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The transcription factor MafB is involved in cellular differentiation and phagocytosis in macrophages. Macrophages phagocytose apoptotic cells in vivo; this process, which is known as efferocytosis, requires Axl receptor tyrosine kinase (Axl) activity. However, the association between MafB and efferocytosis, as well as that between MafB and Axl, in macrophages is unknown. We hypothesized that MafB modulates macrophage efferocytosis by regulating Axl expression. Fluorescent-labeled apoptotic thymocytes were added to RAW264.7-MafB-shRNA and control cells, and the proportion of phagocytosis-positivey fluorescence microscopy and flow cytometry. In addition, Axl mRNA and protein were quantified by real-time PCR and western blotting in each group. RAW264.7-MafB-shRNA cells were transfected with a plasmid expressing green fluorescent protein (GFP)-tagged Axl or a control empty plasmid expressing only GFP. The capacity for phagocytosis of apoptotic cells was assessed in GFP-positive cells gated based on fluorescence intensity. In RAW264.7-MafB-shRNA cells, capacity for phagocytosis of apoptotic thymocytes was significantly reduced compared with that of control cells, as determined by fluorescence microscope and flow cytometry. Axl mRNA and protein expression was significantly reduced in RAW264.7-MafB-shRNA cells relative to control cells. Furthermore, the capacity of RAW264.7-MafB-shRNA cells, transfected with an Axl-expressing plasmid, for phagocytosis of apoptotic thymocytes was significantly greater than that of cells transfected with the control plasmid. Collectively, the present findings indicate that MafB enhances efferocytosis by regulating Axl expression in RAW264.7 macrophages., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Azithromycin (AZM), a 15-member macrolide antibiotic, possesses anti-inflammatory activity. Macrophages are important in innate and acquired immunity, and produce pro-inflammatory cytokines such as interleukin (IL)-12, which are composed of subunit p40 and p35. The key function of IL-12 is the induction and maintenance of T-helper-1 responses, which is associated with the pathogenesis of chronic inflammatory diseases. We investigated the effect of azithromycin on IL-12p40 production in macrophages after lipopolysaccharide (LPS)/interferon (IFN)-gamma stimulation. RAW264.7 macrophage cell line was pre-treated with vehicle or AZM, followed by the stimulation with LPS/IFN-gamma. We measured IL-12 production by RT-PCR and ELISA. IL-12 transcriptional regulation was assessed by electrophoretic mobility shift assay and reporter assay. Phosphorylation of activator protein (AP)-1 and interferon consensus sequence binding protein (ICSBP) was assessed by immunoprecipitation using phosphotyrosine antibody, and immunoblotting using specific antibodies against JunB and ICSBP. AZM reduced the induction of IL-12p40 by LPS/IFN-gamma in a dose dependent manner. AZM inhibited the binding of AP-1, nuclear factor of activated T cells (NFAT), and ICSBP, to the DNA binding site in the IL-12p40 promoter. AZM also reduced LPS/IFN-gamma-induced IL-12p40 promoter activity. Phosphorylation of JunB and ICSBP was inhibited by azithromycin-treatment in stimulated cells. In conclusion, AZM reduced IL-12p40 transcriptional activity by inhibiting the binding of AP-1, NFAT, and ICSBP to the promoter site. This may represent an important mechanism for regulating the anti-inflammatory effects of AZM in macrophages.

Introduction Numerous pathogens invade the lung during respiration. Nonetheless, the lung's innate immune system protects the respiratory system against the development of infectious diseases, such as pneumonia. Alveolar macrophages (AMs) [...], Introduction Pulmonary innate immunity is impaired in cigarette smokers, because the abundant oxidants present in cigarette smoke (CS) cause injury to lung cells. Pulmonary surfactant is a unique material that is important roles in reducing surface tension in the lung and defending against invading pathogens. Oxidants reportedly cleave surfactant phospholipids, resulting in the production of oxidized phospholipids, such as 1-palmitoyl-2-(90-oxo-nonanoyl)-glycerophosphocholine (PON-GPC). Although oxidation of surfactant lipids is thought to be involved in the pathogenesis of smoking-related lung disease, there are no reports on the effect of oxidized surfactant lipid on the immune function of macrophages. We hypothesized that cigarette smoking elevates PON-GPC levels in the lung, and that PON-GPC impairs the innate immune function of macrophages. Methods The levels of PON-GPC in bronchoalveolar lavage fluid (BALF) recovered from mice exposed to CS for 2 weeks (n = 7) were measured by liquid chromatography with electrospray-ionization tandem mass spectrometry. The effects of PON-GPC on inducibility of tumor necrosis factor (TNF)-α, nitric oxide (NO), and nicotinamide adenine dinucleotide phosphate ([NADP.sup.+]) production, as well as bactericidal activity, were investigated in RAW264.7 cells or primary alveolar macrophages. Results The levels of PON-GPC in BALF of mice exposed to CS were significantly elevated, compared with those of control mice. PON-GPC attenuated TNF-α, NO, and [NADP.sup.+] production in macrophages on stimulation with LPS plus IFN-γ. PON-GPC treatment attenuated the phosphorylation of p38 mitogen-activated protein kinase (MAPK). In addition, PON-GPC reduced the bactericidal activity of RAW264.7 cells. Conclusions CS may attenuate innate immunity in the lungs through oxidization of surfactant phospholipids. Keywords Oxidized phospholipid * Macrophage * Innate immunity * Surfactant * Bactericidal activity

Alveolar macrophages (AM) play an important role in the pathogenesis of COPD, and their numbers are significantly increased in patients with COPD. We previously demonstrated that expression of the transcription factor, MafB, was upregulated in AM of mice exposed to cigarette smoke. The aim of this study was to investigate whether the expression of MafB is associated with the degree of airflow limitation (AFL) in smokers. Lung tissue specimens were obtained from male patients undergoing resection of small peripheral lung tumours. The patients were classified into three groups according to smoking index and FEV/FVC: (i) non-smokers or non-heavy ex-smokers without AFL (FEV/FVC ≥0.7, smoking index ≤400) ( n = 8); (ii) heavy current smokers without AFL (FEV/FVC ≥0.7, smoking index ≥800) ( n = 8); and (iii) heavy current smokers with AFL (FEV/FVC <0.6, smoking index ≥800) ( n = 8). The intensity of immunostaining for MafB in AM was quantified by image analysis. Immunostaining for MafB was significantly enhanced in AM of smokers with AFL compared with AM of subjects without AFL. Smoking index, FEV/FVC and FEF (% predicted) were significantly correlated with the intensity of MafB immunostaining. Multiple linear regression analysis revealed that FEV% was also an independent negative predictor of the intensity of MafB immunostaining. The degree of immunostaining for MafB in AM was correlated with the degree of AFL in smokers. MafB may be involved in the pathophysiology of COPD. The expression of the transcription factor, MafB, was investigated in alveolar macrophages of smokers. MafB expression was higher in smokers with airflow limitation than in smokers without airflow limitation, and was related to FEV/FVC. MafB may be involved in pathophysiology of COPD. [ABSTRACT FROM AUTHOR]

This study was designed to investigate whether MafB influences the phagocytic activity of macrophages by modulating the expression of the Fc receptors for IgG (FcγRs), Fcgr2b and Fcgr3. In macrophages, FcγRs are critical for the phagocytosis of opsonized pathogens. Of these receptors, Fcgr3 has been shown to play an important role in host defense. As a model to evaluate the mechanism by which MafB influences phagocytosis, we utilized a macrophage cell-line that constitutively expresses a MafB-specific short hairpin (sh)RNA (RAW264.7- MafB-shRNA ). Specifically, the levels of Fc receptor mediated-phagocytosis and the levels of FcγRs surface expression were evaluated by flow cytometry analysis, while quantitative real-time PCR analysis was utilized to examine the mRNA expression levels of FcγRs. Compared to the control cell population, RAW264.7- MafB-shRNA cells exhibited significant reductions in Fcgr3 expression and Fc receptor-mediated phagocytosis, but no difference in Fcgr2b expression. Likewise, there was markedly decreased surface expression of Fcgr3 antigen, but not Fcgr2b antigen, in RAW264.7- MafB - shRNA , compared to the control cells. Meanwhile, the observed reduction in the phagocytic activity of the MafB-shRNA-expressing cells was attenuated by ectopic expression of Fcgr3. Together, the results presented here indicate that MafB influences the phagocytic activity of macrophages by promoting Fcgr3, but not Fcgr2b, expression. [ABSTRACT FROM AUTHOR]