Study On The Role Of SIGNR1 In Infection By Uropathogenic Escherichia Coli

BackgroundUrinary tract infection（UTI）is a typically persistent infection,which is mainly caused by uropathogenic E.coli（UPEC）.During the interaction between hosts immune responses and UPEC in urinary tract,the bacteria are not completely eliminated.A small number of bacteria exist in the bladder tissue,becoming the source of recurrent infection.Recently,there are plenty of studies that focus on the pathogenesis of persistent UTI.After reaching the mucosa epithelial cells of bladder,UPEC can adhere to and invade these epithelial cells.The infected cells can be lysed by proliferation of bacteria inside cells and releasing toxic substances.The releasing bacteria continue to invade neighboring cells to start a circle anew.Moreover,several studies showed that UPEC could form intracellular bacterial communities（IBCs）and quiescent intracellular reservoirs（QIRs）,which are resistant to bactericidal effects of antibiotic and immune cells,indicating the utilization of epithelial cells as shelters for the persistence of UPEC.Antigen presenting cells（APCs）,including dendritic cells and macrophages,play essential roles in the host immune response to UTI.However,the mechanisms of the interaction between these cells and pathogens are not fully understood.Our previous studies showed that several Gram-negative bacteria can interact with CD209,a C-type lectin receptor expressed by macrophages,through their lipopolysaccharide core structure,thus promoting bacterial infection and dissemination.UPEC may use CD209 as a receptor to invade host cells in the same way as other Gram-negative bacteria do,thereby facilitating its persistence in hosts.In this study,we will investigate the interaction between UPEC and host cells using clinically isolated strains and laboratory strains of UPEC,and study the in vivo role of SIGNR1 receptor in a mouse UTI model.Methods1.Isolation of clinical UPEC strainsThe clinical UPEC strains were isolated from urine samples of patients with cystitis.Polymerase chain reaction（PCR）were performed to confirm the isolated strains.2.O-antigen expression of clinical UPEC isolatesThe lipopolysaccharide（LPS）of UPEC strains was extracted by using LPS-extraction kit,after sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS-PAGE）,the LPS patterns were stained by silver staining to determine the O-antigen expression of these clinical UPEC isolates.3.Invasion assays3.1.UPEC strains were allowed to incubate with Chinese hamster ovary（CHO）cells and CHO cells expressing human DC-SIGN or murine SIGNR1,gentamycin protection treatment was used to determine the phagocytosis of UPEC by host cells,as well as the effect of O-antigen on invasion.3.2.UPEC strains were allowed to incubate with intraperitoneal macrophages derived from wild-type（WT）mice or SIGNR1 knockout(SIGNR1^-/-)mice.Gentamycin protection treatment and immunofluorescence assay were used to determine the effects of SIGNR1 receptor on interaction between UPEC and macrophages.3.3.Inhibition assays with anti-CD209s（anti-hDC-SIGN or anti-m SIGNR1）antibodies or analogues of LPS core to confirm role of CD209 in interaction between host cells and UPEC strains.4.Mouse model of UTI4.1.The WT or SIGNR1^-/-mice were challenged with UPEC though transvesical intubation.Urine or bladder tissues of infected mice were collected for bacterial loads at indicated time points after infection.4.2.In the mouse model,immunohistochemistry（IHC）,immunofluorescence（IF）as well as enzyme-linked immune sorbent assay（ELISA）were exploited to determine the degree of bladder inflammation and related inflammatory indicators in WT and SIGNR^-/-mice,to investigate the role of SIGNR1 in vivo.Results1.Macrophages from SIGNR1^-/-mice have reduced phagocytosis of UPECMacrophages isolated from WT and SIGNR1^-/-were allowed to interact with six UPEC strains.Gentamycin protection treatment and immunofluorescence assay were used to determine the invasion abilities of these strains.The result showed that the phagocytosis of UPEC by macrophages from SIGNR1^-/-was significantly reduced when compared to that of WT mice.2.Human DC-SIGN and murine SIGNR1 are receptors for phagocytosis of UPECUPEC strains were allowed to incubate with CHO cells,CHO-hDC-SIGN cells and CHO-m SIGNR1 cells to determine the role of CD209s（hDC-SIGN and m SIGNR1）in phagocytosis of UPEC.The results showed that CHO cells expressing hDC-SIGN and m SIGNR1 had significantly increased phagocytosis of UPEC,which was inhibited by anti-hDC-SIGN antibody and anti-m SIGNR antibody,respectively,indicating the specificity of binding of UPEC to CD209 receptor.3.Interaction between UPEC and CD209s was independent of LPS coreThe isolate UPEC2 and UPEC4 showed no expression of O-antigen in LPS silver staining,but there were no differences between phagocytosis of these two isolates and that of the other strains tested,when interacted with CHO-hDC-SIGN cells or CHO-m SIGNR1 cells.Moreover,the UPEC-CD209s interaction could not be inhibited by mannan,a LPS core analogue,indicating that the interaction between UPEC and CD209s may not be mediated by LPS core.4.Fewer bacteria were disseminated to the spleen and liver in SIGNR1^-/-mice than in WT miceWT and SIGNR1^-/-mice were challenged with UPEC though intraperitoneal injection,the spleen,liver,and mesenteric lymph nodes（MLNs）of infected mice were collected at 24 h post-infection,homogenates of these organs were plated onto LB agar for bacterial counts.The result showed that less bacteria were disseminated to the spleen and liver in SIGNR1^-/-mice than in WT mice.5.In mouse UTI model,SIGNR1^-/-mice have impaired bacterial clearance ability upon UPEC infectionUrine and bladder of infected mice were collected at indicated time points for measurement of bacterial loads.The infected SIGNR1^-/-mice had higher bacterial titers in urine and bladder tissues than that of WT mice.In histochemical analysis,the bladders from SIGNR1^-/-mice showed more severe inflammatory response upon UPEC infection than that of WT mice.In cytokine expression analysis,there were no differences between the SIGNR1^-/-and WT mice in the local IL-10 response,nor in the levels of IL-6 and TNF-α.Based on this cytokine profile,we could not determine a role for SIGNR1 in the induction of cytokine production to modulate the inflammatory response and the concomitant cellular infiltration upon infection with UPEC.ConclusionIn this study,we first showed that UPEC interacts with CD209-expressing macrophages and transfectants,which is inhibited by anti-CD209 antibody,indicating that CD209s are receptors for UPEC.However,in contrast to previous studies,mice lacking SIGNR1 are more susceptible to infection of this uropathogen,leading to prolonged bacterial persistence.Thus,our study indicates that the innate immune receptor SIGNR1 plays a role in the clearance of UPEC during UTIs.