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

Abstract

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.

CD8+IL-17+T Cells Mediate Neutrophilic Airway Obliteration in T-bet–Deficient Mouse Lung Allograft Recipients

“…Our studies demonstrate an IL-17-dependent mechanism for acute lung rejection pathology and obliterative airway inflammation in anti-CD154-treated T-bet 2/2 recipients. (24). We also treated mouse epithelial cells with murine IL-17 and found increased CXCL1 in the media at 2 hours.…”

NOX2 Activation of Natural Killer T Cells Is Blocked by the Adenosine A2A Receptor to Inhibit Lung Ischemia–Reperfusion Injury

“…Moreover, lung dysfunction and production of proinflammatory cytokines (including IL-17) after IR were restored in Ja18 iNKT cells ( Figures 3B and 3C). Similar to dysfunction, pulmonary edema, neutrophil infiltration, and MPO levels after IR were attenuated by ATL313 treatment of WT mice and Ja18 2/2 mice reconstituted with WT, but not A 2A R To investigate the role of NADPH oxidase activation specifically in iNKT cells, primary murine iNKT cells were exposed to acute HR as a surrogate in vitro model of lung IR, which we previously showed induced IL-17 production by iNKT cells (31). ROS generation, as assessed using dichlorofluorescein dye, was markedly elevated in WT iNKT cells after HR (3 h hypoxia plus 1 h normoxia) compared with normoxia alone, which was significantly attenuated by ATL313 treatment ( Figure 5A).…”

“…In addition, preliminary data and unpublished results suggest that pannexin-1 channel-mediated ATP release by endothelial cells may mediate iNKT cell transmigration and activation after IR (48). Previous studies suggest that NADPH oxidase is critically involved in the redox regulation of pulmonary injury and inflammation after IR (31,(49)(50)(51)(52). A variety of stimuli, including IR, can lead to superoxide production by NADPH oxidase, especially in phagocytes in which a strong response is achieved through ligands that activate Gq-type G protein-coupled receptors (53,54).…”

Models of Lung Transplant Research: a consensus statement from the National Heart, Lung, and Blood Institute workshop

“…neutrophil/mononuclear phagocyte activation and infiltration) (30), and proinflammatory cytokines and chemokines (7,8,15,20,24), all exemplifying multiple complex inflammatory pathways involved in PGD.…”