Person:

Douda, David N.

Rationale: Infection of the lung with Pseudomonas aeruginosa results in upregulation of nitric oxide synthases (NOS) and arginase expression, and both enzymes compete for L-arginine as substrate. Nitric oxide (NO) production may be regulated by arginase as it controls L-arginine availability for NOS. We here studied the effect of systemic arginase inhibition on pulmonary L-arginine metabolism in Pseudomonas pneumonia in the mouse. Methods: Mice (C57BL/6, 8–10 weeks old, female) underwent direct tracheal instillation of Pseudomonas (PAO-1)-coated agar beads and were treated by repeated intra-peritoneal injections of the arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH) or PBS until lungs were harvested on day 3 of the infection. L-arginine metabolites were quantified using liquid chromatography-tandem mass spectrometry, NO metabolites nitrate and nitrite by Griess reagent and cytokines by ELISA. Results: NO metabolite concentrations (48.5±2.9 vs. 10.9±2.3 µM, p<0.0001), as well as L-ornithine (29.6±1.7 vs 2.3±0.4 µM, p<0.0001), the product of arginase activity, were increased in Pseudomonas infected lungs compared to naïve controls. Concentrations of the NOS inhibitor asymmetric dimethylarginine (ADMA) were also increased (0.44±0.02 vs. 0.16±0.01 µM, p<0.0001). Arginase inhibition in the infected animals resulted in a significant decrease in L-ornithine (14.6±1.6 µM, p<0.0001) but increase in L-arginine concentration (p<0.001), L-arginine/ADMA ratio (p<0.001), L-arginine availability for NOS (p<0.001), and NO metabolite concentrations (67.3±5.7 µM, p<0.05). Arginase inhibitor treatment also resulted in an increase in NO metabolite levels in animals following intratracheal injection of LPS (p = 0.015). Arginase inhibition was not associated with an increase in inflammatory markers (IFN-γ, IL-1β, IL-6, MIP-2, KC or TNF-α) in lung. Concentrations of the L-ornithine-dependent polyamines putrescine, spermidine and spermine were increased in Pseudomonas infected lungs (p<0.001, respectively) but were unaffected by ABH treatment. Conclusions: Systemic arginase inhibition with ABH during Pseudomonas pneumonia in mice results in an increase in pulmonary NO formation but no pro-inflammatory effect.

Pulmonary exacerbations in cystic fibrosis airways are accompanied by inflammation, neutrophilia, and mucous thickening. Cystic fibrosis sputum contains a large amount of uncleared DNA contributed by neutrophil extracellular trap (NET) formation from neutrophils. The exact mechanisms of the induction of NETosis in cystic fibrosis airways remain unclear, especially in uninfected lungs of patients with early cystic fibrosis lung disease. Here we show that Hepoxilin A3, a proinflammatory eicosanoid, and the synthetic analog of Hepoxilin B3, PBT-3, directly induce NETosis in human neutrophils. Furthermore, we show that Hepoxilin A3-mediated NETosis is NADPH-oxidase-dependent at lower doses of Hepoxilin A3, while it is NADPH-oxidase-independent at higher doses. Together, these results demonstrate that Hepoxilin A3 is a previously unrecognized inducer of NETosis in cystic fibrosis lungs and may represent a new therapeutic target for treating cystic fibrosis and other inflammatory lung diseases.