Inhibition of p-IκBα Ubiquitylation by Autophagy-Related Gene 7 to Regulate Inflammatory Responses to Bacterial Infection

Background. Klebsiella pneumoniae causes serious infections and healthcare burdens in humans. We have previously reported that the deficiency of autophagy-related gene (Atg) 7 in macrophages (murine alveolar macrophage cell line [MH-S]) induced irregular host immunity against K. pneumoniae and worsened pathologic effects in the lung. In the current study, we investigated the molecular mechanism by which Atg7 influenced K. pneumoniae-induced inflammatory responses. Methods. Expression levels of Atg7, ubiquitin (Ub), and tumor necrosis factor (TNF) a and phosphorylation of I kappa B alpha (p-I kappa B alpha) were determined with immunoblotting. Ubiquitylation of p-I kappa B alpha was determined with immunoprecipitation. Results. We noted an interaction between Atg7 and p-I kappa B alpha, which was decreased in MH-S after K. pneumoniae infection, whereas the interaction between Ub and p-I.Ba was increased. Knock-down of Atg7 with small interfering RNA increased p-I.Ba ubiquitylation, promoted nuclear factor kappa B translocation into the nucleus, and increased the production of TNF-alpha. Moreover, knock-down of Ub with lentivirus-short hairpin RNA Ub particles decreased binding of p-I kappa B alpha to Ub and inhibited TNF-a expression in the primary alveolar macrophages and lung tissue of atg7-knockout mice on K. pneumoniae infection. Conclusions. Loss of Atg7 switched binding of p-I.Ba from Atg7 to Ub, resulting in increased ubiquitylation of p-I.Ba and intensified inflammatory responses against K. pneumoniae. Our findings not only reveal a regulatory role of Atg7 in ubiquitylation of p-I.Ba but also indicate potential therapeutic targets for K. pneumoniae control.