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Macrophages can be alternatively activated by TGF-β1 and high-ambient glucose, in which the role of Smad2 and the crosstalk between ERK and Smad2 pathways are not fully understood. The activation of ERK and Smad2 pathways and the expression of arginase-1 were detected by Western blot. The role of Smad2 and the relationship between ERK and Smad2 pathways were investigated by using biochemical inhibitors. The protein of arginase-1 was significantly overexpressed in RAW264.7 cells stimulated by TGF-β1 and high-ambient glucose, which can be partially blocked by not only U0126 (ERK inhibitor) but also SB431542 (Smad2 inhibitor). Furthermore, simply inhibiting one pathway had no effect on the other pathway. In conclusion, both ERK and Smad2 signal pathways are involved in the activation of macrophages induced by TGF-β1 and high-ambient glucose, while there is no crosstalk shown in the process.

Macrophage can be alternatively activated by TGF-β1, whether high-ambient glucose can enhance the sensitivity of TGF-β1 and the intracellular mechanisms involved in this process are not fully understood. We examined whether the mitogen-activated protein kinase is involved in the activation of macrophage induced by TGF-β1 and high-ambient glucose. The expression of arginase-1, CD206 and TGF-β1 was accessed by Western blot and immunofluorescence in RAW 264.7 cells stimulated with TGF-β1 and high-ambient glucose. The activation of MAPK pathways in the process was investigated by Western blot. The role of MAPK was assessed using biochemical inhibitors. The protein of arginase-1, CD206 and TGF-β1 was significantly overexpressed in RAW264.7 cells stimulated by TGF-β1 and high-ambient glucose. ERK and JNK phosphorylation occurred in 30 min and p38MAPK phosphorylation occurred in 30 min and 24 h after the stimulation. And the expression of arginase-1 and TGF-β1 was partially blocked by the pretreated ERK biochemical inhibitor (U0126) instead of the JNK inhibitor (SP600125) and p38MAPK inhibitor (SB203580). In conclusion, high-ambient glucose can enhance the sensitivity of TGF-β1 in RAW264.7 cells, which resulted in overexpression of TGF-β1 and arginase-1 in macrophages. ERK plays a role in this process.

Background/aims: Since renal fibrosis always predisposes end-stage renal disease, elucidation of the molecular mechanisms that underlie the progression of renal fibrosis may substantially improve the understanding and treatment for renal failure. Previous studies have highlighted an important counteraction between transforming growth factor β 1 (TGFβ1) and bone morphogenic protein 7 (BMP7) in the epithelial-to-mesenchymal transition (EMT) of renal tubular epithelial cells during chronic renal injury. Macrophages are also believed to play a critical role in renal fibrosis. However, the relationship between macrophages and EMT is unknown., Methods: Here, we used a mouse unilateral ureteral obstruction (UUO) model to address to these questions, and analyzed macrophage and its subpopulations purified by flow cytometry., Results: We found that the recruited macrophages are polarized to a M2 subtype after renal injury. M2 macrophages released high levels TGFβ1 to suppress BMP7 to enhance EMT-induced renal fibrosis. Depletion of M2 macrophages, but not of M1 macrophages, specifically inhibited EMT, and subsequently the renal fibrosis. Adoptive transplantation of M2 macrophages deteriorated renal fibrosis., Conclusion: Thus, our study highlights M2 macrophages as a critical target for treating renal fibrosis., (© 2015 S. Karger AG, Basel.)

Alternatively activated macrophages (M2 macrophages) are involved in tissue remodeling and fibrosis, but their effects on peritoneal fibrosis (PF) induced by high glucose peritoneal dialysate have yet to be fully established. In this study, PF was induced in male Sprague-Dawley rats by intraperitoneal injection with Lactate-G4.25% dialysate (20 ml/rat/day) for 4 weeks. Control rats were given an intraperitoneal injection with saline. Establishment of the PF model was verified by Masson's trichrome and H&E staining. M1 macrophages (co-localization of CD68 and CCr7) and M2 macrophages (co-localization of CD68 and CD206) was assayed by immunofluorescence and immunohistochemistry. The levels of dialysate cytokines driving macrophage differentiation (including IFN-γ, IL-2 and IL-4) were detected by ELISA. The expression of transforming growth factor (TGF)-β, p-Smad3, p-Smad2/3 and Smad7 in M2 macrophages (co-localization of CD68, CD206 and TGF-β, or p-Smad3, or p-Smad2/3, or Smad7) was measured by immunofluorescence. We found that PF rats had significantly thicker peritoneal membranes compared to control rats, indicating the successful establishment of our PF model. Compared to controls, PF rats had more peritoneal macrophages (CD68+ cells), more peritoneal M1 macrophages and a greater percentage of peritoneal M2 macrophages. PF rats also had significantly greater levels of dialysate cytokine IL-4, which promotes differentiation to M2 macrophages, higher expression levels of TGF-β in peritoneal M2 macrophages, upregulation of phosphorylated Smad3 and Smad2/3, and downregulation of Smad7 in peritoneal M2 macrophages. Our results indicate that M2 macrophages may play an important role in PF induced by high glucose, and that the cytokine environment in the abdominal cavities of PF rats promotes differentiation to M2 macrophages. The function of M2 macrophages in PF may be related to the TGF-β/Smad signaling pathways.

PD-L1 (programmed death-ligand 1) is the ligand of PD-1 (programmed cell death protein 1) and regulates inhibitory immune responses. It is well known that PD-L1 suppresses T cell function via binding to PD-1. However, little is known about the role of the PD-1/PD-L1 axis in macrophage polarization. According to previous studies, the function of the PD-1/PD-L1 axis in macrophage polarization is controversial, and the underlying mechanism has not been fully elucidated. Thus, we treated THP-1-derived macrophages with human PD-L1 Fc to determine the role of the PD-1/PD-L1 axis in macrophage polarization. To further explore the mechanism, we performed RNA sequencing and used specific inhibitors to identify the implicated signalling pathways. In this study, we found that PD-L1 induces the upregulation of CD206 expression, which is inhibited by nivolumab, LY294002, U0126, and rapamycin. Evaluation of differentially expressed genes (DEGs) and bioinformatics analysis indicated that PD-L1 also induces the upregulation of the expression of genes that maintain mitochondrial function and mediate metabolic switching. In addition, we did not detect PD-L1-induced CD86 alterations, indicating that PD-L1 treatment has no significant influence on M1 polarization. Taken together, these results suggest that PD-L1 binds to PD-1 and promotes M2 polarization accompanied by mitochondrial function enhancement and metabolic reprogramming via Erk/Akt/mTOR. This study elucidates the role of PD-L1 in macrophage polarization and verifies the underlying mechanisms for the first time. Considering that aberrantly upregulated PD-L1 expression contributes to a wide variety of diseases, targeting PD-L1-mediated macrophage polarization is a prospective therapeutic strategy for both neoplastic and nonneoplastic diseases. • PD-L1 treatment upregulates CD206 but not CD86 expression. • PD-L1-induced DEGs are related to the immune response, mitochondrial function, and metabolism. • Nivolumab, LY294002, U0126, and rapamycin inhibit the PD-L1-stimulated CD206 expression. • PD-L1 promotes M2 polarization via Erk/Akt/mTOR. [ABSTRACT FROM AUTHOR]