| Pneumonia represents a major health burden. Previous work demonstrated that although the induction of inflammation is important for adequate host defence against pneumonia, an inability to regulate the host s inflammatory response within the lung later during infection can be detrimental. Intracellular signalling pathways commonly rely on activation of kinases, and kinases play an essential role in the regulation of the inflammatory response of immune cells. The Key Objectives of my proposal are: (1) To obtain insight in the pulmonary expression and activation of kinases during pneumonia, and (2) To inhibit the expression of selected kinases during pneumonia using locally delivered small interfering (si) RNA. In this project I will make use of established mouse models of Gram-positive (Streptococcus pneumoniae) and Gram-negative (Klebsiella pneumoniae) bacterial pneumonia. For Key Objective 1 I will determine the activity of intracellular kinases in the lungs and cellular subsets before and at various time points after infection using peptide arrays and western blot, flowcytometric analysis and/or immunohistochemistry using phosho-specific antibodies. These data will be used for Key Objective 2: local (intranasal) administration of siRNA, which is an excellent tool to selectively silence target genes in the lungs in vivo, will be used to inhibit the expression of selected kinases identified. The effects of siRNA treatment will be evaluated in different settings, including posttreatment in combination with antibiotics, in order to mimic the clinical scenario. Study-endpoints of the pneumonia models include survival, quantitative cultures of lung tissue and blood, and a detailed analysis of the inflammatory response of the lung during the course of the infection. |
