miR-125a-5p regulates differential activation of macrophages and inflammation

Abstract

Macrophage activation is a central event in immune responses. Macrophages undergoing classical activation (M1 macrophages) are proinflammatory, whereas alternatively activated macrophages (M2 macrophages) are generally anti-inflammatory. miRNAs play important regulatory roles in inflammatory response. However, the manner in which miRNAs regulate macrophage activation in response to different environmental cues has not been well defined. In this study, we found that M-BMM macrophages (M2) express greater levels of miR-125a-5p than do GM-BMM macrophages (M1). Stimulation of macrophages through TLR2 and TLR4 but not through TLR3 enhanced miR-125a-5p expression. Up-regulation of miR-125a-5p after TLR2/4 activation requires the adaptor MYD88 but not TRIF. Overexpression of miR-125a-5p diminished M1 phenotype expression induced by LPS but promoted M2 marker expression induced by IL-4. In contrast, knockdown of miR-125a-5p promoted M1 polarization and diminished IL-4-induced M2 marker expression. We found that miR-125a-5p targets KLF13, a transcriptional factor that has an important role in T lymphocyte activation and inflammation. KLF13 knockdown had similar effects on M1 activation as did miR-125a-5p overexpression. In addition, miR-125a-5p regulates phagocytic and bactericidal activities of macrophages. Our data suggest that miR-125a-5p has an important role in suppressing classical activation of macrophages while promoting alternative activation.

Keywords: Inflammation; KLF13; Kruppel-like Factor (KLF); Macrophage Polarization; Macrophages; MicroRNA; Phagocytosis; Toll-like Receptors (TLR).

FIGURE 1.

miR-125a-5p is expressed at a higher level in M-BMM than in GM-BMM. A, M-BMM and GM-BMM were generated by culturing mouse bone marrow cells in 50 ng/ml M-CSF or 20 ng/ml GM-CSF for 7 days. RNA was isolated and miRNA array analysis was performed. Unsupervised hierarchical clustering is presented. B, levels of miR-125a-5p in M-BMM and GM-BMM were determined by real-time PCR. snRNA RNU6B was used as an internal control (n = 3); mean ± S.D.; ***, p < 0.001 compared with GM-BMM. mmu, Mus musculus.

FIGURE 2.

miR-125a-5p is up-regulated in response to TLR2 and TLR4 stimulation. A, GM-BMM were treated with 100 ng/ml LPS for the indicated time. Levels of miR-125a-5p were determined by real-time PCR (n = 3); mean ± S.D.; **, p < 0.01 compared with 0 h. B, experiments were done as in A. Levels of TNF-α were determined by real-time PCR. C, GM-BMM were treated without or with 1 μg/ml Pam(3)CSK(4) or 2 μg/ml poly(I:C) for 24 h. Levels of miR-125a-5p were determined by real-time PCR (n = 3); mean ± S.D.; ***, p < 0.001 compared with control (con). D, peritoneal macrophages isolated from WT, MYD88^−/− or TRIF^−/− mice were treated without or with 100 ng/ml LPS. Levels of miR-125a-5p determined by real-time PCR (n = 3); mean ± S.D.; ***, p < 0.001 compared with the WT group without treatment. E, peritoneal macrophages isolated from MYD88^−/− mice were treated without or with 1 μg/ml Pam(3)CSK(4) (PAM). RNA was isolated, and levels of miR-125a-5p were determined (n = 3); mean ± S.D. F, GM-BMM were treated with LPS for the indicated time. Levels of miR-125b-5p were determined (n = 3); mean ± S.D.; *, p < 0.05 compared with 0 h. The experiments were performed two to three times with similar results.

FIGURE 3.

miR-125a-5p suppresses expression of the M1 phenotype. A–D, GM-BMM were transfected with 20 nm control mimics or miR-125a-5p mimics. At day 3 after transfection, the cells were treated without or with 100 ng/ml LPS for 6 h. Levels of TNF-α, IL-12, and iNOS were determined by ELISA (A and B) or real-time PCR (C and D) (n = 3); mean ± S.D.; *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with the control (con) groups. E, transfection was done as in A–D. Cells were treated without or with LPS for 24 h. Levels of iNOS and GAPDH were determined by Western blotting. A second experiment provided similar results.

FIGURE 4.

Knockdown of miR-125a-5p promotes expression of the M1 phenotype. A–C, M-BMM were transfected with 20 nm control (con) inhibitors or miR-125a-5p inhibitors. At day 3 after transfection, the cells were treated without or with 100 ng/ml LPS for 6 h. Levels of TNF-α, IL-12, and iNOS were determined by ELISA (A) or real-time PCR (B and C) (n = 3); mean ± S.D.; *, p < 0.05; ***, p < 0.001 compared with the control (con) groups. D, levels of miR-125a-5p were determined by real-time PCR in M-BMM that were transfected with control inhibitors or miR-125a-5p inhibitors. E and F, M-BMM were transfected with 20 nm control inhibitors or miR-125a-5p inhibitors. At day 3 after transfection, the cells were treated without or with 100 ng/ml LPS for 24 h. The surface levels of MHCII and CD40 were determined by flow cytometry analysis. Representative results are shown. A second experiment provided similar results.

FIGURE 5.

miR-125a-5p promotes expression of the M2 phenotype. A, GM-BMM were transfected with 20 nm control (con) mimics or miR-125a-5p mimics. After transfection, the cells were treated without or with 10 ng/ml IL-4 for 24 h. Levels of Arg1 were determined by real-time PCR (n = 3); mean ± S.D.; ***, p < 0.001 compared with the control groups. B, GM-BMM were transfected with control mimics or miR-125a-5p mimics. The cells were then treated without or with IL-4 for 24 h. Levels of Arg1 and actin were determined by Western blotting. C, M-BMM were transfected with 20 nm control inhibitors or miR-125a-5p inhibitors. After transfection, the cells were treated without or with IL-4 for 24 h. Levels of Arg1 and actin were determined by Western blotting. The experiments were performed twice with similar results.

FIGURE 6.

miR-125a-5p down-regulates KLF13. A, GM-BMM were transfected with 20 nm control (con) mimics or miR-125a-5p mimics. Levels of KLF13 were determined by real-time PCR (n = 3); mean ± S.D.; **, p < 0.01 compared with the control group. B, GM-BMM were transfected with control inhibitors or miR-125a-5p inhibitors. After transfection, the cells were stimulated with LPS for 24 h. Levels of KLF13 in the cells were determined by real-time PCR (n = 3); mean ± S.D.; *, p < 0.05 compared with the control group. C, 5 ng of pMIR-Report-KLF13 were co-transfected with 20 nm control mimics or miR-125a-5p mimics into HEK-293T cells. 24 h after transfection, luciferase activity in the cells was determined (n = 3); mean ± S.D.; **, p < 0.01 compared with the control group. D, mimics or miR-125a-5p mimics were transfected into HEK-293 cells. 6 h after the transfection, FLAG-KLF13-expressing vector that contains WT or mutant 3′-UTR of the KLF13 gene were transfected into the same HEK-293 cells. Two days after transfection, FLAG-KLF13 levels were determined. E, GM-BMM were treated with LPS for the indicated time. Levels of miR-125a-5p and KLF13 were determined by real-time PCR. F, levels of KLF13 in GM-BMM and M-BMM were determined by real-time PCR. G, GM-BMM were transfected with control siRNAs or KLF13 siRNAs. Levels of KLF13 were determined by real-time PCR. H, GM-BMM were transfected with control siRNAs or KLF13 siRNAs, followed by LPS treatment for 6 h. Levels of TNF-α and IL-12 were determined by ELISA (n = 3); mean ± S.D.; **, p < 0.01; ***, p < 0.001 compared with the control group treated with LPS. Representative results of two experiments are shown.

FIGURE 7.

miR-125a-5p regulates cellular functions associated with the M1 and M2 phenotypes. A, GM-BMM were transfected with 20 nm control mimics or miR-125a-5p mimics. After transfection, live E. coli were added into the media. 1 h after incubation, the supernatants were collected and cultured on Luria broth agar plates at 37 °C for 24 h. The bacteria colonies were counted, and the cfu of E. coli in the supernatants were determined (n = 3); mean ± S.D.; **, p < 0.01 compared with the control group. B, GM-BMM were transfected with 20 nm control (con) siRNAs or KLF13 siRNAs. Bacteria killing assay was performed as in A (n = 3); mean ± S.D.; ***, p < 0.001 compared with the control group. C, GM-BMM were transfected with 20 nm control mimics or miR-125a-5p mimics. After transfection, 0.5 × 10⁶ apoptotic thymocytes were added to the macrophages, and phagocytosis assays were performed (n = 3); mean ± S.D.; **, p < 0.01 compared with the control group. D, M-BMM were transfected with 20 nm control inhibitors or miR-125a-5p inhibitors. After transfection, phagocytosis assays were performed (n = 3); mean ± S.D.; *, p < 0.05 compared with the control group. E, GM-BMM were transfected with 20 nm control siRNAs or KLF13 siRNAs. After transfection, phagocytosis assays were performed (n = 3); mean ± S.D.; ***, p < 0.001 compared with the control group. The experiments were performed twice with similar results.