| Clostridium difficile (C.diff) is a toxin-secreting bacterium and leading cause of infectious colitis in humans. C.diff toxins (TcdA and TcdB) rapidly induce intestinal injury and inflammation via disruption of the intestinal epithelial barrier and induction of proinflammatory mediators. The hypoxia-inducible factor-1 (HIF-1alpha) is a stress-induced transcription factor that impacts the expression of mucosal and barrier protective genes. Initial studies have demonstrated that C.diff toxins alter the expression and activity of the HIF-1alpha. Nitric oxide (NO), reactive oxygen species (ROS), and the factor-inhibiting HIF-1alpha (FIH-1) are well-known modulators of HIF-1alpha stabilization and signaling. Therefore, we hypothesize that NO and ROS produced during C.diff toxin-induced intestinal injury act as an important signal to influence HIF-1alpha and subsequent protection against intestinal damage. In the context of C.diff toxin exposure, it was determined both in vitro and in vivo that stabilization occurred via nitrosylation of HIF-1alpha by NO. Inhibition of iNOS activity by selective inhibitors or genetic iNOS-deficiency in mice attenuated HIF-1alpha stabilization, which identifies iNOS as an essential source of NO in this context. Studies performed with N-acetylcysteine (NAC, a well-known ROS scavenger) in vitro have demonstrated a potential for ROS involvement in the stabilization of HIF-1alpha. Pre-treatment of cells with NAC followed by C.diff toxin exposure attenuated HIF-1alpha expression despite the fact that nitrosylation still occurred. This study is the first to propose a synergistic action between NO and ROS to stabilize HIF-1alpha in the context of C.diff toxin-mediated epithelial damage. |
