Delivery of Periodontopathogenic Extracellular Vesicles to Brain Monocytes and Microglial IL-6 Promotion by RNA Cargo

Abstract

Gram-negative bacterial extracellular vesicles (EVs), also known as outer membrane vesicles (OMVs), are secreted from bacterial cells and have attracted research attention due to their role in cell-to-cell communication. During OMV secretion, a variety of cargo such as extracellular RNA (exRNA) is loaded into the OMV. The involvement of exRNAs from a range of bacteria has been identified in several diseases, however, their mechanism of action has not been elucidated. We have recently demonstrated that OMVs secreted by the periodontopathogen Aggregatibacter actinomycetemcomitans can cross the blood–brain barrier (BBB) and that its exRNA cargo could promote the secretion of proinflammatory cytokines in the brain. However, it was unclear whether the brain immune cells could actually take up bacterial OMVs, which originate outside of the brain, in an appropriate immune response. In the present study, using monocyte-specific live CX3CR1-GFP mice, we visualized OMV-colocalized meningeal macrophages and microglial cells into which bacterial OMVs had been loaded and intravenously injected through tail veins. Our results suggested that meningeal macrophages uptake BBB-crossed OMVs earlier than do cortex microglia. BV2 cells (a murine microglia cell line) and exRNAs were also visualized after OMV treatment and their proinflammatory cytokine levels were observed. Interleukin (IL)-6 and NF-κB of BV2 cells were activated by A. actinomycetemcomitans exRNAs but not by OMV DNA cargo. Altogether, these findings indicate that OMVs can successfully deliver exRNAs into brain monocyte/microglial cells and cause neuroinflammation, implicating a novel pathogenic mechanism in neuroinflammatory diseases.

Role of Microbiota-Derived Extracellular Vesicles in Gut-Brain Communication

“…Although a recent study evidenced that bacterial OMVs could directly cross the BBB [50], the question as to whether they can cross the BBB has not yet been resolved. A recent report demonstrated the transport of OMVs from the circulation to brain microglial cells through the meninges [102] and suggested that the delivery of bacterial EVs to the brain could be associated with infection at any place in the body, which could cause a fatal immune response in the brain [102]. Another recent report evidenced that bacterial EVs could cross the BBB through the vagus nerve.…”

“…Increased TNF-α expression in the mouse brain induced by these OMVs may cause inflammatory diseases such as Alzheimer's disease [50]. Similarly, OMVs secreted by the periodontopathogen Aggregatibacter actinomycetemcomitans can successfully deliver extracellular RNAs to brain monocyte/microglial cells to cause neuroinflammation associated with the up-regulation of IL-6 through NF-κB activation [102]. These effects could be associated with the activation of TLR8 as the OMV RNA of Aggregatibacter actinomycetemcomitans promotes the activation of the NF-κB response via TLR8 in macrophage U937 cells [50].…”

The complex, bidirectional role of extracellular vesicles in infection

“…In terms of placing bEVs at the sites of infection or inflammation in humans, there are still technical and ethical challenges to overcome. Experimental animal models have, however, been exploited to show bEVs travelling to remote sites after injection into the animal, and to demonstrate bEV presence and inflammatory effects, in target organs [31][32][33]. For example, the intraperitoneal injection of Cy7-labelled E. coli bEVs followed by near-infrared imaging in SKH-1 E hairless mice demonstrated travel to liver, lung, spleen, and kidney within 3 h [31].…”

“…More recently, a similar approach was used to follow fluorescently labelled bEV of the oral pathogen A. actinomycetemcomitans where following cardiac injection into monocyte-specific CX3CR1-GFP mice they have been detected in the brain colocalized with meningeal macrophages and microglial cells by intravital imaging techniques. This work suggests that bEVs can cross the blood-brain barrier (BBB) and links bEVs in the brain to elevated production of pro-inflammatory cytokines to support a connection between periodontitis and neuroinflammatory disease [ 32 , 33 ]. The observations made investigating bEV transport in mice add support to a role for bEVs in the aetiology of some instances of sterile inflammation.…”

“…In one example, the commensal gastrointestinal bacterium Bacteroides fragilis selectively packages polysaccharide A (PSA) in bEVs that have been demonstrated to induce immunomodulatory effects and prevent experimental colitis in mice [ 109 ], a finding that suggests some commensal bEVs may be the basis of therapeutic formulations, or even be a rationale for new probiotic approaches. The evidence now emerging to suggest that bEVs may travel between organ systems in the body [ 110 , 111 ] and even cross the BBB [ 33 ] may identify new probiotic approaches and mechanisms by which, e.g. the gut microbiota can influence the health at other sites in the body.…”