<i>Mir223</i> restrains autophagy and promotes CNS inflammation by targeting ATG16L1

<p>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 <i>Mir223</i> 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 <i>Mir223</i>-deficienct mice. Furthermore, it was confirmed that <i>Mir223</i> 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 <i>Atg16l1</i> was decreased in BV2 cells upon <i>Mir223</i> overexpression and increased following the introduction of antagomirs. We also showed that the 3’ UTR of <i>Atg16l1</i> contained functional <i>Mir223</i>-responsive sequences and that overexpression of ATG16L1 returned autophagy to normal levels even in the presence of <i>Mir223</i> mimics. Collectively, these data indicate that <i>Mir223</i> is a novel and important regulator of autophagy and that <i>Atg16l1</i> is a <i>Mir223</i> target in this process, which may have implications for improving our understanding of the neuroinflammatory process of EAE.</p> <p><b>Abbreviations:</b> 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.</p>