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MP20-03 NLRP3 INFLAMMASOME PLAYS A KEY ROLE IN UROTHELIAL DEFENSE AGAINST UROPATHOGENIC E. COLI
THE JOURNAL OF UROLOGYâ
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CONCLUSIONS: Infection and secreted factors induced by infection broadly and persistently alter p16INK4A downregulation, alongside DNMT and EZH2 upregulation. DNMT3B is regulated by LPS alone, while EZH2 and DNMT1 are upregulated by both exosomes and CM from infected cells. Dysregulation of epigenetic machinery by secreted factors may have implications in urothelial maintenance and defence. Source of Funding: CIHR catalyst funding
MP20-03 NLRP3 INFLAMMASOME PLAYS A KEY ROLE IN UROTHELIAL DEFENSE AGAINST UROPATHOGENIC E. COLI Feng He, Yan Liu, Ellen Shapiro, Herbert Lepor, Xue-Ru Wu*, New York, NY INTRODUCTION AND OBJECTIVES: Inflammasomes are relatively recently discovered multi-protein complexes that function to activate proinflammatory cytokines particularly interleukin (IL) 1b and 18 with which to attack pathogens and cause pyroptosis. Initially found in myeloid cells, inflammasomes are also present and operative in certain epithelial cells. Whether inflammasomes are involved in innate immunity of urothelium against uropathgenic E. coli (UPEC) is presently unknown and a subject of this study. METHODS: Primary cultured normal human urothelium and bladder cancer cell lines (UroTsa, RT4 and T24) were surveyed by RTPCR and Western blotting for the expression of the receptors of major inflammasomes (NLRP1, NLRC4, NLRP3 and AIM2). NLRP3 was chosen for additional studies because of its reproducible, high-level expression in urothelial cells. The cultured cells were exposed to NLRP3 ligands (lipopolysaccharide, flagellin and ATP) and to UPEC strains and controls (UTI89, CFT073, NU14 and NU14-1). Activation status of NLRP3 was assessed by ELISA and Western blotting of secreted IL-1b and IL-18, cleavage of pro-caspase-1 and lactate dehydrogenase (LDH) release (marker for pyroptosis). Cultured cells also underwent NLRP3 knockdown or were treated with an NLRP3 inhibitor (glybenclamide) and a caspase-1 inhibitor and their responses to UPEC assessed using ELISA of IL-1b and IL-18. RESULTS: Urothelial cells responded robustly to LPS, flagellin and ATP in a dose-dependent manner by secreting IL-1b and IL-18 and converting pro-caspase 1 to active caspase 1, forming specks of adaptor molecule ASC and releasing LDH, all of which are hallmarks of NLRP3-mediated inflammasome activation. Type 1-piliated UPEC strains (UTI89, CFT073 and NU14) but not the non-type 1-piliated control (NU14-1), also induced rapid activation of NLRP3, peaking at 4 h post-infection. Specific knockdown of NLRP3 by shRNA or addition of NLRP3 inhibitor glybenclamide or caspase-1 inhibitor-I significantly blunted UPEC-induced inflammasome activation. Finally, co-immunoprecipitation (co-IP) and reverse co-IP showed direct binding between NLRP3 and its adaptor molecule ASC in LPS- and UTI89-treated cells but not in untreated controls, further establishing NLRP3 inflammasome activation by UPEC. CONCLUSIONS: Our data demonstrate for the first time that UPEC can activate inflammasomes in the urothelial cells via the NLRP3 pathway and suggest that this activation may play an important role in innate urothelial defenses during urinary tract infection. Source of Funding: NIH
MP20-04 DNA DAMAGE REPAIR GENES REGULATE BLADDER INFLAMMATION Christopher Dru*, Subhash Haldar, Neil Bhowmick, Los Angeles, CA INTRODUCTION AND OBJECTIVES: Chemical cystitis is a prevalent and bothersome inflammatory bladder condition characterized by dysuria and associated with cyclophosphamide and radiation
Vol. 193, No. 4S, Supplement, Saturday, May 16, 2015
therapy. It can progress to life-threatening gross hematuria and ulceration. The bladder mounts a multi-factorial immune response involving various pro- and anti- inflammatory pathways to mediate acute and chronic inflammation. The goal of this project was to determine the mechanisms underlying detrusor smooth muscle responsiveness caused by cyclophosphamide and identify the role of nicotinamide, a nutraceutical well-known for its anti-inflammatory effects, in inflammation attenuation. METHODS: Ovariectomized C57Bl/6 mice received daily intraperitoneal injections of nicotinamide or placebo followed by administration of cyclophosphamide to induce chemical cystitis. Additionally, mouse bladder fibroblasts were cultured in the presence or absence of nicotinamide before induction of inflammation with acrolein. Tissues and cells were collected to determine the degree of biochemical and histological inflammation as well as changes in gene expression and cell death. RESULTS: All mice and cultured cells pre-treated with nicotinamide before administration of cyclophosphamide or acrolein demonstrated a histological and biochemical decrease in inflammation. Nicotinamide increased DNA damage repair gene expression of Neil-1, Parp-1, OGG-1, and RAD-50 to reduce inflammation. FACS analysis using markers for cellular apoptosis demonstrated higher levels of cell survival (74% v 17%) when nicotinamide was added to cell-culture media before addition of acrolein. CONCLUSIONS: Nicotinamide is an important regulator of inflammation and prevention of cell death in both mice treated with cyclophosphamide and cultured mouse bladder fibroblasts. This is evidenced by increased expression of DNA damage repair genes and alterations in cytokine regulation to protect the urothelium from reactive hyperplasia and ulceration. We propose that nicotinamide reduces inflammation through preservation of DNA integrity by maintaining physiologic levels of the DNA damage repair genes Neil-1, Parp-1, OGG-1, and RAD-50. These findings have future implications for the treatment of chemotherapy-induced cystitis as well as in the treatment of other types of inflammation.
Source of Funding: None
MP20-05 BLADDER INSTILLATION THEARPY OF MESENCYMAL STEM CELL AND THE ADDITION OF MANNOSE IN A RAT CYSTITIS MODEL- MULTIPLE SUPPRESSIVE EFFECTS FOR EXCESS CYTOKINES Tokunori Yamamoto*, Yasuto Funahashi, Majima Tsuyoshi, Takai Syun, Yoshihisa Matsukawa, Hideki Mizuno, Momokazu Gotoh, Nagoya, Japan INTRODUCTION AND OBJECTIVES: Mesencymal stem cell(MSC) is used as a clinical regeneration therapeutic agent for various organs. We examined the healing effects of intravesical MSC on damaged urothelium in a rat model of chemically induced cystitis. METHODS: Rat(r) and human(h)-MSC instillation in the bladder of female Sprague Dawleyâ and nude rats to cyclophosphamide induce cystitis respectively. After 8 hour r-MSC(n¼6) ,r and h MSC with mannose(MN) (n¼6),vehicle(n¼6) were administered in the bladder for 48 hour. Histopathology, urothelial permeability, cystometrogram and nociceptive behaviors were evaluated on day 2. Bladder inflammation were evaluated the cytokaines of bladder urine and tissue measured by multipulex system and urine myeloperoxidase(MPO) measured by ELISA.
e224
CONCLUSIONS: Infection and secreted factors induced by infection broadly and persistently alter p16INK4A downregulation, alongside DNMT and EZH2 upregulation. DNMT3B is regulated by LPS alone, while EZH2 and DNMT1 are upregulated by both exosomes and CM from infected cells. Dysregulation of epigenetic machinery by secreted factors may have implications in urothelial maintenance and defence. Source of Funding: CIHR catalyst funding
MP20-03 NLRP3 INFLAMMASOME PLAYS A KEY ROLE IN UROTHELIAL DEFENSE AGAINST UROPATHOGENIC E. COLI Feng He, Yan Liu, Ellen Shapiro, Herbert Lepor, Xue-Ru Wu*, New York, NY INTRODUCTION AND OBJECTIVES: Inflammasomes are relatively recently discovered multi-protein complexes that function to activate proinflammatory cytokines particularly interleukin (IL) 1b and 18 with which to attack pathogens and cause pyroptosis. Initially found in myeloid cells, inflammasomes are also present and operative in certain epithelial cells. Whether inflammasomes are involved in innate immunity of urothelium against uropathgenic E. coli (UPEC) is presently unknown and a subject of this study. METHODS: Primary cultured normal human urothelium and bladder cancer cell lines (UroTsa, RT4 and T24) were surveyed by RTPCR and Western blotting for the expression of the receptors of major inflammasomes (NLRP1, NLRC4, NLRP3 and AIM2). NLRP3 was chosen for additional studies because of its reproducible, high-level expression in urothelial cells. The cultured cells were exposed to NLRP3 ligands (lipopolysaccharide, flagellin and ATP) and to UPEC strains and controls (UTI89, CFT073, NU14 and NU14-1). Activation status of NLRP3 was assessed by ELISA and Western blotting of secreted IL-1b and IL-18, cleavage of pro-caspase-1 and lactate dehydrogenase (LDH) release (marker for pyroptosis). Cultured cells also underwent NLRP3 knockdown or were treated with an NLRP3 inhibitor (glybenclamide) and a caspase-1 inhibitor and their responses to UPEC assessed using ELISA of IL-1b and IL-18. RESULTS: Urothelial cells responded robustly to LPS, flagellin and ATP in a dose-dependent manner by secreting IL-1b and IL-18 and converting pro-caspase 1 to active caspase 1, forming specks of adaptor molecule ASC and releasing LDH, all of which are hallmarks of NLRP3-mediated inflammasome activation. Type 1-piliated UPEC strains (UTI89, CFT073 and NU14) but not the non-type 1-piliated control (NU14-1), also induced rapid activation of NLRP3, peaking at 4 h post-infection. Specific knockdown of NLRP3 by shRNA or addition of NLRP3 inhibitor glybenclamide or caspase-1 inhibitor-I significantly blunted UPEC-induced inflammasome activation. Finally, co-immunoprecipitation (co-IP) and reverse co-IP showed direct binding between NLRP3 and its adaptor molecule ASC in LPS- and UTI89-treated cells but not in untreated controls, further establishing NLRP3 inflammasome activation by UPEC. CONCLUSIONS: Our data demonstrate for the first time that UPEC can activate inflammasomes in the urothelial cells via the NLRP3 pathway and suggest that this activation may play an important role in innate urothelial defenses during urinary tract infection. Source of Funding: NIH
MP20-04 DNA DAMAGE REPAIR GENES REGULATE BLADDER INFLAMMATION Christopher Dru*, Subhash Haldar, Neil Bhowmick, Los Angeles, CA INTRODUCTION AND OBJECTIVES: Chemical cystitis is a prevalent and bothersome inflammatory bladder condition characterized by dysuria and associated with cyclophosphamide and radiation
Vol. 193, No. 4S, Supplement, Saturday, May 16, 2015
therapy. It can progress to life-threatening gross hematuria and ulceration. The bladder mounts a multi-factorial immune response involving various pro- and anti- inflammatory pathways to mediate acute and chronic inflammation. The goal of this project was to determine the mechanisms underlying detrusor smooth muscle responsiveness caused by cyclophosphamide and identify the role of nicotinamide, a nutraceutical well-known for its anti-inflammatory effects, in inflammation attenuation. METHODS: Ovariectomized C57Bl/6 mice received daily intraperitoneal injections of nicotinamide or placebo followed by administration of cyclophosphamide to induce chemical cystitis. Additionally, mouse bladder fibroblasts were cultured in the presence or absence of nicotinamide before induction of inflammation with acrolein. Tissues and cells were collected to determine the degree of biochemical and histological inflammation as well as changes in gene expression and cell death. RESULTS: All mice and cultured cells pre-treated with nicotinamide before administration of cyclophosphamide or acrolein demonstrated a histological and biochemical decrease in inflammation. Nicotinamide increased DNA damage repair gene expression of Neil-1, Parp-1, OGG-1, and RAD-50 to reduce inflammation. FACS analysis using markers for cellular apoptosis demonstrated higher levels of cell survival (74% v 17%) when nicotinamide was added to cell-culture media before addition of acrolein. CONCLUSIONS: Nicotinamide is an important regulator of inflammation and prevention of cell death in both mice treated with cyclophosphamide and cultured mouse bladder fibroblasts. This is evidenced by increased expression of DNA damage repair genes and alterations in cytokine regulation to protect the urothelium from reactive hyperplasia and ulceration. We propose that nicotinamide reduces inflammation through preservation of DNA integrity by maintaining physiologic levels of the DNA damage repair genes Neil-1, Parp-1, OGG-1, and RAD-50. These findings have future implications for the treatment of chemotherapy-induced cystitis as well as in the treatment of other types of inflammation.
Source of Funding: None
MP20-05 BLADDER INSTILLATION THEARPY OF MESENCYMAL STEM CELL AND THE ADDITION OF MANNOSE IN A RAT CYSTITIS MODEL- MULTIPLE SUPPRESSIVE EFFECTS FOR EXCESS CYTOKINES Tokunori Yamamoto*, Yasuto Funahashi, Majima Tsuyoshi, Takai Syun, Yoshihisa Matsukawa, Hideki Mizuno, Momokazu Gotoh, Nagoya, Japan INTRODUCTION AND OBJECTIVES: Mesencymal stem cell(MSC) is used as a clinical regeneration therapeutic agent for various organs. We examined the healing effects of intravesical MSC on damaged urothelium in a rat model of chemically induced cystitis. METHODS: Rat(r) and human(h)-MSC instillation in the bladder of female Sprague Dawleyâ and nude rats to cyclophosphamide induce cystitis respectively. After 8 hour r-MSC(n¼6) ,r and h MSC with mannose(MN) (n¼6),vehicle(n¼6) were administered in the bladder for 48 hour. Histopathology, urothelial permeability, cystometrogram and nociceptive behaviors were evaluated on day 2. Bladder inflammation were evaluated the cytokaines of bladder urine and tissue measured by multipulex system and urine myeloperoxidase(MPO) measured by ELISA.