MP20-02 CONDITIONED MEDIA FROM INFECTED UROTHELIAL CELLS CONTAIN EXOSOMES WITH EPIGENETIC POTENTIAL.

Abstract

You have accessJournal of UrologyInfections/Inflammation of the Genitourinary Tract: Kidney & Bladder II1 Apr 2015MP20-02 CONDITIONED MEDIA FROM INFECTED UROTHELIAL CELLS CONTAIN EXOSOMES WITH EPIGENETIC POTENTIAL. Kenneth Ting, Alaleh Samiei, Karen J. Aitken, Bryce Weber, Fadi Ibrahim, Akshita Kapila, Cornelia Tolg, and Darius Bagli Kenneth TingKenneth Ting More articles by this author , Alaleh SamieiAlaleh Samiei More articles by this author , Karen J. AitkenKaren J. Aitken More articles by this author , Bryce WeberBryce Weber More articles by this author , Fadi IbrahimFadi Ibrahim More articles by this author , Akshita KapilaAkshita Kapila More articles by this author , Cornelia TolgCornelia Tolg More articles by this author , and Darius BagliDarius Bagli More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.974AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Urinary tract infection (UTI) is the second most common infection in children, and results in recurrent/chronic infection in 25–40% of patients of all ages. Chronic in vitro infection of urothelial cell lines leads to increased nuclear localization of DNA methyltransferase 1 (DNMT1) and decreased p16 expression along with a rise in CpG methylation near the first exon (Lab Invest 91:825-36). We noted that uninfected cells neighbouring infected cells upregulated DNMT1. Our objective here is to study if infection-induced secreted factors (e.g. LPS, conditioned media and exosomes) alter the regulation of epigenetic writers (DNMT 1, 3A and 3B, and EZH2) and p16 in naive cells. METHODS HTB9 or normal primary urothelial cells were innoculated with 2 moi of UT189 Escherichia coli, or FimH+/- derivatives or vehicle for 2 hrs. Cells were washed in PBS+50 ug/mL gentamycin and maintained in RPMI+10 ug/mL gentamycin+5% serum. Exosome-free serum was used for exosome isolations. Conditioned media (CM) was collected every 2 days, by centrifugation of cell debris and passing through 0.2 micron filters. Exosomes were isolated from CM using Exoquick TC, and characterized by Nanosight + transmission EM. Factors secreted by host cells (CM at 50% of total media, exosomes at a ratio of 1/2 of starting CM volume and LPS (200 ng/mL)), were added to naive urothelial cells for 2 hours, or 1, 2, 6 days. mRNA of DNMT1, 3a and 3b was examined by QPCR in response to LPS and CM. p16, DNMTs and EZH2 were localized by immunofluorescence. RESULTS CM significantly increased all mRNA expression of DNMT1,3A and 3B at more than one timepoint post-infection (p<0.001). DNMT1, but not DNMT3B, localized to the nucleus during treatment with CM from infected cells, concurrent with p16 protein downregulation. LPS increased DNMTs by immunofluorescence (p<0.0001), yet upregulated mRNA of DNMTs only at discrete time points. Nuclear EZH2 was increased by infection, infection-induced CM and infection-induced exosomes, in primary and HTB9 cells (p<0.005). Exosomes from control cells had the opposite effect, leading to downregulation of EZH2 at day 2. DNMT1 but not DNMT3B was increased by infection-induced exosomes (p<0.005). 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. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e223-e224 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kenneth Ting More articles by this author Alaleh Samiei More articles by this author Karen J. Aitken More articles by this author Bryce Weber More articles by this author Fadi Ibrahim More articles by this author Akshita Kapila More articles by this author Cornelia Tolg More articles by this author Darius Bagli More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...