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. 2016 Dec:14:83-96.
doi: 10.1016/j.ebiom.2016.10.041. Epub 2016 Oct 29.

Reprogramming macrophage orientation by microRNA 146b targeting transcription factor IRF5

Liang Peng  1 , Hui Zhang  1 , Yuanyuan Hao  1 , Feihong Xu  1 , Jianjun Yang  1 , Ruihua Zhang  1 , Geming Lu  1 , Zihan Zheng  1 , Miao Cui  1 , Chen-Feng Qi  2 , Chun Chen  3 , Juan Wang  1 , Yuan Hu  1 , Di Wang  1 , Susan Pierce  2 , Liwu Li  3 , Huabao Xiong  4
Affiliations

Reprogramming macrophage orientation by microRNA 146b targeting transcription factor IRF5

Liang Peng et al. EBioMedicine. 2016 Dec.

Abstract

The regulation of macrophage orientation pathological conditions is important but still incompletely understood. Here, we show that IL-10 and Rag1 double knockout mice spontaneously develop colitis with dominant M1 macrophage phenotype, suggesting that IL-10 regulates macrophage orientation in inflammation. We demonstrate that IL-10 stimulation induced miR-146b expression, and that the expression of miR-146b was impaired in IL-10 deficient macrophages. Our data show that miR-146b targets IRF5, resulting in the regulation of macrophage activation. Furthermore, miR-146b deficient mice developed intestinal inflammation with enhanced M1 macrophage polarization. Finally, miR-146b mimic treatment significantly suppresses M1 macrophage activation and ameliorates colitis development in vivo. Collectively, the results suggest that IL-10 dependent miR-146b plays an important role in the modulation of M1 macrophage orientation.

Keywords: CRISPR/Cas9; Colitis; Interleukin 10; Macrophage; miR-146b.

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Figures

Fig. 1
Fig. 1
IL-10 deficient M1 macrophages drive colitis development. Rag−/− and Rag−/−/IL-10−/− mice were raised until 20 weeks of age and mice were sacrificed. (A) Photographs of intestinal morphology and H&E staining of colon tissues (upper panel scale bar, 100 μm; low panel scale bar, 50 μm); (B) disease score in the colon tissues of Rag−/− and Rag−/−/IL-10−/− mice (*p < 0.05). (C) The mRNA levels of TNFα, IL-12/23 p40, and IL-6 in the colon tissue of the WT, IL-10−/− and IL-10R2−/− mice (n = 5) (*p < 0.05; **p < 0.01; ***p < 0.001). (D) Detection of iNOS-expressing F4/80 positive cells by immunofluorescence in colon tissue of Rag−/− and Rag−/−/IL-10−/− mice (red, F4/80; green, iNOS; blue, DAPI, scale bar, 100 μm). (E, F) The presence of IL-12/23 p40-producing F4/80 positive cells in lamina propria portion of Rag−/− and Rag−/−/IL-10−/− mice, with representative FACS dot plots gated on F4/80+ cells and the percentages of IL-12/23 p40-producing cells shown (*p < 0.05; **p < 0.01; ***p < 0.001). (G) BMDMs cultured from Rag−/− and Rag−/−/IL-10−/− mice were incubated with IFN-γ (20 ng/ml) plus LPS (100 ng/ml) for 24 h, stained for intracellular IL-12/23 p40, and analyzed by flow cytometry. Representative FACS dot plots gated on CD11b+ cells and the percentage of IL-12/23 p40 positive cells are shown (**p < 0.01). (H) ELISA of the serum of the mice detecting the expression of several key pro-inflammatory cytokines, as in (C). All the experiments were repeated three times with similar results.
Fig. 2
Fig. 2
IL-10 deficiency promotes M1 macrophage differentiation. (A) BMDMs from wild type or IL-10−/− mice were stimulated with IFN-γ (20 ng/ml) plus LPS (100 ng/ml) for various time points (0, 2, 4, 8 h) and mRNA was isolated for microarray analysis. Snapshots from heat maps show 72 genes related to macrophage activation. The position of each gene locus is in the same areas of all six heat maps, with 6 loci of interest being highlighted. (B) BMDMs treated as in (A) for 4 h. Total RNA was prepared and qPCR was performed for the analysis of macrophage gene expression (data represent mean ± s.d.). (C). The cells prepared in A were stimulated with IFN-γ (20 ng/ml) plus LPS (100 ng/ml) overnight and analyzed by flow cytometry. Representative FACS dot plots gated on CD11b+ cells and the percentages of IL-12p/23 p40-producing and iNOS-expressing CD11b+ cells are shown. (D). The cells prepared in (A) were stimulated with IFN-γ (20 ng/ml) plus LPS (100 ng/ml) for 24 h and the supernatants were analyzed for the level of IL-12/23 p40, TNFα and IL-6 production by ELISA. *p < 0.05; **p < 0.01. The results are representative of three independent experiments.
Fig. 3
Fig. 3
miR-146b suppresses M1 macrophage activation by targeting IRF5. (A) Schematic representation of wild-type (wt) and mutant (mut) IRF5 3′UTR luciferase reporter constructs. The predicted miR-146b binding region is indicated (left panel). HEK293T cells were co-transfected with either WT or mutant IRF5 luciferase reporter plasmids together the miR-146 mimic for 30 h. The cell lysates were prepared and luciferase activity was detected (Data represent mean ± s.d.). (B) BMDMs from C57BL/6 mice were stimulated with IFN-γ (20 ng/ml) and LPS (100 ng/ml) for 4 h. Total RNA was extracted and immunoprecipitated with anti-Ago2 antibody. The immunoprecipitated RNA was purified and qPCR was performed for the analysis of miR-146b and IRF5 mRNA expression (Data represent mean ± s.d.). (C) BMDMs from C57BL/6 mice were transfected with miR-146b mimic (3 μM) for 48 h and stimulated as in (B) for 24 h. The whole cell lysates were prepared and western blotting was performed for the analysis of IRF5 expression, using GAPDH expression as a control. (D) BMDMs from WT mice were transfected with miR-146b mimic (3 μM) or scramble (3 μM) and stimulated as in B,C for 4 h, followed by ChIP assay. PCR was used to quantify the amount of precipitated DNA with primers flanking the IRF5 binding site in the IL-12 promoter region. (E) Western blot of whole cell lysates from BMDMs of WT and IL-10−/− mice stimulated as before for 24 or 48 h. (F) BMDMs as prepared in A were transfected with either IRF5 siRNA or control siRNA for 48 h and subsequently stimulated as before for 24 h. The cells were stained for intracellular IL-12/23 p40 and analyzed by flow cytometry. Representative FACS dot plots gated on CD11b+ cells and the percentage of IL-12/23 p40-producing CD11b+ cells is shown. (G) BMDMs prepared in A were transfected with IRF5 siRNA or control siRNA and the cells were then stimulated with IFN-γ (20 ng/ml) and LPS (100 ng/ml) for 24 h. The supernatants were collected and the level of IL-12 p40, TNFα and IL-6 production in the supernatants was analyzed for by ELISA. *p < 0.05; **p < 0.01. All results are representative of at least two independent experiments.
Fig. 4
Fig. 4
Overexpression of IRF5 rescued miR-146b mimic-induced suppression of M1 macrophage activation. (A) BMDMs from IL-10−/− mice were transfected with miR-146b mimic (3 μM), miR-nc and IRF5 overexpression plasmid for 48 h and the cells were then stimulated with IFN-γ (20 ng/ml) and LPS (100 ng/ml) for 24 h prior to western blotting for IRF5 expression. (B) BMDMs from C57BL/6 mice were prepared under the same conditions as in A. The supernatants were collected and the level of IL-12 p40, IL-6, and TNFα production in the supernatants was analyzed for by ELISA. ***p < 0.001. (C) BMDMs from C57BL/6 mice treated as in B were stained for intracellular IL-12/23 p40 and analyzed by flow cytometry. Representative FACS dot plots gated on CD11b+ cells and the percentage of IL-12/23 p40-producing CD11b+ cells is shown (*p < 0.05). (D) CD4+ T cells from spleens and lymph nodes of OTII mice were prepared and the cells were incubated with IL-10−/− BMDMs (unstimulated or stimulated with IFN-γ and LPS) with cotransfection of miR-146b mimic (3 μM), miR-nc and IRF5 overexpression plasmid for three days. The cells were stained for intracellular IFN-γ and analyzed by flow cytometry. Representative FACS dot plots gated on CD4+ cells and the percentages of IFN-γ-producing CD4+ cells are shown. (E) The supernatants in (D) were analyzed for IFN-γ production by ELISA. **p < 0.01. All the experiments were repeated three times with similar results.
Fig. 5
Fig. 5
miR-146b deficiency enhanced M1 macrophage orientation. WT and Mir146b−/− mice were raised until 12 weeks of age and mice were sacrificed. (A) Photographs of spleen morphology and HE staining. (B) The presence of Gr+ CD11b+ cells in spleen and Bone marrow cells of WT and Mir146b−/− mice. (C, D) HE staining of colon tissue and qPCR for macrophage genes expression from WT and Mir146b−/− mice. (E) BMDMs from WT and Mir146b−/− were stimulated with IFN-γ (20 ng/ml) and LPS (100 ng/ml) for 24 h, and analyzed by flow cytometry. Representative FACS dot plots gated on CD11b+ cells and the percentage of IL-12/23 p40-producing CD11b+ cells is shown (**p < 0.01). (F) The supernatants from (E) were collected and the level of IL-6, IL-12 p40, and TNFα secretion in the supernatants was analyzed by ELISA. *p < 0.05. (G) Whole cell lysates of BMDMs from WT and Mir146b−/− were treated as before and analyzed via western blot. (H). BMDMs from Mir146b−/− mice were transfected with miR-146b mimic (3 μM) and miR-nc for 48 h and the cells were then stimulated with IFN-γ (20 ng/ml) and LPS (100 ng/ml) for 24 h. The cells were stained for intracellular IL-12/23 p40, NOS2 and analyzed by flow cytometry. Representative FACS dot plots gated on CD11b+ cells and the percentage of IL-12/23 p40-producing and NOS2-producing CD11b+ cells is shown. (I) The supernatants in (G) were collected and the level of IL-6, IL-12 p40, and TNFα production in the supernatants was analyzed for by ELISA. **p < 0.01. (J) The whole cell lysates were prepared and western blotting was performed for the analysis of IRF5 expression after miR146b mimic treatment in Mir146b−/− mice. β-actin expression serves as a control. (K) BMDMs from Mir146b−/− mice were stimulated with IFN-γ (20 ng/ml) and LPS (100 ng/ml) for 24 h in the presence of rIL-10 (10 ng/ml). The supernatants were collected and analyzed for by ELISA. *p < 0.05; **p < 0.01. (L) The whole cell lysates were prepared and western blotting was performed for the analysis of IRF5 expression. β-actin expression serves as a control. All the experiments were repeated three times with similar results.
Fig. 6
Fig. 6
Mir146b−/− macrophages induce enhanced CD4+ T cell activation. (A) CD4+ T cells from spleens and lymph nodes of OTII mice were prepared and the cells were incubated with WT or Mir146b−/− BMDMs for three days. Representative FACS dot plots gated on CD4+ cells of the percentages of IFN-γ positive CD4+ cells are shown (**p < 0.01). (B) The supernatants were analyzed for IFN-γ production by ELISA (**p < 0.01). (C). CD4+ T cells from spleens and lymph nodes of OTII mice were co-cultured as in A and labeled with CFSE. T cell proliferation was analyzed by flow cytometer. (D). CD4+ T cells from spleens and lymph nodes of OTII mice were prepared and the cells were incubated for three days with Mir146b−/− BMDMs already transfected with miR-146b mimic. Representative FACS dot plots gated on CD4+ cells and the percentages of IFN-γ positive CD4+ cells are shown. (E) The supernatants were analyzed for IFN-γ production by ELISA. ***p < 0.001. (F) CD4+ T cells from spleens and lymph nodes of OTII mice were prepared and the cells were incubated with WT and Mir146b−/− BMDMs which were stimulated with IFN-γ (20 ng/ml) and LPS (100 ng/ml) and rIL-10 for 24 h and analyzed as in D. (G) The supernatants were analyzed for IFN-γ production by ELISA. **p < 0.001. All the experiments were repeated three times with similar results.
Fig. 7
Fig. 7
Treatment with miR-146b mimic suppressed M1 macrophage activation in vivo and ameliorated colitis development. 16-week old WT, IL-10−/−, and IL-10R2−/− mice were sacrificed and colons were removed. (A) Total RNA was extracted from colon tissues and qPCR was performed for the expression of miR-146b. (B) In situ hybridization was performed for the detection of miR-146b expression in colon tissues in WT and IL-10−/− mice. The data are representative of two independent experiments. (C) 13-week old IL-10−/− mice were divided into two groups (6 mice/per group). In the treatment group, IL-10−/− mice were treated with miR-146b mimic intraperitoneally twice a week at 10 mg/kg; while in the control group, mice were treated with miR-146b scramble at same dose for 3 weeks. Body weight was monitored every week and mice were sacrificed 3 weeks later. Changes in body weight of IL-10−/− mice (n = 5 mice per group) treated either with miR146b scramble or mimic were recorded. Data are presented as the mean ± s.d. of the percentage of initial body weight. (D) Morphology of intestines, (E) sections of colons with colitis, (F) disease scores from IL-10−/− mice three weeks after treated either with 146b mimic or scramble (n = 5 mice in each group). *p < 0.05. (G) miR146b expression in colon of the scramble and miR146b mimic treatment group (***p < 0.001). (H) The percentages of IL-17- or IFN-γ-producing cells from intestinal lamina propria lymphocytes of IL-10−/− mice (**p < 0.01). (I) The percentages of IL-12/23 p40-producing and MHCII-expressing cells from intestinal lamina propria cells of IL-10−/− mice. Representative FACS dot plots gated on F4/80+ cells and the percentage of IL-12/23 p40-producing and MHCII-expressing cells are shown (n = 6), (*p < 0.05; **p < 0.01).
Fig. 8
Fig. 8
miR-146b mimic ameliorates endotoxin shock in mice. 7 week old IL-10−/− mice were divided into two group (n = 10/per group) and one group of mice were injected (i.p.) with miR-146b scramble and the other group of mice were injected (i.p.) with miR-146b mimic at a dose of 10 mg/kg. (A) 3 h later both groups of mice were challenged (i.p.) with LPS (1000 μg/per mouse) and the survival of mice was monitored. (B, C) 3 h later both groups of mice were challenged (i.p.) with LPS (300 μg/per mouse) and mice were sacrificed 4 h later. Total RNA was extracted from the spleens of mice and qPCR was performed for the analysis of M1 and M2 macrophage signature gene expression. The sera level of IL-6 and TNFα was determined by ELISA. *p < 0.05, **p < 0.01.The results were representative of two independent experiments.
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References

    1. Akira S., Misawa T., Satoh T., Saitoh T. Macrophages control innate inflammation. Diabetes Obes. Metab. 2013;15(Suppl. 3):10–18. - PubMed
    1. Banno K., Yanokura M., Iida M., Adachi M., Nakamura K., Nogami Y., Umene K., Masuda K., Kisu I., Nomura H. Application of MicroRNA in diagnosis and treatment of ovarian cancer. Biomed. Res. Int. 2014;2014:232817. - PMC - PubMed
    1. Bartel D.P. MicroRNAs: target recognition and regulatory functions. Cell. 2009;136:215–233. - PMC - PubMed
    1. Baumjohann D., Ansel K.M. MicroRNA-mediated regulation of T helper cell differentiation and plasticity. Nat. Rev. Immunol. 2013;13:666–678. - PMC - PubMed
    1. Berg D.J., Leach M.W., Kuhn R., Rajewsky K., Muller W., Davidson N.J., Rennick D. Interleukin 10 but not interleukin 4 is a natural suppressant of cutaneous inflammatory responses. J. Exp. Med. 1995;182:99–108. - PMC - PubMed

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