T-cell Immunoglobulin and Mucin (TIM)-family proteins facilitate the clearance of apoptotic cells, are involved in immune regulation, and promote infection of enveloped viruses. These processes are frequently studied in experimental animals such as mice or rhesus macaques, but functional differences among the TIM orthologs from these species have not been described. Previously, we reported that while all three human TIM proteins bind phosphatidylserine (PS), only human TIM1 (hTIM1) binds phosphatidylethanolamine (PE), and that this PE-binding ability contributes to both phagocytic clearance of apoptotic cells and virus infection. Here we show that rhesus macaque TIM1 (rhTIM1) and mouse TIM1 (mTIM1) bind PS but not PE and that their inability to bind PE makes them less efficient than hTIM1. We also show that alteration of only two residues of mTIM1 or rhTIM1 enables them to bind both PE and PS, and that these PE-binding variants are more efficient at phagocytosis and mediating viral entry. Further, we demonstrate that the mucin domain also contributes to the binding of the virions and apoptotic cells, although it does not directly bind phospholipid. Interestingly, contribution of the hTIM1 mucin domain is more pronounced in the presence of a PE-binding head domain. These results demonstrate that rhTIM1 and mTIM1 are inherently less functional than hTIM1, owing to their inability to bind PE and their less functional mucin domains. They also imply that mouse and macaque models underestimate the activity of hTIM1.

BACKGROUND: Atherosclerotic (AS) plaques require a dense necrotic core and a robust fibrous cap to maintain stability. While previous studies have indicated that the traditional Chinese medicine Huang Lian Jie Du Decoction (HLJDD) possesses the capability to stabilize AS plaques, the underlying mechanisms remain obscure. This study aims to delve deeper into the potential mechanisms by which HLJDD improves AS through an integrated research strategy.
METHODS: Leveraging an AS model in ApoE-/- mice exposed to a high-fat diet (HFD), we scrutinized the therapeutic effects of HLJDD using microscopic observations, oil red O staining, HE staining and Masson staining. Employing comprehensive techniques of network pharmacology, bioinformatics, and molecular docking, we elucidated the mechanism by which HLJDD stabilizes AS plaques. In vitro experiments, utilizing ox-LDL-induced macrophages and apoptotic vascular smooth muscle cells (VSMCs), assessed the impact of HLJDD on efferocytosis and the role of SLC2A1.
RESULTS: In vivo experiments showcased the efficacy of HLJDD in reducing the quantity of aortic plaques, diminishing lipid deposition, and enhancing plaque stability in AS mice. Employing network pharmacology and machine learning, we pinpointed SLC2A1 as a crucial regulatory target. Molecular docking further validated the binding of HLJDD components with SLC2A1. The experiments demonstrated a dose-dependent upregulation in SLC2A1 expression by HLJDD, amplifying efferocytosis. Importantly, this effect was reversed by the SLC2A1 inhibitor STF-31, highlighting the pivotal role of SLC2A1 as a target.
CONCLUSIONS: The HLJDD can modulate macrophage efferocytosis by enhancing the expression levels of SLC2A1, thereby improving the stability of atherosclerotic plaques.

Atherosclerotic plaque formation is largely attributed to the impaired efferocytosis, which is known to be associated with the pathologic upregulation of cluster of differentiation 47 (CD47), a key antiphagocytic molecule. By gene expression omnibus (GEO) datasets analysis, we identified that four miRNAs are aberrantly downregulated in atherosclerosis, coronary artery disease, and obesity. Of them, hsa-miR-299-3p (miR-299-3p) was predicted to target the 3\'UTR of human CD47 mRNA by bioinformatics analysis. Further, we demonstrated that miR-299-3p negatively regulates CD47 expression by binding to the target sequence \"CCCACAU\" in the 3\'UTR of CD47 mRNA through luciferase reporter assay and site-directed mutagenesis. Additionally, we found that miR-299-3p was downregulated by ~32% in foam cells in response to oxidized low-density lipoprotein (ox-LDL) stimulation, thus upregulating CD47 and contributing to the impaired efferocytosis. Whereas, restoration of miR-299-3p reversed the ox-LDL-induced upregulation of CD47, thereby facilitating efferocytosis. In high-fat diet (HFD) fed ApoE-/- mice, we discovered that miR-299-3p was downregulated thus leading to upregulation of CD47 in abdominal aorta. Conversely, miR-299-3p restoration potently suppressed HFD-induced upregulation of CD47 and promoted phagocytosis of foam cells by macrophages in atherosclerotic plaques, thereby reducing necrotic core, increasing plaque stability, and mitigating atherosclerosis. Conclusively, we identify miR-299-3p as a negative regulator of CD47, and reveal a molecular mechanism whereby the ox-LDL-induced downregulation of miR-299-3p leads to the upregulation of CD47 in foam cells thus contributing to the impaired efferocytosis in atherosclerosis, and propose miR-299-3p can potentially serve as an inhibitor of CD47 to promote efferocytosis and ameliorate atherosclerosis.

The impact of efferocytosis-related genes (ERGs) on the diagnosis of colorectal cancer (CRC) remains unclear. In this study, efferocytosis-associated biomarkers for the diagnosis of CRC were identified by integrating data from transcriptome sequencing and public databases. Finally, the expression of biomarkers was validated by real-time quantitative polymerase chain reaction (RT-qPCR). Our study may provide a reference for CRC diagnosis.
BACKGROUND: It has been shown that some efferocytosis related genes (ERGs) are associated with the development of cancer. However, it is still uncertain how ERGs may influence the diagnosis of colorectal cancer (CRC).
METHODS: In our study, the CRC cohorts were gained from transcriptome sequencing and the gene expression omnibus (GEO) database (GSE71187). Efferocytosis related biomarkers with diagnostic utility for CRC were identified through combining differentially expressed analysis, machine learning algorithms, and receiver operating characteristic (ROC) analysis. Then, infiltration abundance of immune cells between CRC and control was evaluated. The regulatory networks (including mRNA-miRNA-lncRNA and miRNA/transcription factors (TF)-mRNA networks) were created. Finally, the expression of biomarkers was validated via real-time quantitative polymerase chain reaction (RT-qPCR).
RESULTS: There were 3 biomarkers (ELMO3, P2RY12, and PDK4) related diagnosis for CRC patients gained. ELMO3 was highly expressed in CRC group, while P2RY12 and PDK4 was lowly expressed. Besides, the infiltrating abundance of 3 immune cells between CRC and control groups was significantly differential, namely activated CD4 memory T cells, macrophages M0, and resting mast cells. We then constructed a mRNA-miRNA-lncRNA network containing 3 mRNAs, 33 miRNAs, and 22 lncRNAs, and a miRNA/TF-mRNA network including 3 mRNAs, 33 miRNAs, and 7 TFs. Additionally, RT-qPCR results revealed that the expression trends of all biomarkers were consistent with the transcriptome sequencing data and GSE71187.
CONCLUSIONS: Taken together, this study provides three efferocytosis related biomarkers (ELMO3, P2RY12, and PDK4) for diagnosis of CRC, providing a scientific reference for further studies of CRC.

Alcohol ingestion is a widespread habituation that evolved along with a growing population, altering physiological conditions through immunomodulatory function. There is much research that has reported that consumption of alcohol at low and heavy levels causes different biological impacts, including cellular injury, leading to systemic dysfunction and increased inflammatory markers. In the fate of professional phagocytic cells, efferocytosis is an inevitable mechanism activated by the apoptotic cells, thus eliminating them and preventing the accumulation of cell corpses/debris in the microenvironment. Subsequently, it promotes the tissue repair mechanism and maintains cellular homeostasis. Unfortunately, defective efferocytosis is widely found in several inflammatory and age-related diseases such as atherosclerosis, autoimmune diseases, lung injury, fatty liver disease, and neurodegenerative diseases. Alcohol abuse is one of the factors that provoke an immune response that increases the rate of morbidity and mortality in parallel in systemic disease patients. Information regarding the emergence of immunomodulation during alcoholic pathogenesis and its association with efferocytosis impairment remain elusive. Hence, here in this review, we discussed the mechanism of efferocytosis, the role of defective efferocytosis in inflammatory diseases, and the role of alcohol on efferocytosis impairment.

Apoptosis is crucial for tissue homeostasis and organ development. In bone, apoptosis is recognized to be a main fate of osteoblasts, yet the relevance of this process remains underexplored. Using our murine model with inducible Caspase 9, the enzyme that initiates intrinsic apoptosis, we triggered apoptosis in a proportion of mature osteocalcin (OCN+) osteoblasts and investigated the impact on postnatal bone development. Osteoblast apoptosis stimulated efferocytosis by osteal macrophages. A five-week stimulation of OCN+ osteoblast apoptosis in 3-week-old male and female mice significantly enhanced vertebral bone formation while increasing osteoblast precursors. A similar treatment regimen to stimulate osterix+ cell apoptosis had no impact on bone volume or density. The vertebral bone accrual following stimulation of OCN+ osteoblast apoptosis did not translate in improved mechanical strength due to disruption of the lacunocanalicular network. The observed bone phenotype was not influenced by changes in osteoclasts but was associated with stimulation of macrophage efferocytosis and vasculature formation. Phenotyping of efferocytic macrophages revealed a unique transcriptomic signature and expression of factors including VEGFA. To examine whether macrophages participated in the osteoblast precursor increase following osteoblast apoptosis, macrophage depletion models were employed. Depletion of macrophages via clodronate-liposomes and the CD169-diphtheria toxin receptor mouse model resulted in marked reduction in leptin receptor+ and osterix+ osteoblast precursors. Collectively, this work demonstrates the significance of osteoblast turnover via apoptosis and efferocytosis in postnatal bone formation. Importantly, it exposes the potential of targeting this mechanism to promote bone anabolism in the clinical setting.

Treatment strategies for hard tissue defects aim to establish a mineralized microenvironment that facilitates tissue remodeling. As a mineralized tissue, cementum shares a similar structure with bone and exhibits an excellent capacity to resist resorption under compression. Macrophages are crucial for mineralized remodeling; however, their functional alterations in the microenvironment of cementum remain poorly understood. Therefore, this study explores the mechanisms by which cementum resists resorption under compression and the regulatory roles of cementoblasts in macrophage functions. As a result, extracellular vesicles from compression-loaded cementoblasts (Comp-EVs) promote macrophage M2 polarization and enhance the clearance of apoptotic cells (efferocytosis) by 2- to 3-fold. Local injection of Comp-EVs relieves cementum destruction in mouse root resorption model by activating the tissue repair function of macrophages. Moreover, Comp-EV-loaded hydrogels achieve significant bone healing in calvarial bone defect. Unexpectedly, under compression, EV secretion in cementoblasts is reduced by half. RNA-Seq analysis and verification reveal that Rab35 expression decreases by 60% under compression, thereby hampering the release of EVs. Rab35 overexpression is proposed as a modification of cementoblasts to boost the yield of Comp-EVs. Collectively, Comp-EVs activate the repair function of macrophages, which will be a potential therapeutic strategy for hard tissue repair and regeneration.

Efferocytosis refers to the process by which phagocytes remove apoptotic cells and related apoptotic products. It is essential for the growth and development of the body, the repair of damaged or inflamed tissues, and the balance of the immune system. Damaged efferocytosis will cause a variety of chronic inflammation and immune system diseases. Many studies show that efferocytosis is a process mediated by mitochondria. Mitochondrial metabolism, mitochondrial dynamics, and communication between mitochondria and other organelles can all affect phagocytes\' clearance of apoptotic cells. Therefore, targeting mitochondria to modulate phagocyte efferocytosis is an anticipated strategy to prevent and treat chronic inflammatory diseases and autoimmune diseases. In this review, we introduced the mechanism of efferocytosis and the pivoted role of mitochondria in efferocytosis. In addition, we focused on the therapeutic implication of drugs targeting mitochondria in diseases related to efferocytosis dysfunction.

This study aimed to explore the key efferocytosis-related genes in diabetic retinopathy (DR) and their regulatory mechanisms. Public DR-related gene expression datasets, GSE160306 (training) and GSE60436 (validation), were downloaded. Differentially expressed efferocytosis-related genes (DEERGs) were analyzed using differential expression analysis and weighted gene co-expression network analysis. Functional enrichment analysis was conducted. Moreover, efferocytosis-related signature genes were identified using machine learning analysis, and their expression levels and diagnostic value were analyzed. Furthermore, nomograms were constructed; immune cell infiltration was analyzed; and gene set enrichment analysis, transcriptional regulation analysis, and small-molecule drug (SMD) prediction of efferocytosis-related signature genes were performed. In total, 36 DEERGs were identified in DR, and were markedly enriched in multiple functions, such as visual system development. Through further machine learning analysis, two efferocytosis-related signature genes, Ferritin Light Chain (FTL) and Fc Gamma Binding Protein (FCGBP), were identified, and were found to be upregulated in DR samples and showed high diagnostic performance for DR. A nomogram constructed using FTL and FCGBP accurately predicted the risk of DR. Moreover, the level of infiltration of immature B cells was positively correlated with FTL and FCGBP expression levels. Multiple transcription factors (TFs), such as CCCTC-Binding Factor (CTCF) and KLF Transcription Factor 9 (KLF9), were found to interact with both FTL and FCGBP. In addition, FTL can be targeted by miRNAs, such as miR-22-3p, and FCGBP can be targeted by miR-7973. In addition, both FTL and FCGBP can be targeted by SMDs, such as bisphenol A. Key efferocytosis-related genes, such as FTL and FCGBP, may promote DR development. Detecting or targeting FTL and FCGBP may aid in the prevention, diagnosis, and treatment of DR.

Non-alcoholic fatty liver disease (NAFLD) has become the first major chronic liver disease in developed countries. 10-20% of NAFLD patients will progress to non-alcoholic steatohepatitis (NASH), and up to 25% of NASH patients may develop cirrhosis within 10 years. Therefore, it is critical to find key targets that may treat this disease. Here, we identified C5aR1 as a highly-expressed gene in NASH mouse model through analyzing Gene Expression Omnibus (GEO) database and confirmed its higher expression in livers of NASH patients than that of NAFL patients. Meanwhile, we verified its positive correlation with patients\' serum alanine transaminase (ALT) and aspartate transaminase (AST) levels. In vivo and in vitro experiments revealed that knocking down C5aR1 in liver significantly reduced liver weight ratio and serum ALT and AST levels and attenuated inflammatory cell infiltration and cell apoptosis in the liver of NASH mice as well as enhanced the efferocytotic ability of liver macrophages, suggesting that C5aR1 may play a crucial role in the efferocytosis of liver macrophages. Furthermore, we also found that the expression levels of nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing protein 3 (NLRP3), caspase-1, IL-1β and other inflammation-related factors in the liver were significantly reduced. Our work demonstrates a potential mechanism of how C5aR1 deficiency protects against diet-induced NASH by coordinating the regulation of inflammatory factors and affecting hepatic macrophage efferocytosis.