Cellular and pathogen response to infection of bronchial epithelial cells with pseudomonas aeruginosa and respiratory syncytial virus

Abstract

Cystic fibrosis (CF) is characterised by neutrophilic inflammation of the airways. Whether this inflammatory state is a primary defect due to the cystic fibrosis transmembrane conductance regulator (CFTR) channel dysfunction is unclear, but it is clear that infection with pathogenic bacteria such as Pseudomonas aeruginosa (Pa) perpetuates the cycle of inflammation, resulting in rapid loss of lung function. Anti-inflammatory treatments are being developed for CF although none are yet in widespread clinical use and concerns that highly effective therapies could be detrimental mean that further understanding of inflammatory pathways is required. Alongside bacteria, respiratory viruses are also implicated in up to 60% of exacerbations in CF patients, and of note, respiratory syncytial virus (RSV) has been observed to be followed by bacterial respiratory infections. In vitro data supports the notion that RSV infection is implicated in the acquisition and persistence of Pa in the CF airway, although the mechanism underlying this is incompletely understood. In this thesis I aimed to investigate the molecular mechanism resulting in Pa-induced cytokine and chemokine release from bronchial epithelial cells, the kinetics and mechanism of RSV infection in CFTR deficient CF bronchial epithelial cells compared with wild type (WT), and the effect of RSV infection on Pa attachment to these cells. The use of single kinase inhibitors revealed significant inhibition of Pa induced CXCL8 and IL-6 from the bronchial epithelial cell line, BEAS-2B cells, by a p38 MAPK inhibitor at 0.001 and 0.0001 μg/ml, and a Syk inhibitor at 1 and 0.1 μg/ml, respectively. A Src Kinase inhibitor also showed a trend towards inhibition of Pa induced CXCL8, 83.7% at 1 μg/ml, but not IL-6. These results were confirmed by narrow spectrum kinase inhibitors targeting p38 MAPK, Src and Syk, indicating a significant contribution to Pa induced inflammatory response by p38 MAPK and a partial contribution by Syk and Src kinases. RSV infection of paired bronchial epithelial CFBE41o- cells, expressing either WT or Phe508del CFTR, revealed significantly greater cell death of the latter by day three at a multiplicity of infection of 0.01, which was associated with significantly higher viral burden, 0.663 vs 2.59 log10 PFU/ml, by day one post infection. This was found not to be due to greater viral attachment to the Phe508del cells, but resulted from greater viral replication and/or transcription, as shown by the 2.9 fold higher intracellular RSV levels in the Phe508del cells by eight hours post infection. Baseline levels of the antiviral protein Myxoma resistance protein 1 (Mx1) were significantly lower in the Phe508del cells compared with WT and there was a trend towards lower levels of 2'-5'-oligoadenylate synthetase 1 and 3 (OAS1 and 3). The use of small interfering RNA to knockdown Mx1 expression did not confirm its role so far. No difference in viral kinetics was seen between primary cells from CF and healthy donors used in this project in either a monolayer or air liquid interface (ALI) culture. The greater RSV burden in the Phe508del CFBE41o- cells resulted in significantly higher Pa attachment compared with infected WT cells and uninfected Phe508del cells, 27.8% vs 1.91% and 0.319%, respectively. The attachment was via cell surface expression of the RSV surface glycoprotein (G), which was approximately 4 fold higher on the Phe508del cells. RSV-enhanced Pa attachment to epithelial cells was inhibited by heparin, 82.0%, but not by a specific G-protein antibody, 13.9%, suggesting that the heparin binding domain, not the CX3C motif, is the attachment mechanism. However, the use of heparin does not conclusively prove the role of the RSV G-protein as it may also inhibit Pa attachment to epithelial surface proteins. Further work is therefore required. Thus, greater replication and/or transcription of RSV in a Phe508del epithelial cell line resulted in higher viral burden and cell death, and also greater attachment of Pa compared with WT cells. Pa-induced inflammation was demonstrated to be highly p38 MAPK dependent, and partially dependent on Src and Syk kinases. These results shed further light on the pathways of bacteria simulated inflammation in epithelial cells and potential targets for novel therapies, as well as the mechanism linking RSV and Pa infection.