Increased levels of Gab1 and Gab2 adaptor proteins skew interleukin-4 (IL-4) signaling toward M2 macrophage-driven pulmonary fibrosis in mice

Abstract

M2-polarized macrophages, also known as alternatively activated macrophages, have long been associated with pulmonary fibrosis; however, the mechanism has not been fully defined. Gab1 and Gab2 proteins belong to the Gab family of adaptors and are integral components of the signal specificity in response to various extracellular stimuli. In this report, we found that levels of both Gab1 and Gab2 were elevated in M2-polarized macrophages isolated from bleomycin-induced fibrotic lungs. In vitro Gab1/2 deficiency in bone marrow-derived macrophages abrogated IL-4-mediated M2 polarization. Furthermore, in vivo conditional removal of Gab1 (Gab1^MyKO) and germ line knock-out of Gab2 (Gab2^-/-) in macrophages prevented a bias toward the M2 phenotype and attenuated bleomycin-induced fibrotic lung remodeling. In support of these observations, Gab1/2 were involved in responses predominated by IL-4 signaling, an essential determinant for macrophage M2 polarization. Further investigation revealed that both Gab1 and -2 are recruited to the IL-4 receptor, synergistically enhancing downstream signal amplification but conferring IL-4 signal preference. Mechanistically, the loss of Gab1 attenuated AKT activation, whereas the absence of Gab2 suppressed STAT6 activation in response to IL-4 stimulation, both of which are commonly attributed to M2-driven pulmonary fibrosis in mice. Taken together, these observations define a non-redundant role of Gab docking proteins in M2 polarization, adding critical insights into the pathogenesis of idiopathic pulmonary fibrosis.

Keywords: AKT signaling; Akt PKB; Gabs; M2 polarization; STAT transcription factor; STAT6 signaling; adaptor protein; macrophage; pulmonary fibrosis.

Figure 1.

Elevated levels of Gab1/2 in M2-polarized macrophages. A, the mRNA expression levels of Gab1/2 (relative to β-actin) in alveolar macrophages obtained from SAL- or BLM-treated mice (n = 6/group) were assessed by qPCR. B, Gab1/2 protein levels in alveolar macrophages obtained from SAL- or BLM-treated mice were determined by immunoblotting analysis. Each sample represents three mice. C, representative results of the immunostaining of CD206 (red)/DAPI (blue) in lung sections from SAL- and BLM-challenged mice (n = 6/group). Scale bars, 25 μm. D, analysis of Arg1, FIZZ1, and Ym1 mRNA expression levels by qPCR in alveolar macrophages from SAL- and BLM-treated mice (n = 6/group). E, protein (top) and mRNA (bottom) levels of Gab2 were measured in BMDMs from Gab1^f/f and Gab1^MyKO mice. F, protein (top) and mRNA (bottom) levels of Gab1 were measured in BMDMs from Gab2^+/− and Gab2^−/− mice. Data from three independent experiments are shown. Error bars, S.D. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 2.

Gab1/2-deficient BMDMs exhibit reduced M2 polarization. To obtain BMDMs, BMs from Gab1^f/f, Gab1^MyKO, Gab2^+/−, and Gab2^−/− mice were cultured with M-CSF for 7 days. A and B, after stimulation with IL-4 (20 ng/ml) for 24 h, the Arg1, FIZZ1, and Ym1 mRNA expression levels in BMDMs were measured by qPCR. C and D, expression of Arg1 protein was assessed by immunoblotting analysis in IL-4-stimulated BMDMs. E and F, surface expression of CD206 was evaluated in IL-4-stimulated BMDMs by flow cytometry analysis. A representative graph (left) and statistical graph (right) are shown. Data from three independent experiments are shown. Error bars, S.D.*, p < 0.05; **, p < 0.01; ***, p < 0.001. MFI, mean fluorescence intensity.

Figure 3.

Crucial roles of Gab1/2 in M2 polarization in response to chitin administration. Peritoneal macrophages were harvested from Gab1^f/f, Gab1^MyKO, Gab2^+/−, and Gab2^−/− mice 2 days after chitin administration. A and B, the mRNA levels were determined by qPCR for Arg1, FIZZ1, and Ym1 in peritoneal macrophages. C and D, the protein levels of Arg1 and FIZZ1 in peritoneal macrophages were analyzed by immunoblotting analysis. E and F, surface expression of CD206 on peritoneal macrophages was determined by flow cytometry analysis. Data from three independent experiments are shown. Error bars, S.D. *, p < 0.05; ***, p < 0.001.

Figure 4.

Gab1/2 deficiency attenuates BLM-induced lung fibrosis. Gab1^f/f, Gab1^MyKO, Gab2^+/−, and Gab2^−/− mice were intratracheally injected with BLM and sacrificed 21 days after challenge for functional analysis. A and B, expression levels of M2 markers in alveolar macrophages were measured by qPCR. C and D, the severity of lung fibrosis was analyzed in mice. The left panel presents representative images of Masson's trichrome staining in lung sections as shown at a magnification of ×20. The right panel displays hydroxyproline content in the lungs after acid hydrolysis. Scale bars, 50 μm. E and F, the expression levels of fibrotic markers, including fibronectin, α-SMA, and Col I, in the lungs were determined by immunoblotting analysis. G and H, intratracheally adoptive transfer of IL-4-activated Gab1^f/f, Gab1^MyKO (G), Gab2^+/−, or Gab2^−/− (H) BMDMs into Gab1^f/f (G) or Gab2^+/− mice (H) for functional analysis. The left panel presents representative images of Masson's trichrome staining in lung sections as shown at a magnification of ×20. The right panel displays hydroxyproline content in the lungs after acid hydrolysis. Scale bars, 50 μm. Data from three independent experiments are shown. Error bars, S.D. *, p < 0.05; ***, p < 0.001.

Figure 5.

Distinctive roles of Gab1/2 in IL-4-mediated signaling. BMs from Gab1^f/f, Gab1^MyKO, Gab2^+/−, and Gab2^−/− mice were cultured with M-CSF for 7 days and then stimulated with IL-4 (20 ng/ml) for 0, 5, 30, 60, and 120 min. A and B, left, whole-cell lysates of BMDMs from Gab1^f/f and Gab1^MyKO (A) and Gab2^+/− and Gab2^−/− (B) mice were analyzed by immunoblotting analysis with phosphorylated and total STAT6 antibodies. Right, densitometry analysis of the indicated band intensities from all experiments. C and D, left, whole-cell lysates of BMDMs from Gab1^f/f and Gab1^MyKO (C) and Gab2^+/− and Gab2^−/− (D) mice were analyzed by immunoblotting analysis with phosphorylated and total AKT antibodies. Right, densitometry analysis of the indicated band intensities from all experiments. E, immunoblotting analysis of Gab1^MyKO BMDMs transduced with myr-AKT or EV. F, myr-AKT Gab1^MyKO BMDMs and EV Gab1^MyKO BMDMs were stimulated with IL-4 followed 24 h later by analysis of M2 marker expression. G and H, left, whole-cell lysates of alveolar macrophages from BLM-treated Gab1^f/f and Gab1^MyKO (G) and Gab2^+/− and Gab2^−/− (H) mice were analyzed by immunoblotting analysis with the indicated antibodies. Each sample represents four mice. Right, densitometry analysis of the indicated band intensities from all experiments. Data from three independent experiments are shown. Error bars, S.D. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 6.

Gab1 binds to p85 and Gab2 promotes the association of JAK1 with IL-4Rα. A, RAW264.7 cells were stimulated with IL-4 (20 ng/ml) for 30 min, and total cell lysates were subjected to immunoprecipitation (IP) with anti-Gab1 antibody (left) or anti-p85 antibody (right) followed by immunoblotting analysis with anti-p85 antibody, anti-Gab1 antibody, or anti-Gab2 antibody. B, BMDMs from Gab2^+/− and Gab2^−/− mice were stimulated with IL-4 (20 ng/ml) for the indicated periods, and whole-cell lysates were measured by immunoblotting analysis using anti-phospho-JAK1 and JAK1 antibodies. C, RAW264.7 cells were treated with IL-4 (20 ng/ml) for 30 min. Total cell lysates were prepared and subjected to immunoprecipitation with anti-Gab2 antibody (left) or anti-IL-4Rα antibody (right) followed by immunoblotting analysis with anti-IL-4Rα antibody or anti-Gab2 antibody. D, BMDMs isolated from Gab2^+/− and Gab2^−/− mice were stimulated with IL-4 (20 ng/ml) for 30 min. Whole-cell lysates were immunoprecipitated with anti-JAK1 antibody. Immunoblotting analysis was performed with anti-IL-4Rα antibody. Data from three independent experiments are shown.