The Role And Mechanism Of Nociceptor Neurons In Escherichia Coli Urinary Tract Infections

BackgroundUrinary tract infections(UTIs)are one of the most common bacterial infections with high recurrence.Epidemiological surveys show that more than 130 million people annually worldwide have been impacted urinary tract infections.The main pathogen is uropathogenic Escherichia coli(UPEC).With the increase of antibiotic resistance,the effective treatment of urinary tract infection is facing challenges.UPEC infection can evoke a rapid host response.Its typical immune response is strong cytokine expression and secretion,resulting in the recruitment and aggregation of a large number of neutrophils and macrophages.Although neutrophils and macrophages are essential for the clearance of pathogens,excessive infiltration can also lead to bladder inflammation and epithelial damage.Many studies have shown that there is a close crosstalk between the mucosal immunity and the nervous system.However,the role of nociceptor neurons in bladder host defense is not clear.The purpose of this study is to investigate the role of nociceptor neurons in bladder UTIs.Methods4 weeks,female,C57BL/6J mice were injected with high-dose capsaicin(Caph mice),a high affinity(transient receptor potential vanilloid type 1,TRPV1)agonist,to chemically deplete nociceptor neurons.After 4 weeks,the L5-S1 spinal dorsal root ganglion(DRG)of mouse was embedded in paraffin,and the expression of TRPV1+nerve was observed by immunofluorescence staining.The expression of TRPV1 in bladder was analyzed by polymerase chain reaction(PCR).The L5-S1 DRG of mouse was stimulated by UPEC strain CFT073 in vitro and detected by calcium imaging.8-week-old C57BL/6J mice were infected with UPEC strain CFT073.Bladder inflammation,barrier epithelial function and bladder immune cell were evaluated by H&E staining,trypan blue staining,wheat germ agglutinin fluorescein isothiocyanate(WGA-FITC)and flow cytometry.And urine colony-forming units(CFUs)and bladder invaded bacteria were counted.The level of calcitonin gene-related peptide(CGRP)secreted by nociceptor nerve in infected bladder was detected by ELISA kit.CGRP was used in vitro to evaluate the effects of CGRP on neutrophil phagocytosis,and on macrophage polarization.Botulinum neurotoxin A(BoNT/A)and CGRP receptor antagonist(BIBN4096)were used to evaluate the effect of CGRP on UPEC infection in vivo.8 weeks,female C57BL/6J mice were injected with low-dose capsaicin(CapL mice)intrathecally to excite the nerve of nociceptor.After 24 hours of bladder post-infection with CFT073 strain,the number of bacterial colonies was counted.ResultsResults showed that CFT073 and LPS could immediately promote calcium influx in DRG neurons.Intrathecal injection of high-dose capsaicin reduced TRPV1+nerve in DRG and bladder.After depletion of TRPV1+nerve,we detected the decrease of CGRP content in bladder by ELISA kit.It was found that Caph mice had reduced bladder inflammation,epithelial barrier damage and bacterial load.We found that the number of neutrophils in the infected Caph mice bladder increased,the number of macrophages changed slightly,and the number of M1 macrophages decreased.At the same time,we found that the number and distribution of F4/80+macrophages in the bladder of Caph mice changed slightly by immunofluorescence.In order to verify the effect of CGRP released by TRPV1+nociceptor nerve on innate immunity,we treated neutrophils from peripheral blood with CGRP in vitro.It was found that after adding CGRP,the MPO secretion and the phagocytosis of neutrophils decreased;We treated bone marrow-derived macrophages(BMDM)with CGRP.PCR showed that after adding CGRP,BMDM infected with CFT073 secreted more IL-1 β and IL-6 and less Arg-1.At the same time,mRNA sequencing results also verified the PCR test and suggested that BMDM infected with CFT073 tended to polarization to M1 phenotype after CGRP treatment.In vivo,BoNT/A intraperitoneal injection was used to inhibit the release of CGRP and BIBN4096 intraperitoneal injection was used to antagonize CGRP receptor.We found the reduction of bladder inflammation,epithelial destruction,bacterial load and increased recruitment of neutrophils and decreased M1 macrophages after CGRP signal inhibition.We also found that CFUs changed slightly and invaded bacteria increased in CapL mice.ConclusionAll in all,we found that UPEC and its pathogenic factor lipopolysaccharide(LPS)could directly excite nociceptor neurons,releasing CGRP into infected bladder which suppressed the recruitment of neutrophils,the polarization of macrophages and the killing function to UPEC.Both Botulinum neurotoxin A(BoNT/A)and BIBN4096(CGRP antagonism)blocked neuronal inhibition,and prevented against UPEC infection.The present study showed a novel mechanism by which UPEC stimulated the secretion of CGRP from nociceptor neurons to suppress innate immunity.Thus,blocking neuro-immune suppressive effect may be a potential strategy to treat UTIs.