A new mechanism in rheumatoid arthritis aggravation: Fusobacterium Nucleatum-mediated release of FadA-containing outer membrane vesicles

The findings of a recent study imply that Fusobacterium nucleatum may play a causal role in worsening RA, offering potential therapeutic targets for effectively alleviating RA symptoms.
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What is already known
Several studies have shown the association between the gut microbiota and the rheumatoid arthritis (RA), an autoimmune disease. However, the exact impact and mechanisms by which the gut microbiota contributes to the development of RA have yet to be investigated.

What this research adds
A recent study observed an enrichment of Fusobacterium nucleatum in patients with RA, which was positively correlated with the severity of the disease. In a mouse model of collagen-induced arthritis (CIA), F. nucleatum also contributed to the exacerbation of arthritis. Mechanistically, the outer membrane vesicles (OMVs) of F. nucleatum carry the virulence factor FadA, and migrate to the joints, triggering local inflammatory responses. Moreover, FadA triggers synovial inflammation by activating the Rab5a-YB-1 axis in synovial macrophages.

These findings imply that Fusobacterium nucleatum may play a causal role in worsening RA, offering potential therapeutic targets for effectively alleviating RA symptoms.

Rheumatoid arthritis (RA) is an autoimmune disease affecting approximately 1% of the global population. It is characterized by high levels of inflammatory cytokines in the bloodstream, initiating an inflammatory response within the joints that results in gradual joint deterioration and functional impairment. However, the precise mechanisms responsible for initiating this inflammation remain unknown.

Examinations of stool, serum, and joint synovial fluid samples from individuals with RA have revealed a strong correlation between the gut microbiota and the severity of RA. The gut microbiota has the ability to migrate to distant organs by releasing metabolites, proteins, and outer membrane vesicles (OMVs). A growing body of evidence suggests that microbiota-derived OMVs elicit immune responses by transporting molecules that specifically target immune cells, particularly macrophages and neutrophils.

Based on this, Hong and colleagues, propose that gut microbiota-derived OMVs could mediate the impact of the gut microbiota on the onset and progression of RA. Their recent study, published in Cell & Host Microbe journal, demonstrates that Fusobacterium nucleatum is enriched in the gut of patients affected by RA and aggravates arthritis by delivering the virulence factor FadA to the joints via OMVs. FadA triggers synovial inflammation by activating synovial macrophages.

F. nucleatum is a facultative anaerobe that lives in the oral cavity and gut, and participates in various oral and gastrointestinal diseases and affects various distant organs, such as the liver, brain, and placenta by secreting virulence factors, including proteins and OMVs into circulation, as well as by translocation into organs.

F. nucleatum is enriched in RA patients and positively correlated with serum inflammatory cytokines

Researchers collected serum and stool samples from 49 RA patients and 25 healthy controls to characterize the gut microbiota and the signatures of inflammation, and found higher levels of inflammatory cytokines in the serum of RA patients. 

They also observed difference in microbiota composition, richness and in the abundance of several microbial communities in RA patients compared with healthy individuals, indicating a shift in the composition of gut microbiota in RA. Among others, the genera Fusobacterium was significantly more abundant in RA patients and was correlated with the parameters of RA disease activity, suggesting a relationship between Fusobacterium and RA symptoms. 

In particular, the F. nucleatum strain was more abundant in RA patients and significantly associated with clinical parameters of RA, implying a specific association between F. nucleatum and RA. The positive correlation between the abundance of F. nucleatum and RA disease activities as well as serum inflammatory cytokines indicate the relationship between F. nucleatum and RA.

F. nucleatum aggravates arthritis in CIA mice by secreting OMVs targeting macrophages 

To investigate the potential pathogenic role of F. nucleatum in RA, the authors used mice with induced arthritis and treated them with F. nucleatum and found that its administration induced more severe arthritis compared to the control treatment. Moreover, higher bone erosion and enriched macrophages and neutrophils were observed in the joints of CIA mice, indicating a higher synovial inflammation in mice receiving F. nucleatum

Following antibiotic treatment for Fusobacteria, the authors found that F. nucleatum was almost completely depleted and the depletion significantly ameliorated arthritis by reducing the serum inflammatory cytokines as well as the infiltration of neutrophils and macrophages in the joints. These data imply that F. nucleatum plays a role in mediating the aggravation of arthritis.

In order to understand the underlying mechanisms, the researchers blocked the release of OMVs from F. nucleatum in mice and observed a significant reduction of OMVs secretion. This also prevented the pro-inflammatory effects of F. nucleatum, indicating the crucial role played by OMVs in F. nucleatum-promoted arthritis. Moreover, the OMVs were found to translocate throughout the body and accumulated in the limbs of the mice, with the possibility of OMVs to translocate to the joints.

Indeed, accumulated OMVs in the joints were found to aggravate arthritis, accompanied by higher systemic and synovial inflammation compared to control mice, suggesting that F. nucleatum promotes arthritis by secreting OMVs. Furthermore, OMVs promoted arthritis by specifically targeting macrophages, one of the primary phagocytic cells that identify and internalize bacterial effectors. This was confirmed by observing the internalized OMVs by macrophages in vitro, which promoted macrophage-associated inflammation.

FadA mediates the aggravation effects of OMVs on arthritis through the Rab5a-YB-1 axis

FadA is one of the crucial proteins of F. nucleatum reported to mediate the systemic dissemination and pro-inflammatory cascades of F. nucleatum. Therefore, the authors analyzed OMVs and identified the presence of FadA in the OMVs and in the macrophages in the paws of the mice, suggesting the translocation of F. nucleatum-associated protein FadA into joints in mice. 

The researchers also created a mutant form of F. nucleatum with deletion of FadA and noticed that F. nucleatum mutant could no longer stimulate inflammation, strongly indicating that FadA in the OMVs mediates the pro-inflammation effects of F. nucleatum

One of the most related genes to FadA, Rab5a, was increased in the synovial macrophages in mice treated with OMVs and its expression decreased after antibiotic-mediated depletion of F. nucleatum, revealing that Rab5a might be the target by which FadA-containing OMVs mediate aggravation effects on arthritis. 

Finally, it was also observed that the protein FadA, contained in OMVs, enhances the interaction between Rab5 and YB-1, the most abundant transcription factor interconnected with Rab5a. Overexpression of YB-1 significantly increased inflammatory genes expression. Therefore, OMVs promote inflammation through the FadA-Rab5a-YB-1 axis.

Abundant levels of FadA-Rab5a-YB-1 axis in the synovium of RA patients

In order to translate the findings observed in mice into humans, the authors measured the existence of the aforementioned critical factors in the joint fluid or synovial samples of RA patients and controls. Notably, the FadA protein level in equal-quality OMVs was significantly higher in RA patients than in controls along with higher inflammation, indicating that the translocation of F. nucleatum-associated FadA-containing OMVs from gut to joint specifically underlies RA disease. 

Most importantly, all the critical factors underlying F. nucleatum-aggravated arthritis (FadA, Rab5a, and YB-1), could be detected in the synovialis of humans, and their levels were elevated in RA patients compared with controls. These findings strongly support the occurrence of the translocation of FadA containing OMVs from gut to joint and suggest its causal role in aggravating RA by targeting Rab5a-YB-1 axis.

In conclusion, the present study identified the OMVs-FadA-Rab5a-YB-1 axis in the context of arthritis in mice and observed these critical mediators in the synovium of RA patients, collectively suggesting that F. nucleatum causally aggravates RA by secreting FadA-containing OMVs. 
All together, these findings indicate that gut microbiota could exert pathogenic effects on organs at remote sites by secretion of proteins contained in OMVs. The study provides insights into the causes of inflammation of RA and suggests F. nucleatum and its OMVs as promising therapeutic targets for clinically amelioration of RA disease.