Effect Of Earlier Lipopolysaccharide Intervention On Legionella Pneumophila Infection In A Mouse Model

Objective1.To explore the effect of earlier Lipopolysaccharide intervention on expression of TLR4in animal experiment.2.To explore the effect of earlier Lipopolysaccharide intervention on Legionella pneumophila infection by comparison of pathological changes, TLR4protein and its related cytokines expression after Lipopolysaccharide intervention.Methods1. C3H/HeN mice (6-8weeks) were used as experimental animal. We established three groups including intervention group, non-intervention group and control group. Each group had eight animals, half males and half females. Mices of intervention group were gave Escherichia coli lipopolysaccharide (1mg/kg) by intrapcritoneal injection. Other groups were gave sterile normal saline by intraperitoneal injection. Two infection groups mice were gave50ul1.2×107/ml bacterial suspension liquid by tracheal injection at24h. Mice of Control group were gave50ul sterile normal saline by tracheal injection. All mice were sacrificed at12h,24h,48h respectively.2. About0.02g left lung upper lobe were taken from two bacterial infection groups and control group and fixed to do pathological sections. The levels of TLR4protein expression on PBMC of each group were detected using flow cytometry to analyse the changes of TLR4protein expression level among three groups at each time point.3. The cytokines TNF-α and IL-1β of mouse lung homogenate were assayed by EL1SA.Resultsl.Lung tissue biopsy results showed that lung tissue of infection group had infiltration of inflammatory cells and alveolar vascular bleeding, which were not found in control group.Inflammatory exudate was found in two infection groups24h later.The alveolar vascular bleeding and the degree of inflammatory infiltration in LPS intervention infection group were less than that of LPS non-intervention infection group.The results showed that there was statistical significance of the lung coefficient in three groups (F=5.699, P=0.006), the difference at each time point was statistical significance (F=10.648, P<0.001) and the interaction of grouping and timing was no statistical significance (F=0.668, P>0.05). The lung coefficient of infection group was higher than that of control group(F<0.05).But there were no statistical difference of the lung coefficient between LPS intervention infection group and LPS non-intervention infection group.2.The levels of TLR4protein expression in three groups were significantly difference(F=47.144, P<0.001). The difference of each time was statistically significant (F=141.543, P<0.001), and the interaction of grouping and timing shows statistical significance (F=29.902, P<0.001). TLR4protein expression level in LPS intervention infection group was higher than that of control group at12h and24h (P<0.05).And the level of TLR4protein expression in LPS intervention infection group was higher than that of LPS non-intervention infection group (P<0.05). TLR4protein expression levels of two Infection groups peaked at12h and decreased with time. TLR4protein expression levels between two Infection groups were no statistical difference at24h and48h (P>0.05).3. TNF-a concentration in three groups were statistical difference (F=25.817, P<0.001).The difference of each time point was statistical significance (F=8.3933, P<0.001), but the interaction of group and time showed no statistical significance (F=1.460, P>0.05). Comparison between groups found the TNF-a concentration had no statistical difference between the LPS intervention Infection group and control group (P>0.05), but TNF-α concentration of LPS Non-intervention Infection group was higher than that of LPS intervention Infection group and control group at each time point. TNF-a concentration of two infection groups peaked at24h and then decreased with time. The difference of TNF-α concentration in three groups was statistically significant (F=18.990, P<0.001), the difference of each time point showed no statistical significance (F=1.323,P>0.05), and the interaction of group and time also showed no statistical significance (F=0.382,P>0.05). Comparison between groups found that IL-1β concentration had no difference between the LPS intervention Infection group and control group (P>0.05), but IL-1β concentration of LPS Non-intervention Infection group was higher than that of LPS intervention Infection group and control group (P<0.05).Conclusion1. Earlier LPS intervention can significantly increased TLR4expression level of PBMC in mice and it has time effect. But the effect of earlier LPS intervention on Legionella pneumophila Infection need further study.2. TNF-a and IL-1β concentration of LPS Non-intervention Infection group were significantly higher than that of control group.These results suggested that Legionella infection may increase the expression of TNF-a and IL-1β and be benefitial to antibacterial immunity.While TNF-a and IL-1β concentration were no difference between the LPS intervention Infection group and the control group. This showed that LPS tolerance may alleviate inflammatory symptoms after Legionella infection.