NADPH oxidase mediates synergistic effects of IL-17 and TNF-α on CXCL1 expression by epithelial cells after lung ischemia-reperfusion.

Ischemia-reperfusion (I/R) injury leads to increased mortality and morbidity in lung transplant patients. Lung I/R injury involves inflammation contributed by innate immune responses. IL-17 and TNF-α, from iNKT cells and alveolar macrophages, respectively, contribute importantly to lung I/R injury. This study tests the hypothesis that IL-17 and TNF-α synergistically mediate CXCL1 (a potent neutrophil chemokine) production by alveolar type II epithelial (ATII) cells via an NADPH oxidase-dependent mechanism during lung I/R. Using a hilar clamp model, wild-type and p47(phox-/-) (NADPH oxidase-deficient) mice underwent left lung I/R, with or without recombinant IL-17 and/or TNF-α treatment. Wild-type mice undergoing I/R treated with combined IL-17 and TNF-α had significantly enhanced lung dysfunction, edema, CXCL1 production, and neutrophil infiltration compared with treatment with IL-17 or TNF-α alone. However, p47(phox-/-) mice had significantly less pulmonary dysfunction, CXCL1 production, and lung injury after I/R that was not enhanced by combined IL-17-TNF-α treatment. Moreover, in an acute in vitro hypoxia-reoxygenation model, murine ATII cells showed a multifold synergistic increase in CXCL1 expression after combined IL-17-TNF-α treatment compared with treatment with either cytokine alone, which was significantly attenuated by an NADPH oxidase inhibitor. Conditioned media transfer from hypoxia-reoxygenation-exposed iNKT cells and macrophages, major sources of IL-17 and TNF-α, respectively, to ATII cells significantly enhanced CXCL1 production, which was blocked by NADPH oxidase inhibitor. These results demonstrate that IL-17 and TNF-α synergistically mediate CXCL1 production by ATII cells after I/R, via an NADPH oxidase-dependent mechanism, to induce neutrophil infiltration and lung I/R injury.