A155 THE IMMUNOMODULATOR, LEFLUNOMIDE, REDUCES MITOCHONDRIAL FRAGMENTATION CAUSED BY ADHERENT–INVASIVE E. COLI AND PERSISTENCE OF THE PATHOBIONT WITHIN ENTERIC EPITHELIA

Abstract

Background

Mitochondria can exist as a dynamic network that constantly remodels via the processes of fission and fusion (mitochondrial dynamics) to meet cellular energy demands and allow recycling of damaged mitochondria. Mitochondrial dysfunction can occur in inflammatory bowel disease. Adherent-invasive E. coli (AIEC) are a putative etiological agent in Crohn’s disease. It is unknown if infection with AIEC affects mitochondrial dynamics in intestinal epithelial cells. It was recently discovered that leflunomide, an FDA approved anti-inflammatory and antirheumatic drug, promotes mitochondrial fusion.

Aims

(1) Explore the relationship between mitochondrial dynamics and epithelial function in vitro and determine if, then how, infection with AIEC affects mitochondrial dynamics and any consequence for permeability. (2) Determine if leflunomide blocks any of the effects of AIEC on epithelial function.

Methods

Human colon-derived epithelial lines were cultured with non-invasive E. coli as a control, viable AIEC (LF82: 4h 10⁸ CFU/mL or 16h 10⁴ CFU/mL), fixed (dead) AIEC, spent medium from AIEC cultures ± co-treatment with inhibitors of mitochondrial fission (i.e. P110, Mdivi1) or leflunomide. Epithelia were examined: (a) electron microscopy; (b) ATP; (c) live-cell imaging of mitochondria morphology and membrane potential; (d) immunoblotting for Dynamin-Related Peptide-1 (Drp1); (e) barrier function; (f) cytochrome c release from mitochondria, and (g) intracellular AIEC.

Results

Infection with AIEC resulted in reduced mitochondrial membrane potential and ATP levels, and dramatic Drp1-dependent mitochondrial fragmentation. Fragmentation was dependent on viable AIEC with the ability to adhere and invade epithelia. While P110, Mdivi1 and leflunomide all acutely (2-4h) prevented AIEC-induced mitochondrial fragmentation, this was eventually overcome, such that AIEC-induced loss of the epithelial barrier function and apoptosis eventually emerged (~8h post-treatment with 10⁸ CFU). Leflunomide significantly reduced numbers of AIEC inside cells.

Conclusions

Mitochondrial fragmentation is identified as a novel aspect of AIEC-epithelial interaction that will contribute to loss of function. Precise understanding of the molecular process that facilitate the mitochondrial fragmentation could open new therapeutic avenues relevant to inflammatory disease and/or that due to microbial pathogens. Finally, by virtue of its ability to reduce the intracellular burden of AIEC (possibly linked to its capacity to promote mitochondrial fusion), leflunomide could be a treatment for IBD in which AIEC are implicated.

Funding Agencies

CIHRNSERC, UofC Eyes High, Izaak-Killam