Th17 Cell, the New Player of Neuroinflammatory Process in Multiple Sclerosis


A. Mirshafiey, Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran-14155, P.O. Box: 6446, Iran. E-mail:


Multiple sclerosis (MS) is an autoimmune disease characterized by recurrent episodes of demyelination and axonal lesion mediated by CD4+ T cells with a proinflammatory Th1 and Th17 phenotype, macrophages, and soluble inflammatory mediators. Identification of Th17 cells led to breaking the dichotomy of Th1/Th2 axis in immunopathogenesis of autoimmune diseases such as MS, and its experimental model, experimental autoimmune encephalomyelitis (EAE). Th17 cells are characterized by expression of retinoic acid-related orphan receptor (ROR)γt and signal transducer and activator of transcription 3 (STAT3) factors. Th17-produced cytokine profile including interleukin (IL)-17, IL-6, IL-21, IL-22, IL-23 and tumour necrosis factor (TNF)-α, which have proinflammatory functions, suggests it as an important factor in immunopathogenesis of MS, because the main feature of MS pathophysiology is the neuroinflammatory reaction. The blood brain barrier (BBB) disruption is an early and central event in MS pathogenesis. Autoreactive Th17 cells can migrate through the BBB by the production of cytokines such as IL-17 and IL-22, which disrupt tight junction proteins in the central nervous system (CNS) endothelial cells. Consistent with this observation and regarding the wide range production of proinflammatory cytokines and chemokines by Th17 cells, it is expected that Th17 cell to be as a potent pathogenic factor in disease immunopathophysiology. Th17-mediated inflammation is characterized by neutrophil recruitment into the CNS and neurons killing. However, the majority of our knowledge about the role of Th17 in MS pathogenesis is resulted in investigation into EAE animal models. In this review, we intend to focus on the newest information regarding the precise role of Th17 cells in immunopathogenesis of MS, and its animal model, EAE.


Multiple sclerosis (MS), the principal inflammatory demyelinating disease of the central nervous system (CNS), is believed to have an immunopathological aetiology arising from gene–environment interactions, affecting approximately 0.1% of the northern part of the world. The factors behind the initiation of the inflammatory responses remain unknown at present [1]. The pathological hallmarks of the MS lesion consist of local demyelination, inflammation, scar formation and variable axonal destruction. In spite of classical histopathological study and more recent intensive use of magnetic resonance technology, the MS lesion is incompletely understood [2]. It is shown that patients with MS exhibit various forms of disease with different immunopathology. These different clinical forms of MS are caused by different subsets of T helper (Th) cells, their relative proportion at the sites of inflammation, and their predominant generation of either interleukin (IL)-17 (the hallmark cytokine of Th17 cells) or interferon (IFN)γ (the hallmark cytokine of Th1 cells) [3]. Th17 cells attach to brain endothelial cells better than Th1 cells which is at least in part due to the presence of CD146 on the Th17 cells [4]. Moreover, Th17 cells express high levels of molecules such as CCR6 and CD6, which enhance entry of infiltrating T cells into the CNS and have an important role in the development of experimental autoimmune encephalomyelitis (EAE) and probably MS [5]. These informative data lead to this question: Is MS a Th17-mediated autoimmune disease, or Th1-mediated? However, we try to clarify the precise function of Th17 cells in neuroinflammatory process in immunopathogenesis of MS and its animal experimental model, EAE in this review.

Multiple sclerosis

Multiple sclerosis is a multifactorial disease, and patients with MS may suffer from a variety of clinical symptoms, such as sensory loss, visual problems, muscle weakness and difficulties with coordination and speech. The conventional animal model for MS is EAE [6, 7].

Multiple sclerosis is a heterogeneous disease clinically, pathologically and radiologically. Most patients (∼85%) experience an initial relapsing–remitting (RRMS) course, and the majority will go on to develop secondary progressive MS (SPMS), which is characterized by worsening neurological disability with or without superimposed attacks. Approximately 10% of patients exhibit primary progressive MS (PPMS), which involves continuous disease progression from onset, without relapse or remission. Progressive relapsing MS is a rare subtype that is characterized by neurological progression from onset, with superimposed relapses thereafter [8, 9].

The general population prevalence of MS varies between 60–200/100,000 in Northern Europe and North America, and 6–20/100,000 in low-risk areas such as Japan [10–12].

The aetiology of MS is unknown, but according to current data, the disease develops in genetically susceptible individuals and may require additional environmental triggers. Among the genetic factors, some genes have a significant role in susceptibility to MS, such as genes related to IL-1β and IL-1 receptor (IL-1R), TNF [13], immunoglobulin Fc receptor [14], Apo protein E [15], immunoglobulin heavy chain [16], T cell receptor (TCR) [17], myelin basic protein (MBP) [18], IL-2R, IL-7R (CD127) [10], LAG3 [19] and human leucocyte antigen (HLA) genes [20]. Among these genes, there is significant correlation between HLA locus and susceptibility to MS [11]. Both deleterious and protective alleles of HLA locus have been identified. DR15 haplotype and some alleles of DR2 haplotype, such as DRB1*1501, DRB5*0101 and DQB1*0602, are proposed as susceptible alleles in comparison with other alleles [21–23]. In contrast, some alleles are suggested with protective effects, such as HLADRB1*01, HLA-A*02 and A2Cw7B58DR2DQ1 [24–26].

In general, MS begins with the formation of acute inflammatory lesions characterized by disruption of the blood brain barrier (BBB). Such lesions are often clinically silent and have been estimated to be about ten times more frequent than episodes of clinical worsening. Breakdown of the BBB usually lasts for about a month and then resolves, leaving a site of damage that can be investigated by conventional magnetic resonance imaging (MRI). The pathological features of MS plaques are BBB leakage, destruction of myelin sheaths, oligodendrocyte damage and cell death, axonal damage and axonal loss, glial scar formation and the presence of inflammatory infiltrates that mainly consist of lymphocytes and macrophages [27, 28]. Despite major advances in the current understanding of pathogenesis of MS, exact details of the inflammatory cascade of MS remain unknown. It has been demonstrated that axonal degeneration is the major feature of irreversible neurological disability in patients with MS. Axonal injury initiates at disease onset and correlates with the degree of inflammation within lesions [29]. Four different patterns of pathology with resulting demyelination have been identified in MS lesions: Type I are T cell mediated where demyelination is macrophage mediated, directly or by macrophage toxins. In type II lesions, both T cell and antibody are involved and are the most common pathology observed in MS lesions. In this pattern, demyelination is through specific antibodies and complement. Type III is related to distal oligodendropathy, degenerative changes that occur in distal processes are followed by apoptosis. Type IV results in primary oligodendrocyte damage followed by secondary demyelination. This latter pattern occurred only in a small subset of PPMS patients [30, 31].

The complex process of neuroinflammation involves various components of the immune system [32, 33]. The cellular components involved in the neuroinflammation and neuroimmune activation in the CNS are brain microglial cells, ependymal cells, macrophages, astrocytes and mast cells. Microglial cells, which constitute about 10% of the CNS, are the first to respond to neuronal injury [32]. The inflammatory infiltrates in MS lesions mainly contain T cells, B cells and activated macrophage/microglial [2]. Various subsets of CD4+ T cells have a basic role in immunopathogenesis of MS. Th1 and Th17 cells have a central role in disease development and contrasted to previous view, MS is a Th1/Th17 mediated autoimmune disease and not just Th1 mediated [2, 34, 35]. Although it is reported that Th2 responses lead to amelioration of disease, there is evidence indicating the pathologic function of CD4+ T cells during disease [36]. Another important subset of CD4+ T cells is regulatory T cells (Tregs), which are impaired in patients with MS. Tregs are one of the most important cells in autoimmune disease such as MS, which can control disease and prevent its progression. Although the frequency of Tregs in MS is unaffected, their function are decreased, and this can lead to progression and exacerbation of disease [37]. It is reported that CD8+ T cells can also have encephalopathogenic capacity in vivo, as it is shown that these cells are major components of the inflammatory infiltrate in MS [38–40]. In addition, the role of humoral arm of immune system (B cells and their generated immunoglobulins) in immunopathology of MS is also demonstrated in various studies [41–47].

Th17 cells

The first report on Th17 cells backs to the role of these cells in host immune response to B. burgdorferi which in this study the addition of this pathogen lysate to TCR transgenic T cells induced the production of IL-17 [48]. The IL-17 cytokine family is a recently identified group of cytokines, which includes six members: IL-17A, B, C, D, E (IL-25) and F. IL-17A, the original member of this family was identified in 1995. Different cell types including T cells, γδT cells, natural killer (NK) cells and neutrophils produce IL-17A and IL-17F [49, 50]. The Th17 lineage represents an additional effector CD4+ T cell arm in comparison with Th1 and Th2 (as described in Fig. 1). Th17 cells are characterized by the production of a distinct profile of effector cytokines, including IL-17A, IL-17F, IL-6, IL-9, IL-21, IL-22, IL-23, IL-26 and TNF-α, and probably promote the clearance of a range of pathogens distinct from those targeted for Th1 and Th2 [51–54].

Figure 1.

 Regulatory pattern for various CD4+ T cells differentiations. Differentiation of naïve CD4+ T cell precursors to various CD4+ T cell lineages is controlled through several regulatory factors such as, specific transcription factors and stimulatory and inhibitory cytokines. Thus, it seems that the balance between various stimulatory and inhibitory cytokines in each lineage and local area can determine the fate of naïve CD4+ precursors, and their imbalance can lead to immunopathogenesis of various inflammatory autoimmune diseases such as multiple sclerosis. Treg, regulatory T cell; IL, interleukin; TGF-β, transforming growth factor-β; FoxP3, fork head box protein P3; STAT, signal transducer and activator of transcription; RA, retinoic acid; TNF, tumour necrosis factor; AhR, aryl hydrocarbon receptor; IRF, interferon regulatory factor; IFN, interferon; LT, lymphotoxin.

In addition to CD4+ IL-17+ Th17 cells, a new putative subtype of IL-17 producing CD4+ T cells with CD4+ IL-17+ IFNγ+ (Th17-1 cells) phenotype is also identified. It is revealed that Th17-1 cells mainly populated in memory CD4+ T cells. Interestingly, the pattern of chemokine receptor expression in Th17-1 cells can discriminate them from Th17 cells. Th17 cells express CCR6 and CCR4, whereas Th17-1 cells express CCR6 and CXCR3 [55, 56]. Th17-1 cells are also classified into two subtypes based on their chemokine receptor expression and the levels of cytokine production. It is reported that CCR1+ CCR5 memory Th cells generate a high level of IL-17 and little IFNγ, whereas CCR2+ CCR5+ cells produce a large amount of IFNγ and little IL-17 [57]. However, it should be clarified whether Th17-1 cells represent a stable subtype of Th17 cells or a transitional phenotype between Th17 and Th1 cells [3]. Collectively, the precise origin of Th17-1 cells, their biology and function are unknown, and this field requires more investigation.

Th17 differentiation is promoted by lineage-specific transcription factors, including retinoic acid-related orphan receptor (ROR)γt and RORα and is controlled by the coordinated function of a complex of positive and negative regulators [58]. In the mouse, the differentiation of Th17 cells is initiated by transforming growth factor (TGF)-β, IL-6 and IL-21, which activate signal transducer and activator of transcription 3 (STAT3) and induce the expression of transcription factor RORγt. IL-23, which activates STAT3, apparently serves to maintain Th17 cells in vivo. In human, IL-1, IL-6 and IL-23 promote human Th17 differentiation, but TGF-β1 is reportedly not required [59]. It is demonstrated that during initial Th17 development, IL-6 induces IL-21 in early activated CD4+ T cells, thus acts as a positive amplification loop to enforce Th17 differentiation [60–62]. Although it is reported that the presence of IL-6 is essential for Th17 cell differentiation, it has been shown this lineage can be generated in IL-6-deficient mice [60]. IL-6 signalling requires both STAT3 and to a lower amount STAT1 [63, 64]. It is shown that TGF-β suppresses development of Th1 and Th2 cells by the inhibition of their lineage-specific transcription factors including T-bet and GATA3 [65, 66]. Moreover, it is reported that the absence of IL-21 or IL-21R has no significant effect on Th17 differentiation [67, 68]. IL-21 action is mediated by IL-6 in a STAT3-dependent manner, and STAT3 may directly regulate the IL-21 gene [60, 61, 69]. IL-21 can also induce Th17 differentiation in IL-6-deficient mice [60]. IL-21 binds to a receptor complex composed of unique IL-21Rα chain and the shared common γ chain, which activates the STAT1/STAT3 pathway [70]. IL-23 is another effector cytokine in Th17 fate in the first 5 days after the initiation of the Th17 developmental programme. It seems that IL-23 is not involved in early stage of Th17 differentiation, because naïve T cells express IL-23R in a low level [55]. Possibly, impaired differentiation of Th17 cells in the absence of IL-23R is attributable to impaired expression of the IL-17Rα [71]. IL-23 signalling is mediated through a heterodimeric receptor complex consisting of IL-12Rβ1 and IL-23R [72]. IL-23 signalling in T cells seems to require both STAT3 and to a lesser extent STAT4 [63, 64]. It is shown that although IL-23 is not involved in the initiation of the Th17 development programme, it is required for the full terminal differentiation of Th17 and ultimately their activity [73, 74]. As described in Fig. 2, there is evidence that indicate IL-6, TGF-β and IL-23 do not provide sufficient signals to induce fully qualified Th17 cells. Thus, it is possible that other cytokine(s) may also be involved in the development of Th17 cells [75]. More recently, it is reported that IL-23 promotes Th17 differentiation by inhibiting T-bet and forkhead box protein P3 (FoxP3) and is required for the elevation of IL-22 but not IL-21 [76]. Interestingly, the IL-18Rα but not IL-18 is an important factor in the generation of pathogenic Th17 cells [75].

Figure 2.

 Differentiation and development model of Th17 cells. Stimulation of naïve CD4+ T cell precursors with combination of IL-6 and IL-1β, or IL-21 and TGF-β (as alternative pathway for induction of Th17 lineage in absence of IL-6) leads to induction of commitment naïve CD4+ precursors toward Th17 cells. These commitment cells express specific transcription factors in primary differentiated Th17 cells, such as retinoic acid-related orphan receptor (ROR)γt, RORα, and STAT3 (primary differentiation stage). IL-21 derived from Th17 cells (in autocrine manner) and other IL-21 producer cells then promote Th17 cells proliferation and differentiation (expansion stage). In the final stable state, IL-23 promotes terminal differentiation, development and survival of expanded Th17 cells. IL, interleukin; TGF-β, transforming growth factor-β; STAT, signal transducer and activator of transcription.

In addition to RORγt and RORα, other transcription factors are also identified which affect on differentiation and development of Th17 cells. RORγ is Th17 lineage-specific transcription factor that its presence is critical to IL-17 production [77]. RORγt is a different splice variant of RORγ that has the different promoter. It is demonstrated that RORγt enhances the IL-17 production, and its function is similar to other lineage-specific transcription factors in Th1 (T-bet) and Th2 (GATA-3) cells [78].

RORα is another key transcription factor for Th17 cells, which is induced by IL-6 and TGF-β. RORα in cooperation with RORγt promotes the high levels generation of IL-17A and IL-17F. Moreover, it is shown that double deficiencies of RORγt and RORα lead to complete inhibition of Th17 differentiation [79].

STAT3 is another important transcription factor for Th17 cells. STAT3 mediates the induction of RORγt and RORα in response to IL-6 and IL-23 [80]. Moreover, it is reported that IL-6, IL-21 and IL-23 use the STAT3 transcription factor for their signalling. It is also shown that STAT3 deficiency in T cells leads to large impairment in Th17 differentiation [80, 81]. It should be noted that STAT3 can directly bind to IL-17A/F genes promoter and enhance their transcription [63]. Additionally, STAT3 binds to IL-21 gene promoter and mediates the upregulation of IL-23R through IL-6, IL-21 and IL-23 [55, 61, 62, 69]. However, STAT3 is critical to but not sufficient for IL-17 generation, because the overexpression of active STAT3 in RORγt-deficient mice leads to low-level IL-17 production. Furthermore, the constitutive expression of RORγt cannot induce the IL-17 generation. Thus, it seems that the function of these two transcription factors is cooperative and parallel [62].

The aryl hydrocarbon receptor (AhR) or dioxin receptor, a ligand-activated transcription factor, is another important transcription factor that uniquely expresses in Th17 cells. It is shown that AhR deficiency leads to impaired development of Th17 cells. Moreover, it is appeared that the presence of this transcription factor is necessary for IL-22 generation, whereas Ahr-deficient Th17 cells completely failed to produce IL-22 [82, 83].

Moreover, interferon regulatory factor-4 (IRF-4), and a recently identified transcription factor, BATf, a basic leucine zipper transcription factor, are also other Th17-inducing factors. [73, 84].

Until now, several factors have been identified as inhibitory factors during the Th17 differentiation and development process. It is shown that various hallmark cytokines or lineage-specific transcription factors for Th1, Th2 and Treg cells inhibit commitment to Th17 lineage or decrease the production of Th17-related cytokines and/or prevent Th17 development. Reciprocally, Th17 cytokines and their specific transcription factors promote Th17 differentiation and development through inhibition of other Th subsets including Th1, Th2 and Treg cells.

It is revealed that Th1 hallmark cytokines including IFNγ and IL-12 can promote Th1 differentiation and inhibit Th17 development, because IFNγ can prevent IL-23-triggered expansion of Th17 cells [64]. Moreover, IFNγ increases T-bet expression, and T-bet overexpression leads to robust reduction in IL-17 generation. Surprisingly, T-bet can promote Th17 development, because T-bet can bind to IL-23R promoter and promote its expression [64, 85–88]. STAT1 and STAT4 mediate IFNγ and IL-12 signalling, and it seems that these two transcription factors are also negative regulator of Th17 development, because IL-17 production in STAT1-deficient T cells is increased. Additionally, IFNα signalling through STAT1 leads to the inhibition of Th17 development [64]. Conversely, Th-17 development in STAT1-, STAT4-, and T-bet-deficient mice is unaffected, suggesting that these transcription factors have no significant effects in Th17 development [89, 90]. IL-27, the member of IL-12 family cytokines is also the negative regulator of Th17 cells. Like the IFNγ, IL-27 signalling is through the engagement of STAT1 transcription factor. The producers of this cytokine are macrophages and dendritic cells, and its signalling is mediated through a receptor composed of IL-27R (WSX1 or TCCR) and the gp130 chain [91–94]. In addition, IFNβ inhibits Th17 development through the induction of IL-27 [95].

Like Th1 cells, Th2 cytokines and its transcription factors that promote Th2 development inhibit Th17 differentiation and expansion, so that IL-4 can inhibit both Th1 and Th17 differentiation and expansion [64]. GATA3, c-Maf and STAT6 are the Th2 lineage-specific transcription factors that promote Th2 differentiation and inhibit Th17 development [64, 89, 96]. Moreover, IL-25 (a member of IL-17 cytokine family) can inhibit Th17 development and enhance Th2 differentiation via IL-13 induction. IL-13 inhibits Th17 cell development in dendritic cells via downregulation of Th17 stimulatory cytokines (IL-1, IL-6 and IL-23) [89, 97, 98]. Despite the inhibitory effect of GATA3 on Th17 development, it seems that GATA3 probably promotes Th17 development through inhibition of IL-2, STAT1, and suppressor of cytokine signalling 3 (SOCS3) [99, 100].

IL-2 is a T cell growth factor that is critical to Treg development. It inhibits Th17 cell development effectively. Two pivotal transcription factors that mediate IL-2 signalling are STAT5a/b. IL-2 or STAT5 deficiency is associated with great inhibition of Tregs and expansion of Th17 cells [101–104].

Suppressor of cytokine signalling 3 is another important factor in the downregulation of Th17 cells. SOCS3 deficiency is associated with autoimmune diseases and increased Th17 frequency. Moreover, it is reported that SOCS3-deficient T cells promote phosphorylation of STAT3 in response to IL-23 and increase IL-17 generation [63].

The transcription factor Ets-1, which is positive regulator of Th1 development, is as a negative regulator for Th17 development. Ets-1 deficiency leads to increased Th17 differentiation and promotion of IL-22 and IL-23R mRNA levels in response to IL-6 and TGF-β1. It seems that the inhibitory effect of Ets-1 on Th17 cells is through enhancing IL-2 production [105]. In a recent report, it has been shown that microRNA mir-326 can bind to and prevent translocation of Ets-1 mRNA. Thus, microRNAs can promote Th17 development through the inhibition of Th17 inhibitor, Ets-1 [58, 106].

It should be noted that the transcriptional repressor protein BCL6 regulates T cell differentiation by repressing Th2 cells and enhancing follicular Th cells. It is proposed that BCL6 enhances Th17 differentiation through suppression of Th2 differentiation. Moreover, macrophages from BCL6-deficient mice showed an increased expression of the Th17 promoting cytokines IL-6, IL-23 and TGF-β, and conditioned media from BCL-6-deficient macrophages enhanced the increased IL-17 expression by T cells [107].

Th17 cells and multiple sclerosis

The main feature of MS is neuroinflammation, which leads to neurodegeneration. On the other hand, the central role of Th17 produced cytokines is the induction of inflammatory reactions. Thus, Th17 cells could be as a potent pathogenic cells in neuroinflammatory reactions in CNS [108, 109]. The IL-17-producing T cells (CD4+ or CD8+) have been detected in both active and chronic diseases [35]. The high frequency of CNS autoreactive Th17 cells was detected in the immune periphery before onset of clinical disease, but not in the CNS. In acute EAE, the large number of CNS autoreactive Th17 cells is present in the inflamed CNS. In recovery from an acute EAE, high levels of CNS autoreactive Th17 cells are still present in the immune periphery, but not in the CNS [110, 111]. Moreover, the frequency of Th17 cells is significantly higher in the cerebrospinal fluid (CSF) of RRMS patients during relapse, in comparison with RRMS patients in remission or to patients with other non-inflammatory neurological diseases [4]. Furthermore, RORγt deficiency leads to more susceptibility to EAE induction, which indicating the important role of Th17 cells in EAE and probably MS pathogenesis [77]. Interestingly, IFNβ1a, which has been used during the past 15 years as a primary therapy for RRMS patients, exerts its ameliorating effect in part through Th17 inhibition that indicates the immunopathologic role of Th17 cells in MS [112, 113].

The number one culprit for Th17-mediated encephalopathogenecity is IL-17A cytokine. It is revealed that IL-17 can affect on a wide range of cells such as endothelial cells, epithelial cells, fibroblasts, myeloid cells and synoviocytes. Moreover, IL-17 enhances the secretion of various inflammatory mediators including IL-8, CXCL1, CXCL6, IL-1β, IL-6, TNF-α, granulocyte monocyte-colony stimulating factor (GM-CSF), macrophage inflammatory protein-2 (MIP-2), monocyte chemoattractant protein-1 (MCP-1) and G-CSF. IL-17 is a potent inducer of neutrophil infiltration and inflammatory cytokines [77, 114, 115]. The detection of high levels of IL-17 in both plaques and CSF of patients with MS indicates the role of IL-17 in pathogenesis of MS [116, 117]. Additionally, it is reported that the production of IL-17 correlates with the number of active plaques on MRI [118]. IL-17 levels are higher in circulating leucocytes of patients with MS with active disease in comparison with normal subjects or individuals with inactive disease [112]. It has also been shown that the high expression of IL-17 correlates with MS severity [117]. Moreover, IL-17 generation in CNS infiltrating T cells and glial cells is associated with active disease in MS [35].

It seems that the pathogenic function of IL-17 can be exerted through enhancing the microglia function. It is shown that IL-17 treatment of microglia leads to increased generation of IL-6, MIP-2, nitric oxide, neurotrophic factors and adhesion molecules. Furthermore, addition of IL-1β and IL-23 enhances the production of IL-17 in microglia and may lead to exacerbation of disease [119]. However, it seems that the main function of IL-17 in immunopathogenesis of MS is the breakdown of BBB. Generation of IL-17 enhances the activation of matrix metalloproteinase-3 (MMP-3) and attracts neutrophils to the site of inflammation. Neutrophil-mediated activation of enzymes such as MMPs, proteases and gelatinases participates in BBB disruption. BBB disruption effectively enhances the recruitment of neutrophils. The augmented protease activity, which attracts a large number of monocytes and macrophages to the inflammatory regions, leads to sustained myelin and axonal damage [89, 120, 121]. Recently, the cellular mechanisms of IL-17-mediated BBB breakdown have been revealed. Huppert et al. showed that IL-17 increases the generation of reactive oxygen species (ROS) in brain endothelial cells. The oxidative stress mediates activation of the endothelial contractile machinery. Activation of the contractile apparatus is responsible for the loss and disorganization of tight junction proteins, which consecutively leads to BBB disruption. Moreover, ROS generation leads to upregulation of endothelial adhesion molecules and an increased adhesion and transmigration of inflammatory cells via the BBB. They showed that IL-17 activates the endothelial IL-17R, which is followed by augmented ROS generation mediated by NAD(P)H (nicotinamide adenine dinucleotide phosphate-oxidase) oxidase and xanthine oxidase. The oxidative stress then activates the endothelial contractile machinery through augmenting the amount of phosphorylated myosin light chain (MLC). Phosphorylated MLC associates with the actin cytoskeleton, leading to a cell contraction and increasing the intracellular space of the endothelial cell monolayer. Moreover, they demonstrated that MLC kinase inhibitor ML-7 inhibits the IL-17-induced breakdown of the tight junction molecules, which reinforces the critical role of MLC phosphorylation in IL-17 mediated BBB breakdown [122]. In addition to BBB disruption, Th17 cells enhance EAE (and probably MS) progression through neutrophil activation in the bone marrow and finally triggering the migration of immature monocytes into the blood stream. However, it should be noted that a few neutrophil is usually seen in MS plaques [123].

On the other hand, IL-17-deficient mice showed a delayed onset, decreased severity scores and histological changes with early recovery of EAE [124]. Moreover, IL-17 neutralization attenuated the disease [89]. It has been also reported that IL-17 neutralization in EAE in wild-type mice by monoclonal antibody as well as with a Fc-receptor fusion protein showed effective effects [109].

In contrast, IL-17F knockout mice showed no reduction in EAE severity, with only a mild additional effect of simultaneous IL-17A neutralization as well as T cell-specific IL-17 overexpression did not enhance EAE [110, 125]. Furthermore, it is demonstrated that IL-17-deficient mice are completely susceptible to EAE induction, and overexpression of IL-17 in murine T cells had no effect on the incidence, severity or kinetic of clinical EAE. The fact that IL-17 deficiency partially inhibits EAE could describe Th17 production by other proinflammatory mediators that coordinarily participate to the pathogenic process in parallel to or in synergy with IL-17 [123, 126–128].

IL-6 is an important stimulator for Th17 differentiation, and it is expected that its deficiency leads to Th17 abrogation. Thus, IL-6 targeting can be an efficient protocol in MS therapy. IL-6-deficient mice are protected from EAE induced by myelin antigens [129, 130]. Additionally, the presence of IL-6 is critical to the induction of cerebrovascular adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1), which are pivotal for leucocyte migration into the CNS [131]. Moreover, downregulation of IL-6 and TNF-α is correlated with the attenuated EAE [132]. Enhancing the expression of proinflammatory genes and the activation of astrogliosis and microgliosis by IL-6 shows the important role of IL-6 in immunopathogenesis of EAE and MS [133]. It is recently suggested that the loss of IL-6R expression during inflammation underlines a role for trans-signalling in the local maintenance of Th17 cells. IL-6 signalling is performed through two pathways: via a membrane bound IL-6R, CD126α subunit, (classical pathway), or through a soluble form of this cognate receptor (IL-6 trans-signalling pathway). The classical IL-6R signalling in naïve or central memory CD4+ T cells is required to steer their effector characteristics, whereas local regulation of soluble IL-6R activity might serve to maintain the cytokine profile of the Th cell infiltrate [134]. However, the fact that IL-6-deficient mice exhibit partial inhibition of EAE induced by the myelin-specific Th17 transfer suggests that IL-6 acts downstream of IL-17 in immunopathogenesis of EAE, and probably MS [135].

As mentioned previously, the combination of IL-6 and TGF-β is essential for the initiation of Th17 differentiation in mice. In the mouse model, which TGF-β is expressed under the IL-2 promoter, it could be led to an augmented Th17 differentiation and increased EAE severity [74]. Moreover, the expression of a dominant negative TGF-βRII under a CD4 promoter, which prevents Th17 differentiation and delays the onset of EAE, may prove the role of TGF-β in EAE and MS [136]. Although the TGF-β is essential for Th17 differentiation in mice, it can inhibit the human Th17 differentiation in a dose-dependent manner [137]. Controversially, the recent reports indicate that TGF-β is required for the differentiation of human Th17 cells like to mouse Th17 cells [138–140]. The differentiation of Th17 cells depends on the presence of IL-6 and TGF-β, but this observation that these cells can be developed in IL-6-deficient mice suggests a role for other cytokine(s) besides IL-6 and TGF-β in differentiation process of Th17 cells [60, 73].

Although the presence of IL-21 during Th17 development enhances the expansion of these cells, it seems that this cytokine is not critical to their development, because IL-21 has no important role in immunopathogenesis of EAE and probably MS. It is shown that the absence of IL-21 or IL-21R has no significant difference on the development of Th17 cells and their recruitment to the CNS or the severity of EAE [67, 73]. Vollmer et al. showed that IL-21 administration before MBP-induced EAE enhanced the inflammatory influx into the CNS, as well as the severity of EAE. Autoreactive T cells purified from IL-21-treated mice have shown more severe EAE than did the control encephalitogenic T cells. Such effects were not observed when IL-21 was administered after EAE progressed. Additional studies demonstrated that IL-21 given before the induction of EAE has increased NK cell function, including secretion of IFNγ. Depletion of NK cells abrogated the effect of IL-21 [141]. The results of this study show that IL-21 may affect EAE pathogenesis in indirect manner through enhancing NK cells for IFNγ secretion. In another study, Piao et al. showed that IL-21/IL-21R axis has an important role in the homoeostasis of Tregs in CNS autoimmunity. They demonstrated that blockade of IL-21 in SJL/J mice before and after the induction of EAE promotes the influx of inflammatory cells into the CNS. The blockade of IL-21 leads to proliferation of proteolipid protein (PLP)-autoreactive CD4+ T cells, which can induce severe EAE in adoptively transferred recipient mice. Conversely, Tregs from mice where IL-21 is blocked, lose their capacity to suppress EAE-induced PLP-reactive T cells. The direct effects of IL-21 on Tregs are confirmed by blockade of IL-21 in mice expressing a green fluorescent protein ‘knocked’ in Foxp3 allele, in which a reduction in the number of Tregs and a downregulation of their frequency and expression of Foxp3 are observed [142]. Furthermore, the temporal influence of IL-21 on the activity of immunoregulatory circuits can be effective in the modulation of the course of the autoimmune disease such as EAE and possibly MS. Liu et al. demonstrated that IL-21R-deficient mice developed EAE earlier and more severe neurological impairment than control mice. The impact on EAE initiation by IL-21R deficiency was correlated with a defect of Tregs and a downregulated expression of Foxp3. The recovery in IL-21R-deficient EAE was correlated with an expansion of Tregs as well as an organ-specific redistribution of NK cells [143].

On the other hand, IL-1β, which is one of the important cytokines in the initiation of Th17 differentiation, also has a role in MS pathogenesis, thus IL-1RI-deficient mice are resistant to EAE induction [144]. Furthermore, like the IL-17 and IL-22, IL-1β can enhance BBB disruption. Additionally, IL-1β can also promote microglia and astrocyte activation and stimulate demyelination process [145].

It has been revealed that IL-22 in combination with IL-17 can disrupt BBB integrity by breaking tight junctions. BBB endothelial cells express IL-17R and IL-22R, and treatment with these cytokines disrupts the gap junction of these cells, which leads to increasing their permeability and migration of Th17 cells through BBB. Therefore, IL-22 are also effective in Th17-mediated BBB disruption and severity of MS [146].

There is evidence that IL-9 in combination with TGF-β can promote Th17 cell differentiation, and Th17 cells can generate IL-9 [53]. IL-9 neutralization by monoclonal antibody delays the onset of EAE. Moreover, this observation that IL-9R-deficient mice develop a delayed and milder form of EAE with reduced levels of Th17 cells and IL-6-producer macrophages in comparison with wild-type mice, reinforces the pathogenic function of IL-9 during EAE, and possibly MS [147].

It is suggested that IL-23 has more importance in encephalopathogenecity of Th17 cells rather than IL-6 and TGF-β [148]. It is reported that IL-23-deficient Th17 cells cannot infiltrate into the CNS and remain in lymph nodes [71]. As mentioned previously, IL-23 has an efficient role in terminal differentiation and survival of Th17 cells. IL-23 expression levels in monocyte-derived dendritic cells are higher in patients with MS in comparison with healthy subjects [149]. Moreover, similar to IL-23-deficient mice, IL-23R knockout mice are fully resistant to EAE induction [126, 150]. It has also been reported that IL-23p19 neutralization by monoclonal antibody leads to amelioration of disease [151]. This report raises the possibility that IL-23 neutralization can be considered as an effective protocol in MS treatment. However, a phase II, double-blinded, placebo-controlled, randomized clinical trial of an monoclonal antibody-specific for IL-12p40 (ustekinumab) did not show ameliorating effect during disease [152]. This negative outcome can probably be in part attributable to unavailability of antibody to CNS tissue [128].

It is demonstrated that IL-12 p19 or IL-12/23 p40 deficiency leads to induction of resistance to EAE, whereas IL-12 p35 deficiency increases the susceptibility to disease [85, 85]. Th1 cells mainly express the chemokine receptors CCR5 and CXCR3, whereas Th17 cells preferentially express CCR6 [153]. It is reported that CCR5 deficiency do not affect disease severity, and CXCR3-deficient mice exhibit more severe disease in comparison with wild-type mice [154–156]. Furthermore, Th17 cells produce CCL20 (the ligand of CCR6) in high levels, proposing a possible positive feedback cycle [157–159]. The upregulated expression of both CCR6 and CCL20 was reported in EAE [159]. However, CCR6 deficiency in mouse models exhibits controversial results in susceptibility to EAE [5, 159–161]. Collectively, CCR6 has a crucial role in Th17 infiltration into the CNS [5, 162]. It has been shown that CCR6 expressed Th17 cells infiltrate into the CNS by interacting with CCL20 at the choroid plexus epithelium, which leads to inflammation and increasing BBB permeability and enhancing subsequent infiltration of other lymphocytes in CCR6-independent manner [3, 5]. Moreover, Th17 clones from patients with MS are able to express higher basal levels of the both activation and adhesion molecules such as CD5, CD62, CD2, HLA-DR, CD28, inducible costimulatory molecule (ICOS), CD49d, CD6, and melanoma cell adhesion molecule (CD146) compared to Th1 clones. Furthermore, Th17 cells have a higher proliferative capacity and are less susceptible to suppression by Tregs than Th1 cells [4]. Recently, the cooperative function of both Th1 and Th17 cells in immunopathogenesis of EAE and MS has been suggested, so that mice with defect in either RORγt or T-bet can be resistant to EAE induction [77, 163]. Moreover, the presence of both Th1 and Th17 cells in CNS during EAE has been reported, but their relative proportions were different among various mouse models [126, 164]. Furthermore, investigation into EAE models demonstrated that the pathology induced by Th1 and Th17 cells is distinct [123, 165]. It should be noted that the ratio of Th17 to Th1 is much higher in inflammatory foci in the brain compared with the spinal cord, which indicates the important pathogenic role of Th1 cells in spinal cord autoimmunity [120, 165].

It has been also suggested that the relative proportion of Th17 cells to Tregs might determine whether or not inflammation in the CNS becomes chronic [110]. Thus, the balance between frequencies of Th17 and Tregs is also crucial in immunopathogenesis of disease, which requires more investigation.

The new interesting field in targeting Th17 cells for MS therapy is the use of microRNAs to blocking the Th17 differentiation. Recently, Du et al. showed that miR-326 mediates Th17 differentiation via translational inhibition of Ets-1, a negative regulator of Th17 differentiation. Knockdown or overexpression of miR-326 alleviate or exacerbate EAE, respectively. In patients with MS, miR-326 expression is upregulated, and this correlates with disease severity and IL-17 generation. Thus, miR-326 is a Th17 cell-associated microRNA that acts in the pathogenesis of MS [58, 106].

In addition to Th17 cells, Th17-1 cells are also involved in disease course. It is reported that Th17-1 cells are present in the CNS of EAE mouse models [166, 167]. Moreover, genetic profiling of cloned encephalitogenic T cells from immunized TCR transgenic mice showed both T-bet+/RORγt and T-bet+/RORγt+ cells [3, 166]. Murphy et al. showed that Th17 and Th17-1 cells migrate into the CNS prior to the onset of clinical symptoms of EAE, where they may mediate neuroinflammation. They were demonstrated that Th17-1 cells infiltrate the brain prior to the development of clinical symptoms of EAE and that this coincides with activation of CD11b+ microglia and local production of IL-1β, TNF-α and IL-6 in the CNS. Furthermore, they reported that Th17-1 cells are potent activators of proinflammatory cytokines whereas Th1 cells are less effective [168].


It seems that Th17 cells play an important role in immunopathogenesis of EAE and MS. This claim is proved by several studies in EAE animal models and in patients with MS. Direct role of Th17 cells in demyelination process in EAE animal model of MS is also reported [169]. At now, it is revealed that ameliorating effect of conventional prescribed drugs for patients with MS is in part attributable to the inhibition of Th17 cells [112, 113]. Recently, Hao et al. showed that NK cell enrichment of mouse model of MS had an ameliorative effect on disease status. They reported that the effects of NK cells on CNS pathology were dependent on the activity of CNS resident, but not peripheral NK cells. This activity of CNS resident NK cells was associated with suppression of myelin-reactive Th17 cells. This report and several other reports in this field show that the positive effect of various ameliorative mechanisms in EAE and possibly MS is exerted in part through Th17 modulation [170]. This crucial function of Th17 cells is mainly mediated through disruption of BBB, which leads to large infiltration of various inflammatory cells into the CNS and finally neuroinflammation and demyelination. It is demonstrated that BBB endothelial cells express the high levels of receptors for various inflammatory products of Th17 cells, which more facilitates the BBB breakdown and infiltration of inflammatory cells into the CNS. Among the Th17 produced cytokines, IL-17 and IL-22 have more important role in disruption of BBB. However, IL-23 is the basic Th17-produced cytokine that regulates Th17 functions during disease pathogenesis [3, 89, 120, 146].

The balance between Th17 cells and other CD4+ T cell subsets including Th1 and Tregs in the site of inflammation is an important index for determination of type of pathology, sites of inflammation, and stages of disease. In EAE animal model and possibly in MS, it is demonstrated that Th1 cells are the cause for spinal cord inflammation, whereas the neuroinflammation in brain is mainly mediated through the function of Th17 cells. The frequencies of Th17 cells in brain is higher than Th1 cells, while Th1 cells mainly are populated in large number in the spinal cord in comparison with Th17 cells [120, 165]. Moreover, the balance between Th17 and Tregs can determine the status and disease stages. However, it should be noted that the EAE model is not directly applicable in MS. Thus, before designing any therapeutic method for patients with MS based on the targeting Th17 cells, we need to investigate the precise pathologic function of these cells in neuroinflammatory reaction in the CNS of patients with MS.