Role Of Toll-like Receptors4in Legionella Infection

Objective1. To explore the relationship between TLR4and Legionella infection in a mousemodel.2. To clarify the mechanism of the role of TLR4in Legionella infection bycompairing of TLR4protein and relevant expression of cytokines in its signaltransduction pathway.Methods1. Four male and four female TLR4gene mutant mice of C3H/HeJ and C3H/HeN of6-8weeks were used as experimental group. Infection group of C3H/HeJ andC3H/HeN mice were injected with50ul1.2×10~7/ml bacterial suspension throughtracheal. C3H/HeN mice were injected of50ul sterile saline through tracheal as thecontrol group. All mice were sacrificed at12h,24h,48h and72h respectively.2. About2ml of orbital blood was collected before the mice were sacrificed andPBMC TLR4protein expression was detected by flow cytometry analysis of thethree groups at all time points. About0.02g of the left lung upper lobe was takenfrom infection and control group to do fixed biopsy.3. Concentration of cytokines of TNF-α and IL-1β in lung homogenated was assayedby ELISA.Results①Difference of mortality between C3H/HeJ and C3H/HeN mice was not significant(χ~2=0.686,P=0.408).Pathological examination of lung tissue showed that infiltration ofinflammatory cells and alveolar vascular bleeding were found in C3H/HeJ andC3H/HeN infection group while inflammatory reaction was not found in the controlgroup. Difference of lung organ coefficency among the three group was significant(F=3.765, P=0.028), there was no significant difference between any two time points(F=2.296, P=0.084), and the interaction difference btween group and time points issignificant (F=3.509, P=0.004). The lung coefficient of C3H/HeJ and C3H/HeNinfection group is higher than that of C3H/HeN control group.②Difference of TLR4protein expression among three groups and of any two time piontswere significantly different respectively (F=10.736, P<0.001),(F=55.034, P<0.001),and interaction difference of grouping and timing shows statisticalsignificance(F=8.094, P<0.001).Results from comparison between two groups showedno significant difference was between C3H/HeN infection group and C3H/HeN control group and that statical difference was found in any other two groups compared. TLR4protein expression of C3H/HeJ infection group is relatively low, peaked in24hours.Another two groups were decreased with the pass of time.③TNF-α concentration difference among C3H/HeJ infection group, C3H/HeN infectiongroup and C3H/HeN control group was significant (F=47.215, P<0.001), thedifference of each timing significant (F=21.373, P<0.001), and interaction betweengrouping and timing existed statistically (F=2.816, P=0.021). TNF-α concentration ofC3H/HeJ and C3H/HeN infection group was higher than that of C3H/HeN controlgroup. TNF-α concentration of three group of peaked at24h after the mice wereinfected and then decreased. IL-1β concentration among three groups was statisticallydifferent (F=64.566, P<0.001), and the difference of each timing was significant(F=13.958, P<0.001),and also the interaction between grouping and timing showedstatistical significance (F=3.958, P=0.005). Concentration of IL-1β, at peak at24hafter the mice were infected, in C3H/HeJ infection group was higher than that in theother two groups. Concentration of IL-1β decreased in C3H/HeJ and C3H/HeNinfection group while increased in C3H/HeN control group with the pass of time.Conclusion①No significant difference of the mortality rate between C3H/HeJ mice and C3H/HeNmice suggests that susceptibility of Legionella between them is same.②That the TLR4expression of C3H/HeN infection group was significantly higher thanthat in C3H/HeJ infection group and no statistical difference was found betweenC3H/HeN infection group and C3H/HeN control group shows that TLR4does notplay a role of anti-inflammation against legionella. That TLR4expression ofC3H/HeJ infection group reached peak after24hours of infection indicates that arole of compensation may be palyed by TLR receptors to TLR4.③That TNF-α and IL-1β concentrations of infection groups were higher that those ofcontrol suggests that Legionella infection may increase the expression of TNF-α andIL-1β.