DIABETIC CONDITIONS IMPAIR NEUTROPHIL RECRUITMENT INTO THE UROTHELIUM AND MARKEDLY DELAY THE CLEARANCE OF INTRACELLULAR BACTERIAL COMMUNITY OF UROPATHOGENIC E. COLI

INTRODUCTION AND OBJECTIVES: Hemorrhagic cystitis is the severe clinical manifestation of several systemic chemotherapeutics, most notably cyclophosphamide (CPX), and other nitrogen mustard alkylating agents. 8-oxoguanine DNA glycosylase (Ogg1) is a base excision repair enzyme that edits oxidized nucleotide residues and is essential for initiation of the pyroptotic cell death pathways. We investigated the role of this enzymatic pathway in hemorrhagic cystitis to identify any targets for potential future therapies. METHODS: Several models, including an in-vitro mouse bladder fibroblast model, an in-vivo mouse model, and a cellular Ogg1 knockout model, were treated with either cyclophosphamide or acrolein to induce bladder inflammation and hemorrhagic cystitis. Nicotinamide was also added to treatment groups to limit DNA methylation. Cultured bladder fibroblasts and harvested mouse bladders tissues and cells were subjected to bisulfite sequencing and chromatin immunoprecipitation (ChIP) analysis to evaluate expression of Ogg1 and DNA methylation patterns. RESULTS: We found that Ogg1 was down-regulated with subsequent accumulationof 8-oxoguanineandactivationofNLRP3-mediated pyroptotic cell death signaling pathways when inflammation was induced with cyclophosphamide and acrolein. In the attached graphic, differences incellularadhesionandcell deathareevident in thephasecontrast images and histologic examination of mouse bladder tissues demonstrates characteristic detrusor hypertrophy in response to cyclophosphamide. In bladder fibroblasts treated with acrolein, bisulfite sequence analysis demonstratedCpGmethylation onOgg1 geneRegion III and transcription silencing. Additionally, recruitment of DNMT3b in acrolein-treated cells initiated de novo methylation in ChIP analysis. Knockout of Ogg1 in bladder fibroblasts activated signal I and II in pyroptosis. CONCLUSIONS: Epigenetic regulation of Ogg1 is essential in the pyroptosis cellular pathway in hemorrhagic cystitis. The pyroptosis signaling cascade in detrusor cells and the role of Ogg1 suggest the potential for a novel therapeutic target for prevention and treatment of this highly morbid condition.