Rheumatoid arthritis (RA) is a chronic immune-driven inflammatory disease characterised by synovial inflammation, leading to progressive cartilage and bone destruction, impacting patients’ functional capacity and quality of life. Patients with RA have significant dierences in gut microbiota composition when compared to controls. Intestinal dysbiosis influences the intestinal barrier strength, integrity and function, and diet is considered the main environmental factor impacting gut microbiota. Over the last few years, researchers have focused on the influence of single components of the diet in the modulation of intestinal microbiota in RA rather than whole dietary patterns. Here, we focus on how the Mediterranean diet (MD), a whole dietary pattern, could possibly act as an adjuvant therapeutic approach, modulating intestinal microbiota and intestinal barrier function in order to improve RA-related outcomes. We also review the potential e ects of particular components of the MD, such as n-3 polyunsaturated fatty acids (PUFAs), polyphenols and fibre.
The human gut houses the largest population of non-eukaryotic organisms in the human body. Despite the high variability between individuals’ gut microbiota composition, bacteria are consistently (and by far) the most common microorganisms . Bacteroidetes and Firmicutes are the most abundant phyla . Significant inter-individual differences in the prevalence of certain species within these phylae have been reported, resulting from different genetic backgrounds, dietary habits, lifestyle, hygiene practice, drug use and other environmental factors . The composition of the gut microbiome in people living in distinct geographical areas has a strong association with each population’s dietary habits .
Urbanisation has been closely related to a Western dietary style, antibiotic use and pollution. It induces changes in gut microbiota composition, particularly the loss of intra-individual microbial diversity accompanied by higher inter-individual differences . In non-Westernised communities, people tend to have a more homogeneous and diverse gut microbiome . Their gut microbiota tends to be rich in certain bacteria, such as Bacteroidetes (including Prevotella and Xylanibacter), and poor in Firmicutes .
Three different classes of enterotypes have been proposed according to the abundance of Bacteroides, Prevotella and Ruminococcus, respectively . There is a strong association between the individual enterotype and long-term but not short-term diet .
A healthy microbiota is characterised by the presence of numerous classes of bacteria, with a balanced composition of symbiont and pathobiont organisms . A pathobiont is a permanent resident of the microbiota that does not usually elicit an inflammatory response, but under particular environmentally-induced conditions has the potential to cause dysregulated inflammation and induce disease . A shift in the microbiota composition, with either an increase in pathobionts or a reduction in symbionts, leads to a state of dysbiosis  that disturbs the modulation of the host immune function by the gut microbiota .
The mucosal immune system and intestinal microbiota can influence each other, promoting a balance between tolerance to dietary antigens and protection against harmful pathogens . Antigen-presenting cells located at the mucosal surface, once activated by antigens, can regulate immune tolerance by promoting T cell differentiation into regulatory T cells (Tregs) . Tregs suppress inappropriate activation of effector T cells by secreting anti-inflammatory cytokines . The gut microbiota influences the number and function of colonic Tregs , suggesting that the modulation of gut microbiota may also regulate the mechanism of gut immune tolerance. Most importantly, these interactions may also modulate systemic inflammation, and influence the risk of developing systemic autoimmune diseases , including inflammatory arthropathies .
The production of short-chain fatty acids (SCFAs) is one of the proposed mechanisms by which intestinal microbiota affects Treg cells differentiation and systemic inflammation . SCFAs, particularly butyrate, acetate and propionate, are the key metabolites resulting from the microbial fermentation of dietary fibres . When fermentable fibres are in short supply, bacteria switch to energetically less favourable sources for growth, such as amino acids or dietary fats , resulting in the reduced fermentative activity of the microbiota and SCFA scarcity . SCFAs are an energy source for gut epithelial cells, having an indirect anti-inflammatory effect by improving the assembly of tight junctions and enhancing intestinal barrier function . Butyrate is the preferred energy source for colonocytes and is locally consumed, whereas other absorbed SCFAs drain into the systemic circulation . Histone acetylation is thought to increase accessibility to the transcriptional machinery, to promote gene transcription. Butyrate and propionate are known to act as histone deacetylase inhibitors . Through this mechanism, SCFAs may act as systemic anti-inflammatory or immune-suppressive molecules . Despite the low concentration in the periphery, propionate and butyrate affect peripheral organs indirectly by activation of the hormonal and nervous systems .
The modulation of intestinal permeability is another mechanism that may explain the influence of gut microbiota on the appearance and perpetuation of inflammatory diseases , leading to systemic inflammation. The intestinal lumen is occupied by various exogenous constituents such as microorganisms, toxins and food antigens. The mucosal barrier, which separates the intestinal milieu from the luminal environment, has an essential role in blocking the entry of microorganisms and toxins, while, at the same time, it must allow the absorption of nutrients and water . Intestinal barrier strength and function can be affected by several factors, among which diet, gut microbiota composition and mucosal immune system integrity are key factors . When the mucosal barrier is disturbed, the exogenous luminal constituents invade the intestinal milieu, and immune activation and mucosal inflammation ensue . This process can trigger an abnormal immune response resulting in both local and systemic inflammation, increasing the risk of developing both gut-associated and extra-intestinal diseases .
Over the last few years, emerging evidence has reported the involvement of gut dysbiosis in the onset of autoimmune diseases such as RA [29,30,57], suggesting its role in contributing to a breakdown of immune tolerance .
The natural history of RA includes different stages, including an at-risk pre-symptomatic phase and an early arthritis phase before the classical erosive disease phase . The most noticeable players in the preclinical RA phases are the autoantibodies, a hallmark of RA . RA-associated autoantibodies include rheumatoid factor (RF) , antibodies to citrullinated proteins (ACPA)  and anti-carbamylated peptide antibodies (anti-CarP) . High serum concentrations of RF , ACPA , and anti-CarP  can be detected years before the onset of clinically overt RA. The evolution of the autoantibody profile in the preclinical stage of RA includes isotype switching . The higher prevalence of IgA- than IgG-ACPA in high-RA-risk populations suggests that mucosal immune responses are important in the preclinical phases of the disease . In different groups of individuals who later developed RA, a higher prevalence of serum IgA- and IgM-RF than IgG-RF was found, and IgA-RF appeared earliest .
Growing evidence suggests that the dysbiosis of mucosal microbiota is closely related to local autoimmune processes , and the composition of the microbiota is significantly disturbed in patients with both early and long-standing RA . It is also known that dysbiosis in the oral microbiota induces periodontitis  and that the gingiva of patients with periodontitis contains citrullinated proteins and ACPA . Porphyromonas gingivalis, a common periodontal pathogen, can citrullinate targets for ACPA , suggesting that dysbiosis and periodontitis can play a fundamental role in the early loss of tolerance to self-antigens in RA-susceptible patients .
Different mechanisms can explain the dysbiosis-mediated induction of autoantibodies. The most common theory states that T helper cells can originate from T cell responses to external antigens through molecular mimicry between those antigens and self-antigens . Such activation leads to the positive selection and maturation of self-reactive B cells that produce a variety of RA-associated autoantibodies . It has also been proposed that host cell necrosis and apoptosis, occurring as a consequence of bacterial insult, can result in extracellular exposure of intracellular self-antigens, leading to recognition by B cells and autoantibody production .
Recently, it has been shown that the gut microbiota of RA patients has a significant increase in the class of Bacilli and the order of Lactobacillales compared to healthy controls . This is concordant with data reporting an increase in the Lactobacillaceae family and the Lactobacillus genus in mice susceptible to collagen-induced arthritis . The variety of Lactobacilli is also higher in RA patients . Excess Lactobacillus salivarius in the gut and mouth of RA patients has been described, and a correlation with disease severity has been proposed . Paradoxically, the administration of Lactobacillus acidophilus and Lactobacillus casei (L. casei) seems to be beneficial for RA disease activity, suggesting that different Lactobacilli probably have different roles in RA pathogenesis and disease activity modulation .
On the other hand, significant reductions of the genus Flavobacterium, Faecalibacterium and other butyrate-producing taxa , as well as its related species Faecalibacterium prausnitzii and the species Blautia coccoides were described .
Moreover, a higher prevalence of Euryarchaeota, Gammaproteobacteria, Pasteurellales, and Anaerobranca zavarzinii was correlated with a higher disease activity score-28 (DAS28), whilst Erysipelotrichi, Erysipelotrichales, Coriobacteriales, Coriobacteriaceae, Lactobacillaceae, Collinsella, Bacteroides rodentium, and Collinsella aerofaciens were inversely associated with this score .
Importantly, it has also been shown that RA patients under treatment with etanercept, a fusion protein consisting of a human tumour necrosis factor (TNF) receptor linked to the Fc portion of human immunoglobulin G1 (IgG1), present a partial restoration of a beneficial microbiota . This fact seems to provide further evidence pointing towards gut dysbiosis as a hallmark of the disease.
Pharmacomicrobiomics is an emerging field that investigates the effect of variations within the human gut microbiome on drugs . The variability between individuals in the composition and metabolic competence of their microbiomes has a unique role in determining the bioavailability, clinical efficacy and toxicity of a wide array of drugs, including DMARDs . This variability arises because specific, direct modifications of the chemical structures of ingested drugs are dependent on the composition of the human gut microbiome and its collective enzymatic activity .
Relevant examples include the prodrug sulfasalazine, which requires cleaving by the gut microbiome to become an active drug , as well as cyclophosphamide and methotrexate . Although our basic understanding of how microbiome-dependent biotransformations of xenobiotics affect human health is still incomplete, numerous studies have highlighted the extent to which microbial xenobiotic metabolism varies between individuals, and how these reactions can be manipulated for therapeutic purposes . Diet modifications and probiotics are the biggest candidates to play this modulatory role.