Acetaminophen synthesis MafB a member of the

MafB, a member of the Maf protein family, is essential for terminal differentiation of macrophages (Kelly et al., 2000). The phagocytic activity of polystyrene beads was found to be enhanced in Acetaminophen synthesis with exogenous expression of MafB (Tillmanns et al., 2007). However, in alveolar macrophages of dominant negative-MafB transgenic mice in which MafB activity was only suppressed in macrophages, the capacity for phagocytosis of polystyrene beads was reduced (Sato-Nishiwaki et al., 2013). In addition, MafB has been reported to enhance phagocytic activity against opsonized beads via the promotion of Fcgr3 expression in RAW264.7, a macrophage cell line (Nemoto et al., 2017). These results strongly suggest that MafB promotes phagocytic activity in macrophages. Efferocytosis is a similar phenomenon to phagocytosis; however, to our knowledge, there are no available reports on the relationship between MafB and efferocytosis, or MafB and Axl. In this study, we aimed to determine whether MafB influences efferocytosis capacity and expression of Axl in RAW264.7, a macrophage cell line.
Materials and methods
Results
Discussion The clearance of apoptotic leukocytes following inflammation is critical for maintenance of tissue homeostasis, as apoptotic cells may damage tissues by releasing toxic enzymes, reactive oxygen species, and contents such as proteases and caspases. It has been reported that apoptotic cells release several “find-me” signals such as adenosine triphosphate and CXCL1/fractalkine (Elliott and Ravichandran, 2016). Phagocytes identify apoptotic cells via specific receptors against these signals, e.g. P2Y2 and CX3CR1 recognize extracellular nucleotides and CX3CL1, respectively. Tyro3-Axl-Mer (TAM) receptors on macrophages bind phosphatidylserine on apoptotic cells, resulting in the engulfment of the latter through the activation of the “eat-me” signal. In this study, we observed a marked reduction of efferocytosis in RAW264.7-MafB-shRNA cells relative to that in RAW264.7-control-shRNA cells (Fig. 1, Fig. 2). Furthermore, Axl mRNA and protein expression levels were markedly reduced in RAW264.7-MafB-shRNA cells compared with RAW264.7-control-shRNA cells (Fig. 3). The observed defect of efferocytosis in RAW264.7-MafB-shRNA cells was restored by forced expression of Axl (Fig. 4). To our knowledge, the present study is the first to demonstrate the role of MafB in the regulation of Axl-mediated efferocytosis in macrophages. The mechanism how MafB regulates macrophage phagocytosis has not been fully elucidated. Tillmanns and colleagues reported enhanced phagocytosis in cells transfected with MafB (Tillmanns et al., 2007). Recently, we showed that MafB regulates the phagocytosis of opsonized beads by inducing Fcgr3 expression in RAW264.7 macrophages (Nemoto et al., 2017). However, to date, there have been no studies describing the link between MafB and efferocytosis. Although efferocytosis represents a specific type of phagocytosis in which apoptotic cells are targeted, the underlying pathways are likely to be the same as those involved in the phagocytosis of pathogens. For example, GTPases such as RhoA and Rac regulate changes in cell shape during the phagocytosis of apoptotic cells and pathogens (Weiss and Schaible, 2015). MafB regulates phagocytosis against opsonized beads; accordingly, we hypothesized that MafB additionally regulates efferocytosis against apoptotic cells. The present findings confirm this hypothesis. Axl, a member of the TAM receptor tyrosine kinase subfamily, is expressed ubiquitously in macrophages and dendritic cells (Rothlin et al., 2015). The Axl protein, whose predicted size is 98kDa, is composed of 894 amino acids. However, owing to posttranslational modifications such as glycosylation and phosphorylation (Linger et al., 2008), its actual molecular weight ranges from 100 to 140kDa. Axl possesses an extracellular domain (two immunoglobulin-like motifs at the N-terminal, followed by two fibronectin type 3 motifs) followed by a transmembrane domain and an intracellular protein kinase at the C-terminal. The immunoglobulin-like motifs of Axl bind to growth arrest-specific 6 (Gas6) that recognizes phosphatidylserine on the cellular surface of apoptotic cells. The binding of phosphatidylserine to GAS6/Axl promotes the transmission of signals from the extracellular matrix into the cytoplasm, resulting in actin reorganization and cell migration (Linger et al., 2008). These effects are likely to be mediated by the activation of Gas6/Axl-induced “eat-me” signaling molecules, such as PI3K, Ras, Rac, p38 MAPK, MAPKAP kinase 2, and HSP25, which results in actin reconstruction (Linger et al., 2008, Nielsen-Preiss et al., 2007). It has been reported that efferocytosis is reduced when Axl expression is suppressed in macrophages and dendritic cells (Seitz et al., 2007, Zagorska et al., 2014). In Axl-knockout mice, the termination of inflammation due to bacterial and viral infection is delayed, suggesting impaired efferocytosis (Fujimori et al., 2015). Therefore, Axl plays a key role in tissue homeostasis by promoting efferocytosis by macrophages. In addition, efferocytosis is reported to skew macrophage phenotype towards M2 (Graham et al., 2014). Efferocytosis induces suppressor of cytokine signaling-1 and 3 (SOCS1 and 3) (Graham et al., 2014), and increases the expression of anti-inflammatory cytokines interleukin-10 and transforming growth factor-beta (Zizzo et al., 2012). Therefore, Axl-mediated efferocytosis is considered to promote wound healing and repair of tissue damage following inflammation.