In healthy individuals billions of cells die by apoptosis every day. Clearance of apoptotic cells by phagocytosis (a process called efferocytosis) is very efficient to prevent secondary necrosis and the consequent release of pro-inflammatory cell content that damages the tissue environment and provokes autoimmunity. In addition, apoptotic cells generally induce an anti-inflammatory response, thus removal of apoptotic cells is usually immunologically silent. Since the first discovery that uptake of apoptotic cells leads to transforming growth factor (TGF)-and interleukin (IL)-10 release by engulfing macrophages, numerous anti-inflammatory mechanisms triggered by apoptotic cells have been discovered including release of anti-inflammatory molecules from the apoptotic cells, triggering immediate anti-inflammatory signaling pathways by apoptotic cell surface molecules via phagocyte receptors, activating phagocyte lipid sensing nuclear receptors following uptake and inducing the production of anti-inflammatory soluble mediators by phagocytes that may act in a paracrine or autocrine fashion to amplify and sustain the anti-inflammatory response. Here we summarize our present knowledge about how these anti-inflammatory mechanisms operate during the clearance of apoptotic cells.

In healthy individuals billions of cells die by apoptosis every day. Removal of the dead cells by phagocytosis (a process called efferocytosis) must be efficient to prevent secondary necrosis and the consequent release of proinflammatory cell contents that damages the tissue environment and provokes autoimmunity. In addition, detection and removal of apoptotic cells generally induces an anti-inflammatory response. As a consequence improper clearance of apoptotic cells, being the result of either genetic anomalies and /or a persistent disease state, contributes to the establishment and progression of a number of human chronic inflammatory diseases such as autoimmune and neurological disorders, inflammatory lung diseases, obesity, type 2 diabetes or atherosclerosis. During the past decade our knowledge about the mechanism of efferocytosis has significantly increased, providing therapeutic targets through which impaired phagocytosis of apoptotic cells and the consequent inflammation could be influenced in these diseases.

Transglutaminase 2 (TG2) is an inducible transamidating acyltransferase that catalyzes Ca(2+)-dependent protein modifications. In addition to being an enzyme, TG2 also serves as a G protein for several seven transmembrane receptors and acts as a co-receptor for integrin β1 and β3 integrins distinguishing it from other members of the transglutaminase family. TG2 is ubiquitously expressed in almost all cell types and all cell compartments, and is also present on the cell surface and gets secreted to the extracellular matrix via non-classical mechanisms. TG2 has been associated with various human diseases including inflammation, cancer, fibrosis, cardiovascular disease, neurodegenerative diseases, celiac disease in which it plays either a protective role, or contributes to the pathogenesis. Thus modulating the biological activities of TG2 in these diseases will have a therapeutic value.

TNF-α, a potent proinflammatory cytokine, is generated in a precursor form called transmembrane (m)TNF-α that is expressed as a type II polypeptide on the surface of certain cells. mTNF-α was shown to act both as a ligand by binding to TNF-α receptors, as well as a receptor that transmits outside-to-inside (reverse) signals back into the mTNF-α–bearing cells. In this study, we show that nonactivated macrophages express basal levels of mTNF-α and respond to anti–TNF-α Abs by triggering the MAPK kinase 4 signaling pathway. The pathway induces TGF-β. Based on inhibitory experiments, the production of TGF-β1 is regulated via Jun kinases, whereas that of other TGF-βs is regulated via p38 MAPKs. Exposure to LPS further induced the expression of mTNF-α, and triggering of mTNF-α strongly suppressed the LPS-induced proinflammatory response. Neutralizing TGF-β by Abs prevented the mTNF-α–mediated suppression of LPS-induced proinflammatory cytokine formation, indicating that the immune-suppressive effect of mTNF-α is mediated via TGF-β. Although apoptotic cells are also known to suppress LPS-induced proinflammatory cytokine formation in macrophages by upregulating TGF-β, we show that they do not use the mTNF-α signaling pathway. Because TGF-β possesses a wide range of immune-suppressive effects, our data indicate that upregulation of TGF-β synthesis by those TNF-α–targeting molecules, which are able to trigger mTNF-α, might contribute to their therapeutic effect in the treatment of certain inflammatory diseases such as Crohn’s disease, Wegener’s granulomatosis, or sarcoidosis. Additionally, none of the TNF-α–targeting molecules is expected to interfere with the immune-silencing effects of apoptotic cells.

Skeletal muscle regeneration following injury results from the proliferation and differentiation of myogenic stem cells, called satellite cells, located beneath the basal lamina of the muscle fibers. Infiltrating macrophages play an essential role in the process partly by clearing the necrotic cell debris, partly by producing cytokines that guide myogenesis. Infiltrating macrophages are at the beginning pro-inflammatory, but phagocytosis of dead cells induces a phenotypic change to become healing macrophages that regulate inflammation, myoblast fusion and growth, fibrosis, vascularization and return to homeostasis. The TAM receptor kinases Mer and Axl are known efferocytosis receptors in macrophages functioning in tolerogenic or inflammatory conditions, respectively. Here we investigated their involvement in the muscle regeneration process by studying the muscle repair following cardiotoxin-induced injury in Mer^−/− mice. We found that Axl was the only TAM kinase receptor expressed on the protein level by skeletal muscle and C2C12 myoblast cells, while Mer was the dominant TAM kinase receptor in the CD45⁺ cells, and its expression significantly increased during repair. Mer ablation did not affect the skeletal muscle weight or structure, but following injury it resulted in a delay in the clearance of necrotic muscle cell debris, in the healing phenotype conversion of macrophages and consequently in a significant delay in the full muscle regeneration. Administration of the TAM kinase inhibitor BMS-777607 to wild type mice mimicked the effect of Mer ablation on the muscle regeneration process, but in addition, it resulted in a long-persisting necrotic area. Finally, in vitro inhibition of TAM kinase signaling in C2C12 myoblasts resulted in decreased viability and in impaired myotube growth. Our work identifies Axl as a survival and growth receptor in the mouse myoblasts, and reveals the contribution of TAM kinase-mediated signaling to the skeletal muscle regeneration both in macrophages and in myoblasts.