**4. Discussion**

The dietary habits in industrialized societies have considerable changed in the last years, and concomitant to this changes, the frequency of autoimmune diseases has increased [47]. Western diets include low fiber and high fat consumption, which alters the gu<sup>t</sup> microbiota diversity and function, affecting the mucosal immune system and influencing the development of autoimmune diseases [48]. Berer and colleagues (2018) demonstrated that the supplementation of non-fermentable fiber to transgenic mice of the spontaneous EAE model (opticospinal encephalomyelitis mice) impacted gu<sup>t</sup> microbiota and metabolic profile, increased long-chain fatty acids production, induced polarization to Th2 immune responses, and prevented autoimmune diseases [49]. Furthermore, exercise practice and low-calorie diets based on the consumption of vegetables, fruits, fish, prebiotics, and probiotics induced a decrease in inflammatory mediators and reestablished eubiosis by acting via nuclear receptors [50]. Additionally, Wu and colleagues (2011) showed the influence of diet on the gu<sup>t</sup> microbiota and the prevalence of *Bacteroides* species when animal proteins and saturated fats were consumed, while the presence of *Prevotella* species was associated with carbohydrates and simple sugar intake [51]. In our study, we detected significant differences in the consumption of vegetables, fruits, carbohydrates, animal-derived proteins, and dairy products between patients and controls and, in contrast to Wu et al., we detected an inverse correlation between increased animal-derived protein intake by patients (50% vs. 27.8% in controls) and relative abundance of *Bacteroides vulgatus*. There are no studies evaluating the intestinal microbiota of the Brazilian population as a whole, and it should be noted that the human intestinal microbiota is considered to be variable between individuals and presents geographic variation [52].

Several clinical trials are underway to test the effects of dietary interventions on inflammatory diseases, such as MS (NCT03539094, NCT02580435, NCT04574024, NCT04042415, NCT03451955). So far, protective effects have been proposed for a Mediterranean diet enriched in fibers, vegetables, polyunsaturated fatty acids, and low levels of proteins [51,53]. On the other hand, the consumption of large amounts of milk and derivatives, meat, or

animal fats correlates with an increasing prevalence of MS [54]. We detected differences in dairy products consumption between patients and controls, and inverse correlations with Bacteroidetes members, carbohydrate-degrading, Gram negatives bacteria, including *Bacteroides uniformis* [55]. In MS patients, it has been suggested that dysbiosis caused by an inadequate diet may indirectly influence Tregs/Th17 cell balance in the gu<sup>t</sup> mucosa and activate inflammatory pathways, contributing to intestinal and systemic inflammation and MS pathogenesis [56]. Although we detected differences in diet and alterations in the gu<sup>t</sup> microbiota, the levels of inflammatory IL-17 and IFN*-γ* cytokines, which are involved in MS pathogenesis [57], were similar in patients and controls. However, we detected a significant decrease in IL-6 levels, which are probably associated with DMTs, which impacts the immune response in relapsing–remitting patients [58].

The gu<sup>t</sup> microbiota and the CNS are connected in a bidirectional manner, including neural, endocrine, and immunological interactions [59]. Commensal microbes can interfere with the secretion of neurotransmitters by intestinal cells, stimulate the vagus nerve thus affecting the brain and behavior, produce neuroactive molecules, and modulate mucosal immune cells and systemic populations that can cross the blood–brain barrier (BBB) into the CNS [60]. In turn, the CNS modulates the microbiota by adrenergic signaling and impacts intestinal motility and neurotransmitters actions in immunological cells that shape the gu<sup>t</sup> microbiota composition [60]. Interestingly, a small fraction of metabolites generated by the gu<sup>t</sup> microbiota in response to diet can reach the systemic circulation, cross the blood BBB through vascular epithelial receptors, and modulate CNS inflammation [10,61–63]. Besides that, these metabolites can indirectly act through SCFA receptors in MS patients and through aryl hydrocarbon receptors that influence microglia activation and gene transcription in astrocytes [53,63,64]. In animal models, previous studies showed that germ-free mice with a breakdown of tight-junctions at the BBB had defective permeability, restored when these mice were colonized with conventional microbiota [65]. Therefore, a disbiotic microbiota secretes metabolites that enter the blood stream and impact the development of local and systemic diseases [49]. Moreover, these microbes may influence therapeutic responses by activating or inhibiting exogenous molecules [60].

In the present study, we detected intestinal dysbiosis in RRMS patients receiving DMTs, and our results present some similarities with previous studies in non-treated patients [24–33]. Some of these similarities include decreased *Lactobacillus* spp. (Lactobacillales) and predominance of *Akkermansia muciniphila* and *Methanobrevibacter smithii,* chemilitotrophic specie. *Methanobrevibacter* is involved in inflammatory conditions by recruiting macrophages and activating dendritic cells [66]. *Akkermansia* have immunoregulatory effects by converting mucin into SCFAs [54]; however, they play a role in the degradation of the mucus layer and can promote intestinal inflammation [56]. In addition, we detected a reduced relative abundance of *Bifidobacterium* spp. and Ruminocaceae members including *Faecalibacterium* spp. and *Ruminococcus* spp. *Bifidobacterium* represents one of the first colonizers of the human gu<sup>t</sup> and exerts health-promoting effects [67]. *Faecalibacterium* spp. are butyrate-producing bacteria in the human colon, a bioindicator of human health, and are reduced in inflammatory conditions [68]. *Ruminococcus* spp. re part of the healthy gu<sup>t</sup> microbiota in humans, and some mucus-degrading species are increased in inflammatory diseases [69].

There are few studies evaluating the effect of DMTs on gu<sup>t</sup> microbiota composition, and previous works sugges<sup>t</sup> that these therapies are able to reestablish the gu<sup>t</sup> ecosystem towards a eubiosis condition [19]. Patients on IFN-β or glatiramer acetate treatment showed increased abundance of *Prevotella, Sutterella*, and *Prevotella copri* and decreased *Sarcina* species [29,36,37]. In our MS patients, we also observed an increase in *Prevotella* spp. (Bacteroidales) in treated RRMS patients. The *Prevotella* genus is associated with a high-fiber diet and has regulatory roles via butyrate generation [28]. Butyrate has anti-inflammatory effects, induces Tregs in the gu<sup>t</sup> mucosa, and maintains the epithelial barrier [70]. It is important to note that metabolites produced by the gu<sup>t</sup> microbiota are capable of influencing drug bioavailability, pharmacokinetics, and clinical response, which supports the importance of

studies on the interaction of the gu<sup>t</sup> microbiota with DMTs [38,39]. In our work, the treated RRMS patients had a different microbiota profile when compared with healthy controls, suggesting that the disbiotic microbiota could interfere with the therapeutic response and with intestinal permeability, which was significantly increased in our patients.

In addition to changes in the gu<sup>t</sup> microbiota, recent studies have associated small intestine rupture with the development of MS, and, based on this, Rahman and colleagues hypothesized that a leaky gu<sup>t</sup> is mechanistically linked to BBB disruption through receptors for zonulin [71]. One of the predictors of intestinal permeability in humans is the serum zonulin level. Zonulin is a physiological modulator of tight junctions involved in the traffic of macromolecules and in the maintenance of epithelial barrier integrity and immune tolerance in the gu<sup>t</sup> mucosa. [72]. A leaky gu<sup>t</sup> in mice induces inflammatory cytokines release that promote an increased permeability, establishing a vicious circle favoring the entry of antigens derived from diet and gu<sup>t</sup> microbes, inducing a tolerance breakdown and the activation of immune cells in the gastrointestinal mucosa [73,74]. The activated immune cells can remain in the gu<sup>t</sup> or migrate to distant organs, including the brain [73–75].

Intestinal dysbiosis can activate the zonulin pathway and stimulate cytokines release allowing the leakage of luminal contents through the epithelial barrier [73]. A study from Camara-Lemarroy and colleagues detected an increase in serum zonulin concentrations in RRMS patients, which positively correlated with BBB disruption, confirmed by positive gadolinium images in MRI [76]. In the present study, we detected a significant increase in serum zonulin concentrations in treated RRMS patients, suggesting that increased gu<sup>t</sup> permeability could be a consequence of the intestinal dysbiosis detected in treated RRMS patients.
