HGF-MET Signaling Shifts M1 Macrophages Toward an M2-Like Phenotype Through PI3K-Mediated Induction of Arginase-1 Expression

Abstract

Backgrounds and Aims: Hepatocyte Growth Factor (HGF)-MET signaling is known to promote biological functions such as cell survival, cell motility, and cell proliferation. However, it is unknown if HGF-MET alters the macrophage phenotype. In this study, we aimed to study the effects of HGF-MET signaling on the M1 macrophage phenotype. Methods and Materials: Bone marrow-derived macrophages (BMDMs) isolated from mice were either polarized to an M1 phenotype by IFN-γ and LPS treatment or to an M2 phenotype by IL-4 treatment. Changes in M1 or M2 markers induced by HGF-MET signaling were evaluated. Mechanisms responsible for alternations in the macrophage phenotype and intracellular metabolism were analyzed. Results: c-Met was expressed especially in M1 macrophages polarized by treatment with IFN-γ and LPS. In M1 macrophages, HGF-MET signaling induced the expression of Arg-1 mRNA and secretion of IL-10 and TGF-β1 and downregulated the mRNA expression of iNOS, TNF-α, and IL-6. In addition, activation of the PI3K pathway and inactivation of NFκB were also observed in M1 macrophages treated with HGF. The increased Arg-1 expression and IL-10 secretion were abrogated by PI3K inhibition, whereas, no changes were observed in TNF-α and IL-6 expression. The inactivation of NFκB was found to be independent of the PI3K pathway. HGF-MET signaling shifted the M1 macrophages to an M2-like phenotype, mainly through PI3K-mediated induction of Arg-1 expression. Finally, HGF-MET signaling also shifted the M1 macrophage intracellular metabolism toward an M2 phenotype, especially with respect to fatty acid metabolism. Conclusion: Our results suggested that HGF treatment not only promotes regeneration in epithelial cells, but also leads to tissue repair by altering M1 macrophages to an M2-like phenotype.

Keywords: HGF-MET signaling; PI3K pathway; arginase-1; macrophage; phenotypic alteration.

Figure 1

M1 macrophages induced by IFN-γ and LPS treatment show enhanced expression and phosphorylation of c-Met. (A) Expression of M1 and M2 markers. Bone marrow-derived macrophages (BMDMs; M0 macrophages) were differentiated by treatment of bone-marrow cells with M-CSF (25 ng/mL) for 7 days. Macrophages were polarized to M1 and M2 phenotypes by treatment for 48 h with IFN-γ and LPS or IL-4, respectively. Total RNA extraction and RT-qPCR were used to analyze mRNA expression of the M1 markers iNOS, CD86, TNF-α, and IL-6 and M2 markers Arg-1, CD206, IL-10, and TGF-β1. (B) Concentrations of cytokines in cell culture supernatant. ELISA was used to measure spontaneous secretion of TNF-α, IL-6, IL-10, and TGF-β1 in cell culture supernatant from M0, M1, and M2 macrophages. (C) Expression of c-Met in M0, M1, and M2 macrophages. c-Met expression was compared using RT-qPCR and western blot. The RT-qPCR Ct values were normalized to those of β-actin and have been expressed relative to mean level in M0, which is arbitrarily defined as 1. β-actin was used as a loading control for western blots. The results are represented as the mean and a scatter plot of individual data points (n = 4). One-way ANOVA, *P < 0.05 and ****P < 0.0001.

Figure 2

HGF-MET signaling shifts M1 macrophages to an M2-like phenotype. (A) Effect of HGF treatment on expression of M1 and M2 markers. After differentiation to an M0 phenotype, macrophages were polarized to an M1 phenotype by treatment for 24 h with IFN-γ and LPS and an M2 phenotype by treatment with IL-4. M1 and M2 macrophages were then cultured for 24 h with either PBS or HGF (10 ng/mL). RT-qPCR was used to analyze mRNA expression of the M1 markers iNOS, CD86, TNF-α, and IL-6 and M2 markers Arg-1, CD206, IL-10, and TGF-β1. Ct values were normalized to those of β-actin and are expressed relative to mean level in M0 macrophages not treated with HGF, which was arbitrarily defined as 1. (B) Effect of HGF treatment on secretion of cytokines by M1 and M2 macrophages. ELISA was used to measure the secretion of TNF-α, IL-6, IL-10, and TGF-β1 in cell culture supernatants from M0, M1, and M2 macrophages that were treated or not treated with HGF. The results are represented as the mean and a scatter plot showing individual data points (n = 4). Unpaired Student's t-test (HGF– vs. HGF+), *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 3

HGF-MET signaling induces PI3K-mediated Arg-1 expression. (A) Effect of HGF treatment on activity of the PI3K pathway in macrophages. Bone marrow-derived macrophages (BMDMs; M0 macrophages) were induced through the treatment of bone-marrow cells with M-CSF (25 ng/mL) for 7 days. Macrophages were polarized to M1 and M2 phenotypes with treatment for 48 h using IFN-γ and LPS or IL-4, respectively. Western blotting was used to analyze the levels of total and phosphorylated Akt, GSK-3β, CREB, C/EBPβ, and NF-κB. (B) Effects of HGF treatment on downstream PI3K signaling in the presence and absence of PI3K inhibitor. M1 macrophages were treated with a PI3K inhibitor (LY294002) for 1 h after PBS or HGF treatment for 24 h. Western blotting was used to analyze the levels of total and phosphorylated Akt, GSK-3β, CREB, C/EBPβ, and NF-κB. β-actin was used as a loading control. (C) Effect of PI3K inhibitor treatment on the expression of M1 and M2 markers with or without HGF treatment. RT-qPCR was used to compare mRNA expression of iNOS, TNF-α, IL-6, and Arg-1 in M1 macrophages that were treated with or without PI3K inhibitor and HGF for 24 h. The Ct values were normalized to those of β-actin and are expressed relative to mean levels in M0 macrophages not treated with HGF, which is arbitrarily defined as 1. (D) ELISA was used to measure the secretion of IL-10 and TGF-β1. Data are represented as the mean and a scatterplot showing individual data points (n = 4). One-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Figure 4

HGF-MET signaling shifts M1 macrophage intracellular metabolism to an M2-like phenotype. Effect of HGF treatment on macrophage intracellular metabolism. RT-qPCR was used to analyze mRNA expression of genes involved in glucose metabolism (Slc2a1, hexokinase, and pyruvate kinase), amino acid metabolism (Slc1a5, glutaminase, and glutamate dehydrogenase), and fatty acid metabolism (CD36, fatty acid synthase, and acyl-CoA synthase-1). The Ct values were normalized to those of β-actin and have been expressed relative to mean level in M0 macrophages not treated with HGF, which is arbitrarily defined as 1. The data are represented as the mean and a scatterplot showing individual data points (n = 4). Unpaired Student's t-test, *P < 0.05 and **P < 0.01.