Abstract 16413: MicroRNAs Mediate Cardiac RNA-induced Inflammatory Response via Toll-like Receptor 7

Abstract

Introduction: We have reported that RNA released from necrotic cells induces cytokine production and may contribute to myocardial ischemia/reperfusion (I/R) injury. Our previous study has also demonstrated that RNA isolated from the heart elicits a robust cytokine response in both cardiomyocytes (CMs) and immune cells, but the type of RNA responsible for the inflammatory effect is unknown. We hypothesize that extracellular microRNAs (miRs) mediate the inflammatory effects. Methods: I/R model: mice were subjected to sham procedure or coronary occlusion for 45 min followed by reperfusion. miR array : 68 miRs in the plasma were quantified. Cytokine detection : CMs and macrophages (Mф) were treated with synthetic miRs for 18 h. Cytokines in media were measured by ELISA. miR uptake : Uptake of fluorescent-labeled miR in Mфs was detected by fluorescent microscopy and flow cytometry. miR inhibition : Locked nucleic acid-based inhibitors complementary to 6 target miRs were mixed as anti-miRs-combo. Results: Compared to the sham procedure, 31 out of 68 miRs were significantly increased by > 2-fold at 4 h following I/R. To test whether miR induces inflammatory response, we treated CMs and Mфs with selected 8 miR mimics (0.5 - 1500 nM) and found that 6 of them (miR-34a, -122, -133a, 142a, -146a, -208a) induced cytokine response in a dose-dependent manner. The effects were abolished by pre-treatment of RNase (but not DNase) and by mutations (U→A). Similar to cardiac total RNA, miR-induced cytokines was completely diminished in TLR7 -/- or MyD88 -/- , but not in TLR3 -/- or Trif -/- Mфs, and significantly inhibited by a specific TLR7 inhibitor in CMs. After incubation with fluorescent miR-133a, TLR7 -/- Mфs had similar cellular fluorescence as WT Mфs. Importantly, anti-miRs-combo significantly decreased cardiac RNA-induced cytokine production in both Mфs and CMs. Conclusions: Our data demonstrate that 1) Multiple cellular miRs are released to circulation during I/R; 2) Six miR mimics induce cytokine production specifically via TLR7-MyD88 signaling. 3) Endogenous miRs mediate in part cardiac RNA-induced cytokine response. These data suggest that cellular miRs are potent proinflammatory ligands that act through TLR7-MyD88 signaling.