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.

One of the major roles of professional phagocytes is the removal of dead cells in the body. We know less about the clearance of necrotic cells than apoptotic cell phagocytosis, despite the fact that both types of dead cells need to be cleared together and necrotic cells appear often in pathological settings. In the present study, we examined phagocytosis of heat‐ or H 2 O 2 ‐killed necrotic and apoptotic thymocytes by mouse bone marrow‐derived macrophages ( BMDM s) in vitro and found that the two cell types are engulfed at equal efficiency and compete with each other when added together to BMDM s. Phagocytosis of both apoptotic and necrotic thymocytes was decreased by (a) blocking phosphatidylserine on the surface of dying cells; (b) inhibition of Mer tyrosine kinase, Tim‐4, integrin β3 receptor signaling, or Ras‐related C3 botulinum toxin substrate 1 activity; or (c) using BMDM s deficient for transglutaminase 2. Stimulation of liver X, retinoid X, retinoic acid or glucocorticoid nuclear receptors in BMDM s enhanced not only apoptotic, but also necrotic cell uptake. Electron microscopic analysis of the engulfment process revealed that the morphology of phagosomes and the phagocytic cup formed during the uptake of dying thymocytes is similar for apoptotic and necrotic cells. Our data indicate that apoptotic and necrotic cells are cleared via the same mechanisms, and removal of necrotic cells in vivo can be facilitated by molecules known to enhance the uptake of apoptotic cells.

Phagocytosis of various targets, such as apoptotic cells or opsonized pathogens, by macrophages is coordinated by a complex signaling network initiated by distinct phagocytic receptors. Despite the different initial signaling pathways, each pathway ends up regulating the actin cytoskeletal network, phagosome formation and closure, and phagosome maturation leading to degradation of the engulfed particle. Herein, we describe a new phagocytic function for the nucleoside diphosphate kinase 1 (NDK‐1), the nematode counterpart of the first identified metastasis inhibitor NM23‐H1 (nonmetastatic clone number 23) nonmetastatic clone number 23 or nonmetastatic isoform 1 (NME1). We reveal by coimmunoprecipitation, Duolink proximity ligation assay, and mass spectrometry that NDK‐1/NME1 works in a complex with DYN‐1/Dynamin (Caenorhabditis elegans/human homolog proteins), which is essential for engulf ment and phagosome maturation. Time‐lapse microscopy shows that NDK‐1 is expressed on phagosomal surfaces during cell corpse clearance in the same time window as DYN‐1. Silencing of NM23‐M1 in mouse bone marrow—derived macrophages resulted in decreased phagocytosis of apoptotic thymocytes. In human macrophages, NM23‐H1 and Dynamin are corecruited at sites of phagosome formation in F‐actin—rich cups. In addition, NM23‐H1 was required for efficient phagocytosis. Together, our data demonstrate that NDK‐1/NME1 is an evolutionarily conserved element of successful phagocytosis.—Farkas, Z., Petric, M., Liu, X., Herit, F., Rajnavölgyi, É., Szondy, Z., Budai, Z., Orbán, T. I., Sándor, S., Mehta, A., Bajtay, Z., Kovács, T., Jung, S. Y., Afaq Shakir, M., Qin, J., Zhou, Z., Niedergang, F., Boissan, M., Takács‐Vellai, K. The nucleoside diphosphate kinase NDK‐1/NME1 promotes phagocytosis in concert with DYN‐1/dynamin. FASEB J. 33, 11606–11614 (2019). http://www.fasebj.org

Skeletal muscle regeneration is triggered by local inflammation and is accompanied by phagocytosis of dead cells at the injury site. Efferocytosis regulates the inflammatory program in macrophages by initiating the conversion of their inflammatory phenotype into the healing one. While pro-inflammatory cytokines induce satellite cell proliferation and differentiation into myoblasts, growth factors, such as GDF3, released by healing macrophages drive myoblast fusion and myotube growth. Therefore, improper efferocytosis may lead to impaired muscle regeneration. Transglutaminase 2 (TG2) is a versatile enzyme participating in efferocytosis. Here, we show that TG2 ablation did not alter the skeletal muscle weights or sizes but led to the generation of small size myofibers and to decreased grip force in TG2 null mice. Following cardiotoxin-induced injury, the size of regenerating fibers was smaller, and the myoblast fusion was delayed in the tibialis anterior muscle of TG2 null mice. Loss of TG2 did not affect the efferocytic capacity of muscle macrophages but delayed their conversion to Ly6C−CD206+, GDF3 expressing cells. Finally, TG2 promoted myoblast fusion in differentiating C2C12 myoblasts. These results indicate that TG2 expressed by both macrophages and myoblasts contributes to proper myoblast fusion, and its ablation leads to impaired muscle development and regeneration in mice.

Apoptosis and the proper clearance of apoptotic cells play a central role in maintaining tissue homeostasis. Previous work in our laboratory has shown that when a high number of cells enters apoptosis in a tissue, the macrophages that engulf them produce retinoids to enhance their own phagocytic capacity by upregulating several phagocytic genes. Our data indicated that these retinoids might be dihydroretinoids, which are products of the retinol saturase (RetSat) pathway. In the present study, the efferocytosis of RetSat-null mice was investigated. We show that among the retinoid-sensitive phagocytic genes, only transglutaminase 2 responded in macrophages and in differentiating monocytes to dihydroretinol. Administration of dihydroretinol did not affect the expression of the tested genes differently between differentiating wild type and RetSat-null monocytes, despite the fact that the expression of RetSat was induced. However, in the absence of RetSat, the expression of numerous differentiation-related genes was altered. Among these, impaired production of MFG-E8, a protein that bridges apoptotic cells to the αvβ3/β5 integrin receptors of macrophages, resulted in impaired efferocytosis, very likely causing the development of mild autoimmunity in aged female mice. Our data indicate that RetSat affects monocyte/macrophage differentiation independently of its capability to produce dihydroretinol at this stage.