopolysaccharide-stimulated BMDMs with apoptotic colonic epithelial cell debris dampened the expression of CD86 as well as the pro-inflammatory cytokines in an HO-1-dependent manner. Genetic ablation as well as pharmacologic inhibition of HO-1 significantly reduced the proportion of efferocytic BMDMs expressing the scavenger receptor CD36. Conclusions HO-1 plays a key role in the resolution of experimentally induced colitis by modulating the polarization of macrophages.

OBJECTIVE To investigate the role of omega-3 polyunsaturated fatty acids (Ω-3)–derived proresolving lipid mediators (PRLM) in the resolution of mild airway inflammation in horses. ANIMALS 20 horses with mild airway inflammation. PROCEDURES Horses previously eating hay were fed hay pellets (low Ω-3 content; n = 10) or haylage (high Ω-3 content; 9) for 6 weeks. Dust exposure was measured in the breathing zone with a real-time particulate monitor. Bronchoalveolar lavage (BAL) was performed at baseline, week 3, and week 6. The effect of PRLM on neutrophil apoptosis and efferocytosis was examined in vitro. BAL fluid inflammatory cell proportions, apoptosis of circulating neutrophils, efferocytosis displayed by alveolar macrophages, and plasma lipid concentrations were compared between groups fed low and high amounts of Ω-3 by use of repeated measures of generalized linear models. RESULTS Dust exposure was significantly higher with hay feeding, compared to haylage and pellets, and equivalent between haylage and pellets. BAL fluid neutrophil proportions decreased significantly in horses fed haylage (baseline, 11.8 ± 2.4%; week 6, 2.5 ± 1.1%) but not pellets (baseline, 12.1 ± 2.3%; week 6, 8.5% ± 1.7%). At week 6, horses eating haylage had significantly lower BAL neutrophil proportions than those eating pellets, and a significantly lower concentration of stearic acid than at baseline. PRLM treatments did not affect neutrophil apoptosis or efferocytosis. CLINICAL RELEVANCE Despite similar reduction in dust exposure, horses fed haylage displayed greater resolution of airway inflammation than those fed pellets. This improvement was not associated with increased plasma Ω-3 concentrations. Feeding haylage improves airway inflammation beyond that due to reduced dust exposure, though the mechanism remains unclear.

B cell–activating factor (BAFF), part of a tumor necrosis factor family of cytokines, was recently identified as a regulator of atherosclerosis; however, its role in aortic aneurysm has not been determined. Here, the study examined the effect of selective BAFF antagonism using an anti-BAFF antibody (blocks binding of BAFF to receptors BAFF receptor 3, transmembrane activator and CAML interactor, and B-cell maturation antigen) and mBaffR-mFc (blocks binding of BAFF to BAFF receptor 3) on a murine model of abdominal aortic aneurysm (AAA). In a prevention strategy, the antagonists were injected before the induction of AAA, and in an intervention strategy, the antagonists were injected after the induction of AAA. Both strategies attenuated the formation of AAA. In the intervention group, BAFF antagonism depleted most of the mature B-cell subsets in spleen and circulation, leading to enhanced resolution of inflammation in AAA as indicated by decreased infiltration of B cells and proinflammatory macrophages and a reduced number of apoptotic cells. In AAA tissues, B cells and macrophages were found in close contact. In vitro, B cells, irrespective of treatment with BAFF, impaired the efferocytosis activity of macrophages, suggesting a direct innate role of B cells on macrophage function. Altogether, BAFF antagonism affects survival of the mature B cells, promotes resolution of inflammation in the aorta, and attenuates the growth of AAA in mice.

Resolving tumor-associated inflammation in the tumor microenvironment (TME) may promote antitumor effects. Lipoxin A4 (LXA4) is a short-lived endogenous bioactive lipid with potent anti-inflammatory and pro-resolving properties. Here, a biomimetic of LXA4, NAP1051, was shown to have LXA4-like in vitro properties and antitumor activity in colorectal cancer xenograft models. NAP1051 inhibited neutrophil chemotaxis toward fMLP and dose-dependently promoted dTHP-1 efferocytosis which was equipotent to aspirin-triggered lipoxin A4 (ATLA). In dTHP-1 cells, NAP1051 induced strong phosphorylation on ERK1/2 and AKT similar to formyl peptide receptor 2 (FPR2/ALX) agonists. In two mouse xenograft colorectal cancer models, NAP1051 significantly inhibited tumor growth when given orally at 4.8 to 5 mg/kg/day. Flow cytometric analyses showed that NAP1051 reduced splenic and intratumoral neutrophil and myeloid-derived suppressor cell populations, which correlated to the antitumor effect. In addition, NAP1051 reduced NETosis in the TME while stimulating T-cell recruitment. Overall, these results show that NAP1051 possesses key lipoxin-like properties and has antitumor activity against colorectal cancer via modulation of neutrophils and NETosis in the TME.

Neutrophil-centered inflammation and flawed clearance of neutrophils cause and exuberate multiple pathological conditions. These most abundant leukocytes exhibit very high daily turnover in steady-state and stress conditions. Various armors including oxidative burst, NETs, and proteases function against pathogens, but also dispose neutrophils to spawn pro-inflammatory responses. Neutrophils undergo death through different pathways upon aging, infection, executing the intruder's elimination. These include non-lytic apoptosis and other lytic deaths including NETosis, necroptosis, and pyroptosis with distinct disintegration of the cellular membrane. This causes release and presence of different intracellular cytotoxic, and tissue-damaging content as cell remnants in the extracellular environment. The apoptotic cells and apoptotic bodies get cleared with non-inflammatory outcomes, while lytic deaths associated remnants including histones and cell-free DNA cause pro-inflammatory responses. Indeed, the enhanced frequencies of neutrophil-associated proteases, cell-free DNA, and auto-antibodies in diverse pathologies including sepsis, asthma, lupus, and rheumatoid arthritis, imply disturbed neutrophil resolution programs in inflammatory and autoimmune diseases. Thus, the clearance mechanisms of neutrophils and associated remnants are vital for therapeutics. Though studies focused on clearance mechanisms of senescent or apoptotic neutrophils so far generated a good understanding of the same, but clearance of neutrophils undergoing distinct lytic deaths including NETs, are being the subjects of intense investigations. Here in this review, we are providing the current updates in the clearance mechanisms of apoptotic neutrophils, and focusing on not so well defined recognition, uptake, and degradation of neutrophils undergoing lytic death and associated remnants that may provide new therapeutic approaches in inflammation and autoimmunity.

Myocardial ischemia-reperfusion (IR) generates stress-induced senescent cells (SISCs) that play an important role in the pathophysiology of adverse cardiac remodeling and heart failure via secretion of pro-inflammatory molecules and matrix-degrading proteases. Thus, removal of senescent cells using a senolytic drug could be a potentially effective treatment. However, clinical studies on cancer treatment with a senolytic drug have revealed that systemic administration of a senolytic drug often causes systemic toxicity. Herein we show for the first time that local delivery of a senolytic drug can effectively treat myocardial IR injury. We found that biodegradable poly(lactic-co-glycolic acid) nanoparticle-based local delivery of a senolytic drug (ABT263-PLGA) successfully eliminated SISCs in the IR-injured rat hearts without systemic toxicity. Consequently, the treatment ameliorated inflammatory responses and attenuated adverse remodeling. Surprisingly, the ABT263-PLGA treatment restored the cardiac function over time, whereas the cardiac function decreased over time in the no treatment group. Mechanistically, the ABT263-PLGA treatment not only markedly reduced the expression of pro-inflammatory molecules and matrix-degrading proteases, but also induced macrophage polarization from the inflammatory phase to the reparative phase via efferocytosis of apoptotic SISCs by macrophages. Therefore, the senolytic strategy with ABT263-PLGA in the early stage of myocardial IR injury may be an effective therapeutic option for myocardial infarction. STATEMENT OF SIGNIFICANCE: This study describes a local injection of senolytic drug-loaded nanoparticles that selectively kills stress-induced senescent cells (SISCs) in infarcted heart. Removal of SISCs decreases inflammatory cytokines and normal cell death. We firstly revealed that further efferocytosis of apoptotic senescent cells by macrophages restores cardiac function after myocardial ischemia-reperfusion injury. Importantly, a local injection of senolytic drug did not exhibit systemic toxicity, but a systemic injection did. Our findings not only spotlight the basic understanding of therapeutic potential of senolysis in infarcted myocardium, but also pave the way for the further application of senolytic drug for non-aging related diseases.

Lung diseases, such as cystic fibrosis and COPD, are characterized by mucus obstruction and chronic airway inflammation, but their mechanistic link remains poorly understood. Here, we focus on the function of the mucostatic airway microenvironment on epigenetic reprogramming of airway macrophages (AM) and resulting transcriptomic and phenotypical changes. Using a mouse model of muco-obstructive lung disease (Scnn1b-transgenic), we identify epigenetically controlled, differentially regulated pathways and transcription factors involved in inflammatory responses and macrophage polarization. Functionally, AMs from Scnn1b-transgenic mice have reduced efferocytosis and phagocytosis, and excessive inflammatory responses upon lipopolysaccharide challenge, mediated through enhanced Irf1 function and expression. Ex vivo stimulation of wild-type AMs with native mucus impairs efferocytosis and phagocytosis capacities. In addition, mucus induces gene expression changes, comparable with those observed in AMs from Scnn1b-transgenic mice. Our data show that mucostasis induces epigenetic reprogramming of AMs, leading to changes favoring tissue damage and disease progression. Targeting these altered AMs may support therapeutic approaches in patients with muco-obstructive lung diseases., Muco-obstructive lung diseases are characterised by airway macrophage (AM) populations which may have epigenetic changes. Here using a mouse model the authors show epigenetic alteration of AMs with changes in LPS response, phagocytosis and efferocytosis similar to culture with mucus in vitro.

Multipotent mesenchymal stromal cells (MSCs) ameliorate a wide range of diseases in preclinical models, but the lack of clarity around their mechanisms of action has impeded their clinical utility. The therapeutic effects of MSCs are often attributed to bioactive molecules secreted by viable MSCs. However, we found that MSCs underwent apoptosis in the lung after intravenous administration, even in the absence of host cytotoxic or alloreactive cells. Deletion of the apoptotic effectors BAK and BAX prevented MSC death and attenuated their immunosuppressive effects in disease models used to define MSC potency. Mechanistically, apoptosis of MSCs and their efferocytosis induced changes in metabolic and inflammatory pathways in alveolar macrophages to effect immunosuppression and reduce disease severity. Our data reveal a mode of action whereby the host response to dying MSCs is key to their therapeutic effects; findings that have broad implications for the effective translation of cell-based therapies., Mesenchymal stromal cells (MSCs) demonstrate therapeutic benefits in multiple diseases, but the mechanisms remain unclear as infused MSCs do not persist in the body. Here, the authors show that MSC apoptosis is an important mechanistic element, as MSCs rendered genetically incapable of apoptosis lose their ability to ameliorate disease.

Mechanical ventilation is a life-sustaining therapy for patients with respiratory failure but can cause further lung damage known as ventilator-induced lung injury (VILI). However, the intrinsic molecular mechanisms underlying recovery of VILI remain unknown. Phagocytosis of apoptotic cells (also known as efferocytosis) is a key mechanism orchestrating successful resolution of inflammation. Here we show the positive regulation of macrophage Toll-like receptor (TLR) 4 in efferocytosis and resolution of VILI. Mice were depleted of alveolar macrophages and then subjected to injurious ventilation (tidal volume, 20 mL/kg) for 4 h. On day 1 after mechanical ventilation, Tlr4+/+ or Tlr4−/− bone marrow-derived macrophages (BMDMs) were intratracheally administered to alveolar macrophage-depleted mice. We observed that mice depleted of alveolar macrophages exhibited defective resolution of neutrophilic inflammation, exuded protein, lung edema, and lung tissue injury after ventilation, whereas these delayed responses were reversed by administration of Tlr4+/+ BMDMs. Importantly, these proresolving effects by Tlr4+/+ BMDMs were abolished in mice receiving Tlr4−/− BMDMs. The number of macrophages containing apoptotic cells or bodies in bronchoalveolar lavage fluid was much less in mice receiving Tlr4−/− BMDMs than that in those receiving Tlr4+/+ BMDMs. Macrophage TLR4 deletion facilitated a disintegrin and metalloprotease 17 maturation and enhanced Mer cleavage in response to mechanical ventilation. Heat shock protein 70 dramatically increased Mer tyrosine kinase surface expression, phagocytosis of apoptotic neutrophils, and rescued the inflammatory phenotype in alveolar macrophage-depleted mice receiving Tlr4+/+ BMDMs, but not Tlr4−/− BMDMs. Our results suggest that macrophage TLR4 promotes resolution of VILI via modulation of Mer-mediated efferocytosis.

Patients with cystic fibrosis (CF) have defective macrophage phagocytosis and efferocytosis. Several reports demonstrate that neutrophil elastase (NE), a major inflammatory protease in the CF airway, impairs macrophage phagocytic function. To date, NE-impaired macrophage phagocytic function has been attributed to cleavage of cell surface receptors or opsonins. We applied an unbiased proteomic approach to identify other potential macrophage targets of NE protease activity that may regulate phagocytic function. Using the murine macrophage cell line, RAW 264.7, human blood monocyte-derived macrophages, and primary alveolar macrophages from Cftr-null and wild-type littermate mice, we demonstrated that NE exposure blocked phagocytosis of Escherichia coli bio-particles. We performed liquid chromatography-tandem mass spectroscopy (LC-MS/MS) proteomic analysis of the conditioned media from RAW264.7 treated either with active NE or inactive (boiled) NE as a control. Out of 840 proteins identified in the conditioned media, active NE upregulated 142 proteins and downregulated 211 proteins. NE released not only cell surface proteins into the media but also cytoskeletal, mitochondrial, cytosolic, and nuclear proteins that were detected in the conditioned media. At least 32 proteins were associated with the process of phagocytosis including 11 phagocytic receptors [including lipoprotein receptor-related protein 1 (LRP1)], 7 proteins associated with phagocytic cup formation, and 14 proteins involved in phagocytic maturation (including calpain-2) and phagolysosome formation. NE had a broad effect on the proteome required for regulation of all stages of phagocytosis and phagolysosome formation. Furthermore, the NE sheddome/secretome included proteins from other macrophage cellular domains, suggesting that NE may globally regulate macrophage structure and function.

Cell-associated kidney injury molecule-1 (KIM-1) exerts an anti-inflammatory role following kidney injury by mediating efferocytosis and downregulating the NF-κB pathway. KIM-1 cleavage blunts its anti-inflammatory activities. We reported that Mucin 1 (MUC1) is protective in a mouse model of ischemia-reperfusion injury (IRI). As both KIM-1 and MUC1 are induced in the proximal tubule (PT) during IRI and are ADAM17 substrates, we tested the hypothesis that MUC1 protects KIM-1 activity. Muc1 KO mice and wild-type (WT) littermates were subjected to IRI. KIM-1, MUC1 and ADAM17 levels (and signaling pathways) were assessed by immunoblotting. PT localization was assessed by confocal microscopy and in situ proximity ligation assay. Findings were extended using human kidneys and urine, and KIM-1-mediated efferocytosis assays in mouse PT cultures. In response to tubular injury in mouse and human kidneys, we observed induction and co-expression of KIM-1 and MUC1 in the PT. Compared to WT, Muc1 KO mice had higher urinary KIM-1 and lower kidney KIM-1. KIM-1 was apical in PT of WT kidneys, but predominately with luminal debris in Muc1 KO mice. Efferocytosis was reduced in Muc1 KO PT cultures when compared to WT cells, while inflammation was increased in Muc1 KO kidneys when compared to WT mice. MUC1 was cleaved by ADAM17 in PT cultures, and blocked KIM-1 shedding in MDCK cells. We conclude that KIM-1-mediated efferocytosis and thus anti-inflammatory activity during IRI is preserved in the injured kidney by MUC1 inhibition of KIM-1 shedding.NEW & NOTEWORTHYKIM-1 plays a key role in the recovery of the tubule epithelium during renal IRI by mediating efferocytosis and associated signaling that suppresses inflammation. Excessive cleavage of KIM-1 by ADAM17 provides decoy receptor that aggravates efferocytosis and subsequent signaling. Our data from studies in mice, patients and cultured cells show that MUC1 is also induced during IRI and competes with KIM-1 for cleavage by ADAM17. Consequently, MUC1 protects KIM-1 anti-inflammatory activity in the damaged kidney.

Atherosclerosis is an inflammatory disease with break-down of homeostatic immune regulation of vascular tissues. As a critical initiator of host immunity, dendritic cells (DCs) have also been identified in the aorta of healthy individuals and atherosclerotic patients, whose roles in regulating arterial inflammation aroused great interest. Accumulating evidence has now pointed to the fundamental roles for DCs in every developmental stage of atherosclerosis due to their myriad of functions in immunity and tolerance induction, ranging from lipid uptake, efferocytosis and antigen presentation to pro- and anti-inflammatory cytokine or chemokine secretion. In this study we provide a timely summary of the published works in this field, and comprehensively discuss both the direct and indirect roles of DCs in atherogenesis. Understanding the pathogenic roles of DCs during the development of atherosclerosis in vascular tissues would certainly help to open therapeutic avenue to the treatment of cardiovascular diseases.

Mesenchymal stromal cells (MSCs) are largely known for their immune-suppressive capacity, hence, their common use in the control of unwanted inflammation. However, novel concepts related to their biology, combined with the urgent need to identify MSC subpopulations with enhanced suppressive properties, drive the search for isolation protocols optimized for clinical applications. We show, in this study, that MSCs expressing high CD146 levels exhibit altered surface expression profiles of CD44 and secrete elevated levels of interleukin (IL)-6, amongst other factors. In addition, CD146hi MSCs surpass the polyclonal parental populations in inhibiting alloreactive T cells in vitro, in both a soluble- and cell-contact-dependent manner. Despite the lack of CD146hi MSC-mediated activation of peritoneal macrophages to release the suppressive factor IL-10 in vitro, their administration in animals with graft-versus-host disease alleviates inflammation and leads to 40% survival rate up to 7 weeks post-transplantation. This pronounced inhibitory property is driven by CD146-mediated in situ efferocytosis by myeloid cells. Altogether, this study provides the impetus to adopt an isolation protocol for MSCs based on a CD146 expression profile before their therapeutic use and suggests a major role played by CD146 as a novel “eat-me” signal, capable of enhancing MSC uptake by competent phagocytes.

Regulatory T-cells (Tregs) are central in the maintenance of homeostasis and resolution of inflammation. However, the mechanisms that govern their differentiation and function are not completely understood. Herein, we demonstrate a central role for the lipid mediator biosynthetic enzyme 15-lipoxygenase (ALOX15) in regulating key aspects of Treg biology. Pharmacological inhibition or genetic deletion of ALOX15 in Tregs decreased FOXP3 expression, altered Treg transcriptional profile and shifted their metabolism. This was linked with an impaired ability of Alox15-deficient cells to exert their pro-resolving actions, including a decrease in their ability to upregulate macrophage efferocytosis and a downregulation of interferon gamma expression in Th1 cells. Incubation of Tregs with the ALOX15-derived specilized pro-resolving mediators (SPM)s Resolvin (Rv)D3 and RvD5n-3 DPA rescued FOXP3 expression in cells where ALOX15 activity was inhibited. In vivo, deletion of Alox15 led to increased vascular lipid load and expansion of Th1 cells in mice fed western diet, a phenomenon that was reversed when Alox15-deficient mice were reconstituted with wild type Tregs. Taken together these findings demonstrate a central role of pro-resolving lipid mediators in governing the differentiation of naive T-cells to Tregs.

Phosphatidylserine (PS), a negatively charged phospholipid exclusively located in the inner leaflet of the plasma membrane, is involved in various cellular processes such as blood coagulation, myoblast fusion, mammalian fertilization, and clearance of apoptotic cells. Proteins that specifically interact with PS must be identified to comprehensively understand the cellular processes involving PS. However, only a limited number of proteins are known to associate with PS. To identify PS-associating proteins, we performed a pulldown assay using streptavidin-coated magnetic beads on which biotin-linked PS was immobilized. Using this approach, we identified Hsd17b4, a peroxisomal protein, as a PS-associating protein. Hsd17b4 strongly associated with PS, but not with phosphatidylcholine or sphingomyelin, and the Scp-2-like domain of Hsd17b4 was responsible for this association. The association was disrupted by PS in liposomes, but not by free PS or the components of PS. In addition, translocation of PS to the outer leaflet of the plasma membrane enriched Hsd17b4 in peroxisomes. Collectively, this study suggests an unexpected role of PS as a regulator of the subcellular localization of Hsd17b4.

Fragile X Mental Retardation protein (FMRP), widely known for its role in hereditary intellectual disability, is an RNA-binding protein (RBP) that controls translation of select mRNAs. We discovered that endoplasmic reticulum (ER) stress induces phosphorylation of FMRP on a site that is known to enhance translation inhibition of FMRP-bound mRNAs. We show ER stress-induced activation of Inositol requiring enzyme-1 (IRE1), an ER-resident stress-sensing kinase/endoribonuclease, leads to FMRP phosphorylation and to suppression of macrophage cholesterol efflux and apoptotic cell clearance (efferocytosis). Conversely, FMRP deficiency and pharmacological inhibition of IRE1 kinase activity enhances cholesterol efflux and efferocytosis, reducing atherosclerosis in mice. Our results provide mechanistic insights into how ER stress-induced IRE1 kinase activity contributes to macrophage cholesterol homeostasis and suggests IRE1 inhibition as a promising new way to counteract atherosclerosis., EMBO Molecular Medicine, 14 (4), ISSN:1757-4676, ISSN:1757-4684

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is an overwhelming pulmonary inflammation with limited clinical treatment strategies. Interferon regulatory factor 5 (IRF5) is a crucial regulator of inflammation factors, which can be upregulated under an inflammatory state and related to the efferocytosis of macrophages. Herein, IRF5 was knockdown by small interfering RNA (siIRF5) to promote the anti-inflammatory effect of macrophages. Macrophage-targeting cationic liposome modified by folate (FA-LP) was developed to deliver siIRF5 (FA-LP/siIRF5). Liposomes were characterized for their particle size, zeta potential, protein adsorption and hemolysis of red blood cells. The amount of IRF5 mRNA and the expression of IRF5 were measured using quantitative reverse transcription PCR (RT-qPCR) and western blot, respectively. The phenotype and efferocytosis of macrophages and the regulatory pathway of efferocytosis and biodistribution of liposomes in the ALI mice model were investigated. Data revealed that FA-LP/siIRF5 could obviously downregulate the expression of IRF5 in macrophages, skewing the polarization of macrophages to M2 phenotype (anti-inflammatory state) and thus improving their efferocytosis. Moreover, regulation of efferocytosis of macrophages by siIRF5 is related to the NF- B pathway. The in vivo biodistribution of FA-LP exhibited higher accumulation in the inflammatory lungs, suggesting that FA-LP could be considered as a promising gene delivery system and FA-LP/siIRF5 is an alternative strategy for the treatment of ALI/ARDS. To the best of our knowledge, this is the first study reporting that siIRF5 can be used for the treatment of ALI/ARDS.

Phagocytic clearance of apoptotic cells by the macrophages (efferocytosis) is impaired in sepsis, but its mechanism is poorly understood. Extracellular cold-inducible RNA-binding protein (eCIRP) is a novel damage-associated molecular pattern that fuels inflammation. We identify that eCIRP-induced neutrophil extracellular traps (NETs) impair efferocytosis through a novel mechanism. Coculture of macrophages and apoptotic thymocytes in the presence of recombinant murine CIRP (rmCIRP)–induced NETs significantly inhibited efferocytosis. Efferocytosis was significantly inhibited in the presence of rmCIRP-treated wild-type (WT), but not PAD4−/− neutrophils. Efferocytosis in the peritoneal cavity of rmCIRP-injected PAD4−/− mice was higher than WT mice. Milk fat globule–EGF–factor VIII (MFG-E8), an opsonin, increased macrophage efferocytosis, whereas the inhibition of efferocytosis by NETs was not rescued upon addition of MFG-E8, indicating disruption of MFG-E8’s receptor(s) αvβ3 or αvβ5 integrin by the NETs. We identified neutrophil elastase in the NETs significantly inhibited efferocytosis by cleaving macrophage surface integrins αvβ3 and αvβ5. Using a preclinical model of sepsis, we found that CIRP−/− mice exhibited significantly increased rate of efferocytosis in the peritoneal cavity compared with WT mice. We discovered a novel role of eCIRP-induced NETs to inhibit efferocytosis by the neutrophil elastase–dependent decrease of αvβ3/αvβ5 integrins in macrophages. Targeting eCIRP ameliorates sepsis by enhancing efferocytosis.

Effective and efficient efferocytosis of dead cells and associated cellular debris are critical to tissue homeostasis and healing of injured tissues. This important task was previously thought to be restricted to professional phagocytes (PPs). However, accumulating evidence has revealed another type of phagocyte, the amateur phagocyte (AP), which can also participate in efferocytosis. APs are non-myeloid progenitor/nonimmune cells that include differentiated cells (e.g., epithelial cells, fibroblasts, and endothelial cells [ECs]) and stem cells (e.g., neuronal progenitor cells and mesenchymal cells) and can be found throughout the human body. Studies have shown that APs have two prominent roles: identifying and removing dead cells presumably before PPs reach the site of injury and assisting PPs in the removal of cell corpses and the resolution of inflamed tissue. With respect to the engulfment and degradation of dead cells, APs are slower and less efficient than PPs. However, APs are fundamental to preventing the spread of inflammation over a large area. In this review, we present the diversity and characteristics of healthy and non-neoplastic APs in mammals. We also propose a hypothetical mechanism of the efferocytosis of immunoglobulin G (IgG)-opsonized myelin debris by ECs (APs). Furthermore, the ingestion and clearance of dead cells can induce proinflammatory or anti-inflammatory cytokine production, endothelial activation, and cellular fate transition, which contribute to the progression of disease. An understanding of the role of APs is necessary to develop effective intervention strategies, including potential molecular targets for clinical diagnosis and drug development, for inflammation-related diseases.

BACKGROUND Platelets are increasingly recognized as immune cells. As such, they are commonly seen to induce and perpetuate inflammation; however, anti-inflammatory activities are increasingly attributed to them. Atherosclerosis is a chronic inflammatory condition. Similar to other inflammatory conditions, the resolution of atherosclerosis requires a shift in macrophages to an M2 phenotype, enhancing their efferocytosis and cholesterol efflux capabilities. OBJECTIVES To assess the effect of platelets on macrophage phenotype. METHODS In several in vitro models employing murine (RAW264.7 and bone marrow-derived macrophages) and human (THP-1 and monocyte-derived macrophages) cells, we exposed macrophages to media in which non-agonized human platelets were cultured for 60 minutes (platelet-conditioned media [PCM]) and assessed the impact on macrophage phenotype and function. RESULTS Across models, we demonstrated that PCM from healthy humans induced a pro-resolving phenotype in macrophages. This was independent of signal transducer and activator of transcription 6 (STAT6), the prototypical pathway for M2 macrophage polarization. Stimulation of the EP4 receptor on macrophages by prostaglandin E2 present in PCM, is at least partially responsible for altered gene expression and associated function of the macrophages-specifically reduced peroxynitrite production, increased efferocytosis and cholesterol efflux capacity, and increased production of pro-resolving lipid mediators (ie, 15R-LXA4 ). CONCLUSIONS Platelet-conditioned media induces an anti-inflammatory, pro-resolving phenotype in macrophages. Our findings suggest that therapies targeting hemostatic properties of platelets, while not influencing pro-resolving, immune-related activities, could be beneficial for the treatment of atherothrombotic disease.

Context Naoxintong (NXT), a prescribed traditional Chinese medicine, widely used in cerebrovascular and cardiovascular diseases, could be effective in diabetic wounds. Objective This study evaluates the wound healing activity of NXT by employing an excisional wound splinting model. Materials and methods NXT was dissolved in saline and given daily by gavage. Wounds were induced at the dorsum of non-diabetic (db/+) and diabetic (db/db) mice and treated with saline or 700 mg/kg/d NXT for 16 days. Wound closure was measured every four days. Extracellular matrix (ECM) remodelling, collagen deposition, leukocyte infiltration and expression of Col-3, CK14, CXCL1, CXCL2, MPO, Ly6G, CD68, CCR7, CD206, p-JAK1, p-STAT3 and p-STAT6 was analysed. Results NXT significantly accelerated rate of wound closure increased from 70% to 84%, accompanied by up-regulation of collagen deposition and ECM at days 16 post-injury. Moreover, NXT alleviated neutrophil infiltration, accompanied by down-regulation of CXCL1 and CXCL2 mRNA expression. In addition, NXT markedly augmented neutrophil efferocytosis. In diabetic wounds, the levels of M1 marker gene (CCR7) increased, while M2 marker gene (CD206) decreased, demonstrating a pro-inflammatory shift. Application of NXT increased M2 macrophage phenotype in db/db mice. Mechanistically, NXT treatment increased expression level of p-STAT3 and p-STAT6 at days 3 post-injury, indicating NXT mediated macrophages towards M2 phenotype and alleviated inflammation in diabetic wounds by activation of STAT3 and STAT6. Conclusions Our study provides evidence that NXT accelerates diabetic wound healing by attenuating inflammatory response, which provides an important basis for use of NXT in the treatment of chronic diabetic wound healing.

The accumulation of apoptotic cells is one of the pathological characteristics of systemic lupus erythematosus (SLE). The expression of urokinase-type plasminogen activator receptor (uPAR) has been reported to be increased in SLE patients and to be involved in macrophage efferocytosis. Although the toll-like receptor 7 (TLR7) is also over-expressed in lupus, its relationship to uPAR and its role in macrophage efferocytosis in lupus is still unclear. In the present study, we revealed that apoptotic cells accumulate in the spleen, macrophage efferocytosis is impaired, and uPAR is increased in the spleen and peritoneal macrophages of the TLR7 agonist imiquimod (IMQ)-induced SLE mouse model. Moreover, TLR7 upregulated uPAR expression in the mouse macrophage RAW 264.7 cells in vitro. The same results were also obtained using peritoneal macrophages of female Balb/c mice. When uPAR levels in peritoneal macrophages were knocked down by siRNA or inhibited by the peptide inhibitor UPARANT, and cells further treated with the TLR7 agonist R848, efferocytosis of peritoneal macrophages on apoptotic cells was restored. These results indicated that TLR7 activation impaired efferocytosis via uPAR in mouse peritoneal macrophages. Furthermore, TLR7 regulated uPAR expression via ERK/JNK signaling in macrophages. These results suggest that uPAR may be an important factor related to the accumulation of apoptotic cells in SLE.

Skeletal muscle repair is initiated by local inflammation and involves the engulfment of dead cells (efferocytosis) by infiltrating macrophages at the injury site. Macrophages orchestrate the whole repair program, and efferocytosis is a key event not only for cell clearance but also for triggering the timed polarization of the inflammatory phenotype of macrophages into the healing one. While pro-inflammatory cytokines produced by the inflammatory macrophages induce satellite cell proliferation and differentiation into myoblasts, healing macrophages initiate the resolution of inflammation, angiogenesis, and extracellular matrix formation and drive myoblast fusion and myotube growth. Therefore, improper efferocytosis results in impaired muscle repair. Retinol saturase (RetSat) initiates the formation of various dihydroretinoids, a group of vitamin A derivatives that regulate transcription by activating retinoid receptors. Previous studies from our laboratory have shown that RetSat-null macrophages produce less milk fat globule-epidermal growth factor-factor-8 (MFG-E8), lack neuropeptide Y expression, and are characterized by impaired efferocytosis. Here, we investigated skeletal muscle repair in the tibialis anterior muscle of RetSat-null mice following cardiotoxin injury. Our data presented here demonstrate that, unexpectedly, several cell types participating in skeletal muscle regeneration compensate for the impaired macrophage functions, resulting in normal muscle repair in the RetSat-null mice.

The poor progress of immunotherapy on osteosarcoma patients requires deeper delineation of immune tolerance mechanisms in the osteosarcoma microenvironment and a new therapeutic strategy. Clearance of apoptotic cells by phagocytes, a process termed “efferocytosis,” is ubiquitous in tumors and mediates the suppression of innate immune inflammatory response. Considering the massive infiltrated macrophages in osteosarcoma, efferocytosis probably serves as a potential target, but is rarely studied in osteosarcoma. Here, we verified M2 polarization and PD-L1 expression of macrophages following efferocytosis. Pharmacological inhibition and genetic knockdown were used to explore the underlying pathway. Moreover, tumor progression and immune landscape were evaluated following inhibition of efferocytosis in osteosarcoma model. Our study indicated that efferocytosis promoted PD-L1 expression and M2 polarization of macrophages. Ëfferocytosis was mediated by MerTK receptor in osteosarcoma and regulated the phenotypes of macrophages through the p38/STAT3 pathway. By establishing the murine osteosarcoma model, we emphasized that inhibition of MerTK suppressed tumor growth and enhanced the T cell cytotoxic function by increasing the infiltration of CD8+ T cells and decreasing their exhaustion. Our findings demonstrate that MerTK-mediated efferocytosis promotes osteosarcoma progression by enhancing M2 polarization of macrophages and PD-L1-induced immune tolerance, which were regulated through the p38/STAT3 pathway.

Chronic non-healing wounds are a major complication of diabetes, which affects 1 in 10 people worldwide. Dying cells in the wound perpetuate the inflammation and contribute to dysregulated tissue repair1-3. Here we reveal that the membrane transporter SLC7A11 acts as a molecular brake on efferocytosis, the process by which dying cells are removed, and that inhibiting SLC7A11 function can accelerate wound healing. Transcriptomics of efferocytic dendritic cells in mouse identified upregulation of several SLC7 gene family members. In further analyses, pharmacological inhibition of SLC7A11, or deletion or knockdown of Slc7a11 using small interfering RNA enhanced efferocytosis in dendritic cells. Slc7a11 was highly expressed in dendritic cells in skin, and single-cell RNA sequencing of inflamed skin showed that Slc7a11 was upregulated in innate immune cells. In a mouse model of excisional skin wounding, inhibition or loss of SLC7A11 expression accelerated healing dynamics and reduced the apoptotic cell load in the wound. Mechanistic studies revealed a link between SLC7A11, glucose homeostasis and diabetes. SLC7A11-deficient dendritic cells were dependent on aerobic glycolysis using glucose derived from glycogen stores for increased efferocytosis; also, transcriptomics of efferocytic SLC7A11-deficient dendritic cells identified increased expression of genes linked to gluconeogenesis and diabetes. Further, Slc7a11 expression was higher in the wounds of diabetes-prone db/db mice, and targeting SLC7A11 accelerated their wound healing. The faster healing was also linked to the release of the TGFβ family member GDF15 from efferocytic dendritic cells. In sum, SLC7A11 is a negative regulator of efferocytosis, and removing this brake improves wound healing, with important implications for wound management in diabetes.

Impaired wound healing is a common complication of diabetes. In diabetic wounds, macrophages present dysfunctional efferocytosis and abnormal phenotypes, which could result in excessive neutrophil accumulation and prolonged inflammation, thereby eventually hindering wound repair. ANXA1 N-terminal peptide Ac2-26 exhibits a high potential in mitigating inflammation and improving repair; however, its efficacy in diabetic wound repair remains unclear. In this study, a cutaneous excisional wound model was built in genetically diabetic mice. Ac2-26 or a vehicle solution was employed locally in wound sites. Subsequently, wound zones were measured and sampled at different time intervals post-wounding. Using hematoxylin-eosin and Masson's trichrome staining, we observed the histopathological variations and collagen deposition in wound samples. Based on immunohistochemistry and immunofluorescence, the numbers of neutrophils, macrophages, and CD206-positive macrophages in the wound samples were determined. Cytokine expression in wound samples was studied by immunoblot assay. Results showed that Ac2-26 treatment could facilitate diabetic wound closure, down-regulate the number of neutrophils, and improve angiogenesis and collagen deposition. In addition, Ac2-26 application expedited macrophage recruitment and up-regulated the percentage of macrophages expressing CD206, which is a marker for M2 macrophages. Moreover, Ac2-26 inhibited the expressions of TNF-α and IL-6 and up-regulated the expressions of IL-10, TGF-β, and VEGFA during diabetic wound healing. Hence, based on the aforementioned findings, Ac2-26 application in diabetic wounds could exert anti-inflammatory and pro-repair effects by reducing neutrophil accumulation and facilitating M2 macrophage development.

Neutrophils are polymorphonuclear leukocytes that play a central role in host defense against infection and tissue injury. They are rapidly recruited to the inflamed site and execute a variety of functions to clear invading pathogens and damaged cells. However, many of their defense mechanisms are capable of inflicting collateral tissue damage. Neutrophil-driven inflammation is a unifying mechanism underlying many common diseases. Efficient removal of neutrophils from inflammatory loci is critical for timely resolution of inflammation and return to homeostasis. Accumulating evidence challenges the classical view that neutrophils represent a homogeneous population and that halting neutrophil influx is sufficient to explain their rapid decline within inflamed loci during the resolution of protective inflammation. Hence, understanding the mechanisms that govern neutrophil functions and their removal from the inflammatory locus is critical for minimizing damage to the surrounding tissue and for return to homeostasis. In this review, we briefly address recent advances in characterizing neutrophil phenotypic and functional heterogeneity and the molecular mechanisms that determine the fate of neutrophils within inflammatory loci and the outcome of the inflammatory response. We also discuss how these mechanisms may be harnessed as potential therapeutic targets to facilitate resolution of inflammation.

Efferocytosis triggers cellular reprogramming, including the induction of mRNA transcripts which encode anti-inflammatory cytokines that promote inflammation resolution. Our current understanding of this transcriptional response is largely informed from analysis of bulk phagocyte populations; however, this precludes the resolution of heterogeneity between individual macrophages and macrophage subsets. Moreover, phagocytes may contain so called “passenger” transcripts that originate from engulfed apoptotic bodies, thus obscuring the true transcriptional reprogramming of the phagocyte. To define the transcriptional diversity during efferocytosis, we utilized single-cell mRNA sequencing after co-cultivating macrophages with apoptotic cells. Importantly, transcriptomic analyses were performed after validating the disappearance of apoptotic cell-derived RNA sequences. Our findings reveal new heterogeneity of the efferocytic response at a single-cell resolution, particularly evident between F4/80+ MHCIILO and F4/80− MHCIIHI macrophage sub-populations. After exposure to apoptotic cells, the F4/80+ MHCIILO subset significantly induced pathways associated with tissue and cellular homeostasis, while the F4/80− MHCIIHI subset downregulated these putative signaling axes. Ablation of a canonical efferocytosis receptor, MerTK, blunted efferocytic signatures and led to the escalation of cell death-associated transcriptional signatures in F4/80+ MHCIILO macrophages. Taken together, our results newly elucidate the heterogenous transcriptional response of single-cell peritoneal macrophages after exposure to apoptotic cells.

The phosphatidylserine (PS) receptor Tim-4 mediates phagocytosis of apoptotic cells by binding to PS exposed on the surface of these cells, and thus functions as a PS receptor for apoptotic cells. Some of PS receptors are capable of recognizing other molecules, such as LPS on bacteria, besides PS on apoptotic cells. However, it is unclear whether Tim-4 perceives other molecules like the PS receptors. Here, we report that Tim-4 facilitates the phagocytosis of exogenous particles as well as apoptotic cells. Similar to the process that occurs during Tim-4-mediated efferocytosis, the uptake of exogenous E. coli and S. aureus bioparticles was promoted by overexpression of Tim-4 on phagocytes, whereas phagocytosis of the bioparticles was reduced in Tim-4-deficient cells. A truncation mutant of Tim-4 lacking the cytoplasmic tail promoted phagocytosis of the particles, but a mutant lacking the IgV or the mucin domain failed to enhance phagocytosis. However, expression of Tim-4AAA (a mutant form of Tim-4 that does not bind phosphatidylserine and does not promote efferocytosis) still promoted phagocytosis. Tim-4-mediated phagocytosis was not blocked by expression of the phosphatidylserine-binding protein Anxa5. Furthermore, binding of lipopolysaccharide (LPS), which is found in the outer membrane of Gram-negative bacteria, was higher in Tim-4-overexpressing cells than in Tim-4-deficient cells. In summary, our study suggests that Tim-4 acts as a scavenger receptor and mediates phagocytosis of exogenous particles in a phosphatidylserine-independent manner.

ARTS (Sept4_i2) is a pro-apoptotic protein and a product of the Sept4 gene. ARTS acts upstream of mitochondria to initiate caspase activation. ARTS induces apoptosis by specifically binding XIAP and allowing de-repression of active caspases required for Mitochondrial Outer Membrane Permeabilzation (MOMP). Moreover, ARTS promotes apoptosis by inducing ubiquitin-mediated degradation of both major anti-apoptotic proteins XIAP and Bcl-2. In the resolution phase of inflammation, the infiltrating leukocytes, which execute the acute innate response, undergo apoptosis and are subsequently cleared by phagocytic macrophages (i.e. efferocytosis). In this course, macrophages undergo reprogramming from inflammatory, to anti-inflammatory, and eventually to resolving macrophages that leave the injury sites. Since engulfment of apoptotic leukocytes is a key signaling step in macrophage reprogramming and resolution of inflammation, we hypothesized that a failed apoptosis in leukocytes in vivo would result in an impaired resolution process. To test this hypothesis, we utilized the Sept4/ARTS-/- mice, which exhibit resistance to apoptosis in many cell types. During zymosan A-induced peritonitis, Sept4/ARTS-/- mice exhibited impaired resolution of inflammation, characterized by reduced neutrophil apoptosis, macrophage efferocytosis and expression of pro-resolving mediators. This was associated with increased pro-inflammatory cytokines and reduced anti-inflammatory cytokines, secreted by resolution-phase macrophages. Moreover, ARTS overexpression in leukocytes in vitro promoted an anti-inflammatory behavior. Overall, our results suggest that ARTS is a key master-regulator necessary for neutrophil apoptosis, macrophage efferocytosis and reprogramming to the pro-resolving phenotype during the resolution of inflammation.

Neutrophil death can transpire via diverse pathways and is regulated by interactions with commensal and pathogenic microorganisms, environmental exposures, and cell age. At steady state, neutrophil turnover and replenishment are continually maintained via a delicate balance between host-mediated responses and microbial forces. Disruptions in this equilibrium directly impact neutrophil numbers in circulation, cell trafficking, antimicrobial defenses, and host well-being. How neutrophils meet their end is physiologically important and can result in different immunologic consequences. Whereas nonlytic forms of neutrophil death typically elicit anti-inflammatory responses and promote healing, pathways ending with cell membrane rupture may incite deleterious proinflammatory responses, which can exacerbate local tissue injury, lead to chronic inflammation, or precipitate autoimmunity. This review seeks to provide a contemporary analysis of mechanisms of neutrophil death.

β2 Integrins mediate neutrophil-endothelial adhesion and recruitment of neutrophils to sites of inflammation. The diminished expression of β2 integrins in patients with mutations in the ITGB2 (CD18) gene (leukocyte adhesion deficiency-Type 1; LAD1) results in few or no neutrophils in peripheral tissues. In the periodontium, neutrophil paucity is associated with up-regulation of IL-23 and IL-17, which drive inflammatory bone loss. Using a relevant mouse model, we investigated whether diminished efferocytosis (owing to neutrophil scarcity) is associated with LAD1 periodontitis pathogenesis and aimed to develop approaches to restore the missing efferocytosis signals. We first showed that CD18−/− mice phenocopied human LAD1 in terms of IL-23/IL-17-driven inflammatory bone loss. Ab-mediated blockade of c-Mer tyrosine kinase (Mer), a major efferocytic receptor, mimicked LAD1-associated up-regulation of gingival IL-23 and IL-17 mRNA expression in wild-type (WT) mice. Consistently, soluble Mer-Fc reversed the inhibitory effect of efferocytosis on IL-23 expression in LPS-activated Mϕs. Adoptive transfer of WT neutrophils to CD18−/− mice down-regulated IL-23 and IL-17 expression to normal levels, but not when CD18−/− mice were treated with blocking anti-Mer Ab. Synthetic agonist-induced activation of liver X receptors (LXR) and peroxisome proliferator-activated receptors (PPAR), which link efferocytosis to generation of homeostatic signals, inhibited the expression of IL-23 and IL-17 and favorably affected the bone levels of CD18−/− mice. Therefore, our data link diminished efferocytosis-associated signaling due to impaired neutrophil recruitment to dysregulation of the IL-23–IL-17 axis and, moreover, suggest LXR and PPAR as potential therapeutic targets for treating LAD1 periodontitis.

Nonalcoholic fatty liver disease is the most prevalent liver disease worldwide, affecting 20%-25% of the adult population. In 25% of patients, nonalcoholic fatty liver disease progresses to nonalcoholic steatohepatitis (NASH), which increases the risk for the development of cirrhosis, liver failure, and hepatocellular carcinoma. In patients with NASH, liver fibrosis is the main determinant of mortality. Here, we review how interactions between different liver cells culminate in fibrosis development in NASH, focusing on triggers and consequences of hepatocyte-macrophage-hepatic stellate cell (HSC) crosstalk. We discuss pathways through which stressed and dead hepatocytes instigate the profibrogenic crosstalk with HSC and macrophages, including the reactivation of developmental pathways such as TAZ, Notch, and hedgehog; how clearance of dead cells in NASH via efferocytosis may affect inflammation and fibrogenesis; and insights into HSC and macrophage heterogeneity revealed by single-cell RNA sequencing. Finally, we summarize options to therapeutically interrupt this profibrogenic hepatocyte-macrophage-HSC network in NASH.

Multiple modes of cell death have been identified, each with a unique function and each induced in a setting-dependent manner. As billions of cells die during mammalian embryogenesis and daily in adult organisms, clearing dead cells and associated cellular debris is important in physiology. In this Review, we present an overview of the phagocytosis of dead and dying cells, a process known as efferocytosis. Efferocytosis is performed by macrophages and to a lesser extent by other 'professional' phagocytes (such as monocytes and dendritic cells) and 'non-professional' phagocytes, such as epithelial cells. Recent discoveries have shed light on this process and how it functions to maintain tissue homeostasis, tissue repair and organismal health. Here, we outline the mechanisms of efferocytosis, from the recognition of dying cells through to phagocytic engulfment and homeostatic resolution, and highlight the pathophysiological consequences that can arise when this process is abrogated.

Intercellular adhesion molecule-1 (ICAM-1) is up-regulated during inflammation by several cell types. ICAM-1 is best known for its role in mediating leukocyte adhesion to endothelial cells and guiding leukocytes across the vascular wall. Recently, macrophages have been shown to express ICAM-1, however, their role in macrophage function is unclear. We found that ICAM-1 expression was induced during inflammatory macrophage polarization and high numbers of ICAM-1–expressing macrophages were noted in inflamed colon tissue in a murine colitis model and in human inflammatory bowel disease. Because tissue macrophages play a critical role in removing apoptotic/necrotic cells in inflammation and injury, a process termed efferocytosis, it was examined whether ICAM-1 contributes to this process. Genetic deletion (ICAM-1 knockout mice) or siRNA-mediated knockdown of ICAM-1 in isolated murine and human macrophages significantly impaired apoptotic cell (AC) engulfment. Impairment in the engulfment of Jurkat T cells, neutrophils, and epithelial cells was confirmed ex vivo by inflammatory macrophages and in vivo by thioglycolate-recruited peritoneal macrophages. Decreased efferocytosis was also seen in vitro and in vivo with inhibition of ICAM-1 adhesive interactions, using a function blocking anti–ICAM-1 antibody. Mechanistically, it was found that ICAM-1 actively redistributes to cluster around engulfed ACs to facilitate macrophage–AC binding. Our findings define a new role for ICAM-1 in promoting macrophage efferocytosis, a critical process in the resolution of inflammation and restoration of tissue homeostasis.

Apoptotic cells are rapidly engulfed and degraded by phagocytes through efferocytosis. Efferocytosis is a highly regulated process. It is triggered upon the activation of caspase-dependent apoptosis, which in turn promotes the expression of “eat me” signals on the surface of dying cells and the release of soluble “find me” signals for the recruitment of phagocytes. To date, many “eat me” signals have been recognized, including phosphatidylserine (PS), intercellular adhesion molecule-3, carbohydrates (e.g., amino sugars, mannose) and calreticulin. Among them, PS is the most studied one. PS recognition receptors are different functionally active receptors expressed by phagocytes. Various PS recognition receptors with different structure, cell type expression, and ability to bind to PS have been recognized. Although PS recognition receptors do not fall into a single classification or family of proteins due to their structural differences, they all share the common ability to activate downstream signaling pathways leading to the production of anti-inflammatory mediators. In this review, available evidence regarding molecular mechanisms underlying PS recognition receptor-regulated clearance of apoptotic cells is discussed. In addition, some efferocytosis-independent biological functions of PS recognition receptors are reviewed.

Phosphatidylserine (PS) is an anionic phospholipid found on the membranes of a variety of organelles throughout the cell, most notably the plasma membrane. Under homeostatic conditions, PS is typically restricted to the inner leaflet of the plasma membrane. However, during cellular activation and/or induction of cell death, PS is externalized on the outer surface via the activation of phospholipid scramblases. Externalized PS not only changes the biochemical and biophysical properties of the plasma membrane but also initiates a series of interactions between endogenous extracellular proteins as well as receptors on neighboring cells to stimulate engulfment (efferocytosis) that influence the surrounding immune milieu. In this thematic series published in Cell Communication and Signaling, we feature review articles that highlight recent work in the field of PS biology, including the biochemistry and physiological significance of PS externalization, therapeutic applications and efforts to target PS, as well as posit open questions that remain in the field.

Background and aims Dietary long-chain (≥20 carbons) n-3 polyunsaturated fatty acids (PUFAs) reduce atherosclerosis and enhance macrophage autophagy activation. How macrophage autophagy impacts atherosclerotic progression, particularly when comparing dietary n-3 PUFA supplementation vs. saturated fat feeding, is unknown. Methods We generated myeloid-specific autophagy-deficient and control mice in the Ldlr−/− background by transplanting bone marrow from myeloid-specific autophagy-related (atg) 5 knockout mice and wild type controls into irradiated Ldlr−/− recipients. After 7 weeks for recovery from radiation, mice were fed an atherogenic diet containing 0.2% cholesterol and 20% calories as palm oil (PO diet), or 10% calories as PO plus 10% calories as fish oil (FO diet) for 16 weeks. Results Compared to PO, FO significantly reduced plasma cholesterol, triglyceride, hepatic neutral lipid, and aortic caspase-1 cleavage, but increased aortic efferocytosis, leading to attenuated atherosclerosis in Ldlr−/− mice receiving wild type bone marrow. Myeloid atg5 deletion had little impact on plasma lipid concentrations and hepatic neutral lipid content, regardless of diet. Myeloid atg5 deletion increased aortic caspase-1 cleavage, decreased aortic efferocytosis and worsened atherosclerosis only in the FO-fed Ldlr−/− mice. Conclusions Deficient myeloid autophagy significantly attenuated FO-induced atheroprotection, suggesting that dietary n-3 PUFAs reduce atherosclerosis, in part, by activation of macrophage autophagy.