Figure 6.

miR-192 Overexpression Switches Myocardial-Infiltrating Macrophages to a Predominant M2 Phenotype in AK085865 KO VM Mice

AK085865 KO mice received 1 × 10⁵ PFU of CVB3 i.p. on day 0. For in vivo miR-192 treatment, 30 μL Lipofectamine 2000 reagent was mixed with miR-192 agomir or negative control (10 nmol/site), dissolved in a volume of 50 μL PBS, and the liposome complex was injected into the apex of the left ventricle with a 30-gauge needle at six sites per mouse, 3 days prior to CVB3 infection. Hearts were collected on day 7 postinfection, and myocardial-infiltrating mononuclear cells were isolated from the hearts after enzymatic digestion. (A) The percentages of F4/80⁺iNOS⁺ or F4/80⁺Arg1⁺ cells were analyzed by FACS. (B) The arginase activity of sorted F4/80⁺ macrophages was assessed by an assay of urea production from the arginine substrate and was normalized to cell counts. (C) qRT-PCR was used to determine the NOS2, Arg1, FIZZ1, and YM-1 expression in sorted F4/80⁺ macrophages. (D) For colocalization analysis, sections were costained for CD68 (red, macrophage marker) and iNOS (green, M1 marker) or Arg1 (green, M2 marker). DAPI was used for nucleus staining (blue). The arrows just represent that we magnified the zone (arrows pointing) in the right panel images. Scale bars, 100 μm. Experiments were repeated three times in triplicate with 10–15 mice per group. The data represent the mean ± SEM. The results are representative of at least three independent experiments with n ≥ 3. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.