**4. Discussion**

The first description of the possible involvement of microbiota in the pathology of arthritis was published in 1970s when rats raised in germ-free conditions developed severe joint inflammation with 100% penetrance in an adjuvant-induced arthritis model, while conventionally raised controls showed only mild disease at a very low incidence [56].

A fine equilibrium between 'peace-keeping' and potentially pro-inflammatory intestinal and oral bacteria is necessary to keep gu<sup>t</sup> immunity in check and prevent a state of dysbiosis, which might lead to local and distant deleterious consequences in the host. A crucial driver of changes in the gu<sup>t</sup> and oral environments is the inflammatory response of the host. Intestinal and oral inflammations in people are associated with an imbalance in the microbiota, the dysbiosis, which is characterized by a reduced diversity of microbes, a reduced abundance of obligate anaerobic bacteria, and an expansion of facultative anaerobic bacteria in the phylum Proteobacteria, mostly members of the family Enterobacteriaceae.

In regards to RA and gu<sup>t</sup> microbiota, single microorganisms such as *P. copri* might correlate with the development of RA. Pianta A. et al. reported massive concentrations of antibodies against *P. copri* in the sera of RA patients [31]. Impressive advances in sequencing technologies, compelling animal data, and mounting human evidence have suggested that gu<sup>t</sup> microbiota indeed play a part in the pathogenesis of diseases such as autoimmune arthritis. The few studies addressing potential links between the gu<sup>t</sup> microbiota and human inflammatory joint disease have identified dysbiotic patterns that may contribute to initiate or to perpetuate the disease. Obviously, age can greatly contribute to the increase of systemic inflammation (inflammaging), and the microbiota shaped by aging can negatively modulate the outcome of joint diseases. However, the gu<sup>t</sup> microbiota of RA patients seem to be more dysbiotic than those of healthy patients, thus confirming their role as independent of age. An indirect demonstration of the role of microbiota is that gu<sup>t</sup> microbiome (the same for the oral) is perturbed in rheumatoid arthritis and partly normalized after RA-specific treatment [28].

Dysbiosis occurring, for instance, in jejunoileal-bypass, used as alternative to bariatric banding, seems to be associated with arthritis. In these patients, studies have reported a bacterial overgrowth and a deposition of resultant immune complexes in the synovium [57]. However, a very comprehensive human model fitting with the gut–joint axis and dysbiosis can be represented by Whipple's disease, in which the presence of a single bacterium, *Tropheryma whipplei,* overgrowth in the small intestine is su fficient for the development of joint inflammation in predisposed individuals. Similar results have been appointed by some authors regarding the high quantity of *Streptococci* in milk as a theoretical cause of RA [58,59].

A strong evidence of the gut–bone axis and its role in arthritis outcomes has been reported in germ free mice studies. It has been evidenced that these animals do not show arthritis; however, the mono-colonization of particular intestinal bacterium is su fficient to induce arthritis. Thus, gu<sup>t</sup> microbiota have been further confirmed to be a cause of relevant immunological triggers occurring in arthritis pathogenesis [8,60].

Periodontal disease also correlates with new-onset RA patients, and many bacterial clusters related to this disease have been faced in di fferent studies [22–24]. Gene sequencing studies have investigated the subgingival microbiome of patients with RA and compared the results of subjects with osteoarthritis and healthy controls with or without periodontitis. In both cases, literature revealed that specific bacteria biomarker abundance may influence the severity of the osteoarthritic disease. Unfortunately, no unique microbial oral cluster has been identified so far.

Only one study [28] has reported results on the simultaneous e ffect of oral–gut microbiota in RA patients. By collecting fecal, dental and salivary samples in a cohort of RA and healthy donors, this study demonstrated a rate of dysbiosis in the gu<sup>t</sup> and oral microbiomes of RA patients, suggesting an overlap in the abundance and function of species at di fferent body sites that could be partially resolved after RA treatment.

Despite findings which are suggestive of microbiota–bone axis correlation with inflammatory joint disease, research to date remains inconclusive with regard to the final mechanism. We therefore need to identify the priorities for research in order to substantiate and translate these findings. An important and recent review analyzing nine clinical studies [61] compared changes in diversity and taxa present in the microbiome of RA patients with age, gender and weight-matched controls. Despite microbiome diversity being a generic tool to define whether microbial disturbance in the oral or gu<sup>t</sup> environments has occurred, the study of specific bacterial clusters is of grea<sup>t</sup> interest to establish the possible etiopathogenetic role of microbiota in arthritis. In RA, a correlation between a pro-inflammatory genotype-HLA related bacteria and some bacterial clusters has been strongly postulated. However, well-defined human studies using NGS and metabolomic approaches are needed to better understand if and when intestinal community composition in patients with joint inflammation di ffers (in addition to improving therapies) by looking at specific bacterial markers for disease presence and progression. Prospective studies evaluating the microbiome–host relationship are indeed necessary to establish not only the potential etiology but also the e ffects of immunosuppressive or anti-inflammatory therapies on microbiota. Another final aim will be to establish how the microbiota can influence therapies per se in OA or RA patients and, subsequently, how they may impact the host's well-being. Table 1a,b shows the main taxa abundances in oral and gu<sup>t</sup> microbiota in OA and RA. To date, interesting and exhaustive data have shown that a connection between microbiomes and joint diseases exists in RA. Other diseases, OA in particular, have received little attention so far, despite some promising, suggestive findings. The gu<sup>t</sup> microbiome, indeed, could be the culprit behind arthritis and joint pain for obese people. A recent paper by Schott E.M. et al. [62] demonstrated that changes in the gu<sup>t</sup> microbiomes of the mice coincided with signs of body-wide inflammation, including in their knees, where the authors induced osteoarthritis with a meniscal tear. Compared to lean mice, osteoarthritis progressed much more quickly in the obese mice, with nearly all of their cartilage disappearing within 12 weeks of the tear.

Though studies have specifically investigated the influence of gu<sup>t</sup> microbiota in OA, pre-clinical data and some observational investigations in humans have suggested a potential relationship between the gu<sup>t</sup> and risk factors of OA. The role of some confounding factors (genes, sex, age, diet, living conditions) needs to be better explored to fully understand the role of gu<sup>t</sup> bacterial biomarkers in OA.

Thus, a deeper understanding of the biological complexities of our 'two genomes' (host and microbial) will help elucidate the factors that trigger inflammation and finally bridge the gap in our knowledge regarding the role of gene–environment interactions in other autoimmune and inflammatory processes involved in disease pathogenesis. Next generation sequencing, metatranscriptomic analysis, and metabolomic approaches may provide yet-greater insight and help to further understand these mechanisms.

There is a justified association between oral and gu<sup>t</sup> microbiomes in arthritis, although the current evidence that the microbiome causes arthritis is far from conclusive. Strategic future studies aiming to improve the understanding of the combined role of gut–oral axis in arthritis as well as the use of "microbiome influencers," such as the probiotics, are mandatory.

#### **5. Highlights of Future Perspectives**

Boxes 1–5 report Microbiome definition and its involvement in RA and OA as well as the need for further studies.

#### **Box 1.** Microbiome definition.

The microbiome is defined as the totality of microorganisms and their genes inhabiting a unique environment; the human microbiome outnumbers human genes by several orders of magnitude.

#### **Box 2.** Tools for studying microbiome.

Understanding of the role of microorganisms in modulating health and disease by NGS and metabolomic technologies will be the new era.

#### **Box 3.** Microbiome and RA link.

Despite the fact that precise causation of RA has not ye<sup>t</sup> been established, several clinical investigations have demonstrated the role of some microorganisms in RA pathogenesis, independently of age.

#### **Box 4.** Microbiome and OA link.

OA is the most common disorder of the musculoskeletal system. The literature has considered the microbiome and the use of some selected probiotics as a possible future therapeutic approach.

#### **Box 5.** Need for further studies.

More studies are needed to assess the role of the microbiome in human arthritis and related diseases in the order to finally elucidate their mechanisms and therapeutic targets.

**Author Contributions:** L.D. conceived and write the paper; G.V.Z., C.L.R., R.M., J.H.V. revised the paper; K.G. and J.P. revised the English and improved the manuscript.

**Funding:** This research received no external funding

**Conflicts of Interest:** The authors declare no conflict of interest
