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Extracellular vesicles of Fusobacterium nucleatum compromise intestinal barrier through targeting RIPK1-mediated cell death pathway.

ABSTRACT: Microbial factors that mediate microbes-host interaction in ulcerative colitis (UC), a chronic disease seriously affecting human health, are not fully known. The emerging oncobacterium Fusobacterium nucleatum (Fn) secretes extracellular vesicles carrying several types of harmful molecules in the intestine which can alter microbes-host interaction, especially the epithelial homeostasis in UC. However, the mechanism is not yet clear. Previously, we isolated EVs by the ultracentrifugation of Fn culture media and characterized them as the potent inducer of pro-inflammatory cytokines. Here, we examined the mechanism in detail. We found that in macrophage/Caco-2 co-cultures, FnEVs significantly promoted epithelial barrier loss and oxidative stress damage, which are related to epithelial necroptosis caused by the activation of receptor-interacting protein kinase 1 (RIPK1) and receptor-interacting protein kinase 3 (RIPK3). Furthermore, FnEVs promoted the migration of RIPK1 and RIPK3 into necrosome in Caco2 cells. Notably, these effects were reversed by TNF-α neutralizing antibody or Necrostatin-1 (Nec-1), a RIPK1 inhibitor. This suggested that FADD-RIPK1-caspase-3 signaling is involved in the process. Moreover, the observed effects were verified in the murine colitis model treated with FnEVs or by adoptive transfer of FnEVs-trained macrophages. In conclusion, we propose that RIPK1-mediated epithelial cell death promotes FnEVs-induced gut barrier disruption in UC and the findings can be used as the basis to further investigate this disease.

SUBMITTER: Liu L

PROVIDER: S-EPMC8007154 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:IntroductionMetastasis is an important cause of high mortality and lethality of oral cancer. Fusobacterium nucleatum (Fn) can promote tumour metastasis. Outer membrane vesicles (OMVs) are secreted by Fn. However, the effects of Fn-derived extracellular vesicles on oral cancer metastasis and the underlying mechanisms are unclear.ObjectivesWe aimed to determine whether and how Fn OMVs mediate oral cancer metastasis.MethodsOMVs were isolated from brain heart infusion (BHI) broth supernatant of Fn by ultracentrifugation. Tumour-bearing mice were treated with Fn OMVs to evaluate the effect of OMVs on cancer metastasis. Transwell assays were performed to determine how Fn OMVs affect cancer cell migration and invasion. The differentially expressed genes in Fn OMV-treated/untreated cancer cells were identified by RNA-seq. Transmission electron microscopy, laser confocal microscopy, and lentiviral transduction were used to detect changes in autophagic flux in cancer cells stimulated with Fn OMVs. Western blotting assay was performed to determine changes in EMT-related marker protein levels in cancer cells. Fn OMVs' effects on migration after blocking autophagic flux by autophagy inhibitors were determined by in vitro and in vivo experiments.ResultsFn OMVs were structurally similar to vesicles. In the in vivo experiment, Fn OMVs promoted lung metastasis in tumour-bearing mice, while chloroquine (CHQ, an autophagy inhibitor) treatment reduced the number of pulmonary metastases resulting from the intratumoral Fn OMV injection. Fn OMVs promoted the migration and invasion of cancer cells in vivo, leading to altered expression levels of EMT-related proteins (E-cadherin downregulation; Vimentin/N-cadherin upregulation). RNA-seq showed that Fn OMVs activate intracellular autophagy pathways. Blocking autophagic flux with CHQ reduced in vitro and in vivo migration of cancer cells induced by Fn OMVs as well as reversed changes in EMT-related protein expression.ConclusionFn OMVs not only induced cancer metastasis but also activated autophagic flux. Blocking autophagic flux weakened Fn OMV-stimulated cancer metastasis.

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Extracellular vesicles of Fusobacterium nucleatum compromise intestinal barrier through targeting RIPK1-mediated cell death pathway.

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