Mir223 restrains autophagy and promotes CNS inflammation by targeting ATG16L1

Abstract

Microglia are innate immune cells in the central nervous system (CNS), that supplies neurons with key factors for executing autophagosomal/lysosomal functions. Macroautophagy/autophagy is a cellular catabolic process that maintains cell balance in response to stress-related stimulation. Abnormal autophagy occurs with many pathologies, such as cancer, and autoimmune and neurodegenerative diseases. Hence, clarification of the mechanisms of autophagy regulation is of utmost importance. Recently, researchers presented microRNAs (miRNAs) as novel and potent modulators of autophagic activity. Here, we found that Mir223 deficiency significantly ameliorated CNS inflammation, demyelination and the clinical symptoms of experimental autoimmune encephalomyelitis (EAE) and increased resting microglia and autophagy in brain microglial cells. In contrast, the autophagy inhibitor 3-methylademine (3-MA) aggravated the clinical symptoms of EAE in wild-type (WT) and Mir223-deficienct mice. Furthermore, it was confirmed that Mir223 deficiency in mice increased the protein expression of ATG16L1 (autophagy related 16-like 1 [S. cerevisiae]) and LC3-II in bone marrow-derived macrophage cells compared with cells from WT mice. Indeed, the cellular level of Atg16l1 was decreased in BV2 cells upon Mir223 overexpression and increased following the introduction of antagomirs. We also showed that the 3' UTR of Atg16l1 contained functional Mir223-responsive sequences and that overexpression of ATG16L1 returned autophagy to normal levels even in the presence of Mir223 mimics. Collectively, these data indicate that Mir223 is a novel and important regulator of autophagy and that Atg16l1 is a Mir223 target in this process, which may have implications for improving our understanding of the neuroinflammatory process of EAE. Abbreviations: 3-MA: 3-methylademine; ACTB/β-actin: actin, beta; ATG: autophagy related; ATG16L1: autophagy related 16-like 1 (S. cerevisiae); BECN1: beclin 1, autophagy related; CNR2: cannabinoid receptor 2 (macrophage); CNS: central nervous system; CQ: chloroquine; EAE: experimental autoimmune encephalomyelitis; FOXO3: forkhead box O3; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; H&E: hematoxylin and eosin; ITGAM: integrin alpha M; LPS: lipoplysaccharide; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; miRNAs: microRNAs; MS: multiple sclerosis; PPARG: peroxisome proliferator activated receptor gamma; PTPRC: protein tyrosine phosphatase, receptor type, C; RA: rheumatoid arthritis; SQSTM1: sequestosome 1; TB: tuberculosis; TIMM23: translocase of inner mitochondrial membrane 23; TLR: toll-like receptor.

Keywords: ATG16L1; CNS inflammation; autophagy; experimental autoimmune encephalomyelitis; microglia.

Figure 1.

mir223^−/- mice have alleviated EAE symptoms and decreased spinal cord inflammation and demyelination. EAE was induced with MOG[35–55] in female C57BL/6 mice (n = 12). The clinical scores of all of the EAE mice were assessed daily according to the same criteria for 28 continuous days. Incidence (A), disease onset (B), daily clinical scores (C), peak disease scores (D), and cumulative disease scores (E) were monitored. (A) The incidence of EAE (mice with clinical score ≥ 1 for 2 continuous days). The disease incidence is represented by the percentage of mice suffering from EAE. (B) Line graph depicts the EAE survival rate between WT and mir223^−/- mice. (C) The clinical scores of all of the mice for 28 continuous days. (D) The mean maximum clinical scores. (E) The mean disease cumulative score. (F) H&E staining of representative spinal cord sections from mir223^−/- mice and WT mice shows the infiltration of inflammatory cells in the white matter. (G) Luxol fast blue staining of intact myelin (blue) and demyelination (pink). The data are representative of at least 2 experiments with similar results.

Figure 2.

Mir223 deficiency increased autophagy and resting microglia in the brains of EAE mice. (A) Flow cytometric analysis of microglia, demonstrating PTPRC and ITGAM cells isolated from the CNS of EAE mice (n = 6 mice per group), detected on the 15th day after the induction of EAE. The data are shown in a representative plot. (B) The absolute numbers of the cell subpopulations are shown; the black column is for mir223^−/- mice. Rest, ITGAM⁺ PTPRC^low; mc, ITGAM⁺ PTPRC^{hi and low}. (C) Autophagy was measured in the brains of the mice. LC3 puncta were visualized by confocal imaging of microglia immunostained for LC3 and AIF1, followed by Alexa Fluor 488/555-conjugated secondary antibodies (green/red); nuclei were stained with DAPI. Representative images are shown. Scale bars: 20 µm. (D) and (E) BCL2 and BECN1 expression were visualized by fluorescence imaging of microglia. Scale bars: 50 µm.

Figure 3.

3-MA-mediated blockade of autophagy attenuates the effects of Mir223 on EAE mice. EAE was induced with MOG[35–55] in female C57BL/6 mice (n = 12). Mice were injected with 3-MA every day after immunization. The body weight and clinical scores of all of the EAE mice were assessed daily according to the same criteria for 21 continuous days. Body weight (A), disease onset (B), incidence (C), peak disease scores (D), daily clinical scores (E), and cumulative disease scores (F) were monitored.

Figure 4.

Mir223 deficiency increases autophagy in primary macrophages and BV2 cells. (A) Macrophages were separated from the bone marrow of WT mice and mir223^−/- mice. The cells were stimulated with LPS or starvation. Immunoblot analysis was performed for ATG16L1, SQSTM1, TIMM23, LC3 and ACTB. ATG16L1:ACTB and LC3-II:ACTB ratios are shown below the blots. (B) Deficiency of Mir223 induces autophagic vacuoles in BV2 cells upon LPS stimulation. Scale bars: 0.5 µm. (C) Quantitative analysis of the number of autophagic vacuoles.

Figure 5.

Overexpression of Mir223 blocks lipopolysaccharide-induced autophagy in BV2 cells. (A) Mir223 repressed the formation of starvation-induced GFP-LC3 puncta in BV2 cells. Stable GFP-LC3-expressing cells were cotransfected with mimics or Mim-CN, and autophagy was evaluated after LPS or control treatment for 4 h. Scale bar: 5 μm. (B) Quantitative analysis of the number of GFP-LC3 puncta per cell in (A). The data are presented as the mean± SEM; experiments were performed in triplicate (*p < 0.05, **p < 0.01; NS, not significant). (C) and (D) Western blot analysis of LC3, TIMM23, SQSTM1 and ACTB. (E and F) Autophagy was evaluated in the presence or absence of CQ with LPS or no LPS treatment for 4 h. Western blot analysis of LC3, TIMM23, SQSTM1 and ACTB. Densitometric ratios were quantified using ImageJ software. LC3-II:ACTB and TIMM23:ACTB ratios from immunoblots are shown from 3 independent experiments. ACTB was used as the loading control.

Figure 6.

Inhibition of endogenous Mir223 increases autophagy upon LPS stimulation. (A) Mir223 repressed the formation of starvation-induced GFP-LC3 puncta in BV2 cells. Stable GFP-LC3-expressing cells were cotransfected with inhibitor (In-Mir223) or In-CN, and autophagy was tested after LPS or control treatment for 4 h. Scale bar: 5 μm. (B) Quantitative analysis of the number of GFP-LC3 puncta per cell in (A). The data are presented as the mean± SEM; experiments were performed in triplicate (*p < 0.05, **p < 0.01). (C and D) Western blot analysis of LC3, TIMM23, SQSTM1 and ACTB. (E and F) Autophagy was tested in the presence or absence of CQ with LPS or control treatment for 4 h. Western blot analysis of LC3, TIMM23, SQSTM1 and ACTB. Densitometric ratios were quantified using ImageJ software. LC3-II:ACTB and TIMM23:ACTB ratios from immunoblots are shown from 3 independent experiments. ACTB was used as the loading control.

Figure 7.

Atg16l1 as a direct target of Mir223. (A) The Mir223 target sequence in the 3’ UTR of Atg16l1 in mice. (B) A scheme representing the luciferase constructs with the wild-type (WT) or mutant 3’ UTR Mir223 MRE sequences of Atg16l1. Mutations are marked in lower-case letters and underlined. (C) Normalized luciferase activity in lysates from 293T cells co-transfected with wild-type or mutant Atg16l1-luciferase constructs and Mir223 mimics or Mim-CN (mean± SEM, n = 3, *p < 0.05, **p < 0.01). (D and E) BV2 cells were cotransfected with Mir223 Mim-CN, mimics, In-CN or inhibitor and starved for 4 h. Quantitative PCR (qPCR) analysis of Atg16l1 mRNA levels is shown. (F and G) BV2 cells were cotransfected with Mir223 Mim-CN, mimics, In-CN or inhibitor and starved for 4 h. The ATG16L1 protein level was tested by immunoblotting. ACTB was used as the loading control. (H) Quantitative PCR (qPCR) analysis of Atg16l1 mRNA levels in In-CN- or inhibitor-transfected BV2 cells. The data are expressed as the mean± SEM; experiments were performed in triplicate (*p < 0.05, **p < 0.01). The data were normalized to Gapdh mRNA.

Figure 8.

ATG16L1 protein overexpression abrogates Mir223-mediated autophagy suppression. BV2 cells were cotransfected with Mir223 mimics or Mim-CN and ATG16L1 expression plasmid lacking the Mir223 target region. (A) Cells were starved for 4 h. Immunoblot analysis of ATG16L1 and ACTB is shown. (B) ATG16L1:ACTB ratios. (C) Formation of GFP-LC3 puncta before or after starvation for 4 h. Scale bar: 5 μm. (D) Quantitative analysis of GFP-LC3 puncta (mean± SEM, n = 3, *p < 0.05, **p < 0.01). (E) Model of the effect of Mir223 regulation of autophagy through direct targeting of Atg16l1.