The Role And Mechanism Of Bladder Urothelial Cell Pyroptosis In Urinary Tract Infection

BackgroundUrinary tract infections(UTIs)are among the most common bacterial infections with high recurrent rates.The epidemiological studies suggest that UTIs affect more than 100 million people annually worldwide.The high incidence of UTIs places a huge burden on health and the economy,Approximately 50%of women experience at least one UTIs during their lifetime and 25%of women who have experienced UTIs will contract one or more recurrent infections within 6 months after the initial infection.The majority of UTIs are caused by Uropathogenic Escherichia coli(UPEC).UPEC adheres to and invades bladder epithelial cells and thereby trigger pathological changes in the urinary tract.Because antibiotic resistance rates are increasing,effective treatment of UTIs is becoming more challengingPyroptosis is a unique form of programmed cell death that relies on cytosolic inflammasome activation.The nucleotide-binding domain-like receptor protein 3(NLRP3)inflammasome is a cytosolic receptor that performs a crucial function in innate-immunity-related sensing.Activation of NLRP3 leads to the maturation of caspase-1.Active caspase-1 is a cysteine-dependent protease that converts pro-interleukin(IL)-1? and pro-IL-18 to their biologically active mature forms:cytokines IL-? and IL-18.Recently,growing evidence showed that inflammasome-mediated pyroptosis is involved in the host response to bacterial infection.Mast cells are multifunctional immune cells and are replete with proteases that can maintain and regulate immune function and may disorder urothelial barrier function.During inflammation,mast cells mediate an increase in mucosal permeability by unknown mechanisms.Dysfunction of urothelial barrier is significantly associated with a variety of bladder diseases,including UTIs.Recent studies have highlighted the role of mast cells in the host's response to UPEC,so the purpose of this study is to explore the role of bladder urothelial cells pyroptosis and the interaction between urothelial cells and mast cells during UPEC infection,and to investigate the effect of this interaction on bladder urothelial barrier function.Methods6-8 weeks C57BL/6J female mice were infected with urinary pathogenic Escherichia coli CFT073 for 24 hours,western blot and immunohistochemical staining were used to analyze the expression of pyroptosis-related proteins.The caspase-1 activity kit was used to detect caspase-1 activity;CFT073 was used to stimulate bladder urothelial cells in vitro,Western blot and ELISA were used to detect the expression of pyroptosis-related proteins.The caspase-1 activity kit was used to detect caspase-1 activity.C57BL/6J female mice were given caspase-1 inhibition VX-765 and the bladder was perfused with a urinary pathogenic E.coli CFT073 suspension for 24 hours,followed by tissue homogenization and bacterial culture to observe the number of colony-forming units(CFUs);paraffin-embedded bladder tissue was sectioned by H&E staining and immunofluorescence staining to observe bacteria invasion and integrity of the bladder urothelial barrier;in vitro,we verified the effects of IL-1? and IL-18 released by bladder urothelial cells on mast cells;Western blot and immunofluorescence to detect the effect of tryptase released by mast cells on the bladder epithelial barrier function.Next,C57BL/6J female mouse were,treated with PAR2 inhibitor FSLLRY-NH2 to inhibit the tryptase-PAR2 axis,CFUs was observed after tissue homogenization.H&E staining and immunofluorescence staining were used to observe bacterial invasion and integrity of bladder urothelial barrier.ResultsWestern blot and caspase-1 activity test results shown that the infection of CFT073 activated NLRP3 inflammasome and pyroptosis of bladder urothelial cells in vivo and in vitro.Through H&E staining and immunofluorescence staining,we found that inhibition of caspase-1 can reduce the bladder bacterial load and reduce the bacterial damage to the bladder urothelial barrier function.In addition,the inhibition of pyroptosis reduced the accumulation of mast cells in the bladder tissue.In order to investigate the interaction between bladder urothelial cells and mast cells,through vitro experiments,we found that IL-1? and IL-18 released by bladder urothelial cells induced the migration of mast cells.Tryptase which released by mast cell can bind to the PAR2 receptor of the bladder urothelium and cause the degradation of cell junction proteins ZO-1 and E-cadherin,leading to the destruction of the bladder urothelial barrier function;H&E staining and immunofluorescence staining show that inhibition of the tryptase-PAR2 axis can reduce the disruption of bladder urothelial barrier function and decrease the progression of cystitis.ConclusionThe pyroptosis of bladder urothelial cells and mast cells cause disruption of bladder urothelial barrier function,and plays an important role in the occurrence and development of cystitis.The role of pyroptosis is a potential target for the treatment of bacterial cystitis.