Genetic ablation of iNOS modulates pro-inflammatory cytokine levels after trauma and confers a survival advantage following infectious challenge

Abstract

Introduction: Significant immune changes occur after trauma. Prostaglandin-E2 (PGE2) and nitric oxide (NO ) are key inflammatory mediators involved in trauma-related immune dysfunction. The relative importance of the early response genes nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) after trauma is controversial. This study investigated macrophage cytokine production after trauma and followed survival from subsequent sepsis in COX-2 and iNOS knockout (−/−) and wild-type mice. Methods: Female C57/Bl6 COX-2 and iNOS KO and WT mice were randomized to control or trauma (femur fracture plus 40% blood volume hemorrhage) groups. Expt. 1. Seven days after injury, splenic macrophages were isolated, stimulated with LPS (10ng/mL) for 24 hours and pro- (IL-6 and TNFa) and anti-inflammatory (IL-10 and IL-12) cytokine levels measured by cytometric bead array. Expt. 2. At 7 days after trauma the mice were subjected to cecal ligation and puncture and followed for survival. Results: Macrophages produced significantly decreased TNFa levels in iNOS (−/−) mice compared with WT mice after trauma. iNOS (−/−) mice has a significant survival advantage compared with WT mice following sepsis (10/13 vs 4/12 p < 0.005). IL-10 levels were significantly elevated in COX-2 (−/−) but IL-6, IL-10 and IL-12 levels were unchanged compared with WT mice. Survival was similar between the COX-2 (−/−) and WT mice following septic challenge. Conclusions: These data suggest that inhibition of iNOS is more important than COX-2 in protecting the host from sepsis after trauma. Significantly decreased TNFa production in iNOS (−/−) mice correlated with improved outcome suggesting that NO regulates TNFa production in trauma.