Disease of the holobiont, the example of multiple sclerosis

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Review

Disease of the holobiont, the example of multiple sclerosis夽 Federico Castillo-Álvarez ∗ , María Eugenia Marzo-Sola Servicio de Neurología, Hospital San Pedro, Calle Piqueras n◦ 98, 26006 Logro˜ no, La Rioja, Spain

a r t i c l e

i n f o

Article history: Received 2 July 2018 Accepted 31 August 2018 Available online xxx Keywords: Microbiota Microbiome Holobiont Hologenoma Multiple sclerosis Experimental autoimmune encephalomyelitis

a b s t r a c t In recent years there has been a revolution regarding the role of the microbiota in different diseases, most of them within the spectrum of inflammatory and autoimmune diseases, associated with the development of metagenomics and the concept of holobiont, a large organism together with its microbiota. Specifically, in Multiple Sclerosis, multiple evidence points to the role of the microbiota in experimental autoimmune encephalomyelitis, animal model of the disease, and several articles have been published in recent years about differences in intestinal microbiota among patients with multiple sclerosis and control subjects. We review in this article the concept of holobiont and the gut microbiota functions, as well as the evidence accumulated about the role of the microbiota in experimental autoimmune encephalomyelitis and multiple sclerosis. Nowadays, there is a lot of evidence showing the role of the microbiota in the genesis, prevention and treatment of experimental autoimmune encephalomyelitis based mainly on three immunological pillars, the Th1–Th17/Th2 balance, the Treg cells and the humoral immunity. It is also well documented that there are differences in the microbiota of patients with MS that are associated with a different expression of genes related to inflammation. ˜ S.L.U. All rights reserved. © 2018 Elsevier Espana,

El holobionte enfermo, el ejemplo de la esclerosis múltiple r e s u m e n Palabras clave: Microbiota Microbioma Holobionte Hologenoma Esclerosis múltiple Encefalomielitis autoinmune experimental

˜ En los últimos anos se ha producido una revolución en torno al papel de la microbiota en diferentes enfermedades, la mayoría dentro del espectro de las inflamatorias y autoinmunes, asociado al desarrollo de la metagenómica y al concepto de holobionte, entendido como el conjunto formado por los organismos superiores y su microbiota. Concretamente, en la esclerosis múltiple, existe múltiple evidencia acerca del papel de la microbiota en la encefalomielitis autoinmune experimental, modelo animal de la enfermedad ˜ y se han publicado en los últimos anos diversos artículos acerca de las diferencias en la microbiota intestinal entre pacientes enfermos de esclerosis múltiple y sujetos control. En este artículo revisamos el concepto de holobionte y las funciones de la microbiota dentro del mismo, así como la evidencia acumulada en el papel de la microbiota en la encefalomielitis autoinmune experimental y en la esclerosis múltiple. A día de hoy, existe una amplia evidencia científica del papel de la microbiota en la génesis, prevención y tratamiento de la encefalomielitis autoinmune experimental en base fundamentalmente a tres pilares inmunológicos, el equilibrio Th1-Th17/Th2, las células Treg y la inmunidad humoral. Así mismo está bien documentado que existen diferencias en la microbiota de pacientes con EM que se asocian a una diferente expresión de genes relacionados con la inflamación. ˜ S.L.U. Todos los derechos reservados. © 2018 Elsevier Espana,

Introduction

夽 Please cite this article as: Castillo-Álvarez F, Marzo-Sola ME. El holobionte enfermo, el ejemplo de la esclerosis múltiple. Med Clin (Barc). 2019. https://doi.org/ 10.1016/j.medcli.2018.08.019 ∗ Corresponding author. E-mail address: [email protected] (F. Castillo-Álvarez).

The role assigned to the microbiota, understood as the set of microorganisms present in a certain habitat, in higher organisms, has undergone an important change in recent years, establishing an important role in the normal physiology of the hosts, as well as in their health-disease state. This change has aroused great interest in both the medical community and the general

˜ S.L.U. All rights reserved. 2387-0206/© 2018 Elsevier Espana,

MEDCLE-4646; No. of Pages 7

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population, with abundant scientific and informative literature published regarding the microbiota and the microbiome, together with their genes. Knowledge of the role of the microbiota in higher organisms has undergone a great resolve since approximately 2006 when Rosenberg put forward the coral probiotic hypothesis according to which the Patagonian Oculina, a species of coral that suffered an epidemic produced by a bacterium of the Vibrio genus, managed to overcome the disease thanks to the genetic information provided by its microbiota.1 Two years later, the same group proposed the Hologenome theory of evolution, which studies higher organisms and their microbiota as a whole at functional and evolutionary levels, and that possesses a hologenome, understood as the sum of genetic information of the complex multicellular organism, in our case the human being, and the microbiota that it hosts. This theory can be summarised in four points2 : 1. All animals and plants establish a symbiotic relationship with their microbiota (whose genetic information often exceeds that of the host itself) 2. The cooperation between the host and the microbiota contributes to the strength of the holobion and affects both parties 3. Variations in the hologenome can occur both in the host and in the microbiota, but in situations of environmental stress, the symbiotic microbial community adapts and changes much faster than the host 4. The symbiotic microbiota is transmitted between generations At the same time the conceptual change of the role assigned to the microorganisms occurred, a series of technological advances revolutionised how much knowledge we could obtain from the microbiota through metagenomic techniques. For the study of microbial communities, classical microbiology required the isolation and culture of microorganisms for their subsequent identification, either by staining or growing in certain culture media, or by the subsequent extraction of genetic material and with it the sequencing and identification of species. The limitations for this whole process was the first point, since it was difficult to isolate microorganisms from such complex communities as, for example, intestinal microbiota, and because many microorganisms were, and still are, non-culturable. This limitation has been resolved in recent years with the birth of metagenomics, which consist of the application of modern genomic techniques for the direct study of communities of microorganisms in their natural environment, avoiding the need to isolate and cultivate each of the species that comprise the community. Basically, to date, DNA is extracted from the entire microbial community3 and then the 16S ribosomal DNA sequencing is carried out, especially in certain hypervariable regions. This DNA has the characteristic of being universally found in all bacterial and archaea organisms, but not in eukaryotic cells, which makes it possible to exclude host cells from the study of the holobion. Subsequently, the obtained sequences can be treated in different ways to increase yield, and are quantified and identified by comparison with certain databases of known sequences in order to assign them an ‘operational taxonomic unit’, that is, to identify them as a determined taxon, at any level of the phylogenetic scale, from phylum (the highest level in which each kingdom can be separated) to species.4–7 The global RDP (Ribosomal Database Project) was specifically created for the analysis of the metagenomic data of the gene that codifies for the 16S and which hosts approximately one million sequences, comprising part of GenBank, the USA NIH (National Institute of Health) genetic sequences database.8 As a result of these technological and knowledge changes, in recent years scientific production has increased exponentially in

terms of microbiota. When the term ‘microbiota” was entered into PubMed in 2006, it would give 289 results; in 2017 it would give 6683. And a search for the term ‘metagenomic’, has gone from 65 publications in 2006 to 1659 in 2017. The human holobion Recently, in 2016, the relationship between the number of human and bacterial cells in the human holobion was revised. Thus, it is estimated that a male human body weighing approximately 70 kg comprises 3.0 × 1013 cells, most of them corresponding to erythrocytes, which total a weight of approximately 46 kg. The numbers do not vary much for a female of equal weight. For this same model, bacteria provide a much lower weight, around 200 grams, but the number of bacteria is around 3.8 × 1013 (somewhat higher, 4.4 × 1013 for women), that is, a number slightly higher than that of human cells, but in a ratio close to 1:1.9 If instead of number of cells we study the gene, considering that human cells share the same genetic load, but the microorganisms of the microbiota do not, the ratio shoots up in favour of the bacteria, which contribute 3.3 × 106 genes to the holobiont, exceeding human genes by about 150 times.10 We also know today that 10% of the metabolites found in the blood of mammals is bacterial.11 Until recently, it was assumed that newborns were sterile, and that the first contact with microorganisms occurred in the birth canal. Today it is suggested that the implantation of the microbiota in the holobiont occurs before birth, having been shown that there are microbial communities in the uterus, semen, placenta, amniotic fluid, umbilical cord and meconium. However, it is during vaginal delivery and during lactation that large bacterial communities are introduced into the intestine of the new human being. At the end of lactation, and coinciding with solid feeding, the ideal conditions for the growth of bacteria that degrade plant polysaccharides, such as Bacteroidetes, prepare the way for the implantation of the second dominant phyla in the human intestinal microbiota, Firmicutes. Thus, at age three, the microbiota is already mature, diverse and stable, and it will remain that way, except for changes that are maintained over time, until old age, where an impoverishment in bacterial diversity is again observed with an increase in anaerobes, which subtracts adaptation capacity from the microbiota, increasing the risk of dysbiosis.12 Functions of the microbiota Nowadays, fundamentally thanks to studies carried out with axenic mice (those reared in sterile conditions that ensure the absence of germs in their organism), we can confirm that the microbiota is necessary, but not vital, for adequate development and metabolic functioning. Table 1 summarises the functions that the aforementioned microbiota contribute to the holobion, and in turn for the study and understanding of multiple sclerosis (MS), the maturation and development of the central nervous system (CNS) and the development and modulation of the immune response.13 Table 1 Biological functions of the gut microbiota in humans. Integrity of the epithelial barrier Barrier to the colonisation of pathogens Development of the immune response Energy source Biosynthesis of vitamins Transformation of bile salts Metabolism of xenobiotics Maturation and development of the central nervous system

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The presence of cerebral anomalies in axenic mice, and their reversibility through early recolonisation – not when it is carried out in adult life – has revealed the influence of the microbiota on brain development.14,15 The term bowel–brain axis is used to describe the close relationship between the bowel and the CNS, both at afferent and efferent levels. Bidirectional modulation occurs at the following levels: hormonal, mediated by adrenocorticotropin and cortisol; nervous, mainly through the vagus nerve; immunological, and through neurotransmitters, where the role of serotonin, but also of adrenaline and dopamine, stand out.16 In addition to MS, the microbiota has been implicated in various pathologies that affect the normal functioning of the CNS, such as mood alterations, autism spectrum disorders, attention deficit hyperactivity disorder, and optic neuromyelitis.17 Within this last autoimmune disease, belonging to the demyelinating spectrum, in addition to the role played by the microbiota in the CNS and in the immunological regulation, we now know that those positive aquaporin patients are capable of developing a cross-reactivity with a homologous sequence in an epitope present in Clostridium perfringes, representative of the commensal flora, suggesting that a mechanism of cellular mimicry may be involved in the activation of a Th17 response.18 Regarding the role of the intestinal microbiota in the immune response, it has been implicated in various mechanisms of the development and maturation of the immune system, both innate and adaptive, thus, to cite some examples, it has been involved in the regulation and maturation of the immune systems, Peyer’s patches, mesenteric lymph nodes and germinal centres, in the regulation of the number of plasma cells that produce IgA, intestinal T lymphocytes and CD4+, with the same type of expression in the lamina propria or intraepithelial cells, as well as in the gene expression of TLR and Major histocompatibility complex II.19 On the other hand, the microbiota can modulate the immune system through numerous metabolites, which gives it a preponderant role in autoimmune diseases, and therefore in MS. First, bacteria can metabolise dietary components, such as dietary fibre, tryptophan or arginine, resulting in polyamines, indoles and short chain fatty acids, including propionic, butyric or acetic. The latter present a central role in the regulation of the immune response, increasing the expansion of regulatory T cells (tolerogenic), decreasing and favouring an anti-inflammatory phenotype in dendritic cells and decreasing the production of proinflammatory cytokines in neutrophils and macrophages,20 in addition to promoting the integrity of the blood–brain barrier.21 Other mechanisms by which the microbiota regulates the immune response is through metabolites produced by the host and modified by bacteria, as is the case of secondary bile acids that regulate dendritic cells, macrophages and natural killers, or through metabolites produced de novo by bacteria; the protective role of polysaccharide A of Bacteroides fragilis, and the autoimmune progen of ATP produced by segmented filamentous bacteria have been much studied.20

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Escherichia coli presented a clinically milder, histologically less inflammatory disease, with less differentiation to T lymphocytes producers of IFN-Y, and greater than TregFoxP3+, lymphocytes, and overexpression of IL-4, IL-10, IL-13 and TGF-␤.23–25 These studies showed that certain microbial components influence the development of EAE. To implicate the microbiota directly in the development of the EAE, mice were treated with broad-spectrum antibiotics prior to the induction of the disease, which hindered their development, reducing the production of proinflammatory cytokines and mesenteric Th17 cells,26 also being implicated in the process to TregFoxP3+ lymphocytes and dendritic cells CD103+. In addition, the reintroduction of the microbiota in the mice reversed the effects of the antibiotics.27 From these experiments it is clear that the presence or absence of intestinal microbiota affects the course of EAE. The next step was to try to involve certain specific microorganisms, or their products in the EAE. Thus, the polysaccharide A (PSA) of the Bacteroides fragilis capsule, known for its protective role in inflammatory bowel disease was tested for its ability to correct deviations between Th1 proinflammatory and Th2 more tolerogenic, lymphocytes. For this, mice were treated with antibiotics and re-colonised with B. fragilis PSA+ or PSA− were used, and it was seen that the first maintained an EAE resistance, presenting a greater ratio of TregFoxP3+ lymphocytes producing IL-10 (tolerogenic). A similar effect was obtained when treating only with purified PSA administered orally. These benefits were lost if the mice in the experiment were deficient for the production of IL-10.28–31 In the opposite direction, and now working with axenic mice, it was demonstrated that monocolonisation with segmented filamentous bacteria generated EAE in predisposed mice, implicating the Th17 lymphocytes (pro-autoimmune) and the IL-17 produced by them in the process, which increased at both gastrointestinal and CNS levels.32 This demonstrated that certain bacteria or bacterial products alone could have a protective or inducer role in EAE. Also working with axenic mice, capable of developing EAE spontaneously, and after the administration of the self-antigen target, the MOG (myelin oligodendrocyte glycoprotein), a lower production of antibodies was observed. The replacement of the microbiota in conjunction with MOG produced an inflammatory response and a rapid increase in the production of antibodies against MOG.33 With this, the implication of humoral immunity was added to the already known mechanisms by which the microbiota intervenes in the EAE. Since then, numerous studies have been published that implicate multiple microorganisms – some used as probiotics – and various immunological mechanisms in the modulation of the immune response in EAE.34–41 Today we can state that there is important evidence showing the modulation of the immune response in the EAE by the microbiota, which influences the genesis, prevention and treatment of the EAE, based mainly on three immunological pillars, the Th1-Th17/Th2 equilibrium, Treg cells and humoural immunity.

The microbiota in experimental autoimmune encephalomyelitis

The microbiota in multiple sclerosis

In the last decade, a large amount of scientific evidence has been produced that relates the experimental autoimmune encephalomyelitis (EAE), animal model of multiple sclerosis, with the intestinal microbiota.22 Thus, in 2007, based on the studies carried out by Ochoa-Repáraz, intestinal bacterial began to be associated with peripheral tolerance and the prevention or treatment of EAE. It was shown that mice ‘vaccinated’ with the antigenic colonisation factor I (CFA/I) fimbriae of enterotoxigenic

The first studies linking MS with the microbiota were presented in the form of poster communications in congresses, during the 2014 American Academy of Neurology (AAN) meeting. The first was presented by the Jangi group, and presented the results of a yet unpublished study – which we will explore further below – that showed an increase in archeas (microorganisms straddling bacteria and eukaryotes) in patients with MS and a decrease in butyrate producing microorganisms.42 The second of the papers

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presented in this congress, a modest study with seven patients and eight controls, also published later, showed differences between patients with MS and controls at the phyla level, but that were not statistically significant.43 That same year, the following data were presented at the ACTRIMS-ECTRIMS meeting in Boston: a preliminary study carried out on a paediatric population showing differences in certain bacterial genres; a second poster that further explored the immunological mechanisms presented by Jangi, and a third study outlining the role of human Treg lymphocytes and IL-10 in lymphocyte cultures in the presence of B. fragilis PSA.44 Later in 2015, the study presented in the AAN meeting by Mowry was published; this was a substudy of another study carried out whose objectives were to demonstrate the influence of treatment with vitamin D on patients with MS. They presented differences between eight healthy control subjects and seven with MS – two without treatment and five treated with glatiramer acetate – which were mainly associated with sex and changes in treatment; however, the study weakness, sample size and methodological problems where three of the treated patients gave stool samples outside the initially defined period, limited theses results.45 Subsequently, also in 2015, Miyake et al. published the first descriptive and comparative study with a significant number of patients – working with a Japanese population, the authors managed to recruit 20 patients with relapsing-remitting MS (RRMS) and 40 healthy controls. The first observation, and one which has been maintained throughout the different published studies, is that patients with RRMS did not present differences compared to healthy patients in the alpha diversity, i.e. the bacterial richness, the differences resided in specific taxa and not in the total number of species that comprised individuals’ intestinal microbiota. Apropos specific taxa: they reveal differences at the phyla level, although not statistically significance differences. They are, however, statistically significant in the abundance of 21 species, belonging mainly to taxa included in the clostridia XIVa and IV clusters and in the phylum Bacteroidetes, taxa involved in the production of short chain fatty acids.46 One year later, a study by Chen et al. into a North American population was published comparing 36 controls and 31 patients with RRMS. This study focused on a difference in species richness between patients with active MS and those in remission and the control group, although this difference may be caused by the fact that patients undergoing corticosteroid treatment were not excluded. This group also revealed changes in certain bacterial genera, reinforcing the idea that the profile of microbial communities in MS varies specific taxa.47 Tremlett et al. carried out a study with 18 patients with RRMS and 17 healthy controls also in the North American population, but in this case the sample was from the paediatric age. Concurring with what had been published until then, the authors found no differences in alpha diversity, but they did find differences in certain genres, with an emphasis in the enrichment in MS patients of genres involved in the metabolism of glutathione.48 In another study, this same group found an inverse relationship between the Bacteroidetes phylum and Th17, and a direct relationship between richness and Th17 in patients with MS, but not in controls.49 Going one step further, the Jangi group, which had already submitted partial results in the 2014 ANA, published an article using a North American adult population, 60 patients with MS and 43 control subjects, and, in addition to looking for differences at the metagenomic level in faeces by means of two different techniques, they took serum from the subjects and performed a profile analysis of gene expression in mononuclear cells. Thus, in addition to demonstrating differences in the abundance of certain genera, they

found a correlation between their abundance and the expression of genes related to the maturation of dendritic cells, and the signalling of interferons or NF-kB in T lymphocytes and circulating monocytes, coinciding with the fact that these alterations in the abundance of taxa are associated with immunological changes that point to pro or anti-inflammatory profiles.50 Recently, in October 2017, two studies were published by the same group combining the studies carried out in humans, in vitro and in animal models. Thus, in a first phase of the study, intestinal microbiota samples obtained from control subjects and patients with MS were compared using metagenomics, and colonisation of animal models was subsequently carried out with the microbiota obtained from each of the groups to explore the ‘clinical evolution’ in EAE. In the first of these, 71 patients with MS and 71 controls were chosen, and those bacteria that were increased and decreased in subjects with MS were identified. Subsequently, it was observed that those taxa that were increased in patients with MS were able to increase the inflammatory response both in vitro, in lymphocyte colonies, and in vivo, in axenic mice, which were subsequently monocolonised with these bacteria. Likewise, the diminished bacteria in patients with MS were able to stimulate the differentiation to IL-10+ T lymphocytes in vitro, and to stimulate Treg in monocolonised mice. Finally, they demonstrated that mice colonised with microbiota MS showed severe symptoms of EAE and a decreased proportion of IL-10+ Treg lymphocytes.51 In the second series, first carried out with a European population, in Germany, an almost perfect control group was sought. Thus, they took 34 pairs of twins that differed in the disease, that is, one that was healthy and the other who had it. Increased bacteria were identified in the subjects affected by MS who were not treated compared to the controls, and it was subsequently observed that the mice that had been colonised with the faeces of the patients, generated in mice models for spontaneous cerebral autoimmunity a higher incidence of autoimmunity and a lower production of IL10.52 Finally, and already in 2018, a study carried out in a Spanish population with 15 patients with RRMS who were not treated, 15 who were treated with interferon-␤1b and 14 control subjects, showed statistically significant differences in the ␤ diversity of MS patients compared to controls, specifically in four phyla and 17 taxa at the species level. In addition this study showed statistically significant differences between patients who were not treated and those treated with interferon-␤-1b in Prevotella copri, a species previous studies have implicated in the protection against MS, which opens the door to investigating a possible role of treatment of the disease in the normalisation of the changes produced in the microbiota of patients affected by MS and possible clinical benefits associated with this event.53 The results of the main studies described into patients with multiple sclerosis are presented in Table 2

Unanswered questions There are still many questions surrounding the role of the microbiota in MS. First, none of the studies carried out so far in humans has been able to demonstrate causality, and answer the question of what came first, whether it was the chicken or the egg; i.e. is it the alterations in the microbiota that condition a susceptibility to MS? Or vice versa, does the disease condition changes in the microbiota? Second, the revolution that metagenomics has supposed, based on the study of 16s ribosomal DNA, leaves out by definition other microorganisms that do not possess said DNA, such as fungi. There

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Table 2 Summary of studies into microbiota in multiple sclerosis. Medullary

Sample

Results

Ref

Cases and controls

7 patients 8 controls American adults 20 patients 40 controls 18 long-term controls Japanese adults 31 patients 36 controls American adults 20 patients 16 controls American children

Differences in Firmicutes, Bacteroidetes and Proteobacteria but not statistically significant

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Not statistically significant differences in Actinobacteria, Bacteroidetes and Firmicutes phyla Statistically significant differences in 21 species, 14 belonging to Clostridia XIVa and IV groupings Increase among patients in the Pseudomonas, Mycoplana, Haemophilus, Blautia and Dorea genera. Increase in controls of Parabacteroides, Adlercreutzia and Prevotella genera Enrichment in patients in the relative abundance of genera belonging to Desulfovibrionaceae (Bilophila, Desulfovibrio and Christensenellaceae) and between controls in Lachnospiraceae and Ruminococcaceae. Increase in bacteria involved in glutathione metabolism Relationship in the direct cases between richness and Th17 and inverse between Bacteroidetes and Th17 Relationship in the controls between Fusobacteria and the Tregs Increase in MS of Methanobrevibacteriaceae and Akkermansia, and decrease of Butyricimonas, and correlation with gene expression of interleukins of T cells and monocytes Increase in patients treated for Prevotella and Sutterella, and decrease in Sarcina Increase of exhaled methane in patients with MS Differences in the relative abundance of 25 bacterial genera Induction of proinflammatory response in mononuclear cells and in monocolonised mice with Akkermansia muciniphila and Acinetobacter calcoaceticus, increased in MS; induction of anti-inflammatory response by Parabacteroides distasonis, increased in controls. More severe EAE in mice colonised with the patient’s microbiota. Increase in certain taxa increased in patients with MS who were not treated versus controls. Mice colonised with patients’ faeces had a higher incidence of autoimmunity and lower production of IL-10. Differences in 4 phyla and 17 taxa at the species level. Statistically significant differences between patients who were not treated and those treated with interferon-␤1b in Prevotella copri

46

Cases and controls

Cases and controls

Cases and controls

Cases and controls

Cases and controls

Cases and controls In vitro

15 patients 9 controls American children 60 patients 43 controls American adults

71 patients 71 controls American adults

Animal models Cases and controls Animal models Cases and controls

34 pairs of twins one with the disease, the other control German adults 14 controls 15 patients who were not treated 15 patients treated with IFN-␤1b Spanish Adults

47

48

49

50

51

52

53

MS: multiple sclerosis; EAE: experimental autoimmune encephalomyelitis; IFN: interferon.

are certain indications – studies that associate antigens or antibodies against certain fungi with MS, or the fact that dimethyl fumarate was initially used as an antifungal – that suggests that fungi may have a role to study in MS.54,55 For the same reason, we also need to study the possible role of viruses in the disease, beyond the well-known role of the Epstein Barr virus.56 The possible therapeutic utility of interventions related to the microbiota in MS has also been pointed out in very modest studies, such as stool transplantation,57 administration to probiotic patients,58 or dietary interventions aimed at increasing the amount of non-digestible polysaccharides available that can act as prebiotics and that are associated with changes in the microbiota, in addition to immunological and clinical changes in patients.59

To date, there is extensive evidence showing the role of the microbiota in the genesis, prevention and treatment of EAE, based mainly on three immunological pillars, the Th1–Th17/Th2 equilibrium, Treg cells and humoural immunity. It is also well documented that there are differences in the microbiota of patients with MS which are associated with a different expression of genes related to inflammation. There are still many open questions – such as those that demonstrate causality, i.e. whether it is the microbiota that influences the disease or vice versa, the role of viruses or fungi and the usefulness of probiotics, dietary interventions and stool transplants, etc. – which when answered may be able to give a totally novel perspective to the understanding of the physiopathological and therapeutic mechanisms of certain autoimmune diseases, among them MS.

Conclusions The role of the microbiota in autoimmune diseases has gained much interest in the last 15 years, due to the development of metagenomics and the concept of the holobiont. It could be said that certain pathologies cause the holobiont to fall ill – a group comprising the human being and its microbiota – rather than the human being, the former having the greater number of genes, a 150:1 ratio, that play a fundamental role in the maturation and immune regulation, this fact being what allows the microbiota to influence the health-disease state, especially notable in the field of autoimmune diseases.

Conflict of interest The authors declare no conflict of interest. References 1. Reshef L, Koren O, Loya Y, Zilber-Rosenberg I, Rosenberg E. The coral probiotic hypothesis. Environ Microbiol. 2006;8:2068–73. 2. Zilber-Rosenberg I, Rosenberg E. Role of microorganisms in the evolution of animals and plants: the hologenome theory of evolution. FEMS Microbiol Rev. 2008;32:723–35.

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