Outer membrane vesicles of avian pathogenic Escherichia coli induce necroptosis and NF-κB activation in chicken macrophages via RIPK1 mediation

Outer membrane vesicles (OMVs) are soluble mediators secreted by Gram-negative bacteria that are involved in communication. They can carry a variety of harmful molecules, which induce cytotoxic responses and inflammatory reactions in the absence of direct host cell-bacterium interactions. We previously reported the isolation of OMVs from avian pathogenic Escherichia coli (APEC) culture medium by ultracentrifugation, and characterized them as a substance capable of inducing the production of pro-inflammatory cytokines and causing tissue damage. However, the specific mechanisms by which APEC-secreted OMVs activate host cell death signaling and inflammation are poorly understood. Here, we show that OMVs are involved in the pathogenesis of APEC disease. In an APEC/chicken macrophage (HD11) coculture system, APEC significantly promoted HD11 cell death and inflammatory responses by secreting OMVs. Using western blotting analysis and specific pathway inhibitors, we demonstrated that the induction of HD11 death by APEC OMVs is associated with the activation of receptor interacting serine/threonine kinase 1 (RIPK1)-, receptor interacting serine/threonine kinase 3 (RIPK3)-, and mixed lineage kinase like pseudokinase (MLKL)-induced necroptosis. Notably, necroptosis inhibitor-1 (Nec-1), an RIPK1 inhibitor, reversed these effects. We also showed that APEC OMVs promote the activation of the NF-kappa B signaling pathway, leading to the phosphorylation of I kappa B-alpha and p65, the increased nuclear translocation of p65, and the significant upregulation of interleukin 1 beta (IL-1 beta) and IL-6 transcription. Importantly, APEC OMVsinduced IL-1 beta and IL-6 mRNA expression and the activation of the NF-kappa B signaling pathway were similarly significantly inhibited by a RIPK1-specific inhibitor. Based on these findings, we have established that RIPK1 plays a dual role in HD11 cells necroptosis and the proinflammatory cytokine (IL-1 beta and IL-6) expression induced by APEC OMVs. RIPK1 mediated the induction of necroptosis and the activation of the NF-kappa B in HD11 cells via APEC OMVs. The results of this study provide a basis for further investigation of the contribution of OMVs to the pathogenesis of APEC.