| Objective: Urinary tract infection(UTI) is a common infectious disease,and the majority(>80%) of which are caused by the urinary tract pathogenic E. coli (UPEC). FimH is the adhesive subunit of type 1 fimbriae of the Escherichia coli,and is a key factor in where bacteria infects urinary tract cells.FimH can specifically bind with mannosylated protein which is on the surface of bladder epithelial cell, lead to local rearrangement of host cell and directly mediate the invasion of UPEC, cause cystitis and recurrent urinary tract infections (UTIs). Now, FimH adhesin has become the candidate target protein preventing bacterial urinary tract infections. After immunized with FimH protein, Animals could produce high levels of specific antibodies(IgG and SIgA) anti-FimH. The antibodies had cross reaction to more than 90% of uropathogenic strains who expressed FimH, and had protective effects to urinary tract infection. Our privious research obtained similar with this.Research showed: FimH adhesin is a novel pathogen-associated molecular pattern of TLR4, which bound directly to TLR4 on the suface of macrophages, promote the production of TNF-αand other cytokines through MyD88-dependent signaling pathway activation, and,increase the antiviral activity of peritoneal macrophages of mice. In addition, FimH activated NK cells via TLR4- dependent pathway to produce IFN-γand antiviral cytotoxic- ity. It is thus clear that FimH adhesin has an effect of regulating the innate immunity. TLR4 is also expressed on the urothelial cells and renal tubular epithelial cells, and is involved in anti-inflammatory response of urinary tract cells. And the effects to the urinary tract cells and TLR4 activated by FimH protein has not been reported.The research aims to research the effectiveness of FimH protein regulating TLR4 and related cytokines expression in urinary tract cells, to investigate the antimicrobial effects of FimH protein in urinary tract cells, and inflammatory response intensity in bladders of infected mice. It would be to provide experimental evidence for researching FimH regulating mucosal innate immunity.Methods:1 Purification of FimH: The fimH gene from E. coli strain J96 had cloned into pGEX-4T-2 and expressed in BL-21-competent E. coli, and induced by IPTG. The expression products were analyzed by SDS-PAGE. The cytotox- icity of the fusion protein were detected by MTT assay. LPS levels in FimH preparations were determined using a Limulus amebocyte lysate LPS detect- ion kit.2 Human urothelial carcinoma cells 5637, renal carcinoma cells A498 and macrophages Thp-1 were stimulated with different concentrations of FimH preparations.Then, the expression of TLR4 on the cells were detected by flow cytometry, the levels of cytokines expressions including IL-6, IL-8 and TNF-a were detected by Real-Time PCR.3 After different concentrations of FimH preparations stimulated 5637 cells for 48 hours, E. coli U30 was added and cultured togther with the cells for 2 hour. Then extracelluar bacteriums were washed out, the cells were dissolved with 0.2% Triton X-100, the lysate were diluted and inoculated in LB plate overnight, cultured at 37℃, the colonies were counted.4 6-8 weeks Balb/C female mice were randomly divided into two groups, 15 mice in each. Mice of one group were injected with the FimH proteins (100ug/per) via peritoneal approach, another group was injected with 0.9% Nacl. After 7 days, IL-6, TNF–a mRNA expression in bladder and kidney tissue of mice were detected by Real-time PCR and the expression of TLR4 on the surface of the tissues'were detected by immunohistochemical test.5 The E. coli U30 were injected the bladder of mice through the urethra at concentration 108CFU/50μl. 6 hours after infection, the control group mice were injected with 0.9% Nacl intraperitoneally(100μl/per); the experimental group mice were injected FimH proteins intraperitoneally(100ug/per), once a day for 3 days. The cytokine expression of IL-6 and TNF-αin bladders and kidneys of the infected mice were detected by Real-time PCR. Bladder HE stains were observed to evaluate the degree of bladder inflammatory cell infiltration.Results:1 FimH concentrations were 166.5ug/ml. The LPS level in FimH preparations was determined using a Limulus amebocyte lysate LPS detection kit, and was 1030pg/ml. It means 1mg FimH preparations content 6.18pg. Reference reporter, LPS had no activity on FimH when it was 4~7pg/ug. The MTT assay proved that the FimH protein had no cytotoxicity to cells when the concentration≤25ug/ml.2After 5637, A498 and THP-1 cells were stimulated by FimH proteins for 48 hours, the expressions of TLR4 on the surface of the cells detected by flow cytometry were increased significantly (P<0.05).3 FimH proteins stimulated 5637, A498 and THP-1 cells for 24 hours, Real-time PCR analysis showed: In 5637and A498 cells, the IL-6 and IL-8 mRNA expression were increased consistency, but the TNF-αand MyD88 mRNA expression had no changes; compare with the blank control group and the LPS control group, the IL-6, TNF-αand MyD88 mRNA expression in THP-1 cells were increased significantly (P<0.05).4 After 48h stimulated with FimH 0.5μg/ml, 2.5μg/ml,12.5μg / ml, the numbers of U30 in 5637 cells were all significantly lower than controls (230±69) (P<0.05), and the bacteria in group of 2.5μg/ml was significantly lower (77±20).5 After FimH protein injected mice for 7 days, IL-6, TNF-αmRNA expression were significantly higher (P<0.01) in the bladder and kidney, and mRNA of TNF-αin bladders was significantly higher than that of kidneys. Immunohistochemistry showed that FimH protein effected group, expression of TLR4 in bladder and kidney were higher than control.6After application of FimH protein for three days, IL-6, TNF-αmRNA expression in the bladder and kidney of infected mice were all significantly higher than controls (P<0.05).The results of HE stains showed: Infiltration of inflammatory cells in bladder tissues of mice injected with FimH were significantly lower than the control bladders, and the mucous were more smooth and complete than controls.Conclusion:1 FimH adhesin of urinary pathogenic E. coli, was able to significantly increased the expression of TLR4 on the surface of 5637, A498 and THP-1 cells. In vivo, FimH could also promoted TLR4 expression on bladder and renal of mice.2 FimH protein was able to increased the expression of IL-6, IL-8 mRNA in both 5637 and A498 cells, and it may be through MyD88-independent pathway. The expression of IL-6 and TNF-αmRNA in THP-1 cells stimulated by FimH protein were enhanced significantly,and via MyD88 dependent pathway.3 FimH protein can significantly enhanced ability of the 5637 cells resistant to E.coil U30.4 FimH protein was able to increased the IL-6 and TNF-αmRNA expression of in the bladders and kidneys of infected mice, it also reduced the intensity of bladder inflammation after urinary tract had been infected. |
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