Exploration of inflammasomes as targets for therapy of Pseudomonas aeruginosa infection
Doctor of Philosophy
Small molecule inhibitor
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Pseudomonas aeruginosa is a common Gram-negative opportunistic bacterial pathogen capable of infecting humans with compromised natural defenses and causing severe pulmonary disease. It is the major cause of severe chronic pulmonary disease in cystic fibrosis (CF) patients subsequently resulting in progressive deterioration of lung function. Interaction between P. aeruginosa and host induces a number of marked inflammatory responses and is associated with complex therapeutic problems. NOD-like receptors (NLRs) can recognize a variety of endogenous and exogenous ligands and its activation initiate inflammasome formation that induces maturation of the proinflammatory cytokine interleukin (IL)-1β through activation of caspase-1. Through a literature search, no prior research on mutant strains as well as clinical isolates of P. aeruginosa from CF patients at different stages of infection has been conducted to explore NLR-mediated innate immune responses to this bacterial infection. All the work presented in this thesis focuses on the exploration of inflammasomes as targets for therapy of P. aeruginosa infection. We hypothesized that genetic alterations of P. aeruginosa affect the innate immune response of human monocytes. THP-1 human monocytic cells were infected with clinical P. aeruginosa isolates from CF patients, or with P. aeruginosa mutant strains lacking flagella, pili, lipopolysaccharide, or pyocyanin. The overall involvement of NLRs in innate immune recognition of P. aeruginosa was addressed through demonstrating of NLR-mediated caspase-1 activation or P. aeruginosa-induced IL-1β secretion. Our findings suggest that P. aeruginosa, which lost certain virulence factors during pulmonary infection, may fail to induce caspase-1 activation and secretion of IL-1β in the process of host-pathogen interactions. This may reveal novel mechanism of the pathogen adaptation to avoid detection by NLR(s). As P. aeruginosa infections are characterized by strong inflammation of infected tissues anti-inflammatory therapies in combination with antibiotics have been considered for the treatment of associated diseases. Spleen tyrosine kinase (SYK), a non-receptor tyrosine kinase, is an important regulator of inflammatory responses. Several studies have highlighted SYK as a key player in the pathogenesis of a multitude of diseases. Inhibition of SYK activity was explored as a therapeutic option in several inflammatory conditions; however, this has not been studied in bacterial infections. We used a model of an in vitro infection of human monocytic cell line THP-1 and lung epithelial cell line H292 with both wild type and flagella-deficient mutant of P. aeruginosa strain K, as well as with clinical isolates from CF patients, to study the effect of a small molecule SYK inhibitor R406 on inflammatory responses induced by this pathogen. The role of SYK in regulation of inflammasome activation was also determined by evaluating the effect of SYK inhibitor on innate immune responses in P. aeruginosa infected cells. The results suggest that SYK is involved in the regulation of inflammatory responses to P. aeruginosa, and R406 may potentially be useful in dampening the damage caused by severe inflammation associated with this infection.