TNFR1 shedding by mitochondrial RISP in lung microvascular endothelium

Abstract

In sepsis, ligation of the endothelial (EC) TNFα receptor 1 (TNFR1) tumor necrosis factor alpha (TNFα) promotes lung inflammation. However, the role of EC TNFR1 shedding in lung inflammation is unknown. To determine real‐time, in‐situ TNFR1 dynamics and inflammation, we imaged ECs of single microvessels of inflated isolated, blood‐perfused mouse lungs by live confocal microscopy. By micropuncture, we infused microvessels with a fluorophore‐conjugated TNFR1 or E‐selectin mAb followed by buffer wash. At baseline, TNFR1 expression was stable for >1h. Following 10 min infusion of TNFα (1 ng/ml), EC fluorescence decreased by 72±3% (P<0.05 n=3) in 30 min. TNFR1 shedding was blocked in lungs from mice injected with siRNA to mitochondrial complex III Rieske Iron‐Sulphur protein (siRISP). Infusion of TNFα for 2h increased EC E‐selectin expression and leukocyte adhesion two‐fold (P<0.05). By contrast, TNFα infusion in lungs from siRISP‐mice resulted in a three‐fold increase in E‐selectin expression and leukocyte adhesion relative to control (P<0.05). To determine shedding in sepsis, we instilled mice with lipopolysaccharide (LPS 1 mg/kg, 4h). TNFR1 shedding was inhibited >50% (P<0.05). We interpret, TNFR1 ligation promotes RISP‐dependent TNFR1 shedding, possibly limiting inflammation. In sepsis, mitochondrial dysfunction may impair TNFR1 shedding thus exacerbating inflammation (support: HL57556).