Effects Of TcpC On Differentiation And Maturation Of Kidney Dendritic Cells In Mouse With Pyelonephritis

Background and ObjectivesUrinary tract infection is one of the most common bacterial infections in clinical,and it is mainly caused by bacteria from the urethral retrograde.Part of the severe patients can lead to kidney failure.E.coli is the most common pathogen of urinary tract infection and occupies nearly 80%.Uropathogenic E.coli strain CFT073 can secrete Toll/interleukin 1 receptor domain containing protein(TcpC)that interferes directly with the Toll-like receptor(TLR)function.Toll-like receptors(TLRs)are a kind of pattern recognition receptors that expressed on macrophages and DCs,and TLR signaling plays an important role in the initiation of local inflammation.It was found that TcpC could bind directly with myeloid differentiation factor 88(MyD88),hereby blocking the TLR signaling pathway,leading to inhibition of NF-?B activation and inhibiting bactericidal activity of macrophages.Therefore,TcpC,serving as an important virulence factor,can promote the survival of the bacteria in macrophages,and is closely related to the pathogenesis of pyelonephritis.Dendritic cells(DCs)are the most potent antigen presenting cells(APCs),and play a central role in the processing and presentation of antigens to T cells during an immune response.Immature DCs capture antigens,but weakly stimulate T cells.Mature DCs have strong antigen presenting function,mainly involved in cellular immunity and T cells dependent humoral immune response.DCs serve as the bridge and link between the innate and adaptive immunity.The immunoregulatory effects of DCs are closelyVII related to its surface receptor molecules,such as TLRs and C type lectin.TLRs are important pattern recognition receptors(PRR),and play a key role in differentiation and maturation of DCs.TLRs can specifically identify the conservative pathogen-associated molecular patterns(PAMPs)of bacteria,fungi and viruses,and capture the pathogen,regulate the maturation of DCs.An extensive network of resident DCs populates the kidney tubulointerstitium.DCs can eliminate pathogens by adjusting the natural immune and adaptive immune response,and affect the occurrence and development of pyelonephritis.In order to further elucidate the pathogenesis of acute pyelonephritis,the regulatory effects of TcpC on differentiation and maturation of kidney DCs in mouse with pyelonephritis are examined.Methods1 The establishment of pyelonephritis mouse model:the mice were anesthetized with Avertin and were infected by transurethral instillation of 1×1010 UPEC using a indwelling needle with curved 35°.Three hours later,the procedure was repeated to induce bacterial pyelonephritis.Mice were sacrificed three days later and the kidneys were removed for subsequent analysis.This experiment was set to 3 groups:NS,normal saline;TcpCwt,the wild type CFT073 strains;TcpCdel,tcpc deleted CFT073 strains.2 Paraffin section:fixed specimens,rinse,dehydration,transparent,paraffin,embedding,slice,patches.HE staining:dewaxing,nuclear staining(hematoxylin staining for 10 min;tap water washing;hydrochloric acid alcohol 3-5s;tap water washing),Cytoplasmic staining(eosin staining 15-20 min.70%alcohol,80%alcohol),dehydration,transparent,sealing piece.3 Isolating kidney DCs using the CDllc microbeads:first excise the kidneys,removing the capsule and place them in a dish.Kidneys were sliced with a scalpel into small pieces and digested for 25 min at 37 ? with 0.5 mg/ml collagenase and 0.1 mg/ml DNAse in RPMI 1640 medium containing 10%FBS.Single cell suspensions were filtered through a 40 ?m nylon mesh and washed with buffer(2 mM EDTA,0.5%BSA in PBS).Centrifuge cell suspension at 800r for 5 min.Aspirate supernatant completely.Resuspend cell pellet in 400 ?l of buffer per 108 total cells.Add 100 ?l of CD11c microbeads per 108 total cells.Mix well and incubate for 15min at 4?.Wash cells by adding 1-2 ml of buffer per 107 cells and centrifuge at 800 r for 5min.Place column in the magnetic field of a MACS Separator.Prepare column by rinsing with 0.5 ml buffer.Wash column three times each with 0.5 ml buffer.Remove column from the sepatator and pipette the appropriate amount of buffer onto the column.Immediately flush out the magnetically labeled cells by firmly pushing the plunger into the column.4 Phenotypic analyses by flow cytometry:expression of CD40,CD54,CD80,CD86,MHC-I and MHC-II on the surface of kidney DCs was detected by flow cytometry.5 DC cell culture:kidney DCs were resuspended in complete medium(RPMI-1640 containing 10%FBS,100 U/ml penicillin-streptomycin solution)at concentration of 5×105 cells/ml,and plated into 24-well plates(1 ml/well).The plate was cultured at 37?,5%CO2 atmosphere.Culture supernatants were collected after 48 h.6 Isolating CD4~+ T cells from OT-? mice and CD8~+ T cells from OT-? mice using microbeads:prepare single-cell suspensions,pass cells through 30?m nylon mesh,determine cell number,centrifuge cell suspension,add microbeads and incubate for 15min,and then magnetic separation.7 Effects of TcpC on antigen presenting function of DCs(1)Coculture of DCs with wild-type CFT073 strain or with tcpc deleted CFT073,strain:DCs were cocultured with wild-type CFT073 strain or with tcpc deleted CFT073 strain in Transwell.3×105 DC2.4 were added into the lower chamber of the 24-well Transwell plate,3×106 CFT073 or tcpc deleted CFT073 were added into the upper chamber.The following 3 treatments were set:blank control group,i.e.DC2.4;DC2.4+TcpCwt group;DC2.4+TcpCdel group.The plates were incubated at 37?,5%CO2 for 20 h.Cells were collected for coculture with T cells.(2)The activation and proliferation response of CD8~+T or CD4~+T cells:the collected cells were cocultured with OT-I derived CD8~+T or OT-? derived CD4~+T cells at 1:10 in RPMI 1640 medium containing 2 ?g/ml OVA257-264 or OVA323-339.The 96-well plate was cultured at 37?,5%CO2 atmosphere for 72 h.Culture supernatants were collected for measurement of IL-2 and IFN-?.8 ELISA:CXCL-2,IL-1?,IL-6,IL-12,IL-23,IL-2 and IFN-? concentrations in the culture supernatants were measured by ELISA.Results1 TcpC can increase the bacteria burden and the neutrophilic infiltrates in the mouse UTI model.The results of kidney homogenate germiculture showed that the bacteria amount in TcpCwt group was 3.5 times as many as TcpCdel group,while no bacterial growth in NS group.A large number of neutrophil infiltration in TcpCwt group and less infiltration in TcpCdel group were observed in paraffin section.2 TcpC can significantly increase the concentration of CXCL-2 and has inhibitory effect on the secretion of IL-6,while has no significant impact on IL-1? in kidney with pyelonephritis.The concentrations of cytokines CXCL-2,IL-1? and IL-6 in NS group were 155.4±23.3(ng/ml)?17600.0±763.7(pg/ml)and 465.6±13.8(pg/ml),respectively;the concentrations of cytokines CXCL-2,IL-1? and IL-6 in TcpCwt group were 327.9±20.9(ng/ml),19016.0±503.5(pg/ml)and 616.4±26.5(pg/ml),respectively;and the concentrations of CXCL-2,IL-1? and IL-6 in TcpCdel group were 206.6±17.1(ng/ml),20600.0±480.8(pg/ml)and 740.2±17.1(pg/ml),respectively.The data indicated that TcpC significantly increase the amount of CXCL-2 and has inhibitory effect on the secretion of IL-6,while has no significant impact on IL-1? in kidney with pyelonephritis.3 TcpC inhibits the expression of CD40,CD80,CD86 and MHC II on the surface of kidney DCs.The mean fluorescence intensity(MFI)of CD40,CD54,CD80,CD86,MHC-I and MHC-II on the surface of kidney DCs in NS group was 49.94±4.80,43.99±1.90,39.43±1.37,58.56±2.63,65.58±5.78 and 138.20±10.95,respectively;the MFI of CD40,CD54,CD80,CD86,MHC-I and MHC-II on the surface of kidney DCs in TcpCwt group was 26.72±4.24,39.90±3.01,33.16±2.60,51.43±3.33,67/78±4.45 and 85.88±3.34,respectively;while the MFI of CD40,CD54,CD80,CD86,MHC-? and MHC-? on the surface of kidney DCs in TcpCdel group was 92.88±10.20,52.12±5.58,47.26±1.84,86.94±4.16,88.28±8.29 and 139.69±6.75,respctively.These data showed that kidney DCs expressed higher membrane surface molecules after stimulation,and TcpC inhibits the expression of CD40,CD80,CD86 and MHC II on the surface of kidney DCs.4 TcpC has inhibitory effect on the function of kidney DCs.4.1 TcpC inhibits the secretion of IL-1?,IL-6,IL-12 and IL-23 of kidney DCsThe concentrations of IL-1?,IL-6,IL-12 and IL-23 in the culture supernatant in NS group were 340.5±12.0,51.3±3.5,29.0±4.2 and 67.6±12.8(pg/ml),respectively;the concentrations of IL-1?,IL-6,IL-12 and IL-23 in the culture supernatant in TcpCwt group were 185.0±26.9,34.2±7.6,40.4±5.7 and 93.4±8.1(pg/ml),respectively,and the concentrations of IL-1?,IL-6,IL-12 and IL-23 in the culture supernatant in TcpCdel group were 427.5±34.7,72.6±5.4,61.0±3.4 and 148.9±13.3(pg/ml),respectively,indicating that kidney DCs secreted high level cytokines after stimulation,and that TcpC inhibits the secretion of IL-1?,IL-6,IL-12 and IL-23 of kidney DCs.4.2 TcpC inhibits antigen presentation of DCs by MHC class II molecule pathwayCD8~+ T cells secreted low level IL-2 and IFN-?,and there was no significant differences between the TcpCwt and TcpCdel groups(P>0.05).The concentration of IL-2 and IFN-? secreted by CD4~+T cells in TcpCwt group(1870.0±216.5,10581.3±914.9(pg/ml))was significantly lower than that in TcpCdel group(2699.4± 134.1,15520.2±780.2(pg/ml)),P<0.05.The data indicated that the antigen presenting function through MHC class II molecule pathway in TcpCwt-treated DCs is inhibited..ConclusionTcpC has inhibitory effect on differentiation and maturation of kidney DCs in mouse with pyelonephritis.