Mitochondrial DNA Activates NLRP3 Inflammasome and Contributes to Endothelial Dysfunction and Inflammation in Type 1 Diabetic Mice

Abstract

IntroductionNLRP3 inflammasome is a molecular platform of innate immune system that regulates the inflammatory response through the activation of caspase‐1 and IL‐1β. It can be activated by several ligands, for example, mitochondrial DNA (mDNA). Some studies have shown that circulating mDNA is increased in patients with diabetes and play a role in the development of the disease; however, its role in vascular changes is still unknown. In this sense, we tested the hypothesis that mDNA contributes to vascular inflammatory/oxidative processes associated to type 1 diabetes (T1D) via NLRP3 inflammasome activation.MethodsWild‐type and NLRP3‐deficient (NLRP3−/−) mice were treated with vehicle (Veh) or streptozotocin (40 mg/kg) (T1D) for 5 days. Vascular reactivity was determined in mesenteric arteries. Caspase‐1 and IL‐1β expression were evaluated by western blot. Pancreatic mDNA was extracted from control (cmDNA) and diabetic animals (dmDNA) for endothelial cells stimulation. ROS generation was determined by chemiluminescence. Hydrogen peroxide (H2O2) production and calcium influx were determined by fluorescence. Data are presented as mean ± standard error of the mean.ResultsDiabetes increased vascular caspase‐1 and IL‐1β activation [arbitrary units (a.u.), 1.2±0.1 vs. 0.8±0.1; 4.8±1.1 vs. 0.8±0.5 vs. the Veh, respectively, p <0.05]. However, this activation was attenuated in T1D NLRP3−/−. Mesenteric arteries from T1D exhibited decreased ACh‐induced vasodilatation vs. Veh [(Emax), 46.6±4.0 vs. 91.5±2.8, n=4–5, p<0.05], which was not observed in T1D NLRP3−/−. The NLRP3 inhibitor MCC950 acutely improved endothelium‐dependent vasodilation in T1D [(Emax), 62.5±3.8 vs. 85.6±3.7, n=4–5, p<0.05]. ROS generation [(Relative luminescence unit, RLU), 1.5×107±2.3 vs. 48.9×103±1.1, n=4–5, p<0.05] as well as H2O2 production [(Relative fluorescence unit, RFU), 1082±242.6 vs. 232.0±42.8, n=4–5, p<0.05] were lower in mesenteric bed from T1D NLRP3−/− than T1D. Only incubation with dmDNA reduced ACh‐induced vasodilation [(Emax), 48.4 ± 4.1 vs. 90.7 ± 3.4, n=4–5, p<0.05], which was attenuated by the presence of an antioxidant [(Emax), 48.4 ± 4.1 vs. 71.5 ± 3.1, n=4–5, p<0.05]. Similarly, only LPS‐primed cells incubated with dmDNA had significant NLRP3 activation (caspase‐1 and IL‐1β activation, respectively) [(u.a.), 1.6 ± 0.2 vs. 0.9 ± 0.1; and 1.6 ± 0.2 vs. 1.0 ± 0.1, n=4–5, p<0.05]. The dmDNA induced in a time‐dependent way both calcium influx and ROS generation in endothelial cells, being the last reversed by the presence of an antioxidant. Likewise, T1D patients had increased circulating mDNA vs. healthy volunteers and also higher NLRP3 serum expression, and caspase‐1/IL‐1β activation.ConclusionThe T1D increases mDNA release which in turn stimulates increased calcium influx, induces ROS generation, and triggers vascular NLRP3 inflammasome activation contributing to inflammatory response and endothelial dysfunction in diabetes.Support or Funding InformationFinancial Support: FAPESP and CAPES. Ethics Committee number (026/2015).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.