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MP24-06 IN VIVO ROLE OF NLRP3 INFLAMMASOME IN INNATE UROTHELIAL DEFENSES AGAINST UROPATHOGENIC E. COLI
THE JOURNAL OF UROLOGYâ
e272
understanding of defence mechanisms against such infections remains limited. The urothelium is a primary component of the innate defence to urinary tract infection, providing both a physical barrier and producing a cocktail of antimicrobials and immunomodulatory molecules. Our aim has been to establish a normal human urothelial (NHU) cell culture system to investigate the composition of the human urothelial 0 defendome0 and here we report on the urothelial expression of antimicrobial peptides in response to bacterial challenge. METHODS: NHU cells were exposed to uropathogenic E. coli (UPEC) and bacterial pathogen-associated molecular pattern (PAMP) molecules. RNA and protein lysates were collected. Antimicrobial peptide expression was assessed by reverse transcriptase polymerase chain reaction (RT-PCR), quantitative RT-PCR and western blotting. RESULTS: Challenge of NHU cells with UTI89, a UPEC cystitis isolate, induced expression of b-defensins 2 and 4 at 6 and 24 h postchallenge, alongside constitutive, transcript-level expression of RNASE7 and b-defensin 1. Exposure of urothelial cells to lipopolysaccharide, the prototypic proinflammatory molecule expressed by UPEC, did not induce AMP expression. Treatment of NHU cells with UPECderived flagellin, a Toll-like receptor (TLR) 5 ligand, induced a similar response to UPEC challenge, inducing both b-defensin 2 and 4 at both 6 and 24 h time points. CONCLUSIONS: Our findings indicate that several peptides with recognised antimicrobial activity are produced by the urothelium, both constitutively and in response to flagellin challenge. The absence of an AMP response to LPS confirms our previous findings that human urothelial cells are non-responsive to ligands that activate TLR4. Our findings highlight the importance of the TLR5-flagellin axis in the urothelial acute antimicrobial defensive response to E. coli UTI. Source of Funding: CE was supported by a Wellcome Trust PhD scholarship. JS holds a research chair funded by York against Cancer.
MP24-06 IN VIVO ROLE OF NLRP3 INFLAMMASOME IN INNATE UROTHELIAL DEFENSES AGAINST UROPATHOGENIC E. COLI Yan Liu, Feng He, Ellen Shapiro, Herbert Lepor, Xue-Ru Wu*, New York, NY INTRODUCTION AND OBJECTIVES: The NLRP3 inflammasome is a multi-protein complex consisting of a pattern recognition receptor (NLRP3), an adaptor (ASC) and caspase 1. It is a critical integrator of innate host defenses in myeloid and non-myeloid cells by promoting the maturation of proinflammatory cytokines such as interleukin 1beta and 18. To date, no study has been reported regarding the in vivo role of NLRP3 inflammasome in urinary tract infection (UTI) caused by uropathogenic E. coli (UPEC). METHODS: Experimental bladder infection was elicited in wildtype female mice through transurethral inoculation of clinical cystitis isolates, UTI89 and NU14. Formation of speck in urothelium, urine IL 1beta and cleaved caspase 1 in urothelium, hallmarks of NLRP3 inflammasome activation, were determined using immunofluorescence staining, ELISA and Western blotting, respectively. Studies were extended into NLRP3 and caspase 1 knockout (KO) mice, using UPEC strains as well as E. coli strains isolated from human asymptomatic bacteriuria (ABU; ABU83972 and ABU35) and recurrent UTI (C055 and C001). Additionally, Toll-like receptor 4 (TLR4) KO mice were analyzed to ascertain whether TLR4 status affects NLRP3 inflammasome activation. RESULTS: Transurethral inoculation of UPEC strains UTI89 and NU14, but not laboratory strain AAEC191A or FimH-deletion strain NU14-1, led to urothelial speck formation, increased urine IL 1beta secretion and urothelial pro-caspase 1 cleavage. These three NLRP3 inflammasome activation markers peaked by 6 hours and subsided by 12 hours after UPEC inoculation. Such responses were significantly blunted in NLRP3 KO mice and even more so in caspase 1 KO mice. The decreased NLRP3 inflammasome response in these
Vol. 195, No. 4S, Supplement, Saturday, May 7, 2016
KO mice inversely correlated with significantly increased UPEC retention in the bladder. Whereas the two ABU strains elicited only 1/5 of urinary IL 1beta in wild-type mice, compared with the UPEC strains, the two strains from recurrent UTI elicited as much IL 1beta response as the two cystitis strains. Finally, markedly decreased IL 1beta production and secretion by urothelium was observed in TLR4 KO mice compared with wild-type mice, suggesting that lipopolysaccharide/ TLR4 interaction is crucial for NLRP3 inflammasome activation in the urothelium. CONCLUSIONS: Our data demonstrate for the first time that UPEC can activate inflammasomes in the urothelial cells via the NLRP3 pathway and suggest that reduced activity of this signaling pathway may predispose the urinary tract to recurrent infections by uropathogenic E. coli. Source of Funding: NIH
MP24-07 MULTINUCLEATED UROTHELIAL UMBRELLA CELLS AS INITIATORS OF INNATE HOST DEFENSES AGAINST UROPATHOGENIC E. COLI Yan Liu, feng he, Ellen Shapiro, Herbert Lepor, Xue-Ru Wu*, New York, NY INTRODUCTION AND OBJECTIVES: Mammalian urothelial surface is covered by highly differentiated and morphologically distinct superficial cells, also named umbrella cells. These cells contain multiple nuclei but, with the exception of a role in permeability barrier, no other function has been attributed to these specialized cells. This study was undertaken to explore the in vivo role of urothelial umbrella cells in innate host defenses against uropathogenic E. coli (UPEC). METHODS: Female mice (7-9 weeks) were inoculated via the transurethral route with type 1 piliated UPEC strains (UTI89, NU14, CFT073), isogenic mutant strains lacking FimH adhesin (UTI89-1, NU14-1) or expressing a mutated FimH (UTI89-Q133N), or a nonUPEC laboratory strain (HB101). Translocation of p65 subunit of NFkB from cytoplasm to nucleus, an indicator of NF-kB activation, was assessed by confocal immunofluorescent microscopy of cross sections and whole-mount bladders. Production and urinary secretion of proinflammatory cytokines by urothelial cells was determined by multiplex ELISA. The effects of TLR4 status on p65 translocation were determined using TLR4 mutant mice and isogenic control mice. RESULTS: Inoculation of mouse bladders with UPEC induced rapid translocation of p65 from the cytoplasm into the nucleus, peaking at 1 hour and diminishing after 6 hours post-inoculation. The nuclear translocation of p65 was confined to the umbrella cells, sparing intermediate and basal cells of the urothelium completely. When one nucleus had nuclear p65, all other nuclei (from 2-10) within a cell also did so, indicating a highly synchronous nature of NF-kB activation in multinucleated umbrella cells. The p65 nuclear translocation depended on urothelial adhesion of UPEC, because nonUPEC laboratory strain and mutated UPEC strains lacking FimH or expressing an adhesion-deficient FimH mutant or UPEC strains in the presence of D-mannose failed to adhere to urothelial surface and failed to trigger p65 nuclear translocation. The p65 nuclear translocation coincided with a marked increase of urothelial and urine proinflammatory cytokines (TNF-alpha, IL-1alpha and IL-1beta). Finally, TLR mutant mice had 3 times less p65 nuclear translocation than the isogenic controls. CONCLUSIONS: These results represent the first demonstration that the umbrella cells of the urothelium play a critical role in triggering innate host defenses against UPEC, and suggest that the multiple nucleation of these cells serves an important function in amplifying mRNAs encoding proinflammatory signals to combat invading pathogens. Source of Funding: NIH
e272
understanding of defence mechanisms against such infections remains limited. The urothelium is a primary component of the innate defence to urinary tract infection, providing both a physical barrier and producing a cocktail of antimicrobials and immunomodulatory molecules. Our aim has been to establish a normal human urothelial (NHU) cell culture system to investigate the composition of the human urothelial 0 defendome0 and here we report on the urothelial expression of antimicrobial peptides in response to bacterial challenge. METHODS: NHU cells were exposed to uropathogenic E. coli (UPEC) and bacterial pathogen-associated molecular pattern (PAMP) molecules. RNA and protein lysates were collected. Antimicrobial peptide expression was assessed by reverse transcriptase polymerase chain reaction (RT-PCR), quantitative RT-PCR and western blotting. RESULTS: Challenge of NHU cells with UTI89, a UPEC cystitis isolate, induced expression of b-defensins 2 and 4 at 6 and 24 h postchallenge, alongside constitutive, transcript-level expression of RNASE7 and b-defensin 1. Exposure of urothelial cells to lipopolysaccharide, the prototypic proinflammatory molecule expressed by UPEC, did not induce AMP expression. Treatment of NHU cells with UPECderived flagellin, a Toll-like receptor (TLR) 5 ligand, induced a similar response to UPEC challenge, inducing both b-defensin 2 and 4 at both 6 and 24 h time points. CONCLUSIONS: Our findings indicate that several peptides with recognised antimicrobial activity are produced by the urothelium, both constitutively and in response to flagellin challenge. The absence of an AMP response to LPS confirms our previous findings that human urothelial cells are non-responsive to ligands that activate TLR4. Our findings highlight the importance of the TLR5-flagellin axis in the urothelial acute antimicrobial defensive response to E. coli UTI. Source of Funding: CE was supported by a Wellcome Trust PhD scholarship. JS holds a research chair funded by York against Cancer.
MP24-06 IN VIVO ROLE OF NLRP3 INFLAMMASOME IN INNATE UROTHELIAL DEFENSES AGAINST UROPATHOGENIC E. COLI Yan Liu, Feng He, Ellen Shapiro, Herbert Lepor, Xue-Ru Wu*, New York, NY INTRODUCTION AND OBJECTIVES: The NLRP3 inflammasome is a multi-protein complex consisting of a pattern recognition receptor (NLRP3), an adaptor (ASC) and caspase 1. It is a critical integrator of innate host defenses in myeloid and non-myeloid cells by promoting the maturation of proinflammatory cytokines such as interleukin 1beta and 18. To date, no study has been reported regarding the in vivo role of NLRP3 inflammasome in urinary tract infection (UTI) caused by uropathogenic E. coli (UPEC). METHODS: Experimental bladder infection was elicited in wildtype female mice through transurethral inoculation of clinical cystitis isolates, UTI89 and NU14. Formation of speck in urothelium, urine IL 1beta and cleaved caspase 1 in urothelium, hallmarks of NLRP3 inflammasome activation, were determined using immunofluorescence staining, ELISA and Western blotting, respectively. Studies were extended into NLRP3 and caspase 1 knockout (KO) mice, using UPEC strains as well as E. coli strains isolated from human asymptomatic bacteriuria (ABU; ABU83972 and ABU35) and recurrent UTI (C055 and C001). Additionally, Toll-like receptor 4 (TLR4) KO mice were analyzed to ascertain whether TLR4 status affects NLRP3 inflammasome activation. RESULTS: Transurethral inoculation of UPEC strains UTI89 and NU14, but not laboratory strain AAEC191A or FimH-deletion strain NU14-1, led to urothelial speck formation, increased urine IL 1beta secretion and urothelial pro-caspase 1 cleavage. These three NLRP3 inflammasome activation markers peaked by 6 hours and subsided by 12 hours after UPEC inoculation. Such responses were significantly blunted in NLRP3 KO mice and even more so in caspase 1 KO mice. The decreased NLRP3 inflammasome response in these
Vol. 195, No. 4S, Supplement, Saturday, May 7, 2016
KO mice inversely correlated with significantly increased UPEC retention in the bladder. Whereas the two ABU strains elicited only 1/5 of urinary IL 1beta in wild-type mice, compared with the UPEC strains, the two strains from recurrent UTI elicited as much IL 1beta response as the two cystitis strains. Finally, markedly decreased IL 1beta production and secretion by urothelium was observed in TLR4 KO mice compared with wild-type mice, suggesting that lipopolysaccharide/ TLR4 interaction is crucial for NLRP3 inflammasome activation in the urothelium. CONCLUSIONS: Our data demonstrate for the first time that UPEC can activate inflammasomes in the urothelial cells via the NLRP3 pathway and suggest that reduced activity of this signaling pathway may predispose the urinary tract to recurrent infections by uropathogenic E. coli. Source of Funding: NIH
MP24-07 MULTINUCLEATED UROTHELIAL UMBRELLA CELLS AS INITIATORS OF INNATE HOST DEFENSES AGAINST UROPATHOGENIC E. COLI Yan Liu, feng he, Ellen Shapiro, Herbert Lepor, Xue-Ru Wu*, New York, NY INTRODUCTION AND OBJECTIVES: Mammalian urothelial surface is covered by highly differentiated and morphologically distinct superficial cells, also named umbrella cells. These cells contain multiple nuclei but, with the exception of a role in permeability barrier, no other function has been attributed to these specialized cells. This study was undertaken to explore the in vivo role of urothelial umbrella cells in innate host defenses against uropathogenic E. coli (UPEC). METHODS: Female mice (7-9 weeks) were inoculated via the transurethral route with type 1 piliated UPEC strains (UTI89, NU14, CFT073), isogenic mutant strains lacking FimH adhesin (UTI89-1, NU14-1) or expressing a mutated FimH (UTI89-Q133N), or a nonUPEC laboratory strain (HB101). Translocation of p65 subunit of NFkB from cytoplasm to nucleus, an indicator of NF-kB activation, was assessed by confocal immunofluorescent microscopy of cross sections and whole-mount bladders. Production and urinary secretion of proinflammatory cytokines by urothelial cells was determined by multiplex ELISA. The effects of TLR4 status on p65 translocation were determined using TLR4 mutant mice and isogenic control mice. RESULTS: Inoculation of mouse bladders with UPEC induced rapid translocation of p65 from the cytoplasm into the nucleus, peaking at 1 hour and diminishing after 6 hours post-inoculation. The nuclear translocation of p65 was confined to the umbrella cells, sparing intermediate and basal cells of the urothelium completely. When one nucleus had nuclear p65, all other nuclei (from 2-10) within a cell also did so, indicating a highly synchronous nature of NF-kB activation in multinucleated umbrella cells. The p65 nuclear translocation depended on urothelial adhesion of UPEC, because nonUPEC laboratory strain and mutated UPEC strains lacking FimH or expressing an adhesion-deficient FimH mutant or UPEC strains in the presence of D-mannose failed to adhere to urothelial surface and failed to trigger p65 nuclear translocation. The p65 nuclear translocation coincided with a marked increase of urothelial and urine proinflammatory cytokines (TNF-alpha, IL-1alpha and IL-1beta). Finally, TLR mutant mice had 3 times less p65 nuclear translocation than the isogenic controls. CONCLUSIONS: These results represent the first demonstration that the umbrella cells of the urothelium play a critical role in triggering innate host defenses against UPEC, and suggest that the multiple nucleation of these cells serves an important function in amplifying mRNAs encoding proinflammatory signals to combat invading pathogens. Source of Funding: NIH