A230 DEATH, MITOCHONDRIA, AND INFLAMMATION IN E. COLI-LF82 INFECTION

Abstract Background The Crohn’s disease-associated adherent invasive E. coli (AIEC) (strain LF82) decreases mitochondrial membrane potential and fragments the epithelial mitochondrial network. Other groups have also reported activation of NF-κB-mediated inflammation and generation of reactive oxygen species in AIEC-LF82 infected epithelia. It remains unclear how these findings integrate over the course of AIEC-LF82 infection and how they influence each other over the course of infection. Aims To characterize the sequence of events following AIEC-LF82 infection in a non-cancer derived epithelial cell model focusing on mitochondrial disruption, cell death, mtDNA release, and inflammatory gene expression. Methods The fetal derived human colon epithelial cell line (FHC) were infected with E. coli-LF82 or the commensal E. coli-HB101 at multiplicity of infection (MOI) of 100 or uninfected over the course of various experiments. FHCs were stained with SYTOX green and imaged on a CellCyte X system for 24h to measure cell death or stained with MitoTracker Deep Red and imaged on a spinning disc-confocal microscope for 16h to assess mitochondrial morphology. Also, in fixed-cell preparations mitochondrial networks were imaged by staining with anti-TOMM20 antibodies. Imaging of cytosolic DNA was performed via immunofluorescent staining of mtDNA and the mitochondrial network. As markers of inflammation, CXCL10 and IL-6 mRNA were assessed via qRT-PCR. Results Epithelia infected with E. coli-LF82 but not E. coli-HB101 displayed dramatic fragmentation of their mitochondrial network at 6h (p < 0.0001) (n=6), while SYTOX green imaging revealed significant cell death beginning at 9h (p < 0.001) (n=3). Furthermore, our preliminary data indicates that transcription of inflammatory cytokines including CXCL10, and IL-6 occurs at 8h while cytosolic escape of mtDNA occurs at 6h, after mitochondrial network disruption, but prior to cell death. Conclusions The data begins to unravel the sequence of events in a non-cancer derived epithelium after infection with E. coli-LF82, integrating mitochondrial function, inflammatory gene expression, and cell viability and providing a more fulsome understanding of the course of E. coli-LF82 infection. Together, these data advance understanding of relationship between mitochondrial fragmentation and cell death in the context of a Crohn’s disease-relevant pathobiont, AIEC-LF82. Funding Agencies TRIANGLE