Targeting M2 Macrophages Alleviates Airway Inflammation and Remodeling in Asthmatic Mice via miR-378a-3p/GRB2 Pathway

Abstract

Background: Asthma is a complex respiratory disease characterized by airway inflammation and remodeling. MicroRNAs (miRNAs) mediate various cellular processes including macrophage polarization and play an important role in the pathogenesis of asthma. In present study, we aimed to screen miRNA profiling involved in macrophage polarization and investigate its possible functions and mechanisms. Methods: An OVA-sensitized mouse model was established and 2-chloroadenosine (2-CA) was used to interfere with macrophages. The airway inflammation and remodeling were assessed. The identification and function of M2 alveolar macrophages were assessed by flow cytometry, RT-qPCR, arginase activity and co-culture experiment. Microarray screening was used to select miRNAs which were related to macrophage polarization and RNA interference (RNAi) technique was performed to confirm the function of the selected miRNA and its target gene. Results: Alveolar macrophages of asthmatic mice showed significant M2 polarization. 2-CA alleviated airway inflammation and remodeling as well as M2 polarization. In vitro, IL-4-induced M2 macrophages promoted the proliferation of α-SMA-positive cells. And miRNA profiling showed a remarkable increased expression of miR-378a-3p in IL-4 induced M2 macrophages. Dual luciferase reporter assay confirmed growth factor receptor binding protein 2 (GRB2) was a target gene of miR-378a-3p. A miR-378a-3p inhibitor and knockdown of GRB2 repolarized alveolar macrophages from M1 to M2 phenotype. Conclusion: Our findings suggest that miR-378a-3p/GRB2 pathway regulates the polarization of alveolar macrophages which acts as a potential therapeutic target for airway inflammation and remodeling in asthma.

Keywords: GRB2; M2 macrophages; airway inflammation; airway remodeling; asthma; miR-378a-3p.

FIGURE 1

Airway inflammation and remodeling along with M2 macrophage polarization in OVA-sensitized mice with chronic asthma (A). Timeline of establishment of the chronic asthma model which was sensitized and challenged with OVA for 8 weeks (B). Representative images of HE, PAS, Masson, ECP-1, and α-SMA immunohistochemistry staining of lung sections from control and OVA-sensitized mice as shown at a magnification of 20X. Scale bar: 50 μm. Bar graph represent morphometric semi-quantitative analysis of histopathological data from HE, PAS, and Masson staining. Quantification of ECP-1⁺ cells per 20X field and percentage of α-SMA⁺/total muscular layer area (C). IL-4 levels in plasma and BALF from control and OVA-sensitized mice assessed by ELISA (D). Expression of CCR3 in BALF from control and OVA-sensitized mice as determined by flow cytometry analysis. The representative histograms and quantitation of the MFI are shown (E). Representative images of F4/80 immunohistochemistry staining of lung sections from control and OVA-sensitized mice as shown at a magnification of 40X. Scale bar: 50 μm. Quantification of F4/80⁺ cells per 40X field (F). Expression of CD80 and CD206 in alveolar macro-phages of BALF from control and OVA-sensitized mice as determined by flow cytometry analysis. The representative histograms and quantitation of the MFI are shown (G). Expression of IL-6, IL-12, iNOS, FIZZ1, Arg1, and IL-10 in lungs from control and OVA-sensitized mice evaluated by RT-qPCR. (n = 8, *p < 0.05, **p < 0.01, ***p < 0.001 vs. control mice).

FIGURE 2

Inhibition of M2 macrophage polarization by 2-CA is accompanied by alleviation of airway inflammation and remodeling (A). Timeline of mice with chronic asthma treated with 2-CA before every subsequent three challenges of OVA (B). Representative images of F4/80 immunohistochemistry staining of lung sections from OVA-sensitized and 2-CA-treated mice as shown at a magnification of 40X. Scale bar: 50 μm. Quantification of F4/80⁺ cells per 40X field (C). Expression of CD80 and CD206 in alveolar macrophages of BALF from OVA-sensitized and 2-CA-treated mice as determined by flow cytometry analysis. The representative histograms and quantitation of the MFI are shown (D). Expression of IL-6, IL-12, iNOS, FIZZ1, Arg1, and IL-10 in lungs from OVA-sensitized and 2-CA-treated mice evaluated by RT-qPCR (E). Representative images of HE, PAS, Masson, ECP-1, and α-SMA immunohistochemistry staining of lung sections from OVA-sensitized and 2-CA-treated mice as shown at a magnification of 20X. Scale bar: 50 μm. Bar graph represent morphometric semi-quantitative analysis of histopathological data from HE, PAS and Masson staining. Quantification of ECP-1⁺ cells per 20X field and percentage of α-SMA⁺/total muscular layer area (F). IL-4 levels in plasma and BALF from OVA-sensitized and 2-CA-treated mice assessed by ELISA (G). Expression of CCR3 in BALF from OVA-sensitized and 2-CA-treated mice as determined by flow cytometry analysis. The representative histograms and quantitation of the MFI are shown. (n = 8, *p < 0.05, **p < 0.01, ***p < 0.001 vs. asthmatic mice).

FIGURE 3

IL-4-induced M2 macrophages promote the proliferation of α-SMA-positive cells in vitro. MH-S cells were treated for 48 h with 20 ng/ml IL-4, and the identification and function of IL-4-induced M2 macrophages were analyzed (A–C) (A). Expression of CD80 and CD206 in IL-4-induced M2 macrophages evaluated by flow cytometry analysis. The representative histograms and quantitation of the MFI are shown (B). Expression of IL-6, CCL5, iNOS, FIZZ1, Arg1, and CCL24 in IL-4-induced M2 macrophages evaluated by RT-qPCR (C). Arginase in IL-4-induced M2 macrophages measured by the arginase assay (D). Representative image of α-SMA-positive cells under light microscope and immunofluorescent staining of α-SMA as shown at a magnification of 40X. Scale bar: 50 μm. The solid arrow points to the hill and the dotted arrow points to the valley (E). MH-S cells were stimulated with 20 ng/ml IL-4 for 48 h and co-cultured with primary α-SMA-positive cells for another 48 h. M(C) means MH-S cells treated without IL-4 and M(IL-4) means with IL-4. The proliferation of α-SMA-positive cells detected by EdU incorporation assay as shown at a magnification of 20X. Scale bar: 10 μm. Quantitation of percentage of EdU⁺ cells per 20X field. (*p < 0.05, ***p < 0.001 vs. control).

FIGURE 4

MiR-378a-3p is upregulated in IL-4-induced M2 macrophages (A). Differential expression levels of miRNAs (fold changes ≥2 and p-value < 0.05) in MH-S cells treated with or without 20 ng/ml IL-4 for 48 h were presented in a heatmap. The mean fluorescence intensity was calculated as the average for three replicates (B). Expression levels of the candidate miRNAs in MH-S cells following 20 ng/ml IL-4 for 48 h treatment and in the lungs of control and OVA-sensitized mice as determined by RT-qPCR (C). Representative images of FISH for miR-378a-3p in the lung tissues of control and OVA-sensitized mice as shown at a magnification of 40X. Scale bar: 20 μm. Quantitation of percentage of miR-378a-3p⁺ cells per 40X field (D). Scatter plots of CD80, CD206 MFI in BALF and relative miR-378a-3p expression from control and OVA-sensitized mice. Individual data points are shown with p value and linear regression. (n = 8, *p < 0.05, **p < 0.01, ***p < 0.001 vs. control).

FIGURE 5

miR-378a-3p directly targets GRB2 in murine alveolar macrophages (A). Target gene prediction of miR-378a-3p with three bioinformatics tools and the results are shown in the Venn diagram (B). KEGG pathway analysis of target genes of miR-378a-3p (C). The binding sites between miR-378a-3p and target gene GRB2 (D). RT-qPCR and Western blotting assay of GRB2 expression in MH-S cells treated with miR-378a-3p mimics and miR-378a-3p inhibitor or control (E). Diagram of the wild-type and a mutated-type of binding site between miR-378a-3p and GRB2 and luciferase reporter assays for 293T cells transfected with GV272 vectors carrying GRB2-3′ UTR versus GRB2-MUT-3′ UTR in the absence or presence of miR-378a-3p mimics. (*p < 0.05, **p < 0.01, ***p < 0.001 vs. control).

FIGURE 6

(A) MiR-378a-3p contributes to alveolar macrophage polarization by targeting GRB2 Expression of IL-6, IL-12, iNOS, FIZZ1, Arg1, and IL-10 in MH-S cells treated with 100 pmol miR-378a-3p mimics or negative control for 48 h valuated by RT-qPCR (B). Expression of IL-6, IL-12, iNOS, FIZZ1, Arg1, and IL-10 in MH-S cells treated with 100 pmol miR-378a-3p inhibitor or negative control for 48 h evaluated by RT-qPCR (C). GRB2 mRNA and protein in MH-S cells treated with 100 pmol si-GRB2 for 48 h measured by RT-qPCR and Western Blot (D). Expression of IL-6, IL-12, iNOS, FIZZ1, Arg1, and IL-10 in MH-S cells treated with 100 pmol si-GRB2 for 48 h evaluated by RT-qPCR. (*p < 0.05, **p < 0.01, ***p < 0.001 vs. control).