Multiple Sclerosis, Gut Microbiota and Permeability

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REVIEW ARTICLE

Multiple Sclerosis, Gut Microbiota and Permeability: Role of Tryptophan Catabolites, Depression and the Driving Down of Local Melatonin Moses Rodriguez1,2,4, Bharath Wootla1,2,3 and George Anderson5 1

Department of Neurology, Mayo Clinic College of Medicine, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA; 2Mayo Clinic Center for Multiple Sclerosis and Autoimmune Neurology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA; 3 Center for Regenerative Medicine, Neuroregeneration, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA; 4Department of Immunology, Mayo Clinic College of Medicine, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA; 5CRC Scotland & London, Eccleston Sqaure, London, UK Abstract: Background: Alterations in gut microbiota, coupled to increased gut permeability are now widely recognized as having a role in the etiology, course and treatment of many medical conditions, including autoimmune and neurodegenerative disorders. Methods: In this review, the role that such gut changes play over the course of multiple sclerosis (MS) is detailed. ARTICLEHISTORY Received: July 26, 2016 Accepted: September 8, 2016 DOI: 10.2174/1381612822666160915 160520

Results: Given the wide array of biological factors and processes that have been shown to be altered in MS, including changes in the gut, this allows for a better integration of the diverse array of pathophysiological processes linked to MS. Such pathophysiological processes include increases in oxidative and nitrosative stress, pro-inflammatory immune responses, especially T helper (Th)17 cell proliferation and activation, tryptophan catabolites, Moses Rodriguez pain, fatigue and increased levels of depression. By raising levels of immune activation, increased gut permeability and alterations in gut microbiota impact on all of these MS-associated processes. Alterations in the regulation of local melatonergic pathway activation is proposed to be an important hub for such pathophysiological processes in MS, allowing for the increased frequency of depression that may be prodromal in MS, both in the first episode as well as in relapses, to become more intimately associated with the etiology and course of MS. We propose this occurs by decreasing serotonin availability as a precursor for the melatoninergic pathways. Conclusions: Changes in the gut are evident in the early stages of MS, including in paediatric MS, and may interact with pro-inflammatory genetic susceptibility genes to drive the biological underpinnings of MS. Such a conceptualization of the biological underpinnings of MS also has treatment implications.

Keywords: Multiple sclerosis, gut microbiota, gut permeability, melatonin, serotonin, pathophysiology, immune inflammation, oxidative stress, Th17, depression. INTRODUCTION The pathophysiological processes known to be altered in the etiology and course of multiple sclerosis (MS) are manifold. MS has been linked to increased levels of pro-inflammatory cytokine production, especially interleukin (IL)-17, which is predominantly derived from T helper (Th)17 cells. MS symptomatology is highly correlated with white matter loss, ultimately leading to the malfunctioning of neurons and significant changes in the strength and consistency of brain inter-area communication. The correlation of MS symptoms with white matter lesions has been central to conceptualizations of MS that emphasized central changes, including the site of actions of MS susceptibility genes [1]. MS is associated with many other pathophysiological processes, including increases in Th17 cells and IL-17 levels, increased levels of reactive oxygen and nitrogen species that drive raised levels of oxidative and nitrosative stress (O&NS), mitochondrial dysfunction and increased systemic immune-inflammatory processes. MS is also highly associated with a wide array of symptoms, including: depression, which may more likely be prodromal, either *Address correspondence to this author at the M.D., Guggenheim 4-42B, 200 1st Street SW, Mayo Clinic, Rochester, MN 55905, USA; Tel: 507-2844663; Fax: 507-284-1087; E-mail: [email protected] 1381-6128/16 $58.00+.00

to the first episode and/or to subsequent relapses in the case of relapse-remitting MS (RRMS); fatigue, which is subjectively the most disabling consequence of the early stages of MS; loss of circadian rhythm and associated sleep disruption; increased pain sensitivity, a contributory factor to sleep and fatigue symptoms. A consequence of enhanced levels of pro-inflammatory cytokines in MS, is their induction of indoleamine 2, 3-dioxygenase (IDO), which drives tryptophan away from serotonin synthesis, instead leading to the synthesis of kynurenine and subsequently other tryptophan catabolites (TRYCATs), such as kynurenine acid and the excitotoxic quinolinic acid (QUIN). TRYCATs such as QUIN and kynurenic acid are neuroregulatory, and likely to impact on many emerging, classically conceived MS comorbidities, such as emergent seizures [2]. The activation of the TRYCATs pathway is likely to contribute to the many wider aspects of MS symptomatology, including the loss of cognition driven by neurodegeneration and decreased neurogenesis. By decreasing serotonin, TRYCATs pathway activation deprives every cell in the body of the serotonin that is necessary as a precursor for the synthesis of Nacetylserotonin (NAS) and melatonin [3, 4]. As detailed below, changes in the melatonergic pathways in MS may be relevant to many pathophysiological processes associated with MS that encompass changes in gut permeability and gut microbiota.

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Fig. (1). Healthy and abnormal gut-brain axis. Under healthy conditions a gut-brain axis exists, in association with normal behavior, cognition and emotion, including healthy levels of inflammatory cells and normal gut microbiota. However, this becomes dysregulated under the influence of factors such as stress, cortisol, alcohol and dietary factors, which increase gut permeability, possibly by impacting on local melatonin synthesis. Increased gut permeability raises levels of circulating lipopolysaccharide and tiny fragments of partially digested food that trigger a low level inflammatory response, which associates with increased depression and depression-associated neurodegenerative and CNS disorders. Such changes centrally are likely to reciprocally interact with gut permeability, via influences on vagal nerve and neuronal sensory connectivity.

Here we review the available data on MS pathophysiological processes, with an emphasis on the role of gut permeability and changes in gut microbiota. Given the increased immune inflammatory activation that follows increased gut permeability, it is likely that genetic and epigenetic factors that modulate MS susceptibility may be acting in the gut, leading to a conceptualization of MS that emphasizes whole body changes and not limited to a solely central focus. We shall first review some of the classical pathophysiological underpinnings of MS, before looking at changes in the gut. MS: CLASSICAL PATHOPHYSIOLOGY In this section, we briefly review some of the key pathophysiological processes in MS. MS Phenotypes MS is usually described as having four phenotypes, viz: relapsing-remitting (RRMS), secondary progressive (SPMS), primary progressive (PPMS), progressive relapsing (PRMS) [5]. A later classification review added clinically isolated syndrome (CIS) and radiologically isolated syndrome (RIS) to these four phenotypes [6], although these additions are not thought to represent a high percentage of MS presentations. RRMS is the phenotype that is most common, occurring in about 80% of MS patients. RRMS is characterized by unpredictable relapses following variably lengthed periods of remission. Functional deficits arising over the course of a relapse can be fully resolved or, especially as the in later stages, leave impairments. The first MS episode is usually classified as CIS, where an attack occurs that is suggestive of a demyelination episode, but does not fulfill full MS criteria. About 65% of those presenting with RRMS will go on to develop SPMS, which is characterized by progressive neurological and functional decline, with no clear periods of remission. A shift in diagnosis from RRMS to SPMS is typically around 20 years. PPMS is evident in about 15% initial MS presentations, being characterized by no obvious periods of remission after initial presentation, with no remission after the

initial symptoms. PRMS patients generally have a steady neurological and functional decline, with no obvious evidence of periods of attack. MS can have very distinct presentations, highly suggestive of, as yet, undiscovered distinct pathophysiological features. Most evidence has been acquired in RRMS patients, with an acute attack in RRMS thought to represent the core biological underpinnings of MS. Active MS Lesion Features An active MS attack features: myelin sheath destruction; axonal degeneration to a varying degree; pro-inflammatory immune cells, including macrophages that inhibit cell proliferation and cause tissue damage (M1 phenotype), and leukocytes, predominantly CD8 + and CD4+ T cells, as well as B cell infiltration [7]. Of the infiltrating T cells, Th17 cells produce IL-17 and Th1 cells produce a range of pro-inflammatory cytokines. There is a relative paucity of antiinflammatory regulatory T cells (Tregs) around active lesions. The immune infiltrates around active MS plaques are indicative of local pro-inflammatory immune activation. T cell receptor analysis on infiltrating CD8+ T cells clearly indicates clonal expansion, driven, presumably, by lesion associated autoantigen. CD8+ T cells are associated with progressive clinical deficits and axonal destruction. Although T cells clearly predominate around active MS plaques, B cells also contribute significantly to the classical central pathophysiology of active MS lesions. Accordingly, depletion of B lymphocytes is associated with suppression of inflammatory activity in MS, with therapy targeting antibodies against B cell markers, such as rituximab or ocrelizumab, showing treatment efficacy in MS. However, when atacicept, a recombinant fusion protein that suppresses B-cell function and antibody production, effective in several models of autoimmunity, including the animal model of multiple sclerosis experimental autoimmune encephalomyelitis (EAE) [8] was tested in a clinical trial (ATAMS), it increased clinical disease activity suggesting that the role of B cells and humoral immunity in MS is complex. In contrast to anti-CD20 monoclonal

Multiple Sclerosis, Gut Microbiota and Permeability

antibodies that completely deplete B cells from the circulation, atacicept causes an incomplete reduction of B cells, i.e., memory B cells persist. Theoretically, the atacicept-associated proinflammatory response was perhaps due to the preferential attenuation of regulatory B cells [9]. Recent work shows that the presumed B cell marker, CD20, is also expressed in a subset of T cells, suggesting efficacy of rituximab, at least in part, via T cell regulation [10]. Another study showed that atacicept also effectively reduces the pathogenic Th1 and Th17 cells, with no effect on memory T cells in EAE mice [11]. B cells are likely to have an array of effects in active MS lesions, including presenting (auto-)antigen to T cells, thereby influencing T cell activation and differentiation; their release of soluble factors at the lesion site [12]; releasing immunoglobulins (Ig), with candidate autoantigens in MS, including potassium channel (KIR)4A and myelin oligodendrocyte glycoprotein (MOG) [13]. Such data supports a conception of MS as an immune -mediated disorder, although an autoantigen for active T cells in MS remains elusive. The EAE model indicated a number of autoantigens, usually myelin proteins, which are not confirmed in MS patients. Given patient responses to anti-inflammatory therapies, genetics, serology and pathology studies, MS appears to be primarily an inflammatory demyelinating disease of the CNS with varied clinical presentations and heterogeneous histopathological features. While there is relatively strong evidence to consider MS an immune-mediated disease, the authors of this review consider the evidence to classify MS as a classical autoimmune disease weak and circumstantial. Data derived from EAE model was suggestive that MS is an autoimmune disorder. Consequently, studies with the EAE model of MS played an important role in identifying and delineating several aspects of MS biology: inflammation, immune surveillance and immune-mediated tissue injury. This experimental model directly led to the development of the following medications approved for MS: glatiramer acetate, IFN-β, mitoxantrone and natalizumab [14]. However a number of available treatments that showed incredible promise in this animal model [15-20] did not translate to a successful therapy in MS, questioning the validity of this model to be used as the only drug discovery platform for MS. Other Pathophysiological Processes in MS Blood-Brain Barrier Permeability In line with the role of systemic immune cell infiltration into the CNS, it has long been proposed that pro-inflammatory cytokine induced increases in the blood-brain barrier (BBB) permeability have a role in the etiology and course of MS [21, 22]. Recent work suggests that increases in BBB permeability are mediated via the activation of astrocytes and microglia [23], suggesting local variations in glia pro-inflammatory cytokine production, perhaps especially tumour necrosis factor-alpha (TNF-α) actions at the TNR receptor 2 (TNFr2), is important to the local regulation of BBB permeability [22]. Laterality and Vitamin D MS is more common as one moves from the equator to the poles. This has been interpreted as an indication of the relevance of decreased sunlight-derived vitamin D in the etiology of MS [24]. Work in recent decades showing the importance of vitamin D in the regulation of the immune system further supports its role in MS via the regulation of optimized immune-inflammatory responses [25]. Vitamin D and its metabolites stabilize the endothelium [26], which suggests that it is likely to impact on BBB permeability. To date, most data points to a role for decreased vitamin D in the etiology of MS, but there is no convincing data showing vitamin D to strongly modulate the course of any MS subtypes [24, 27]. Gut Permeability and Microbiota A growing body of research indicates a role for variations in gut microbiota and gut permeability in a host of medical conditions, perhaps especially in depression-associated conditions, such as

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Alzheimer's disease and Parkinson's disease, as well as MS [28]. Such work led to conceptualizations of a gut-brain axis, allowing a two-way interaction that acts to modulate levels of immuneinflammatory activity [28]. We shall now look at factors acting to regulate gut microbiota and gut permeability, before looking at their role in MS. Gut Permeability There is now considerable interest in the role of gut permeability across a diverse array of medical conditions, including in CNSassociated conditions. The effects of increased gut permeability are mediated via bacteria and tiny fragments of partially digested food that drive an immune response in the host. The resulting immune response may have direct effects in the CNS and/or may have indirect effects via the regulation of functioning in other organs. Other tissues and organs that have direct environmental contact, such as the lung and periodontal tissue may also allow bacteria to have a wide range of impacts, including centrally [28]. Enhanced levels of gut permeability arise from the loosening of tight junctions that tightly bind the cells forming the gut. A growing number of factors have been shown to enhance gut permeability: dietary fats [29], stress/cortisol [30] and alcohol [31] as well as alcohol binge drinking [32]. Whilst a variety of factors maintain gut integrity, including dietary whole grains [33] and melatonin [34], with melatonin preventing alcohol-induced increases in gut permeability [31]. Increased gut permeability associates with increased levels of depression, which is also driven, in part, by immuneinflammation [35]. The association of gut permeability with classically conceived central conditions such as Alzheimer's disease and MS may mediate increased levels of depression [36]. Given the high levels of depression in MS patients [37], it is likely that increased gut permeability contributes to such depression, and thereby in the course of MS per se. MS with depression is associated with elevations in peripheral inflammation, disability and disease progression as well as gastrointestinal and visual symptoms [38], indicating that the processes driving, as well as occurring during, depression impact on the course of MS. Given that a high percentage of MS patients receiving high dose corticosteroid therapy have resultant increases in depression [39], it requires investigation as to what degree such medication-linked depression is driven by increased gut permeability, since stress/cortisol increases gut permeability [30]. This is of some importance to levels of central inflammation, given that changes in microglia and glia-neuronal interactions have been shown in EAEassociated depression [40]. It is also of note that increased levels of depression in MS patients are associated with elevated levels of alcohol abuse [41], with alcohol known to increase gut permeability [34]. With depression, alcohol abuse and MS per se being associated with increased levels of O&NS, which can then act to further increase gut permeability, it is likely that a vicious cycle may be formed involving the interactions of O&NS with immuneinflammatory processes in the regulation of gut permeability as well as MS symptomatology and the presence of classically conceived comorbidities, such as depression and alcohol abuse [42]. As elaborated on below, such increases in cortisol/stress and proinflammatory cytokines will increase the activation of tryptophan 2, 3-dioxygenase (TDO) and IDO in MS, in turn decreasing tryptophan availability for serotonin and melatonin [43] at different sites and in different cells. This is of some relevance to changes in gut permeability, as melatonin significantly decreases gut permeability, including when induced by alcohol [34]. We now look at the role for alterations in gut microbiota, including as to how this impacts on gut permeability. Microbiota and the Gut Human gut harbours a community of over 100 trillion microbial cells that forms a complex community, from which the host usually

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benefits. It was estimated that the gut contains in the range of 1000 bacterial species and 100-fold more genes than are found in the human genome [44]. Gut microbiota help the host by: aiding the fermentation of undigested carbohydrates, allowing short-chain fatty acid absorption; vitamins B and K synthesis; metabolizing bile acids, sterols and xenobiotics [45]. Factors that enhance gut permeability, such as stress, alcohol and dietary fats, lead to the transfer of an array of commensal, gram-negative bacteria as well as partially digested food fragments that can initiate an immune-inflammatory response. Intestinal inflammation is associated with a reduced bacterial diversity and, in particular, a lower abundance of, and a reduced complexity in, the Bacteroidetes and Firmicutes phyla with a specific reduction of abundance in the Clostridium leptum and Clostridium coccoides groups [46]. The driving of immune-inflammation is one way that depression-associated stress and dietary factors, allow changes in gut permeability and microbiota to associate with MS. Lipopolysaccharide (LPS), a component of the bacterial wall of gram negative bacteria induces an innate immune response, which is driven by the activation of toll-like-receptor-2/4 (TLR2/4)-CD14 complex. An increased transfer of such bacteria leads to enhanced levels of plasma IgA and/or IgM, which is evident in depression among other conditions [42]. It is the activation of the complex formed by CD14-TLR2/4 that induces an inflammatory response, often involving the activation of the transcription factor nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB). NF-κB is a major mediator of many inflammatory processes, including the induction of pro-inflammatory cytokines, including TNF-α and IL1β, as well as the inflammation-associated cyclo-oxygenase-2 (COX-2) [47]. IgM and IgA responses can be induced in the blood, with IgA responses also able to take place when bacteria only translocate to the mesenteric lymph nodes. Gut microbial composition and function partly provides for the nutritional value of food. Inversely, the composition of gut microbiota is also influenced by the ingested food [48]. This interrelationship was strengthened by further studies of the faecal microbiomes of obese and lean humans, as well as monozygotic and dizygotic twin pairs concordant for leanness or obesity [49]. Caloric restriction-induced alterations in the host’s immune system have been studied in detail, including the role played by the sirutin and mammalian target of rapamycin (mTOR) pathways [50]. Sequence analysis from human adult microbiota of microbial ribosomal RNA-encoding genes (16S ribosomal DNA) pointed towards five bacterial phyla: Firmicutes and Bacteroidetes predominate, with Actinobacteria, Proteobacteria, and Verrucomicrobia comprising just 2% of organisms. Most belong to the genera Faecalibacterium, Bacteroides, Roseburia, Ruminococcus, Eubacterium, Coprabacillus, and Bifidobacterium [51]. While animal protein and fat favours abundance of Bacteroides, a monosaccharide rich diet favours abundance of Prevotella species [52]. An oligosaccharide-rich diet favours growth of Bifidobacteria, which is the dominant genus of breast-fed infants [53]. It was reported that the aryl hydrocarbon receptor (AhR) is a crucial regulator in maintaining intraepithelial lymphocyte (IEL) numbers in both the skin and the intestine. In the intestine, AhR deficiency or the lack of AhR ligands compromises the maintenance of IELs and the control of the microbial load and composition, resulting in heightened immune activation and increased vulnerability to epithelial damage [54]. When the animal diet was changed from a standard murine diet to a synthetic feed that lacks ingredients of vegetable origin, the modified diet caused a decline in IELs, enhanced susceptibility to epithelial damage, and increased numbers of intestinal Bacteroides, a Gram-negative genus prevalent in mammalian intestines that is able to induce colitis in a host-genotype-specific manner [55]. Although enhanced levels of gut permeability allow gut microbiota to drive immune-inflammatory processes in the host, gut microbiota may also influence the host by other means. Microbiota

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can synthesize and release, as well as respond to, a variety of compounds, including neuroregulatory factors [56]. The array of neuroregulatory factors produced by microbiota include: dopamine, acetylcholine, somatostatin, norepinephrine and progesterone. Gut microbiota can directly activate neuronal activity, including central autonomic centres' projections to and from the gut. Given the high levels of melatonin produced in the gut, primarily from enterochromaffin cells, and the presence of activated melatonin pathways in some bacteria [3], it will be important to determine whether there are variations in melatonin synthesis and release by different strains of gut bacteria and/or whether there are releases that influence endogenous gut melatonin synthesis. This is important as melatonin acts to maintain the integrity of the gut barrier [57], when challenged by alcohol [34]. Gut microbiota modulate gut functioning, barrier permeability and the two-interactions of the gut and brain in different ways [28, 58]. The gut-brain axis is now accepted as a significant modulator of many medical conditions, including MS, but also Parkinson's disease [59] and the autistic spectrum [60]. It is worth noting that variations in melatonin, both in the brain and gut, may be a hub upon which many factors that regulate the gut-brain axis act. Although predominantly known for its release by the pineal gland in the driving of circadian rhythms, melatonin production in the gut may be 100-fold higher than the night-time pineal peak [61]. Melatonin is now known to be produced by many, if not all mitochondria-containing cells [3], suggesting that its regulation locally is likely to be a modulator of inter-area interactions, such as occur in the gut-brain axis [4]. Melatonin is a powerful antioxidant, anti-inflammatory and anti-nociceptive, as well as an optimizer of mitochondrial oxidative phosphorylation and wider mitochondrial functioning [62]. An immune-pineal axis was proposed by Regina Markus and colleagues, whereby systemic inflammation induced cytokines, such as TNF-α, act on pinealocytes to suppress pineal melatonin synthesis, with LPS acting on pineal microglia TLR4, leading to TNF-α release, which then acts on pinealocytes to decrease circadian melatonin synthesis and release [63]. This is important for immune regulation, as pineal melatonin suppresses the adhesion of leukocytes to endothelial cells of different organs, including the CNS, thereby impacting on the levels of immune-inflammatory responses in a variety of organs. Changes in gut and brain inflammatory processes via driving tryptophan away from serotonin and melatonin synthesis to neuroregulatory TRYCATs production will then act on pineal processes that can regulate leukocyte entry to different organs. The immune-pineal axis may therefore be intimately associated with the gut-brain axis, and the mechanism by which gut microbiota and gut permeability modulate the etiology and course of MS. Tryptophan availability for the host can also be significantly determined by the specificity of gut bacteria [64], thereby allowing gut bacteria to influence levels of serotonin availability, including as a precursor for the production of melatonin. This is likely to be of wider relevance to a host of other medical conditions, including glioblastoma [65] as well as psychiatric and neurodegenerative conditions, such as bipolar disorder [66] and Alzheimer's disease [67]. Given the potentially important role of variations in melatonin at different sites in the regulation of gut permeability, it should be noted that melatonin closely interacts with the alpha 7 nicotinic receptor (α7nAChr), with melatonin increasing the levels and activity of the α7nAChr [68] and the α7nAChr mediating some of the effects of melatonin [69]. This is of some importance, as the α7nAChr has a number of effects that are relevant in MS and its regulation by gut microbiota and gut permeability. The α7nAChr can: decrease gut permeability under challenge [70]; decrease gut mast cell-induced elevations in gut permeability [30]; and have wider immune-suppressive effects [71, 72]. Agonists of the α7nAChr are currently in clinical trials for the treatment of cogni-

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tive deficits in Alzheimer's disease and schizophrenia [73], which suggests that they may also have efficacy in the management of decrements in cognitive function in MS [74]. This also suggests that the release of acetylcholine by microbiota, may be interacting with the levels of gut melatonin, in the regulation and activation of the α7nAChr, with consequences for gut permeability and immune cell activation. Finally, variations in gut microbiota are likely to be in close interaction with alterations in melatonin and the α7nAChr. GUT CHANGES IN MS Data accumulated from the utilization of the EAE model have indicated the relevance of gut microbiota alterations in the development of demyelinating disease, with an oral antibiotic shown to decrease the severity of EAE [75]. In an EAE variation, using a murine T cell receptor transgenic EAE model, it was shown that EAE-resistant germ-free mice will go on to develop EAE when recolonized with bacteria indigenous to this species or when transfected with segmented filamentous bacteria [76, 77]. Such induction of EAE initiation was temporally linked to the production of enteric Th17 cells. Such data highlighted the potential for the modulation of gut bacteria and gut permeability in the etiology and course of MS. As well as promoting the development of EAE, variations in specific gut bacteria can suppress such inflammatory conditions. Interestingly, Clostridia clusters XIVa, IV, and Bacteroides fragilis microbiota that have been derived from human feces are able to induce immune-suppressive T-regs, thereby contributing to the inhibition of EAE pro-inflammatory processes [78]. Such data indicates the relevance of variations in gut bacteria to the induction of pro-inflammatory and autoimmune-associated Th17 cells as well as to the induction of anti-inflammatory processes via upregulation of T-regs. As indicated above, variations in melatonin, such as bacteria-derived melatonin, may impact on these processes. In support of this, recent data shows that melatonin is able to suppress the development of Th17 cells, as well as modulate T-regs in the EAE model [79]. The work by Farez and colleagues [79], suggests that melatonin may play a role in the seasonality of MS, by seasonal variations in pineal melatonin synthesis that are night-length determined. These authors showed that melatonin, via the induction of the transcriptional repressor, nuclear factor, interleukin 3 regulated (Nfil3), blocks Th17 differentiation whilst also enhancing the generation of T-regs via extracellular signal-related kinases 1/2, coupled to the transactivation of the IL-10 promoter by retinoid-related orphan receptor alpha. As indicated previously, the activation of low level inflammatory activity, including possibly by gut bacteria, can act to switch off pineal melatonin synthesis [63], suggesting that one way in which gut bacteria may contribute to increased Th17 cells is by the suppression of pineal melatonin by pro-inflammatory cytokines such as TNF-α. As reported [61], gut melatonin levels are far higher than those released by the pineal gland, suggesting that such local melatonin regulation may be relevant to the induction of Th17 cells, including enteric Th17 cells. Such data indicates a significant role for local melatonin synthesis in the etiology and course of MS, as recently proposed [80], in the regulation of wider MS comorbidities, and also in the emergence of seizures [2]. Further work in the EAE model shows that LPS effects via the TLRs not only activate the innate immune system but also increase Th17 cells, where TLR4 are more highly expressed versus Th1 and Th2 cells [81]. These authors reported that LPS increased the production of IL-17 producing cells in the inguinal lymph nodes, significantly exacerbating EAE [81]. An increase in gut permeability and the crossing over of LPS from gram-negative bacteria will heighten the production of Th17 cells, in contrast to the effects of melatonin. Further data in the EAE model shows that increased gut permeability develops very early, during EAE onset, with a leaky gut being induced by the adoptive transfer of auto-reactive T cells [82]. Work by Berer and colleagues looked at the role of the gut in

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modulating Th17 responses in the EAE model [83]. These authors compared TCR transgenic mice that were genetically disease susceptible versus those that were genetically disease resistant, showing that Th17 cells are sequestered within the gut of EAE resistant mice. These authors also showed that transferred Th17 cells migrate to the gut lymphoid organs of resistant mice, concluding that the gut is a check-point for controlling pathogenic, organ-specific T cells [83]. This would also suggest that variations in gut permeability, LPS and melatonin will be key gut factors that act to regulate the proposed gut checkpoint. A further elaboration of the work on the role of gut microbiota in EAE, is seen in the work by Miller and colleagues, who showed that gut microbiota may interact with the TNF receptor 2 (TNFR2) to modulate levels of immune responses [84]. It should be noted that the activation of TNFR2 can induce an anti-inflammatory immune response in some organs [85], indicating that this receptor may have wider immune-regulatory effects, in addition to its modulation of the interactions of gut microbiota and levels and type of immune-inflammatory activation. However, given the inadequacies of the EAE model as a representation of the complex presentations in human MS [14], we will now review the limited data collected on gut changes in the human condition. Recent work by Miyake and colleagues compared the gut microbiota of MS patients with healthy participants, by using a highthroughput culture-independent pyrosequencing method [86]. Bacterial gene analysis of DNA that was isolated from the MS patients versus healthy participants showed a relatively moderate dysbiosis to be evident in the gut microbiota of MS patients. These authors found the composition of gut microbiota to be altered in MS patients, although not to the same extent as the gut microbiota changes evident in inflammatory bowel disease [86]. We have recently published some data on the gut microbiota changes in paediatric MS [87]. In this study, a comparison was made between microbial community profiles in early onset paediatric MS and control children that were matched for age and sex, although having no evidence of an autoimmune disorder. All paediatric cases were 18 years of age or less at time of MS onset, with a mean age of 13 years. Stools were shipped on ice, stored at -80C and analysed by 16S ribosomal sequencing. The 18 MS patients (10 girls, 8 boys) had RRMS and short disease duration, with a mean of 11 months. Half of the patient samples were on immunomodulatory drug treatment. MS patients, in comparison to controls, showed significant differences in the patterning of the gut microbiota community, irrespective of the presence of immunomodulatory therapy. The data on this pediatric MS sample indicate that the composition of the gut microbiome is significantly altered, with a shift towards a pro-inflammatory community. Such data are reminiscent of studies by Miller and colleagues in adult MS presentations and suggests that gut microbiota changes are evident in MS, regardless of age of first symptoms. The body of data collected in the EAE model is highly suggestive of a role for gut permeability and variations in gut microbiota in the etiology and course of MS. The limited data collected in MS patients, both paediatric and adult, suggest that some of the changes seen in the EAE model may be paralleled in human presentations of MS. As indicated throughout, the role of depression and stress in MS may be at least partly mediated by increases in gut permeability and alterations in levels and patterning of gut microbiota, perhaps in interaction with pro-inflammatory genetic susceptibility factors. It is also clear that the gut-brain axis is likely to interact with the immune-pineal axis, with consequences for levels of immuneinflammatory processes and wider circadian regulation. An increase in the pro-inflammatory milieu, via the activation of TDO and IDO will decrease the availability of serotonin and therefore of local, as well as pineal, melatonin synthesis. It is likely that alterations in

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melatonin, including in glia and immune cells, but perhaps especially in the gut, will significantly interact with the nature of the two-way communications between the gut and brain, with consequences for the etiology and course of MS. FUTURE RESEARCH AND THERAPEUTIC DIRECTIONS More data has to be collected on gut changes in MS patients, especially as this may have readily applicable treatment implications. This requires investigation as to whether gut microbiota produce melatonin, and/or release factors that impact on the levels of endogenous melatonin production in the gut. This may have some important consequences for levels of Th17 induction and the balance of Th17/T-regs, perhaps especially around the gut. In this context, it will be important to link the plethora of genetic susceptibility factors that were linked to MS, especially as to whether some of the genetic susceptibility pro-inflammatory factors are mediating their effects in the gut, rather than in the brain. Based on genomewide association studies, there are now 110 established MS risk variants in 103 discrete loci outside of the major histocompatibility complex (MHC) [88]. The work of Toivanen et al., [89] reported that bacterial composition of faecal flora is influenced by MHC. In light of this, we suspect that some of the identified MHC risk variants may act in the gut, however direct evidence is lacking in support of this. On the basis of the data collected in the EAE model, it was proposed that modulating gut microbiota with pre- and pro-biotics could have therapeutic efficacy in MS. Similarly, dietary factors that improve the maintenance of gut integrity were also proposed. However, it is likely that collection of some readily available data in this paradigm, as indicated above, will considerably clarify the utility of the array of factors that may impinge on gut microbiota and gut permeability in MS. It will also be important to look at the role of the gut in the currently defined MS subtypes. CONCLUSION Gut changes have been highly investigated in the EAE model of MS, however, there is very limited data on the gut changes occurring in MS patients. There are some important investigations that are still to be carried out, which should better clarify gut changes in the etiology, course and management of MS. Available data would suggest that alterations in levels of serotonin availability for melatonin synthesis is likely to be a significant aspect in the gut-brain changes that may contribute to the biological underpinnings of MS. CONFLICT OF INTEREST The authors confirm that this article content has no conflict of interest. ACKNOWLEDGEMENTS Declared none. LIST OF ABBREVIATIONS α7nAChr = Alpha 7 nicotine acetylcholine receptor BBB = Blood brain barrier CIS = Clinically isolated syndrome COX = Cyclo-oxygenase EAE = Experimental autoimmune encephalomyelitis IDO = Indoleamine 2, 3-dioxygenase IFN-γ = Interferon-gamma Ig = Immunoglobulin IL = Interleukin iNOS = inducible nitric oxide LPS = Lipopolysaccharide MAO = Monoamine oxidase

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MOG MS NADPH NAS NF-κB

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NMDAr NO O&NS PPMS PRMS QUIN RRMS SPMS TDO Th TLR TNF-α Tregs TRYCAT

= = = = = = = = = = = = = =

Myelin oligodendrocyte glycoprotein Multiple sclerosis nicotinamide adenine dinucleotide phosphate N-acetylserotonin nuclear factor κ-light-chain-enhancer of activated B cells N-methyl d-aspartate receptor Nitric oxide Oxidative and nitrosative stress Primary progressive Progressive relapsing Quinolinic acid Relapse-remitting MS Secondary progressive Tryptophan 2, 3-dioxygenase T-helper Toll-like-receptor Tumor necrosis factor-alpha Regulatory T cells Tryptophan catabolites

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