Nox1-Derived ROS Amplifies Calcium Entry and Enhances Pneumolysin-Induced Lung Endothelial Barrier Dysfunction in Hyperglycemia

<b>Background:</b><i>Streptococcus pneumonia</i> is the primary etiological agent of community-acquired pneumonia (CAP). Pneumococci promote severe lung injury through the release of virulence factors, including pneumolysin (PLY). Obesity/diabetes increases pneumonia-associated mortality, but the mechanisms remain elusive. We found that obese db/db mice have increased pulmonary barrier disruption to PLY. Previously we showed that upregulation of NOX1 in endothelial cells (EC) of db/db mice drives endothelial dysfunction, but a role for NOX1 in PLY-induced lung injury, especially in diabetic conditions, has not yet been described. <b>Results:</b> Increased NOX1 in lung ECs dose-dependently increased superoxide and EC barrier disruption (<i>p</i> < 0.05). Even at low activity levels, NOX1 greatly potentiated PLY-induced EC barrier disruption, whereas loss of NOX1 activity, either pharmacological or genetic, reduced barrier disruption (<i>p</i> < 0.05). Blockade of calcium entry protected the EC barrier from combined PLY and NOX1, indicating a key role for calcium. Hyperglycemia amplified PLY-enduced EC barrier disruption and intracellular calcium and these effects were mitigated by NOX1 inhibition and silencing (<i>p</i> < 0.05). NOX1-enhanced calcium entry was reduced by knockout of calcium sensor STIM1, and PLY-induced barrier disruption was reduced by STIM1 inhibition. Levels of STIM1, Orai1, TRPV4, or TRPC4 were unchanged by HG, but TRPC1 significantly increased (<i>p</i> < 0.05). NOX1 and HG promoted increased STIM1 and TRPC1 binding, and silencing TRPC1 ameliorated PLY-induced barrier disruption (<i>p</i> < 0.05). Increased calcium promoted mitochondrial permeability transition pore (MPTP) opening and PPIF inhibition protected EC barrier function (<i>p</i> < 0.05). <b>Conclusions:</b> These results suggest that elevated glucose levels in obesity primes EC barrier disruption by amplifying PLY-induced calcium influx via a novel NOX1, STIM1, TRPC1 and MPTP signaling axis.