Fusobacterium nucleatum Extracellular Vesicles Promote Experimental Colitis by Modulating Autophagy via the miR-574-5p/CARD3 Axis

Abstract

Background Ulcerative colitis (UC) may be exacerbated by Fusobacterium nucleatum (Fn) infection. However, the mechanism underlying Fn-mediated progression of UC has yet to be established. Here, we aimed to establish whether and how Fn-derived extracellular vesicles (Fn-EVs) participate in the development of experimental colitis through microRNAs (miRNAs). Methods EVs were isolated and purified by ultracentrifugation from Fn and Escherichia coli culture supernatants. Differentially expressed miRNAs in control intestinal epithelial cells (IECs) and Fn-EV–treated IECs were identified by miRNA sequencing. EVs were cocultured with IECs or administered to CARD3wt/CARD3–/– mice by gavage to assess inflammatory responses to and the mechanism of action of Fn-EVs. Results Fn-EVs promoted upregulation of proinflammatory cytokines (interleukin [IL]-1β, IL-6, tumor necrosis factor α), downregulation of anti-inflammatory IL-10 and intercellular tight junction proteins ZO-1 and occludin, and epithelial barrier dysfunction in IECs. Fn-EVs significantly aggravated experimental colitis in mice associated with Fn-EV–mediated downregulation of miR-574-5p expression and autophagy activation. Blockade of autophagy using chloroquine alleviates barrier damage exacerbated by Fn-EVs in vitro and in vivo. Inhibition of the miR-574-5p/CARD3 axis reduced the severity of colitis, epithelial barrier damage, and autophagy activation induced by Fn-EVs. Conclusions Here, we describe a new mechanism by which Fn-EVs mediate experimental colitis severity through miR-574-5p/CARD3–dependent autophagy activation, providing a novel target for UC monitoring and targeted therapy.

Bacterial membrane vesicles in the pathogenesis and treatment of inflammatory bowel disease

“… 136 MVs from Fn promoted the secretion of proinflammatory cytokines IL-8 and TNF-α in vitro in colonic epithelial cells. 27 Fn -MVs triggered an upregulation of the proinflammatory cytokines IL-1β, IL-6, TNF-α, and iNOS 28 , 134 and downregulation of anti-inflammatory IL-10 in vitro in intestinal epithelial cells and in vivo in colitis mice. 134 Increased levels of F4/80+ iNOS+M1-like macrophages were also reported; thus, Fn -MVs enhanced apoptosis of intestinal epithelial cells in vivo by inducing the pro-inflammatory M1 phenotype resulting in intestinal barrier dysfunction in UC ( Figure 6 ).…”

“… 28 Another study reported that Fn -derived MVs downregulated tight junction proteins ZO-1 and occludin, resulting in epithelial barrier dysfunction both in vitro and in vivo . The exacerbation of colitis by the MVs was linked with Fn -MVs facilitated downregulation of miR-574-5p expression and activation of autophagy 134 ( Figure 6 ).

Figure 6.

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“… 146 , 147 Additionally, as MVs interact with host cells and modulate their signaling pathways, they can suppress immune responses, ultimately creating a more favorable environment for colonization by harmful bacteria. 28 , 134 …”

“…The downregulation or upregulation of certain miRNAs in the presence of MVs tends to monitor not only the progression of IBD but also the alleviation of inflammation. For instance, the downregulation of miR-574-5p expression by Fn -MVs 134 and restoration of miR-199a-3p expression by Cb -MVs 154 demonstrate that miRNAs are intricately associated with MV exposure in IBD cases. With this knowledge, some specific miRNAs could serve as both diagnostic and potential targets for IBD treatment.…”

“… 71 The blockade of autophagy using chloroquine and inhibition of miR-574-5p/CARD3 axis ameliorated epithelial barrier dysfunction, autophagy activation, and subsequently, colitis severity mediated by Fn -MVs in vitro and in vivo . 134 Hickey et al reported that deletion of the anaerobic sulfatase maturating enzyme (anSME) from the wild-type Bt remarkably eliminated the ability of the MVs to stimulate colitis in dnKO mice. This revealed that access of Bt -MVs to host immune cells was sulfatase-dependent.…”