20 ELUCIDATING THE ROLE OF FUSOBACTERIUM NUCLEATUM IN INTESTINAL INFLAMMATION

Abstract Background While the direct cause of inflammatory bowel disease (IBD) is unknown, the gut microbiota is speculated to play a key role. The complexity of the microbiome has made it difficult to pinpoint whether bacterial species are specifically associated with IBD exacerbations; although select microbes have emerged as compelling candidates. Several groups have identified increased abundance in Fusobacterium in IBD patients. Fusobacterium nucleatum drives inflammation in the oral cavity, but few studies have examined the potential for F. nucleatum to promote intestinal inflammation. We hypothesize that F. nucleatum secretes Outer Membrane Vesicles (OMVs) that activate epithelial Toll-like receptor 4 (TLR4) to drive inflammation. Methods & Results Given the prevalence of F. nucleatum in IBD specimens, we sought to determine if this pathobiont could promote pro-inflammatory responses in human epithelial cultures. Using fluorescently tagged F. nucleatum, we demonstrate that F. nucleatum subspecies polymorphum adheres to the mucus layer of human colonic HT29-MTX cells. Application of F. nucleatum metabolites to HT29-MTX cells resulted in upregulation of pro-inflammatory cytokines IL-8 and TNF by qPCR and ELISA. Purified OMVs from F. nucleatum alone were able to stimulate IL-8 and TNF production. This demonstrates the robust response of colonic epithelial cells to F. nucleatum. Additionally, we used human jejunum and colon enteroid monolayers treated with F. nucleatum metabolites in an anti-oxidant free enteroid media and found that F. nucleatum secreted products promoted TNF secretion by ELISA. Using the Luminex Magpix Platform we further queried the enteroid system to assess which pathways were activated. Enteroid monolayers treated with F. nucleatum metabolites exhibited increased phosphorylated ERK and CREB, downstream effectors of TLRs. We next sought to address whether F. nucleatum alone could elicit pro-inflammatory responses in a mouse model. Mice harboring a human microbiota, or humanized mice, were treated for 5 days with a cocktail of antibiotics and treated with F. nucleatum (108 CFU) by oral gavage; a regimen designed to mimic IBD patient treatment. Compared to control mice that received antibiotics and PBS vehicle control, mice treated with F. nucleatum exhibited disruption of the colonic architecture, with increased immune infiltrate and depleted mucus layer, resulting in a closer proximity of luminal contents to the epithelium. Analysis of mucosal gene expression revealed increased levels of epithelial TNF and KC (mouse homolog of IL-8), in addition to immune cell derived IL-6 in F. nucleatum-treated mice compared for controls. Conclusions These data provide evidence that F. nucleatum is capable of driving a pro-inflammatory signaling cascade in vitro and in vivo and F. nucleatum may represent a specific target for drug therapy.