Role of the acid ceramidase and sphingosine for the interplay of viral and bacterial pulmonary infections

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In the previous grant period we demonstrated that ceramide in CF bronchial epithelial cells mediates an ectopic expression, trapping and clustering of β1-integrin in the luminal plasma membrane of CF tracheal and bronchial epithelial cells. Ectopic β1-integrin triggers up-regulation of IRF8, which in turn mediates a down-regulation of acid ceramidase thereby promoting further ceramide accumulation via a vicious cycle of ceramide-mediated clustering of β1-integrin in the luminal membrane of epithelial cells. The down-regulation of acid ceramidase results in reduced sphingosine levels in CF cells, which results in the high susceptibility to acute P. aeruginosa infection. Reconstitution of sphingosine, normalization of ceramide levels and correction of the ectopic β1-integrin expression in CF lungs normalizes the infection susceptibility of CF mice. We extended these studies to viral infections and demonstrated that the acid sphingomyelinase/ ceramide/acid ceramidase system also plays an important role in several viral infections.Viral infections often sensitize, via unknown molecular mechanisms, the respiratory tract to subsequent bacterial infections or induce severe exacerbations in cystic fibrosis. Preliminary data indicate that infections with rhinovirus induce a dramatic down-regulation of the already reduced acid ceramidase in CF mice, while infection only moderately down-regulates the enzyme in wildtype mice. Down-regulation of the acid ceramidase resulted in a further increase of susceptibility of CF mice to P. aeruginosa infections due to reduced sphingosine formation, which was prevented by pulmonary application of acid ceramidase. The present proposal aims to characterize the acid ceramidase as master player in viral and bacterial infections in CF lungs. We aim to provide a molecular mechanism for the hyper-susceptibility of CF patients to bacterial infections as consequence of viral infections. Based on our previous data, we now aim to 1. identify molecular mechanisms how pulmonary infections with rhinovirus induce a down-regulation of the acid ceramidase,2. define whether down-regulation of the acid ceramidase by rhinoviral infections sensitizes CF mice and human bronchial cells to P. aeruginosa and S. aureus infections and whether reconstitution of acid ceramidase in CF cells prevents bacterial super-infections upon pulmonary infection of CF mice with rhinovirus, 3. characterize the regulation and function of sphingosine and ceramide for bacterial infections of CF lungs upon a primary rhinovirus infection.