Serum-dependent processing of late apoptotic cells for enhanced efferocytosis

Binding of the serum protein complement component C1q to the surface of dying cells facilitates their clearance by phagocytes in a process termed efferocytosis. Here, we investigate during which phase of apoptotic cell death progression C1q binding takes place. Purified C1q was found to bind to all dying cells and, albeit weaker, also to viable cells. The presence of serum abrogated completely the binding to viable cells. In addition, C1q binding to dying cells was limited to a specific subpopulation of late apoptotic/secondary necrotic cells. Co-culturing serum-treated apoptotic cells with human monocytes revealed a much higher phagocytosis of C1q-positive than of C1q-negative late apoptotic/secondary necrotic cells. But this phagocytosis-promoting activity could not be observed with purified C1q. Serum-treated C1q-positive late apoptotic/secondary necrotic cells exhibited a similar volume, a similar degraded protein composition, but a much lower DNA content in comparison with the remaining late apoptotic/secondary necrotic cells. This was mediated by a serum-bound nuclease activity that could be abrogated by G-actin, which is a specific inhibitor of serum DNase I. These results show that serum factors are involved in the prevention of C1q binding to viable cells and in the processing of late apoptotic/secondary necrotic cells promoting cell death progression toward apoptotic bodies. This process leads to the exposure of C1q-binding structures and facilitates efferocytosis.     

Reduction of TMAO level enhances the stability of carotid atherosclerotic plaque through promoting macrophage M2 polarization and efferocytosis

It has been demonstrated that trimethylamine N-oxide (TMAO) serves as a driver of atherosclerosis, suggesting that reduction of TMAO level might be a potent method to prevent the progression of atherosclerosis. Herein, we explored the role of TMAO in the stability of carotid atherosclerotic plaques and disclosed the underlying mechanisms. The unstable carotid artery plaque models were established in C57/BL6 mice. L-carnitine (LCA) and methimazole (MMI) administration were applied to increase and reduce TMAO levels. Hematoxylin and eosin (H&E) staining, Sirius red, Perl’s staining, Masson trichrome staining and immunohistochemical staining with CD68 staining were used for histopathology analysis of the carotid artery plaque. M1 and M2 macrophagocyte markers were assessed by RT-PCR to determine the polarization of RAW264.7 cells. MMI administration for 2 weeks significantly decreased the plaque area, increased the thickness of the fibrous cap and reduced the size of the necrotic lipid cores, whereas 5-week of administration of MMI induced intraplate hemorrhage. LCA treatment further deteriorated the carotid atherosclerotic plaque but with no significant difference. In mechanism, we found that TMAO treatment impaired the M2 polarization and efferocytosis of RAW264.7 cells with no obvious effect on the M1 polarization. In conclusion, the present study demonstrated that TMAO reduction enhanced the stability of carotid atherosclerotic plaque through promoting macrophage M2 polarization and efferocytosis.     

Efferocytosis Fuels Requirements of Fatty Acid Oxidation and the Electron Transport Chain to Polarize Macrophages for Tissue Repair

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ATG proteins mediate efferocytosis and suppress inflammation in mammary involution

Involution is the process of post-lactational mammary gland regression to quiescence and it involves secretory epithelial cell death, stroma remodeling and gland repopulation by adipocytes. Though reportedly accompanying apoptosis, the role of autophagy in involution has not yet been determined. We now report that autophagy-related (ATG) proteins mediate dead cell clearance and suppress inflammation during mammary involution. In vivo, Becn1^+/? and Atg7-deficient mammary epithelial cells (MECs) produced ‘competent’ apoptotic bodies, but were defective phagocytes in association with reduced expression of the MERTK and ITGB5 receptors, thus pointing to defective apoptotic body engulfment. Atg-deficient tissues exhibited higher levels of involution-associated inflammation, which could be indicative of a tumor-modulating microenvironment, and developed ductal ectasia, a manifestation of deregulated post-involution gland remodeling. In vitro, ATG (BECN1 or ATG7) knockdown compromised MEC-mediated apoptotic body clearance in association with decreased RAC1 activation, thus confirming that, in addition to the defective phagocytic processing reported by other studies, ATG protein defects also impair dead cell engulfment.

Using two different mouse models with mammary gland-associated Atg deficiencies, our studies shed light on the essential role of ATG proteins in MEC-mediated efferocytosis during mammary involution and provide novel insights into this important developmental process. This work also raises the possibility that a regulatory feedback loop exists, by which the efficacy of phagocytic cargo processing in turn regulates the rate of engulfment and ultimately determines the kinetics of phagocytosis and dead cell clearance.     

Gene expression signature-based approach identifies a pro-resolving mechanism of action for histone deacetylase inhibitors

Despite several therapies being currently available to treat inflammatory diseases, new drugs to treat chronic conditions with less side effects and lower production costs are still needed. An innovative approach to drug discovery, the Connectivity Map (CMap), shows how integrating genome-wide gene expression data of drugs and diseases can accelerate this process. Comparison of genome-wide gene expression data generated with annexin A1 (AnxA1) with the CMap revealed significant alignment with gene profiles elicited by histone deacetylase inhibitors (HDACIs), what made us to hypothesize that AnxA1 might mediate the anti-inflammatory actions of HDACIs. Addition of HDACIs (valproic acid, sodium butyrate and thricostatin A) to mouse macrophages caused externalization of AnxA1 with concomitant inhibition of cytokine gene expression and release, events that occurred independently as this inhibition was retained in AnxA1 null macrophages. In contrast, novel AnxA1-mediated functions for HDACIs could be unveiled, including promotion of neutrophil apoptosis and macrophage phagocytosis, both steps crucial for effective resolution of inflammation. In a model of acute resolving inflammation, administration of valproic acid and sodium butyrate to mice at the peak of disease accelerated resolution processes in wild type, but much more modestly in AnxA1 null mice. Deeper analyses revealed a role for endogenous AnxA1 in the induction of neutrophil death in vivo by HDACIs. In summary, interrogation of the CMap revealed an unexpected association between HDACIs and AnxA1 that translated in mechanistic findings with particular impact on the processes that regulate the resolution of inflammation. We propose non-genomic modulation of AnxA1 in immune cells as a novel mechanism of action for HDACIs, which may underlie their reported efficacy in models of chronic inflammatory pathologies.     

Macrophage Metabolism of Apoptotic Cell-Derived Arginine Promotes Continual Efferocytosis and Resolution of Injury

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How apoptotic cells aid in the removal of their own cold dead bodies

Apoptotic cell clearance facilitates the removal of aged, damaged, infected or dangerous cells although minimizing perturbation of surrounding tissues, and is a vital process in the development and homeostasis of multicellular organisms. Importantly, failure to correctly execute programmed cell death and subsequent corpse clearance is broadly associated with chronic inflammatory and/or autoimmune diseases such as systemic lupus erythematosus. Apoptotic cells develop dramatic morphological changes including contraction, membrane blebbing and apoptotic body formation, which were among the first and most readily identifiable features of cellular suicide. However, understanding the purpose of apoptotic cell morphological changes has proven to be elusive, and recent studies have made somewhat surprising, and occasionally opposing, conclusions about the contribution of blebbing to phagocytic clearance and prevention of inflammatory/autoimmune disease. We review the evidence indicating how apoptotic blebs actively promote corpse recognition, uptake, and generation of auto-reactive antibodies.