Diss. Linköping : Univ., 2003.

Indiana University-Purdue University Indianapolis (IUPUI) / The patients with Cystic fibrosis (CF), an inherent genetic disorder, suffer from chronic bacterial infection in the lung. In CF, modification of epithelial cells leads to alteration of the lung environment, such as inhibition of ciliary bacterial clearance and accumulation of thickened mucus in the airways. Exploiting these conditions, opportunistic pathogens like Pseudomonas aeruginosa cause lifelong persistent infection in the CF lung by forming into antibiotic-resistant aggregated communities called biofilms. Airway infections as well as inflammation are the two major presentations of CF lung disease. P. aeruginosa strains isolated from CF lungs often contain mutations in the mucA gene, and this mutation results in higher level expression of bacterial polysaccharides and toxic lipoproteins. In a previous work, we have found a putative lipoprotein gene (PA4326) which is overexpressed in antibiotic-induced biofilm formed on cultured CF-derived airway cells. In the current work, we speculated that this particular putative lipoprotein affects cellular cytotoxicity and immune-stimulation in the epithelial cells. We found that mutation of this gene (ΔPA4326) results in reduced airway cell killing without affecting other common virulence factors.Moreover, we observed that this gene was able to stimulate secretion of the proinflammatory cytokine IL-8 from host cells. Interestingly, we also found that ΔPA4326 mutant strains produced less pyocyanin exotoxin compared to the wild type. Furthermore, our results suggest that PA4326 regulates expression of the pyocyanin biosynthesis gene phzM, leading to the reduced pyocyanin phenotype. Overall, these findings implicate PA4326 as a virulence factor in Pseudomonas aeruginosa. In the future, understating the molecular interplay between the epithelial cells and putative lipoproteins like PA4326 may lead to development of novel anti-inflammatory therapies that would lessen the suffering of CF patients.

Indiana University-Purdue University Indianapolis (IUPUI) / More than 300 million people world-wide suffer from breathlessness, wheezing, chest tightness, and coughing characteristic of chronic bronchial asthma, the global incidence of which is on the rise. Allergen-sensitization and challenge elicits pulmonary expression of chemoattractants that promote a chronic eosinophil-rich infiltrate. Eosinophils are increasingly recognized as important myeloid effectors in chronic inflammation characteristic of asthma, although few eosinophil molecular signaling pathways have successfully been targeted in asthma therapy. p21 activated kinases (PAKs), members of the Ste-20 family of serine/threonine kinases, act as molecular switches in cytoskeletal-dependent processes involved in cellular motility. We hypothesized that PAK1 modulated eosinophil infiltration in an allergic airway disease (AAD) murine model. In this model, Pak1 deficient mice developed reduced inflammatory AAD responses in vivo with notable decreases in eosinophil infiltration in the lungs and broncho-alveolar lavage fluids (BALF). To test the importance of PAK1 in hematopoietic cells in AAD we used complementary bone marrow transplant experiments that demonstrated decreased eosinophil inflammation in hosts transplanted with Pak1 deficient bone marrow. In in vitro studies, we show that eotaxin-signaling through PAK1 facilitated eotaxin-mediated eosinophil migration. Ablating PAK1 expression by genetic deletion in hematopoietic progenitors or siRNA treatment in derived human eosinophils impaired eotaxin-mediated eosinophil migration, while ectopic PAK1 expression promoted this migration. Together these data suggest a key role for PAK1 in the development of atopic eosinophil inflammation and eotaxin-mediated eosinophil migration.