Contributions Of Innate Immune System In Pseudomonas Aeruginosa Pulmonary Infection

Backgroud and Obiective:Pseudomonas aeruginosa pulmonary infection is one of the leading causes ofmortality in nosocomial infections. As an opportunistic pathogen, P. aeruginosa hasmultiple bacterial virulence factors and genetic flexibility enabling it to survive invaried environments. The interaction of bacteria and host immunity determined thepathogenesis and outcome of infection. Innate immune system in pulmonary plays animportant role in P. aeruginosa infection. Therefore, in this study we will investigatethe expressions and effects of innate immune system (TLRs and NOD2) whenpulmonary infections were caused by planktonic and biofilm strains of ATCC 27853,respectively. The results may provide helps to improve the levels of clinical diagnosisand treatment.Methods:1. Routinely grown in Luria-Bertani (LB) broth culture method and themodified plate culture method were used to establish the planktonic strain and biofilmstrain of P. aeruginosa ATCC 27853 respectively. To challenge the airway epithelialcell lines A549 and HBE with planktonic and biofilm strains of ATCC 27853.Semi-quantitative RT-PCR and western blot were used to determine the expressionlevels of TLR2, TLR4, TLR5 and NOD2 in respiratory epithelial cells. To measuredthe levels of IL-6, IL-8 in supernatant by ELISA. The levels of TRAF6 and NF- K Bprotein expression were determined by western blot.2. Rat models of pulmonary infection were established by intratrachealchallenge with planktonic and biofilm strains of ATCC 27853. Sixty-six SD rats wererandomly divided into normal control groups, planktonic strain infection groups andbiofilm strain infection groups. 4 hours, 1 and 3 days after challenging in groupsplanktonic strain infection, 4 hours, 7 andl4days after challenging in groups biofilmstrain infection, lung tissue were collected. Pathological features of lung tissue wereobserved under optical microscope. Semi-quantitative RT-PCR and western blot wereused to determine the expression levels of TLR2, TLR4, TLR5 and NOD2 in lung tissue.3. Extract the secreted proteins in the supernatant after 4hours challenged withplanktonic strain; immobilized pH gradient 2-DE was applied to separate the totalproteins. The selected differential protein spot was identified by peptide massfingerprint (PMF) based on matrix-assisted laser desorption/ionization-time of flightmass spectrometry (MALDI-OF-MS) and database searching was used to identifyprotein.Results:1. In vitro, the expressions of TLR2 had no significant difference in the groups ofplanktonic and biofilm strains infectious models (P＞0.05); the expressions of TLR4were up-regulated in both planktonic and biofilm strains infectious models 4h groups(P＜0.05); the levels of TLR5mRNA in planktonic model 15minutes group wereobserved to up-regulated significantly, and the expressions went to the highest in 30minutes group then declined gradually (P＜0.05); the expressions of TRAF6 and NF-κB were observed the same tendency as that of TLR5(P＜0.05); the levels of IL-6and IL-8 were up-regulated with the extension of challenge time(P＜0.05).2. In rat pulmonary infection models, the pathomorphological changes ofplanktonic strain infection groups were different significantly from biofilm straininfection groups. In the groups of planktonic strain infectious models, the expressionsof TLR2 had no significant difference in the three groups (P＜0.05); the expressions ofTLR4 increased significantly in day 3 group (P＜0.05); the expressions of TLR5increased significantly in 4h group then declined gradually (P＜0.05); the expressionsof NOD2 increased significantly in 4h group then came to normal levels (P＜0.05). Inthe groups of biofilm strain infection models, the expressions of TLR2, TLR5, NOD2increased significantly gradually(P＜0.05); the expressions of TLR4 increasedsignificantly in day 14 group(P＜0.05).3. A good 2-DE pattern with high resolution and reproducibility was obtained.Database searching was used to identify protein. The protein was named Notchlessprotein homolog 1, while the function of it was still not illuminated.Conclusion:1. The same isolate in planktonic and biofilm state respectively lead to infectionswith different pathogenesis and prognosis.2. Flagellin of Pseudomonas aeruginosa contributes to the inflammatory responses of respiratory epithelium in a TLR5/TRAF6/NF-κB signalingpathway-dependent manner in the early stage of infection. And TLR2, TLR4 are notinvolved in hypersusceptibility to acute Pseudomonas aeruginosa lung infections.3. In chronic Pseudomonas aeruginosa pulmonary infections the innate immunesystem was activated dramatically.