<i>Fusobacterium nucleatum</i> Secretes Outer Membrane Vesicles and Promotes Intestinal Inflammation

Abstract

Multiple studies have implicated microbes in the development of inflammation, but the mechanisms remain unknown. Bacteria in the genus <i>Fusobacterium</i> have been identified in the intestinal mucosa of patients with digestive diseases; thus, we hypothesized that <i>Fusobacterium nucleatum</i> promotes intestinal inflammation. The addition of >50 kDa <i>F. nucleatum</i> conditioned media, which contain outer membrane vesicles (OMVs), to colonic epithelial cells stimulated secretion of the proinflammatory cytokines interleukin-8 (IL-8) and tumor necrosis factor (TNF). In addition, purified <i>F. nucleatum</i> OMVs, but not compounds <50 kDa, stimulated IL-8 and TNF production; which was decreased by pharmacological inhibition of Toll-like receptor 4 (TLR4). These effects were linked to downstream effectors p-ERK, p-CREB, and NF-κB. <i>F. nucleatum</i> >50-kDa compounds also stimulated TNF secretion, p-ERK, p-CREB, and NF-κB activation in human colonoid monolayers. In mice harboring a human microbiota, pretreatment with antibiotics and a single oral gavage of <i>F. nucleatum</i> resulted in inflammation. Compared to mice receiving vehicle control, mice treated with <i>F. nucleatum</i> showed disruption of the colonic architecture, with increased immune cell infiltration and depleted mucus layers. Analysis of mucosal gene expression revealed increased levels of proinflammatory cytokines (KC, TNF, IL-6, IFN-γ, and MCP-1) at day 3 and day 5 in <i>F. nucleatum</i>-treated mice compared to controls. These proinflammatory effects were absent in mice who received <i>F. nucleatum</i> without pretreatment with antibiotics, suggesting that an intact microbiome is protective against <i>F. nucleatum</i>-mediated immune responses. These data provide evidence that <i>F. nucleatum</i> promotes proinflammatory signaling cascade<i>s</i> in the context of a depleted intestinal microbiome.<b>IMPORTANCE</b> Several studies have identified an increased abundance of <i>Fusobacterium</i> in the intestinal tracts of patients with colon cancer, liver cirrhosis, primary sclerosing cholangitis, gastroesophageal reflux disease, HIV infection, and alcoholism. However, the direct mechanism(s) of action of <i>Fusobacterium</i> on pathophysiological within the gastrointestinal tract is unclear. These studies have identified that <i>F. nucleatum</i> subsp. <i>polymorphum</i> releases outer membrane vesicles which activate TLR4 and NF-κB to stimulate proinflammatory signals <i>in vitro</i> Using mice harboring a human microbiome, we demonstrate that <i>F. nucleatum</i> can promote inflammation, an effect which required antibiotic-mediated alterations in the gut microbiome. Collectively, these results suggest a mechanism by which <i>F. nucleatum</i> may contribute to intestinal inflammation.