Study On The Mechanisms That Toll/Interleukin-1 Receptor Containing Protein Encoded By E.coli Regulates The Function Of Dendritic Cells

Background and objectivesDendritic cells(DCs)are the most powerful professional antigen presenting cells in vivo which are widely distributed in the peripheral tissue.Most DCs are in immature state under normal circumstance and they can accomplish antigen uptake by receptor-mediated endocytosis,macropinocytosis and phagocytosis.After the uptake of antigen or stimulated by certain inflammatory signals such as LPS,IL-1β and TNF-α,the immature DCs become to differentiate and mature.The antigen presentation ability of DCs can directly or indirectly activate T,B cells,and thus DCs have occupied a unique position in the immune response.The study of DCs will help us elucidate the mechanisms of immune response and immune regulation.Toll-like receptors(TLRs)are a class of structurally and functionally well-defined pattern recognition receptors that are widely distributed in innate tissues and cells,which are highly expressed in innate immune cells such as macrophages and DCs.TLRs can recognize pathogen associated molecular patterns(PAMPs)which casue immune cells activation and immune effector molecules expression.Therefore,TLRs play an important role in immune response and inflammatory reaction.Urinary tract infection(UTI)is a urethral tract inflammation which caused by a variety of pathogens invading the urinary tract,urinary tract mucosa or tissues.The clinical signals of UTI are osphyalgia,frequent micturition,urgent urination and odynuria.The most common pathogenic bacteria causing the urinary tract inflammation is E.coli,we can take antibiotics or anti-fungal drugs for treatment according to the different characteristics of these pathogens.But with the improper use of these antimicrobial agents,drug-resistance of pathogen also increases,which has become a great challenge for the clinical treatment.Toll/interleukin-1 receptor containing protein encoded by E.coli(TcpC)is a TIR(Toll/interleukin-1 receptor)domain containing protein secreted by the uropathogenic Escherichia coli CFT073 strain,and the TIR domain of TcpC is similar to the TIR domain of the TLRs,so TcpC can directly bind to the myeloid differentiation factor 88(MyD88)which is an adaptor protein in the downstream of TIR,thus blocking the TLR signaling pathway to interfere with the normal immune response.It was demonstrated that TcpC palys an important role in the pathogenesis of pyelonephritis.At present,the research of TcpC disturbing the immune response mostly focuses on macrophages,neutrophil and epithelial cells,but the influence of TcpC on DC differentiation and function has not been reported.Our previous study showed that TcpC can inhibit the maturation and antigen presentation through MHC calss II pathway of DC.Therefore,the present study aims to explore the mechanism of TcpC inhibiting DC and provide experimental evidence for further research.Methods1 The inducible expression of TcpC The engineered strain E.coli BL21pET42a-TepC was identified by freeze-thawing and inoculated into kanamycin LB solid medium with culturing overnight at 37℃.Then the single well-separated,semitransparent white colonies were collected and incubated overnight at 37℃,220rpm with kanamycin LB liquid medium.Then 1ml bacterial solution was inoculated in LB liquid medium containing kanamycin.When the density of bacteria in medium reached 0.6-0.8 at OD(optical density)--nm,IPTG was added at 37℃ and 220rpm for oscillation induction.2 The purification and concentration of TcpC We used nickel column to purify the target protein and the ultrafiltration tube to concentrate the target protein.3 Preparation of polyclonal rabbit antibody aganist TcpC The concentrated TcpC protein was mixed with Freund’s complete adjuvant and Freund’s incomplete adjuvant,and emulsified to a "water-in-oil" state.Before the first immunization the ear vein blood should be collected as negative control,two weeks after the first immunization we should repeat three times injection to strengthen immunity with an interval of one week.The titer was measured 10 days after the last immunization.4 Titration of the antibody against TcpC The titer of the antibody against TcpC was detected by double agar gel immune diffusion test.5 The purification of rabbit polyclonal antibody against TcpC When the titer met the requirements,the blood was collected from rabbit carotid artery in order to acquire the whole blood and separate serum.Then,the immunoglobulin IgG in the serum was purified by saturated ammonium sulfate method.6 The specificity detection of rabbit polyclonal antibody against TcpC Western blotting of the purified TcpC and the native expression TcpC in E.coli CFT073 by using the TcpC-/-strain as negative control.7 The detection of whether DCs can uptake TcpC Co-culture of DCs with different strains in transwell chamber;the upper chamber contained 3×106 bacteria and the lower chamber contained 3 × 105 cells(multiplicity of infection(MOI)=10:1).The following three treatments were set:DC2.4 + TcpCwt group,DC2.4 + TcpC-/-group,DC2.4 group(the blank control).Triplicate wells in each group were set.The cells were cultured at 37℃ and harvested at different time points,and the intracellular proteins were prepared by lysis buffer.The rabbit anti-TcpC polyclonal antibody was used for western blotting detection.8 The mechanism of TcpC in DCs Co-culture of DCs with different strains in transwell chamber:the upper chamber contained 3 × 106 bacteria and the lower chamber contained 3 × 105 cells(MOI=10:1).The following three treatments were set:DC2.4 + TcpCwt group,DC2.4 + TcpC-/-group,DC2.4 group.Each group was set in triplicate wells.The cells were harvested at 16 hours,and the intracellular proteins were obtained by lysis buffer.The antibody against MyD88 and the antibody against p-p65 were used for western blotting detection.Results1 With adding IPTG to the final concentration of 0.1mM and in culture 6-7h at 37℃and 220rpm oscillation,TcpC protein was expressed abundantly,which mainly expressed in the form of inclusion bodies.2 When the imidazole concentration was 2mM,the target protein can be eluted down.After purification,the TcpC concentration was elevated to about 2mg/ml which could be used to immune rabbits.3 The titer of antibody against TcpC which detected by double agar gel immunodiffusion test was 1:8.4 Western blotting of the purified TcpC and the native expression TcpC in E.coli CFT073 both showed a single clear band,and but no band was seen when the control strain(the TcpC-/-strain)was used.5 Co-culture of DCs with different strains in transwell chamber and the cells were harvested at different time points to detect the accumulation of TcpC in DCs.We found that the TcpC accumulation in DCs began to be detected after 8h co-culture,with time longer TcpC protein accumulation in DCs gradually increased,and reached the highest level at 16h,but at 20h and 24h of co-culture the intracellular TcpC protein accumulation reduced.6 Co-culture of DCs with different strains in transwell chamber and the cells were harvested at 16h.The results showed that the amount of MyD88 protein and p-p65 protein in DCs co-culture with CFT073 strain was significantly lower than that in the blank control group and the TcpC-/-strain control group.ConclusionsTcpC can be taken up by DCs.Accumulation of TcpC in DC can result in MyD88 protein degradation and inhibition of NF-κB p65 protein phosphorylation,which might be one of the mechanisms that TcpC employs to interfere with TLR signaling pathway,hereby inhibiting DC phenotypic and functional maturation.