Cytotoxic Necrotizing Factor 1 Downregulates CD36 Transcription In Macrophages To Induce Inflammation During Acute Urinary Tract Infections

Urinary tract infections(UTIs)caused by uropathogenic Escherichia coli(UPEC)induce cystitis,pyelonephritis,and can cause kidney scarring and failure if inflammation is not under control.The discovery of the major virulence factor of uropathogenic Escherichia coli(UPEC)and the investigation of the pathogenic mechanism of urinary tract infections are particularly important for the prevention and treatment of urinary tract infections.The detailed effects of cytotoxic necrotizing factor 1(CNF1),the key UPEC toxin,on pathogenicity of UPEC remain unclear.Innate immune response is the main way for the host to effectively resist UPEC infection,in addition,neutrophils and macrophages play a major role in acute urinary tract infections for bacterial clearance.However,the natural immune response must be effectively regulated,otherwise it will cause serious tissue damage.CD36 is one of the important scavenger receptors,responsible for clearance of pathogens and apoptotic cells in macrophages,and therefore plays an important role in host immune defense and homeostasis.Regulation of CD36 by bacterial toxins has not been reported.This paper intends to solve the following three problems: firstly,systematic study the effects of CNF1 on bacterial survival and tissue inflammatory lesions in acute urinary tract infections;secondly,to study the effect of CNF1 on the expression and transcription of macrophage CD36;lastly,to explore the molecular regulation mechanism of CNF1 on CD36.Methods:1.The cnf1 gene was expressed in UPEC strain CFT073 and the constitutive expression strain of CNF1(CFT073-CNF1,CFT073-vector used as a control)or the cnf1 gene was knocked out in UPEC strain UTI89 to construct the deletion strain of CNF1(?cnf1,wild UTI89 used as a control).We used a mouse model of pyelonephritis and caused mouse urinary tract infections.The mice were sacrificed at different times and the kidney and bladder tissues of the mice were homogenized.The number of tissue bacteria was counted by serial dilution plating.2.The numbers of neutrophils and macrophages in the kidney and bladder of mice were detected by flow cytometry.The neutrophils were labeled with CD11 b and Ly6 G.Mouse kidney and bladder tissue paraffin-embedded sections were taken and H&E staining was used to observe tissue damage,edema and inflammatory cell infiltration.3.Immunofluorescence staining was performed on frozen sections of kidney and bladder of mice to detect CD36 expression level of macrophages and phagocytosis UPEC of macrophages and neutrophils.4.The CNF1 expression plasmid was constructed and CNF1 protein was purified by nickel column method.We used flow cytometry,immunofluorescence microscopy and confocal microscopy to detect CNF1 treated RAW264.7 macrophages,THP1-differentiated macrophages,peritoneal macrophages and BMDM macrophages phagocytosis of bacteria and fluorescent-labeled latex beads.5.Western blotting,real-time fluorescence quantitative PCR,flow cytometry and confocal microscopy were performed to detect CD36 expression of CNF1 treated macrophages.6.We constructed CD36 knockout shRNA,packaged lentivirus and transfered to RAW264.7 cells.The CD36 knockout macrophages were selected by Puromycin.CD36 overexpression pcDH-MSCV-MCS-EF1-copGFP vector was constructed,the lentivirus was packaged and then transfected into RAW264.7 cells.RAW264.7 cells stably expressing CD36 were screened by flow cytometry.RAW264.7 was treated with different concentrations of CD36 inhibitor(SAB)and the effec t on phagocytosis was examined using confocal microscopy.7.Western blotting,qRT-PCR and confocal microscopy were used to detect the expression of LXR? and C/EBP? in CNF1 treated macrophages.8.We used CHIP-qPCR to detect whether CNF1 influences the binding of LXR? and C/EBP? to the CD36 promoter.9.We performed western blotting assay to detect whether CNF1 protein enters the cell and Pull downing assay to detect whether CNF1 influences RhoA,Cdc42 and Rac1 activation.10.The effects of Cdc42 inhibitor,RhoA inhibitor and Rac1 inhibitor on CD36,LXR? and C/EBP? expression in macrophages were detected by western blotting,qRT-PCR and confocal microscopy.Results:1.Mouse urinary tract infection CFT073-CNF1 significantly inhibited bacterial clearance in bladder and kidney tissues compared to the control group(CFT073-vehicle);mouse urinary tract infection ?cnf1 significantly promoted bladder and kidney removal of bacteria in the tissue compared to the control group(wild UTI89).2.Mice infected with CFT073-CNF1 significantly increased neutrophils infiltration relative to the control group;Mice infected with ?cnf1 decreased neutrophils infiltration relative to the control group.More infiltrated neutrophils,serious edema and urothelial damage were identified in bladder and kidney tissues infected with CNF1-expressing CFT073 by H&E staining.3.CNF1-expressing CFT073 inhibited CD36 expression in murine macrophages and inhibited macrophage phagocytosis.4.CNF1 reduced macrophage nonopsonic phagocytosis of UPEC in vitro.5.CNF1 decreased CD36 expression in macrophages.6.Downregulation of CD36 inhibited nonopsonic macrophage phagocytosis.7.CNF1 decreased CD36 transcription via impacting C/EBP? and LXR? expression.8.CNF1 impacted LXR? and C/EBP? recruitment to the CD36 promotor.9.CNF1 can enter and translocates into cytoplasm,and activated the three Rho GTPases including RhoA,Cdc42 and Rac1.10.CNF1 impacted LXR? and CD36 expression through activating Cdc42.Conclusion:1.The CNF1 expression bacteria caused urinary tract infection in mice by using a Pyelonephritis Mouse Model.The results showed that CNF1 expressing bacteria inhibited urinary tract bacterial clearance,promoted inflammation,and aggravated tissue damage.2.CD36 functions as macrophage surface scavenger receptor,mainly mediating phagocytosis pathogens and apoptotic neutrophils.We found that both in vitro and in vivo experiments CNF1 reduced the expression of macrop hage surface receptor CD36,thus inhibited the clearance of UPEC and neutrophils.3.We found that CNF1 inhibits the transcription of CD36 by decreasing the expression of its upstream transcription factors LXR? and C/EBP? and inhibiting the transcription factor binding to the CD36 promoter.4.CNF1 activated RhoA,Cdc42 and Rac1 in macrophages,and Cdc42 was found involved in CNF1-mediated downregulation of LXR?.