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

在之前的资助期间，我们证明了CF支气管上皮细胞中的神经酰胺介导了β1-整合素在CF气管和支气管上皮细胞的管腔质膜上的异位表达、捕获和聚集。异位β1整合素触发β8的上调，进而介导酸性神经酰胺酶的下调，从而通过神经酰胺介导的IRF1整合素在上皮细胞管腔膜上聚集的恶性循环促进神经酰胺的进一步积累。酸性神经酰胺酶的下调导致CF细胞中的鞘氨醇水平降低，从而导致对急性铜绿假单胞菌感染的高易感性。鞘氨醇的重建、神经酰胺水平的正常化以及异位β1-整合素在CF肺中的表达纠正使CF小鼠的感染易感性正常化。我们将这些研究扩展到病毒感染，并证明酸性鞘磷脂酶/神经酰胺/酸性神经酰胺酶系统在几种病毒感染中也起着重要作用。病毒感染通常通过未知的分子机制使呼吸道对随后的细菌感染敏感，或导致囊性纤维化的严重恶化。初步数据表明，感染鼻病毒会导致CF小鼠体内已经还原的酸性神经酰胺酶显著下调，而感染只会适度下调野生型小鼠的酸性神经酰胺酶。酸性神经酰胺酶的下调通过减少鞘氨醇的形成而进一步增加了CF小鼠对铜绿假单胞菌感染的易感性，这一点可通过肺部应用酸性神经酰胺酶来防止。本研究旨在探讨酸性神经酰胺酶在慢性阻塞性肺疾病肺部病毒和细菌感染中的作用。我们的目标是提供一种分子机制，使CF患者对病毒感染所致的细菌感染高度敏感。根据我们以前的数据，我们现在的目标是：1.确定鼻病毒肺部感染诱导酸性神经酰胺酶下调的分子机制；2.确定鼻病毒感染下调酸性神经酰胺酶是否使CF小鼠和人支气管细胞对铜绿假单胞菌和金黄色葡萄球菌感染敏感，以及CF细胞中酸性神经酰胺酶的重建是否防止鼻病毒感染的CF小鼠肺部感染时细菌重叠感染；3.表征鞘氨醇和神经酰胺在原发鼻病毒感染后CF肺细菌感染中的调节和作用。

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.