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P. aeruginosa induced TNF-[Alpha] production in macrophages

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Title: P. aeruginosa induced TNF-[Alpha] production in macrophages
Author: Miao, Li
Degree: Master of Science - MSc
Program: Microbiology and Immunology
Copyright Date: 1996
Issue Date: 2009-02-14
Series/Report no. UBC Retrospective Theses Digitization Project [http://www.library.ubc.ca/archives/retro_theses/]
Abstract: Pseudomonas aeruginosa has shown itself to be particularly adept at colonizing CF lungs and intractable to treatment even with the most aggressive therapy. Failure to control colonization with P. aeruginosa is a major cause of pulmonary debilitation that ultimately leads to almost all of the deaths in CF patients. During bacterial infections, macrophages produce many proinflammatory cytokines, among which TNF-a is believed to be the principal cytokine mediating many of the catastrophic host responses to bacterial infections. Little is known about the virulence factor(s) of P. aeruginosa in the pathogenesis of CF lung infections in terms of TNF-a production. The aim of this study was to determine the virulence factor(s) of P. aeruginosa with regard to TNF-a production. The kinetics of TNF-a production by live P. aeruginosa coincubated with RAW 264.7 macrophages was studied. RAW 264.7 macrophages were challenged with P. aeruginosa at concentrations of 104,106 and 108 CFU/ ml respectively. The production of TNF-a by macrophages increased in a time-dependent fashion with peak levels occurring at about 8 hours, after which the amount of TNF-a decreased, possibly related to TNF-a degradation by proteases from macrophages as well as P. aeruginosa itself. However, the data suggested a possible inverse relationship between the peak values of TNF-a produced and the number of bacteria added into macrophages, in that macrophages challenged by lower numbers of P. aeruginosa produced higher levels of TNF-a, accompanied by a slower rate of decline in TNF-a content. To understand what was occurring with both the P. aeruginosa and the macrophages during the production of TNF-a, the growth of P. aeruginosa and the viability of macrophages during their coincubation were studied in a time course. Depending on the starting bacterial number, the stationary phase of bacterial growth occurred between 8 and 12 hours. Eight hours after challenge with P. aeruginosa, macrophage viability started to decrease. The decrease of macrophage viability suggested P. aeruginosa had cytotoxic effect on macrophages. TNF-a production appeared to be related to both the growth stage of the P. aeruginosa culture and the presence of functioning macrophages. TNF-a production increased during the log growth phase of P. aeruginosa, after which the presence of TNF-a in the supernatants as well as macrophage viability decreased. These findings suggested the growth of P. aeruginosa and the viability of macrophages were important factors in P. aeruginosa induced TNF-a production. Macrophage-bacteria association is the initial step of macrophage phagocytosis. The relationship between the macrophage-bacteria association and TNF-a production by macrophages is not known. The effect of macrophage-P. aeruginosa association on TNF-a production was assessed by increasing their physical contact. The results showed that direct association of RAW 264.7 macrophages with P. aeruginosa significantly reduced TNF-a production, indicating the association of macrophages with P. aeruginosa may down-regulate the function of macrophage TNF-a production. This hypothesis was supported by further experiments which used transwell filter inserts. Transwell filter inserts keep the bacteria from direct contact with macrophages while allowing factors released from the bacteria to come into contact with the macrophages. These experiments demonstrated that the production of TNF-a was higher when P. aeruginosa were incubated in transwell filter inserts than when incubated directly in association with macrophages. These findings suggested that factors released from P. aeruginosa might play a major role in TNF-a production whereas the direct interaction of bacteria with macrophages may suppress TNF-a production. Presuming that released LPS might be the major virulence factor in the production of TNF-a by P. aeruginosa, the inhibition of P. aeruginosa induced TNFa production with different LPS antagonists was investigated. Eight P. aeruginosa LPS specific antibodies were acquired, and none of them were able to block TNF-a production by P. aeruginosa LPS even though they could bind specific epitopes of P. aeruginosa LPS by Western irnmunoblotting and ELISA. Polymyxin B (PMB) was shown to be cytotoxic to RAW 264.7 macrophages in this study and, therefore, is not a suitable antagonist for LPS-induced TNF-a production. However, Rhodopseudomonas sphaeroides diphosphoryl lipid A (RsDPLA) could inhibit P. aeruginosa induced TNF-a production by 70-75%. CEME, an cecropm-melittin hybrid, inhibited more than 90% of P. aeruginosa induced TNF-a production. In addition, inactivation of the released products of P. aeruginosa by heat-treatment resulted in 20% reduction of TNF-a production, indicating that heat stable products such as LPS must be responsible for 80% of TNF-a production by P. aeruginosa. Taken together the results suggest that LPS is the major virulence factor in the production of TNF-a by P. aeruginosa. The reasons behind the predilection and chronic persistence of mucoid P. aeruginosa in respiratory tract of CF patients are not completely understood. The ability of mucoid P. aeruginosa to induce TNF-a in murine alveolar macrophage was, therefore, compared with their nonmucoid counterparts. All the mucoid strains studied induced less TNF-a than their nonmucoid counterparts. The reduced TNF-a production seen with the mucoid form of P. aeruginosa may be attributed to the partial obstruction of LPS release by copious alginate coating around bacteria. This is consistent with the clinical findings that mucoid strains of P. aeruginosa are more suited to chronic rather than to acute respiratory infection in that reduced TNF-a as well as other virulence factors could temper the severity of infections.
Affiliation: Science, Faculty of
URI: http://hdl.handle.net/2429/4596
Scholarly Level: Graduate

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