PcrV Passive And Active Immunization Attenuate Multi-drug Resistant Pseudomonas Aeruginosa Induced Acute Lung Injury

Part1:The Effect of PcrV passive immunization to multi-drug resistant pseudomonas aeruginosa induced cytotoxicityObjective:Compare the expression level of the Tye Ⅲ secretion system (T3SS) between the multi-drug resistant strains and susceptible strains. Then, using the anti-PcrV antibody, to investigate whether it cloud protect multi-drug resistant pseudomonas aeruginosa induced cytotoxicity.Methods:We collected the clinical isolated PA strains randomly, and it was confirmed non-clonal by RAPD analysis. We analyze the MIC of PA to clinical routinely used antibiotics and then divided the strains into multi-drug resistant group and susceptible group. We compare the genotype of T3SS between the two groups by PCR of pcrV, multi-plex PCR of four exo-toxin genes (exoU, exoS, exoY and exoT), then we analyze the phenotype of T3SS between the two groups by real-time PCR of pcrV, western blot analysis of ExoU and ExoS. The protective effect of anti-PcrV IgG against each strain was determined using an epithelial cell line cytotoxicity assay.Results:We collected53strains including25multi-drug resistant strains and28susceptible strains, which shown to be non-clonal by RAPD PCR analysis. We analyze the distribution pattern of four exotoxin genes, the expression level of pcrV mRNA, and the expression of ExoU and ExoS proteins in two groups, we found that there are no significant differences between the two groups. After the PA infection, the LDH in the culture medium were increased, and anti-PcrV antibody could decreased it by about20%, and the difference was statistically significant.Conclusion:These data suggest that the type Ⅲ secretion expression level in multid-rug reisitant strains is comparable to susceptible strains, and anti-PcrV antibody can protect multi-drug resistant Pseudomonas aeruginsa induced cytotoxicity. So we conclude that even in multi-drug resistant PA strains, type Ⅲ secretion system can also be a treating target. Part2:PcrV passive and active immunization could protect against multi-drug resistant pseudomonas aeruginosa induced acute lung injuryObjective:Choose a multi-drug resistant PA strain that secret ExoU in vitro to establish the model of multi-drug resistant PA induced acute lung injury, then observe whether PcrV active and passive immunization could improve the mice survival and attenuate the lung injury, and whether block PcrV affect the bacteria antibiotic susceptibility and biofilm production.Methods:R4is a multi-drug resistant PA strain, which only susceptible to imipenem in vitro. We chose R4to infect mice using one lethal dose and divided the mice into four groups, control, ceftazidime treated group and PcrV active and passive immunized group. Then we compare the survival rate between the four groups. Lung injury was measured by lung wet-to-dry weight ration, total protein and histological analysis. IL-6, MIP-2and TNF-α levels were measured in BALF and plasma. Then we analyzed the MIC and biofilm of bacteria that cultured to investigate if block PcrV could affect the antibiotic susceptibility and the biofilm production.Results:Compared to the other two groups, PcrV passive and active immunization improved survival of mice significantly. Furthermore, PcrV passive immunization attenuated the lung injury. The underlying mechanism of the anti-PcrV appears to involve a protection against lung injury and ultimately preventing bacterial dissemination to blood. It could also inhibit the expression of cytokines like IL-6, TNF-α, and MIP-2. Furthermore, blocking PcrV would not affect the antibiotic susceptibility, however, it could inhibit biofilm production.Conclusion:PcrV passive immunization could improve mice survival and attenuate lung injury. Four weeks after PcrV active immunization, the antibody titer came to the peak, and the mice survival rate also improved greatly. So anti-PcrV provide a new more effective strategy against PA infection even in multi-drug resistant strains, furthermore, it can be a method to the increasing antibiotic resistance. On the other hand, it would not affect the antibiotic susceptibility, but it could inhibit biofilm production, making it easier to eliminate. Part3KGF-2could attenuate pseudomonas aeruginosa induced acute lung injury partly through enhanced macrophage phagocytosisObjective:To investigate whether KGF-2had a beneficial effect on pseudomonas aeruginosa induced acute lung injury, and the effect of KGF-2on macrophage cells was also investigated to provide insight into the possible mechanisms involved in the action of KGF-2.Methods:PAO1was administered intratracheally to induce acute lung injury in male Balb/C mice. Single dose of rhKGF-2(5mg/kg) was intratracheally instilled72hours before PAO1challenge. Then we compare the survival rate between the two groups. Six and twelve hours after infection, we collected the blood and lung to detect blood dissemination. Lung injury was measured by lung wet-to-dry weight ratio, total protein and histological analysis. IL-6, MIP-2and TNF-a levels were measured in BALF and plasma. Then we analyze the p38MAPK/NF-κB signal channel activity. We culture the macrophage cells and pretreat it with rhKGF-2for24hours. And we compare the phagocytosis rate in those groups.Results:After PAO1infection,90%of the mice were all died in the first48hours, and pretreatment with rhKGF-2(5mg/kg) could improve the survival rate significantly. Furthermore, pretreatment with rhKGF-2could decrease the bacteria dissemination and attenuate acute lung injury score. It could also inhibit the p38MAPK/NF-κB pathway activation. In vitro, pretreatment with rhKGF-2could enhance the macrophage phagocytosis.Conclusion:Keratinocyte growth factor-2pre-treatment could protect pseudomonas aeruginosa induced acute lung injury. The protective effects of Keratinocyte growth factor-2may partly because inhibited activation p38MAPK/NF-κB pathway, and partly contribute to enhanced macrophage cell’s phagocytosis.