ECM1 is an essential factor for the determination of M1 macrophage polarization in IBD in response to LPS stimulation

Abstract

Inflammatory bowel disease (IBD) comprises chronic relapsing disorders of the gastrointestinal tract characterized pathologically by intestinal inflammation and epithelial injury. Here, we uncover a function of extracellular matrix protein 1 (ECM1) in promoting the pathogenesis of human and mouse IBD. ECM1 was highly expressed in macrophages, particularly tissue-infiltrated macrophages under inflammatory conditions, and ECM1 expression was significantly induced during IBD progression. The macrophage-specific knockout of ECM1 resulted in increased arginase 1 (ARG1) expression and impaired polarization into the M1 macrophage phenotype after lipopolysaccharide (LPS) treatment. A mechanistic study showed that ECM1 can regulate M1 macrophage polarization through the granulocyte-macrophage colony-stimulating factor/STAT5 signaling pathway. Pathological changes in mice with dextran sodium sulfate-induced IBD were alleviated by the specific knockout of the ECM1 gene in macrophages. Taken together, our findings show that ECM1 has an important function in promoting M1 macrophage polarization, which is critical for controlling inflammation and tissue repair in the intestine.

Keywords: ARG1; ECM1; GM-CSF; IBD; STAT5.

Fig. 1.

Ecm1 expression is strongly correlated with the pathogenic process of IBD in both humans and mice. (A and B) Real-time quantitative PCR analysis of TNF-α and ECM1 expression in colonic mucosal tissue samples from healthy subjects (n = 10) and patients with inactive (TNF-αlo, n = 7) and active (TNF-αhi, n = 8) ulcerative colitis. GAPDH served as an internal reference. Different groups are indicated as diamonds (healthy individuals), circles (UC patients with low TNF-α), or squares (UC patients with low TNF-α). ***P < 0.001; not significant (ns), P > 0.05 (one-way ANOVA with Tukey’s multiple comparisons test). (C) Analysis of the correlation between ECM1 and TNF-α gene expression (n = 8). Each circle indicates a tissue sample from one individual. The correlation was assessed with Pearson’s test. (D) IHC staining of human colonic mucosal tissue sections from healthy (Top) subjects and patients with active UC (Bottom), using an anti-ECM1 antibody. Right is from the enlarged Left. (Scale bars: Left, 400 μm; Right, 100 μm.) (E) Quantification of ECM1 expression in the samples from healthy subjects (n = 5) and patients with active UC (n = 8) in D. Different groups are indicated as circles (healthy individuals) or squares (active UC patients). ***P < 0.001 (Student’s t test) (F) Diagram (Top) showing the process of establishing a DSS-induced IBD mouse model. The percentage change in body weight (Bottom) in DSS-induced IBD mice (n = 5) is shown. (G) H&E staining (Top) and IHC staining with an anti-ECM1 antibody (Bottom) of mouse colon samples with DSS-induced IBD at different time. (Scale bars: Top, 200 μm; Bottom, 100 μm.) The data are representative of three independent experiments. (H) Quantification of ECM1 expression in the DSS-induced IBD mouse colon tissue samples collected at different points in G (n = 4). Different groups are indicated as circles (Day 0), squares (Day 5), up triangle (Day 10), or down triangle (Day 14). **P < 0.01; ***P < 0.001; ns, P > 0.05 (one-way ANOVA with Tukey’s multiple comparisons test). (I) Immunoblot analysis of total protein extracts from DSS-induced IBD mouse colon tissue samples harvested at different points. The data are representative of three independent experiments. Actin served as a loading control throughout these experiments.

Fig. 2.

ECM1 does not directly affect conventional CD4⁺ T and Treg cell functions in the pathogenesis of IBD. (A) Real-time quantitative PCR analysis of ECM1 expression in colonic tissue samples, lamina propria lymphocytes (LPLs), and intestinal epithelial cells (IECs) from naive and DSS-induced IBD mice. The data are representative of three independent experiments. ***P < 0.001; **P < 0.01; not significant (ns), P > 0.05 (one-way ANOVA with Tukey’s multiple comparisons test). (B) Naïve CD4⁺ T cells from ECM1^+/+ or ECM1^−/− mice were adoptively transferred into Rag1^−/− mice to establish a T cell-dependent IBD mouse model. n = 5. (C) H&E staining of colon samples from the mice in B. (Scale bars: 200 μm.) (D) Mixtures of wild-type naive CD4⁺ T cells and Treg cells from ECM1^+/+ or ECM1^−/− mice were adoptively transferred into Rag1^−/− mice to establish the T cell-dependent IBD mouse model. n = 5. (E) H&E staining of colon samples from the mice in D. (Scale bars: 200 μm.)

Fig. 3.

ECM1 is mainly produced by macrophages in DSS-induced IBD. (A) Immunofluorescence analysis of ECM1 and F4/80 expression in colon tissue from DSS-induced IBD mice. (Scale bars: 100 µm.) ECM1 and F4/80 are shown in green and red, respectively. Nuclei were stained with DAPI and are shown in blue. (B) Immunoblot analysis of ECM1 in CD4⁺ helper T cell subsets after in vitro differentiation and in macrophages isolated from the peritoneal cavity (PM: peritoneal macrophage). Actin served as a loading control throughout these experiments. The data are representative of three independent experiments. Flow cytometric analysis of ECM1 in macrophages (CD11B⁺F4/80⁺) and nonmacrophage cells (CD11B⁻F4/80⁻) isolated from the peritoneal cavity of ECM1^+/+ and ECM1^−/− mice (C) and the lamina propria of ECM1^+/+ mice with DSS-induced colitis on day 10 (D). The data are representative of three independent experiments. (E) Quantification of the ECM1 mean fluorescence intensity in D (n = 4). ***P < 0.001 (Student’s t test).

Fig. 4.

Macrophages from ECM1-deficient mice exhibit enhanced M2 polarization and impaired proinflammatory function. (A) GO analysis of genes showing up-regulated expression in Lyz-Cre/ECM1 f/f macrophages, as demonstrated in the RNA-Seq data. (B) GSEA analysis of arginine and proline metabolism-related genes in ECM1 f/f and Lyz-Cre/ECM1 f/f macrophages. (C) Immunoblot analysis of ECM1 and ARG1 expression in peritoneal macrophages stimulated with LPS for 0, 4, and 20 h. The data are representative of three independent experiments. Actin served as a loading control throughout this experiment. (D) Flow cytometric analysis of CD206 in peritoneal macrophages from ECM1 f/f and Lyz-Cre/ECM1 f/f mice. The data are representative of three independent experiments. (E) Quantification of the percentage of CD206⁺ cells in D (n = 3). **P < 0.01 (Student’s t test). (F) ELISA of the levels of TNF-α and IL-6 secreted by ECM1 f/f and Lyz-Cre/ECM1 f/f peritoneal macrophages stimulated with LPS for 0, 4, and 20 h. ***P < 0.001; not significant (ns), P > 0.05 (Student’s t test). The data are representative of three independent experiments.

Fig. 5.

Autocrine GM-CSF is responsible for enhanced arginase 1 expression in ECM1-deficient macrophages. (A) Quantitative analysis of ARG1 expression in ECM1 f/f and Lyz-Cre/ECM1 f/f peritoneal macrophages stimulated with LPS (200 ng/mL) or IL-4 (10 ng/mL). **P < 0.01; not significant (ns), P > 0.05 (Student’s t test). (B) Quantitative analysis of ARG1 expression in GM-CSF-differentiated BMMs and M-CSF-differentiated BMMs stimulated with LPS (200 ng/mL) and/or IFN-γ (50 ng/mL). **P < 0.01; ***P < 0.001 (Student’s t test). (C) Quantitative analysis of GM-CSF expression in ECM1 f/f and Lyz-Cre/ECM1 f/f peritoneal macrophages stimulated with LPS (200 ng/mL) or IL-4 (10 ng/mL). **P < 0.01 (Student’s t test). Quantitative analysis of the ARG1 (D) and GM-CSF (E) levels in ECM1 f/f and Lyz-Cre/ECM1 f/f peritoneal macrophages treated with anti-CD25 antibody (10 µg/mL) or anti–GM-CSF antibody (10 µg/mL) and then stimulated with LPS. ***P < 0.001; *P < 0.05 (Student’s t test). (F) Quantitative analysis of the ARG1, IL-6, and TNF-α levels in peritoneal macrophages stimulated with LPS (200 ng/mL) or GM-CSF (10 ng/mL). (G) Quantitative analysis of ARG1 expression in ECM1 f/f and Lyz-Cre/ECM1 f/f peritoneal macrophages stimulated with GM-CSF (10 ng/mL). ns, P >0.05. All the cells were stimulated for 20 h, and all the data are representative of three independent experiments.

Fig. 6.

ECM1 inhibits ARG1 expression through the GM-CSF/STAT5 pathway during M1 macrophage polarization. (A) Immunoblot analysis of STAT3/6, NF-κB, MAPK (Jnk/Erk/P38), and PI3K/Akt signaling activation in ECM1 f/f and Lyz-Cre/ECM1 f/f peritoneal macrophages stimulated with LPS. (B) Immunoblot analysis of STAT5 signaling activation in ECM1 f/f and Lyz-Cre/ECM1 f/f peritoneal macrophages treated with or without anti–GM-CSF antibody and then stimulated with LPS. (C) Quantification of the results from the Western blot analysis of STAT5 activation (phosphorylated STAT5/total STAT5) in B. ***P < 0.001; not significant (ns), P > 0.05 (Student’s t test). (D) Quantitative PCR analysis of ECM1, ARG1, GM-CSF, TNF-α, and IL-6 expression in ECM1 f/f and Lyz-Cre/ECM1 f/f peritoneal macrophages treated with DMSO/STAT5-IN-1 for 3 h and then stimulated with LPS for 20 h. ***P < 0.001; **P < 0.01; *P < 0.05; ns, P > 0.05 (Student’s t test). (E) Quantitative PCR analysis of GM-CSF, ARG1, IL-6, IL-12p40, and TNF-α expression in ECM1 f/f and Lyz-Cre/ECM1 f/f GM-CSF-differentiated BMMs treated with recombinant Fc-ECM1 protein (20 µg/mL) for 7 d and then stimulated with LPS for 20 h. ***P < 0.001; **P < 0.01; *P < 0.05; ns, P > 0.05 (one-way ANOVA with Tukey’s multiple comparisons test). All the data are representative of three independent experiments.

Fig. 7.

Ecm1-aggravated colitis through macrophages in vivo. (A) Percentage changes in the body weights of ECM1 f/f and Lyz-Cre/ECM1 f/f mice with colitis. The small horizontal lines indicate the means ± SEMs. ***P < 0.001 (Student’s t test). (B) Percentage survival of the ECM1 f/f and Lyz-Cre/ECM1 f/f mice with DSS-induced IBD in A. **P < 0.01 (Student’s t test). Representative images (C) and length (D) of the colon (on day 13) of the ECM1 f/f and Lyz-Cre/ECM1 f/f mice with colitis in A. Different groups are indicated as circles (ECM1 f/f + H₂O), up triangle (ECM1 f/f + 3% DSS), or down triangle (Lyz-Cre/ECM1 f/f + 3% DSS). *P < 0.05 (Student’s t test). (E) H&E staining of colonic tissue samples harvested on days 7, 10, and 13 from the ECM1 f/f and Lyz-Cre/ECM1 f/f mice with colitis. (Scale bars: 200 µm.) (F) Flow cytometric analysis of macrophage counts in the colonic lamina propria of ECM1 f/f and Lyz-Cre/ECM1 f/f mice with colitis on day 13. Not significant (ns), P > 0.05 (Student’s t test). (G) Flow cytometric analysis of CD206 in macrophages from the colonic lamina propria of ECM1 f/f and Lyz-Cre/ECM1 f/f mice with DSS-induced colitis on day 10. (H) Quantification of the percentage of CD206⁺ cells in F. Different groups are indicated as circles (ECM1 f/f + 3% DSS) or squares (Lyz-Cre/ECM1 f/f + 3% DSS). **P < 0.01 (Student’s t test). (I) ELISA of the IL-6, TNF-α z, and IL-10 levels in the supernatants of peritoneal macrophages collected from ECM1 f/f and Lyz-Cre/ECM1 f/f mice with colitis after stimulation with LPS for 20 h. **P < 0.01; *P < 0.05 (Student’s t test).

Fig. 8.

Schematic of our working mechanism. During the process of colitis, a large number of macrophages migrate into the inflammatory sites of intestinal tissue and are stimulated with components (e.g., lipopolysaccharides) from the invading microbiota. Those colonic macrophages can express extracellular matrix protein 1 (ECM1), which promotes inflammation and suppresses tissue repair by inhibiting TLR-mediated granulocyte-macrophage colony-stimulating factor (GM-CSF) expression. After ECM1 deletion in macrophages, a higher amount of autocrine GM-CSF is secreted from LPS (lipopolysaccharides)-stimulated macrophages, and the secreted GM-CSF in turn activates signal transducer and activation of transcription 5 (STAT5). Phosphorylated STAT5 can suppress inflammatory cytokine transcription and promote expression of the M2 macrophage-related gene ARG1, which functions in tissue repair. Enhanced ARG1 expression and arginine metabolism synergize with impaired inflammation to alleviate colitis when ECM1 is deleted from macrophages. White circles indicate vesicles; colored circles indicate nucleus; red bacteria indicate gram-negative bacteria; green bacteria indicate gram-positive bacteria.