Interplay between Mediterranean Diet and Gut Microbiota in the Interface of Autoimmunity: An Overview

Laboratory of Microbiology, Medical School, Democritus University of Thrace, University Hospital,68100-Dragana,Alexandroupolis,Greece Laboratory of Molecular Cell Biology, Cell Cycle and Proteomics,Department of Molecular Biology and Genetics,Democritus University of Thrace, 68100-Dragana,Alexandroupolis, Greece Department of Dermatology, Faculty of Medicine, School of Health Sciences, University of Thessaly, Larissa, Greece Department of Neurology, Faculty of Medicine, School of Health Sciences, University of Thessaly, Larissa, Greece Department of Immunology-Histocompatibility, Evangelismos General Hospital, Athens, Greece Department of Rheumatology and Clinical Immunology, Faculty of Medicine, School of Health Sciences, University of Thessaly, Larissa, Greece


Introduction
It is widely accepted that the Mediterranean Sea offers an ideal temperate environment, with regards to temperature, humidity and sunlight to the surrounding countries and nations. Most of the inhabitants of the Mediterranean Sea basin have specific dietary trends, which scientists believe that support a healthy state of life. Since the early 1960's, it has become apparent that Mediterranean people follow a habitual way of eating with slight differences, the so called "Mediterranean Diet" (MedDiet). But it was not until 1993, when the Harvard School of Public Health, Ordway's Preservation and Exchange Trust and the European Office of World Health Organization, together with Greek researchers introduced the MedDiet Pyramid, based mainly on the eating patterns of Crete island and Southern Italy [1,2]. MedDiet is plant-orientated with high intake of vegetables, cereals, nuts and fruits -meaning lots of fiber-, and smaller quantities of animal products preferentially seafood and fish.
Over the last 15 years, research has yielded a wealth of knowledge regarding human microbiome. The inconceivable diversity and abundance of all living organisms (microbes, fungi, viruses, parasites), which co-evolved with humans for thousands of years and inhabit the human body was revealed by next generation technologies.
Observations in both experimental models and in humans struggle to define the precise relations and interactions between microbiota and health, as well as their correlation to different diseases. Symbionts and pathobionts are in a constant battle within gut microbiota contributing to a final state of symbiosis or dysbiosis. Different external and internal factors affect the composition of gut microbiome and diet appears to be one of the most important. Dietary habits have figurative results in intestinal microbiota and MedDiet seems to favor specific phyla while suppressing others [3]. 7 inhabitants from different body sites and concluded that the gastrointestinal tract (especially the gut) is, by far, the most populated site of the human body [24]. The genome of these gut microorganisms forms the gut microbiome, a very abundant and diverse ecosystem with complicated interactions among them as well as with the host, by affecting different aspects and maintaining human health throughout life. Largescale studies have dictated that different eating patterns promote a variety of gut microbiota composition and diversity and highlight diet as one of the most significant influencing factors [3,6,25,26].
Different eating habits, alterations in dietary components -such as fats, proteins and carbohydrates-, food complements as probiotics and prebiotics, even salt composition, can all modulate the gut microbiota [27]. However, such changes can exert an effect not just at microbial species level but also at phyla differences [28]. Diets rich in carbohydrates or fats alter the numbers of various microbial species, increasing some and decreasing others, and as a whole they may reduce the microbiota diversity [29][30][31][32][33]. However, addition of prebiotics may restore population alterations caused by highfat diet in obese mice [34]. Moreover, the gut microbiota of mice fed with increased amount of protein also showed significant increase or decrease of particular microbial genera and these alterations were dose-dependent [35]. In humans, gut microbiota varies at the species level, depending on fat and fiber or carbohydrate consumption, between omnivores and vegetarians, or even among vegetarian subgroups [3]. In general, eating habits have an effect on gut microbiota diversity, with meat consumption -in addition to fibers-allowing increased bacterial species diversity. This diversity lies on higher taxonomy levels, as phylum, family and genus, rather than at the species level. Alterations in single dietary components, such as gluten, may also change composition and function of the gut microbiome and host physiology of healthy 8 individuals [36]. Effects of diet on gut microbiota are demonstrated by numerous studies, comparing different food habits, dependent on the geographical and socioeconomical differences seen in different areas [37][38][39]. Interestingly, a large-cohort study on US immigration from nonwestern countries showed a relatively fast (within 9 months of immigration) and long-term loss of gut microbiome diversity, loss of bacterial enzymes related to plant fiber degradation, displacement of native strains and functions and, as a result, predisposition of individuals to obesity and metabolic diseases. These profound perturbations in gut microbiome can be partially explained by dietary variations [40].

Gut microbiome's link to autoimmunity
It seems that apart from the identification of all taxa of microbes, the ultimate challenge is to define the benefits and drawbacks of the 'healthy gut'. The human microbiota, and the gut in particular, has been deemed an "essential organ", containing approximately 1000 different microbial species and counting for over 150 times more genes than those of the whole human genome [41,42]. The vital (essential/fundamental) role of human microbiota in health and disease includes a variety of mechanisms. The microbiota assists energy extraction from food, supplies unique enzymes and biochemical pathways to the human body, and acts as a physical barrier against foreign pathogens through antimicrobial substances or regional competition [43][44][45]. Last but not least, the gut microbiota is indispensable for the normal development of both the intestinal mucosa and the host humoral and cellular immune system, as signals and metabolites from the microorganisms are sensed by cells of the innate immune system and are recognized as physiological responses [46][47][48]. The term "autoimmunity" refers to activation of adaptive immune responses, with the involvement of T and B lymphocytes against self-antigens, i.e. against its own healthy cells and tissues. The actual causes of 9 autoimmunity are not fully understood, but various environmental factors, including diet, lifestyle and infections, along with the genetic background of the host, seem to play a role [49,50].The principle of initiating the adaptive immune responses lies in the innate-adaptive immune system connection. It is the innate immune system that can discriminate between self and non-self-antigens, whereas the adaptive immune system recognizes either native antigens or peptides connected to major histocompatibility complex molecules [51]. As a result, it is the adaptive immune system that responds to invasion of the host by a microbial pathogen or parasite. Studies in both humans and animal models designate the involvement of commensal microbiota in autoimmunity.
Effects of the microbiota on autoimmune diseases that require the innate-adaptive immune system connection may vary from being neutral to being requisite for the induction of autoimmunity. The loss of immune tolerance to self-antigens can be a result of microbial composition alterations, and, thus, the human microbiota can be a key participant in the development and perpetuation of autoreactive immune responses which lead to self-tissue destruction and overt autoimmune disease [52,53]. There are several reasons for loss of immune tolerance and induction of autoimmunity by microorganisms, including molecular mimicry, bystander activation, and viral persistence with or without epitope spreading [54][55][56][57][58].
Epitope spreading refers to autoimmune responses to self-epitopes, when, at an inflammatory response self-antigen are released. These self-antigens can be a result of slightly changed antigens, even at an amino acid residue. As a result, the immune reaction will affect both the "wild" protein or the altered one [59].
In molecular mimicry, a shared immunologic epitope between a microbe and the host is the prerequisite for the initiation of cross-reactive immune responses. One of the finest examples of molecular mimicry as a mechanism responsible for the development of autoimmune disease is that in individuals with rheumatic fever after infections with group A beta-hemolytic streptococci. Analysis of infected hosts' sera demonstrated the presence of antibodies reactive with heart, joints, brain, and skin. Moreover, patients' antibodies are found to cross-react with streptococcal antigens, like the group A carbohydrate antigen, the M protein (a streptococcus-related virulence factor) and to cross-react with myosin. Cross-reactive peptides from M protein and cardiac myosin may provoke the onset of autoimmune disease in mice with rheumatic heart disease [60]. The mechanism of molecular mimicry can also operate at the T-cell level involving antigenic epitopes of human and foreign origin which serve as targets of CD4 and CD8 T-cell responses. Previous studies of our group has meticulously addressed the role of molecular mimicry in the induction of autoimmune diseases, primarily affecting the liver and the gastrointestinal tract [61][62][63][64][65][66][67][68][69][70][71][72][73][74][75][76][77][78][79].
Bystander activation/killing is another mechanism resulting in autoimmune diseases.

Adherence in MedDiet and its effects on microbiome related to autoimmune diseases: the case of rheumatoid arthritis
The Mediterranean diet is rich in fiber, antioxidants and vitamins and encompasses antiinflammatory properties [81]. Various data have demonstrated that consumption of cereals, fruits and vegetables, nuts and legumes, omega-3 polyunsaturated fatty acids in olive oil and moderate consumption of red wine flavonoids leads to the reduction of pro-inflammatory cytokines, the increase of anti-inflammatory cytokines and the decrease of oxidative stress [82,83]. It has been shown, that adherence to a Mediterranean pattern diet leads to the reduction of CRP and TNF-α levels [84,85]. Another study claims that adherence to MedDiet is associated with decreased disease activity, better physical activity, as well as increased viability in RA individuals [86].
Subsequent studies have produced inconsistent results [86][87][88][89][90][91][92][93]. A recent systemic review analyzing the data so far provided from prospective human studies, concluded that MedDiet has beneficial effects in reducing pain and increasing physical function in people with RA but underline that so far there is unsatisfactory evidence to support widespread recommendation of MedDiet for the prevention and management of RA [7].
Data from studies on short term MedDiet, as well as fasting, found no effect of these diets on the microbiologically assessed intestinal flora of RA or fibromyalgia patients [94]. Recent studies have underlined the long term adherence (more than 3 12 months) to MedDiet is needed to produce significant diversity in the gut microbiome of overweight omnivores [95].  [96].
An association between MD and increased SCFAs production is well known [97].Rich fiber foods such as fruit, vegetables, and legumes which are repeatedly consumed by those adherent to MedDiet are degraded by Firmicutes and Bacteroidetes bacteria, leading to the production of high levels of feacal SCFA [98,99], which is beneficial for the intestinal barrier integrity; for example butyrate, the most extensively investigated functional SCFA produced during the fermentation of dietary fiber by the anaerobic intestinal microbiota, increases the expression of tight junction proteins and prevents from deleterious intestinal permeability and bacterial translocation, which have a blocking impact on the initiation of pro-inflammatory responses [100,101,98].
To relevance in collagen-induced arthritis, an animal model of RA, butyrate is able to suppress RA features and this is achieved via a butyrate-mediated increase of IL-10 producing Tregs and a decrease of Th17 [102].However, not all Mediterranean type of diets increase SCFAs, and butyrate in particular at the same extent, and this may have 13 an impact in their ability to suppress anti-inflammatory immune responses or to influence intestinal microbiota-related influence of the immune system. Modified Mediterranean type enriched for SCFA production are increasingly popular but their effect in RA has not been assessed. Omnivores who consume a MedDiet-pattern diet rich in fruit, legumes and vegetablesnot only have increased SCFAs [98] but also decreased trimethylamine N-oxide (TMAO), a microbial metabolite the precursors of which are carnitine and choline which are primarily found in foods of animal origin [98,103]. Several microbial genera, like L-Ruminococcus, have been linked to the intake of animal proteins such as a diet plenty in red meat consumption and increased TMAO levels. This is very interesting, in view of recent data demonstrating a twofold to threefold increased abundance of Ruminococcus gnavusin patients with spondylarthritis and to a lesser extent in RA patients compared to healthy controls [104].
However, the most notable association over the last few years is that linking P.copri the TMAO-producing anaerobic, Gram-negative Bacteriodetes, with the development of RA, an association thoroughly reviewed elsewhere [105,106].Prevotella spp are abundant in the periodontium, the intestine, and the respiratory system and its enriched presence is considered a risk factor for RA and features associated with RA, such as cardiovascular risk-events [105,[107][108][109][110]. Furthermore, it may alter the metabolism of the microbiota to reduce the effectiveness of the common disease-modifying antirheumatic drug (DMARD) methotrexate [111]. The question arises as to whether adherence to MedDiet alone or in combination with other diet supplementation can alter gut dysbiosis to a state that Prevotella spp. are not dominant [61,112,113]. This event would stop the vicious circle of immunological events that take place, Prevotella being in the center of it and could prevent from RA. Thus, there is evidence that a molecular mimicry mechanism is in operation, which could account for the induction of those autoantibodies [115].Of relevance, molecular mimicry involving an oral Prevotella sp. and collagen I, have been previously reported and has been considered a likely trigger for chronic periodontitis and possibly inflammatory [116].
The GNS peptidyl sequence was highly homologous to a sequence from sulfatase proteins of the Prevotella sp. and Parabacteroides sp. Finding marked homologies between human and microbial highly conserved proteins is extremely common and the homology reported by Pianta et al belongs to this category. In a similar vein, homologies between human and microbial heat shock proteins and human and microbial 2-oxo-acid dehydrogenase complexes have been identified and suggested as triggers of various organ and non-organ specific immune-mediated and autoimmune diseases. Because they are extremely common, several investigators, including authorities in the field, suggested that molecular mimicry involving such homologues must not be regarded as a perpetuator of autoimmunity.
We carefully examined the reported similarities between human GNS and Prevotella. Pianta  We went a step further and postulated that gut dysbiosis leads to immunologic alterations, which are pivotal for the loss of immunological tolerance to RA-specific autoantigens. We considered that this is achieved in a stepwise manner and that several immunological mechanisms are involved, molecular mimicry being just one of those.
Firstly, we have an external parameter, which leads to changes in the gut microbiome  [117].
Of indirect relevance to the topic we discuss, amongst the American indigenous populations, the Canadian Inuit population has the lowest age-adjusted prevalence of rheumatoid arthritis (at 0.65%, with an incidence of 48.2 per 100,000 per year) [118]. This is of interest because a microbiome study has found that Prevotella spp., were enriched among the Inuit consuming a Western diet. However, the gut microbiomes of Inuit consuming a traditional high-fiber diet [119] had significantly less genetic diversity within the Prevotella genus, compared to the Inuit consuming a Western diet further, indicating that a low-fiber diet might not only select against Prevotella but also decrease its diversity, a factor which could be relevant to the induction of autoreactive responses implicating Prevotella in inflammatory arthritis.
A very recent study has shown that berberine modulates gut microflora and exerts an anti-inflammatory effect on collagen-induced arthritis [120]. This is achieved because the abundance of Prevotella is diminished and the abundance of butyrate-producing bacteria in CIA rats is increased [120].
However, a recent study investigating changes on gut microbiota associated to MedDiet found a higher presence of Bacteroidetes and a lower Firmicutes-Bacteroidetes ratio, who had a higher MedDiet score but failed to report significant differences in levels of Bacteroides and Prevotella, genus included in Bacteroidetes phyla [121]. Bowel Disease [132][133][134].
Taken together, future studies on the role of Mediterranean diet components or as a whole on improvement of microbial dysbiosis, as well as reduction of autoimmunerelated inflammatory reactions would be of high importance. The diet appears to influence components of the innate and adaptive immunity, via a plethora of mechanisms, which act in isolation or in concert.

Restoring microbiota through 'nutraceuticals'
Nutraceuticals, a term coined by their "Godfather" Dr Stephen De Felice in 1989, contains both nutrition and pharmaceutical concepts and include products isolated or purified from foods. Established nutraceuticals include probiotics, prebiotics, omega-3 and -6 fatty acids, and others like polyphenols, phytoestrogens, flavonoids and antioxidants, with already recognized favourable effects under specific conditions [135,136]. Nutraceuticals are "related" to the human microbiota that includes 6 taxonomic bacterial phyla with Firmicutes and Bacteriodetes occupying the 90% of the host's colonized areas [137]. Nutraceuticals include probiotics, which WHO defines as "live micro-organisms" which, when administered in adequate amounts, confer a health benefit on the host. Probiotics produce short chain fatty acids (SCFAs), which are able to restore both population numbers and diversity of microbiota. Lactobacillus species may decrease or even prevent the symptoms of antibiotic associated diarrhea (AAD) [138], while a meta-analysis study showed positive effect of probiotics on AAD [139].
Furthermore, Lactobacillus, Bifidobacterium or Escherichia coli species have positive impact on host against metabolic diseases or gastrointestinal disorders [137].  [144]. Phytoestrogens include flavonoids, which regulate the intestinal barrier and own antimicrobial effect against pathogens, being characterized as alternative antibiotics [145]. Anthocyanins (also flavonoids), found in fruits like grapes and apples, may prevent a wide range of diseases [146]. Polyphenols, as quercetin, found in apples, grapes, onions, tomatoes, nuts and seeds, alters the gut microbiota in overweight mice fed with high-fat sucrose diet [147].
Resveratrol, another polyphenol taken as a food supplement regulates gut microbiota dysbiosis caused by high-fat diet, by enhancing growth of Lactobacillus and Bifidobacterium, raising the ratio of Bacteroidetes/Firmicutes and by hampering growth of E. faecalis [148]. Carvacrol and thymol, phenols in the aromatic plant Oregano vulgare, have antibacterial properties and ability to affect the gut microbiota and the immune status in animal models [149]. Use of omega-3 and -6 polyunsaturated fatty acids, as supplements, alter gut microbiota composition, by increasing Bacteroidetes/Firmicutes ratio, restrains growth of pathogenic bacteria like Helicobacter, Firmicutes, Pseudomonas sp, thus, avoiding immunological disturbances [144].

Conclusions
Taken together, there seems to be a close link among dietary habits, -both as foods or dietary supplements like nutraceuticals-, modulation of gut microbiota and, in turn, a critical role of human commensals on disease prevention/regulation, onset/development of immunological disturbances, as autoimmune, metabolic and other diseases. The Mediterranean diet is based on fruits, vegetables, seeds, nuts, fish, whose composition is rich in prebiotics, phytoestrogens as flavonoids, polyphenols and omega -3 and -6 polyunsaturated fats. Numerous studies have correlated Mediterranean dietary habits with beneficial effects on gut microbiota composition and possible positive connection with inflammatory diseases' risk of appearance and/or progression.
Thus, it could be of critical importance to introduce or recommend/ or amplify the need for adopting Mediterranean dietary habits that may modulate microbial homeostasis, decrease the effects of pathogen invasions, and influence the inflammation pathway, for health's benefit. However, further studies, on its individual components and/or total/overall compounds of the Mediterranean diet "philosophy" on health's benefit via microbiota homeostasis should be conducted, to address the effect of the diet and its constituents in the prevention and clinical management of patients affect with specific autoimmune disorders.

Figure 1
The nutritional habits regulate the gut microbiota and may provoke or prevent autoimmunity. The Med Diet activate gut microbial symbiosis, on the other hand Western diet seems to be a critical player for microbial dysbiosis and that may develop