IL-21 and IL-21 receptor in the immunopathogenesis of multiple sclerosis

Abstract

Cytokines are considered important factors in the modulation of various immune responses. Among them, interleukin (IL)-21 is one of the major immune modulators, adjusting various immune responses by affecting various immune cells. It has been suggested that IL-21 may enhance autoimmunity through different mechanisms, such as development and activation of helper T (T_H)-17 and follicular helper T (T_FH) cells, activation of natural killer (NK) cells, enhancing B-cell differentiation and antibody secretion and suppression of regulatory T (T_reg) cells. Moreover, IL-21 has also been suggested to be an inducer of autoimmunity when following treatment of MS patients with some therapeutics such as alemtuzumab. This review will seek to clarify the precise role of IL-21/IL-21R in the pathogenesis of MS and, in its animal model, experimental autoimmune encephalomyelitis (EAE).

Keywords:

• Interleukin (IL)-21
• IL-21 receptor
• multiple sclerosis
• pathogenesis

Introduction

Cytokines play an important role in the modulation of several immune and non-immune related processes. Moreover, some cytokines have critical roles in the induction, initiation, progression and attenuation of a variety of diseases (Ghalamfarsa et al. 2013; Tangye 2015). Thus, it seems that identification of the precise mechanism(s) by which cytokines modulate disease status may help design new therapeutic methods of treatment.

Interleukin-21 (IL-21), a member of the common γ-chain cytokine family, has attracted much attention among investigators because of its complex structure and multi-functionality. Several cell types, such as helper T (T_H)-17, follicular helper T (T_FH), Th2 and natural killer T (NKT) cells, can produce IL-21. Moreover, a wide variety of immune and non-immune cells express the IL-21 receptor (IL-21R) on their surface (Nurieva et al. 2007; Zhou et al. 2007; King et al. 2008; Suto et al. 2008). Thus, it seems that IL-21 may play a critical role in the immuno-pathogenesis of several types of cancers and autoimmune diseases.

Multiple sclerosis (MS), a central nervous system (CNS)-related chronic inflammatory demyelinating disease, is characterized by re-occurring episodes of demyelination and axonal lesion induced by T_H (particularly T_H1 and T_H17) cells, macrophages and soluble inflammatory mediators (Jadidi-Niaragh and Mirshafiey 2010, 2011b, 2011c). There is also evidence implying the role of other immune cells such as T_FH, NK and B-cells in the immunopathogenesis of MS (Chanvillard et al. 2013; Plantone et al. 2013; Romme Christensen et al. 2013; Haugen et al. 2014). Although the precise etiology of MS is unknown, it has been is suggested that genetic and environmental factors have great influences on the onset and progression of MS (Mirshafiey and Jadidi-Niaragh 2010a, b).

Increased levels of IL-21 have been observed during the progression of several auto-immune diseases; this implies a pathologic function for this cytokine in autoimmunity (Zanin-Zhorov et al. 2014). Moreover, it has been reported that IL-21 acts as a pro-inflammatory agent during MS progression and so it has been suggested that IL-21 may promote disease advancement (Vollmer et al. 2005). Since T_H17 cells play an important role in the neuro-inflammatory process of MS, with regards to the stimulatory effect of IL-21 on development and expansion of T_H17 cells, it has been proposed that IL-21 and IL-21R may be considered key targets in the treatment of MS. This review intends to discuss the precise roles for IL-21/IL-21R in the immunopathogenesis/treatment of MS and in its animal model, experimental autoimmune encephalomyelitis (EAE).

Multiple sclerosis

MS is a complex heterogenic autoimmune disease of the CNS in which the myelin sheaths of the neurons are relatively destroyed by self-reactive immune responses (Mirshafiey et al. 2014). MS patients may express several manifestations such as paresthesia, diplopia, visual impairment, numbness or weakness of the limbs, bowel or bladder dysfunction, spasticity, ataxia, fatigue and altered mental functions (Gaby 2013). Although the precise etiology of MS is unknown, genetic and environmental factors play key roles in its etiology (Hoglund and Maghazachi 2014). There are four main phenotypes of MS including relapsing remitting (RR), primary progressive (PP), secondary progressive (SP) and progressive relapsing (PR) (Gaby 2013). It seems that destruction of the blood–brain barrier (BBB) and migration of autoreactive T-cells from the blood to the CNS are the main processes involved in the development of MS (Cheng and Chen 2014). Following the breakdown of the BBB (due to several triggers such as oxidative stress), peripheral activated lymphocytes infiltrate the CNS and induce local immune responses, ultimately destroying the myelin and even the axons themselves (Ortiz et al. 2014). Injured cells in the BBB secrete various inflammatory cytokines and chemokines that play an important role in the recruitment of inflammatory cells to the CNS (Cheng and Chen 2014). It seems that autoreactive lymphocytes are initially activated in the systemic lymphoid organs by various mechanisms like molecular mimicry and bystander activation. Subsequently, they transmigrate to the CNS where they are re-activated following the encounter of their specific antigen, which eventually leads to demyelination and axonal damage (Hartung et al. 2014).

Although autoreactive T (particularly T_H1 and T_H17)-cells are the main players in the immunopathogenesis of MS, innate immune responses are also involved in its initiation and progression (Roozbeh et al. 2014). In addition to T_H cells, CD8⁺ T-cells also contribute to the development of the disease and clonally expanded CD8⁺ T-cells are observed within the MS lesions (Denic et al. 2013). The invariant natural killer T (iNKT)-cells are another lymphocyte sub-type which can secrete various cytokines (such as interferon [IFN]-γ), interleukin (IL)-10, IL-4) and may be involved in disease progression (Roozbeh et al. 2014). Regulatory T (T_reg) cells play an important role in the maintenance of self-tolerance in the immune system. It has been reported that, although the frequency of T_reg cells is intact in MS patients, their functionality is rather perturbed (Jadidi-Niaragh and Mirshafiey 2011a). A skewed balance between T_reg and T_H17 cells during MS progression has also been demonstrated (Jadidi-Niaragh and Mirshafiey 2012).

In addition to T-cells, B-cells are also supposed to have a critical effect in the immunopathogenesis of MS. B-cells secrete various antibodies and cytokines which can enhance disease furtherance. They can also act as antigen presenting cells for T-cells. Moreover, identification of regulatory B-cells with anti-inflammatory function further complicates the identification of the precise role of B-cells in MS development (Krumbholz and Meinl 2014).

Immunobiology of IL-21

IL-21 is a member of the Class I cytokine family, which includes IL-2, -4, -7, -9 and -15 (Spolski and Leonard 2008). The IL-21R exhibits homology with the β-chain of IL-2/IL-15Rs and constitutes a complex with the common γ-chain (Collins et al. 2003; Parrish-Novak et al. 2000). Several types of immune cells, such as activated T_H, NKT, T_FH and T_H17 cells, can produce IL-21 (Parrish-Novak et al. 2000; Mehta et al. 2004; Leonard and Spolski 2005; Spolski and Leonard 2008). This cytokine regulates the differentiation and function of various immune cells. Interaction of IL-21 with its receptor leads to regulation of activation, proliferation and survival of both T_H and B-cells. It can also inhibit the differentiation and function of inducible T_reg cells (Monteleone et al. 2009). Moreover, IL-21 plays an important role in the development of T_FH (Shekhar and Yang 2012), B-cell somatic hypermutation and immunoglobulin class switching (Eto et al. 2011), T_H2 cell function (Pesce et al. 2006), expansion and activity of CD8⁺ T-cells (Zeng et al. 2005), cytotoxic activity of NK cells (Strengell et al. 2003) and the differentiation and function of NKT cells (Smyth et al. 2005; Coquet et al. 2007). It is reported that, while the IL-21 enhances the cytotoxic function and IFNγ secretion in murine-derived activated NK cells, it inhibits IL-15-mediated expansion of resting NK cells (Kasaian et al. 2002). IL-21 also promotes the terminal differentiation of mouse NK cells (Brady et al. 2004; Li et al. 2015). Further, IL-21 enhances the proliferation, IFNγ generation and cytotoxic activity of CD8⁺ effector T-cells in allogeneic mixed-lymphocyte responses (MLR) (Kasaian et al., 2002).

IL-21 can potently affect the B-cell proliferation, survival, differentiation and immuno-globulin responses (Ghalamfarsa et al. 2013, 2015; Spolski and Leonard 2014) While the IL-21 induces apoptosis of B-cells, it enhances IL-4 mediated B-cell proliferation and differentiation toward memory or plasma cells (Ettinger et al. 2005). These contradictory functions of IL-21 on B-cells depend on the interplay with co-stimulatory signals and the developmental stage of B-cells (Konforte et al. 2009). IL-21 also stimulates antibody production and, thereby, enhances host defense against various malignances and infectious diseases (Ettinger et al. 2008).

IL-21 also increases resistance of effector T-cells against the suppressive function of T_reg cells (Peluso et al. 2007). It can also inhibit granulocyte–macrophage colony-stimulating factor (GM-CSF)-mediated dendritic cell (DC) maturation. Thus, IL-21 shifts DC toward immature phenotype, which inhibits T-cell responses and induces T_reg cells (Brandt et al. 2003). In addition, IL-21 decreases the expression of MHC Class II, CD80, CD86 and CCR7 on the surface of DC and inhibits the secretion of IL-6, IL-12, IL-1β and tumor necrosis factor (TNF)-α cytokines by DC (Brandt et al. 2003; Strengell et al. 2006). It can also induce apoptosis of conventional DC in a STAT3- and BIM-dependent manner (Wan et al. 2013).

T_H17 cells generate IL-21 in a STAT3-dependent manner and IL-21 induces expression of RAR-related orphan receptor γ-t (RORγt), IL-17A and IL-17F in a positive autocrine loop that leads to expansion of T_H17 cells (Wei et al. 2007). IL-21 can promote differentiation of T_H17 cells, even in the absence of IL-6 (Deenick and Tangye 2007). Moreover, IL-21 skews the balance between T_H17 and T_reg cells toward T_H17 cells through STAT3 and RORγt transcription factors (Deenick and Tangye 2007; Korn et al. 2007; Nurieva et al. 2007). It was also demonstrated that the frequency of T_H17 cells was significantly reduced when the IL-21/IL-21R signaling was blocked (Deenick and Tangye 2007; Korn et al. 2007; Nurieva et al. 2007). Secretion of IL-21 and IL-17 from T-cells can be controlled via ROCK2 through STAT3, IRF-4 and RORγt. It is reported that inhibition of ROCK2 increases the suppressive function of T_reg cells via up-regulation of STAT5 phosphorylation. Thus, the use of ROCK2 antagonist shifts the T_H17/T_reg balance toward the T_reg phenotype (Zanin-Zhorov et al. 2014).

T_FH cells express B-cell CLL/lymphoma-6 (BCL6) transcription factor and can produce high levels of IL-21 (Chtanova et al. 2004). IL-21 enhances the secretion of TNFα by conventional T-cells, which leads to up-regulation of CXCL9 on DC in the draining lymph nodes and facilitates T_FH differentiation (Yoo and Braciale 2014). As IL-21 affects the differentiation, function and survival of a wide variety of immune cells, it may be considered as a worthy target for treatment of several diseases.

IL-21 and IL-21R in CNS-related disorders

There is evidence implying IL-21 modulates the self-reactive immune responses in various CNS-related autoimmune diseases (Petrelli et al. 2011; Yoshizaki et al. 2012; Linhares et al. 2013; Liu et al., 2015). Geri et al. (2011) have reported that IL-21 promotes inflammatory lesions in Behçet disease (BD) through the expansion of T_H17 and suppression of T_reg cells. BD is chronic systemic inflammatory disease that can also involve the CNS. They showed that, while the serum of patients with active BD enhances the production of IL-17 in purified CD4⁺ T-cells of healthy donors, it reduces the expression of forkhead box protein 3 (FoxP3) in vitro. Moreover, they could detect IL-21- and IL-17A-producing T-cells within cerebrospinal fluid, brain parenchyma inflammatory infiltrates and intra-cerebral blood vessels of patients with active BD with CNS involvement. Further, treatment of isolated CD4⁺ T-cells with IL-21 led to the up-regulation of T_H17 and T_H1 and down-regulation of T_reg cells. On the other hand, blocking IL-21 via an IL-21R-Fc restored the T_H17 and T_reg cell balance in BD patients by suppressing IL-17A production and increasing FoxP3 expression (Geri et al. 2011). Thus, it was proposed that IL-21 enhanced disease progression, in part, through promotion of inflammatory responses by expansion of T_H17 cells and also interfered with self-tolerance by down-regulation of T_reg cells.

In another study, Linhares et al. (2013) investigated the function of T-cells in patients with neuromyelitis optica (NMO), which is a type of CNS-related autoimmune disease. Those authors showed that, while T_H1 cytokines were decreased in cultured T-cells isolated from NMO patients, T_H17 and related cytokines (i.e. IL-23 and IL-6) were significantly increased. Moreover, polyclonal stimulation of CD4⁺ T-cells led to the secretion of IL-21 and IL-6 that was positively correlated to neurological disability and was resistant to glucocorticoid inhibition in NMO patients. Based on that previous study, it seemed that the presence of a positive autocrine loop between T_H17 and IL-21 might play a pivotal role in the immunopathogenesis of CNS-related autoimmunity.

There is also evidence which indicates an important role of IL-21 in CD8⁺ T-cell survival in the CNS during acute mortal JHMV and West Nile (WNV) viral infections (Stohlman et al. 1998; Janssen et al. 2005; Sitati and Diamond 2006; Fröhlich et al. 2009; Barker et al. 2010). It is suggested that CD4⁺ T-cells are the main source of IL-21 in the CNS, since it was observed that its mRNA level was significantly reduced in the CNS of CD4⁺ T-cell-depleted mice (Phares et al. 2011). It is also suggested that IL-21 producing CD4⁺ T-cells promote the peripheral activation of CD8⁺ T-cells and increase their antiviral function within the CNS (Phares et al. 2012). Moreover, IL-21R deficiency was associated with the down-regulation of IFNγ and IL-10 in CNS-derived CD4⁺ T-cells and disturbed peripheral B-cell activation and impaired CNS humoral responses (Phares et al. 2013). Thus, IL-21 secreting T-cells may enhance antibody production by B-cells, which may promote autoimmune responses (Metcalf et al. 2013). Similar results regarding the stimulatory and pathologic effects of IL-21, on both T- and B-cells in the CNS, have been reached using toxoplasma gondii-infected IL-21-deficient mice (Stumhofer et al. 2013).

An increased frequency of IL-21- and IL-9-producing CD4⁺ T-cells has also been noted in Alzheimer’s disease (Saresella et al. 2011). The extent of brain atrophy was also highly correlated with IL-21 producing CD4⁺ T-cells (Baglio et al. 2013). Moreover, using IL-21R-deficient mice with experimental autoimmune uveitis, it was demonstrated that blocking IL-21 signaling could lead to attenuation of CNS auto-inflammatory diseases (Wang et al. 2011b). It was also reported that IL-21 was increased in the injured mouse brain after cerebral ischemia. Moreover, IL-21-deficient mice exhibited smaller infarcts, better neurological function and decreased lymphocyte infiltration into the brain. The brain infiltrated CD4⁺ T-cells were the main source of IL-21. Administration of IL-21 receptor F_c fusion protein to these mice protected them from re-perfusion injury. Further, IL-21 was identified as a mediator of brain injury in post-mortem human brain tissue, since IL-21 was detected in perivascular CD4⁺ T-cells in the area surrounding acute stroke lesions (Clarkson et al. 2014). Lastly, Daga et al. (2007) was able to successfully eradicate glioblastoma (a tumor of the CNS) in mice through local delivery of IL-21 by IL-21 gene-transduced cells or using recombinant IL-21. These investigators showed that IL-21 exerted its anti-tumor effects mainly through the activation of anti-tumor antibody-secreting B-cells. That study aimed to prove the ability of IL-21 in the activation of B-cells in the CNS, which has been little researched until now.

Thus, it seems IL-21 is an important factor for the modulation of immune responses and inflammatory reaction in the CNS. In some disease such as BD and brain stroke, inhibition of IL-21 led to disease attenuation. On the other hand, IL-21 protected hosts against viral encephalitis and glioblastoma through stimulation of anti-viral and anti-tumor immune responses. The above-mentioned studies demonstrated that IL-21 exerted its effects, in part, through effects on the expansion of T_H17 and T_H1 cells, suppression of T_reg cells and modulation of humoral immune responses. The skewed balance between T_H17 and T_reg cells has frequently been noted in various cancers and autoimmune diseases and known to be an important factor in the immuno-pathogenesis of several diseases (Gol-Ara et al. 2012; Azizi et al. 2013; Jadidi-Niaragh and Mirshafiey 2012; Jadidi-Niaragh et al. 2013a, b, c). Thus, it is obvious to us that IL-21 was an essential immune factor for the modulation of neuro-inflammatory reactions in CNS-related disorders.

Expression of IL-21 and IL-21R in multiple sclerosis

It has been suggested that the expression of IL-21/IL-21R has high associations with different autoimmune diseases (Collins et al. 2003; Vollmer et al. 2005). Moreover, the polymorphism of IL-21 showed a high association rate with the susceptibility to autoimmune disease in Caucasians (Liu et al., 2015). Consistently, the gene and surface expressions of IL-21 and IL-21R were significantly increased in the CD4⁺ T-cells from patients with progressive MS (Romme Christensen et al. 2013). On the other hand, it has been reported that IL-21 was only over-expressed on infiltrating CD4⁺ T-cells in the acute and chronic white matter of MS lesions, whereas IL-21R was mainly expressed on CD4, CD19 and CD8 lymphocytes, but not MHC II-expressing macrophages/microglia. Both of the IL-21 and IL-21R were also expressed in neurons in the cortical area (Tzartos et al. 2011). There are similar results regarding the association of IL-21R gene polymorphism and MS disease (Nohra et al. 2010). Further, increased expression of IL-21 mRNA was detected during MS relapses (Tegla et al. 2013). Oddly, there was no significant change in the cerebrospinal fluid (CSF) concentration of IL-21 in MS patients (Wu et al. 2012). In addition, it was reported that IL-21 was not considered a major risk factor for MS in Spanish and Swedish populations (Fedetz et al. 2009; Linden et al. 2011). Another study demonstrated there were no significant differences in serum levels of IL-21 between MS patients and normal subjects (Wang et al. 2011a). These discrepancies (as shown in Table 1

Table 1. Studies related to expression of IL-21/IL-21R in MS patients.

Source of sample	Outcome	Reference
Peripheral blood	Increased expression of IL-21 and IL-21R in CD4^+ T cells from progressive MS	Romme Christensen et al. (2013)
MS lesion	(1) Increased expression of IL-21 only in infiltrating CD4^+ cells in MS lesions (2) IL-21R was broadly distributed on CD4^+, CD19^+ and CD8^+ cells	Tzartos et al. (2011)
CSF	Concentration of IL-21 in CSF of MS patients was not remarkably raised	Wu et al. (2012)
DNA	Association of IL-21R genetic polymorphisms with MS in Nordic population	Nohra et al. (2010)
DNA	No association of IL-21 gene with MS in Swedish population	Linden et al. (2011)
DNA	No association of TENR-IL-2-IL-21 locus on susceptibility or disease progression  in MS in Spanish population	Fedetz et al. (2009)
Serum	No difference of IL-21 in MS patients compared to control subject	Wang et al. (2011a)
Peripheral blood	Increased expression of IL-21 mRNA during MS relapses	Tegla et al. (2013)

CSF, Cerebrospinal Fluid; MS, multiple sclerosis; IL-21R, interleukin-21 receptor.

) may be due in part to the different sample sources, assessment techniques and sample sizes. It seems further studies are required to clarify precise relations between expression of IL-21/IL-21R and MS (Fedetz et al. 2009).

IL-21/IL-21R in EAE

Several studies have been demonstrated that prove IL-21/IL-21R can exacerbate EAE in part through the development of T_H17 cells (Table 2

Table 2. Studies related to the role of IL-21/IL-21R in EAE.

Main claim	Reference
EAE
Diminished EAE disease progression in IL-21 KO mice, which was associated with the expansion of Treg cells	Nurieva et al. (2007)
Administration of IL-21 before initiation of EAE enhanced autoimmune symptoms, which was associated with the expansion of Treg and down-regulation of TH17 cells	Korn et al. (2007)
Treatment of EAE mice with salmonella-CFA/IIC greatly attenuated disease, in part through suppression of IL-17 and IL-21	Ochoa-Reparaz et al. (2008)
Suppressed EAE by DNA vaccination through down-regulation of IL-21	Andersson et al. (2008)
Suppressed EAE induction via LXR agonist T0901317 through inhibition of IL-21	Xu et al. (2009)
Curcumin can attenuate EAE via inhibition of TH17 cells and IL-21 formation	Xie et al. (2009)
Treatment of EAE mice with IFNβ decreases IL-17 and IL-21	Chen et al. (2009)
Decreased TH17/IL-21 and increased Treg cells in E-FABP-deficient EAE mice	Li et al. (2009)
Tolerogenic DC prevent EAE development via down-regulation of RORγt, IL-17A, IL-17F, IL-21 and IL-22	Zhou et al. (2014)
Salmon cartilage Proteoglycans reduce EAE progression via down-regulation of IL-6, IL-21, IL-23R and RORγt and up-regulation of Foxp3	Sashinami et al. (2012)
Blockage of NR4A2 expression inhibits EAE through suppression of IL-21 production and IL-23R expression	Raveney et al. (2013)
Aggravated EAE in BLIMP-1-deficient mice was associated with increased numbers of CNS-infiltrating TH1, TH17, IFNγ^+IL-17A^+ and IL-21^+IL-17A^+ CD4^+ T-cells in brain and spinal cord.	Lin et al. (2014)
IL-21/IL-21R deficient mice are susceptible to EAE	Coquet et al. (2008), Sonderegger et al. (2008)
Inhibition of IL-21 was associated with increased entry of inflammatory cells into the CNS	Piao et al. (2008)
Severe neurological impairment in IL-21R deficient EAE mice	Piao et al. (2008), Liu et al. (2008)

). Consistently, IL-21 or IL-21R deficient mice showed attenuated EAE that was associated with a significant reduction of T_H17 (Korn et al. 2007; Nurieva et al. 2007; Spolski and Leonard 2008) and expansion of T_reg cells (Nurieva et al. 2007). Administration of IL-21 to mice before EAE induction was also associated with an increase in T_H17 and a decrease in T_reg cells (Korn et al. 2007). Treatment of proteo-lipid protein (PLP)-induced EAE mice with salmonella-CFA/IIC was also associated with disease attenuation and reduction of IL-17 and IL-21 cytokine levels, which implies the pathologic role of IL-21 in EAE (Ochoa-Reparaz et al. 2008). Similar results were observed when EAE mice were treated with DNA-carrying vaccines (Andersson et al. 2008). Regarding the studies mentioned above, it seems that IL-21/IL-21R could be considered as a pathologic axis in EAE. Thus, some investigators have tried to block this axis in order to attenuate EAE progression. Consistently it has been reported that administration of the LXR agonist T0901317 (which suppresses expression of IL-21 and IL-22 mRNA) can inhibit EAE induction (Xu et al. 2009). Moreover, treatment of EAE mice with curcumin could ameliorate disease (in part]) through the suppression of IL-21, IL-6, RORγt and STAT3-mediated signaling (Xie et al. 2009). Similarly, treatment of EAE mice with IFNβ was associated with down-regulation of IL-17, osteopontin and IL-21 (Chen et al. 2009).

Li et al. (2009) suggested that the expression of epidermal fatty acid-binding protein (E-FABP) on T-cells could enhance development of T_H17 and suppress that of T_reg cells. Those investigators showed that the myelin oligodendrocyte glycol-protein peptide (MOG)-mediated induction of EAE in E-FABP-deficient mice was associated with down-regulation of T_H17 and up-regulation of T_reg cells, an outcome that was due in part to significant decreases in IL-21 levels. It has also been shown that tolerogenic DC can suppress EAE development, in part through down-regulation of RORγt, IL-17A, IL-17F, IL-21 and IL-22 (Zhou et al. 2014). It has also been reported that treatment of EAE mice with salmon cartilage proteoglycans could lead to down-regulated IL-6, IL-21, IL-23R and RORγt, and up-regulated Foxp3 in both draining lymph nodes and spinal cords (Sashinami et al. 2012).

NR4A2 (nuclear receptor sub-family 4, group A, member 2) plays an important role in the differentiation of T_H17 cells and production of IL-21 and IL-17 cytokines. Consistently, in vivo inhibition of NR4A2 expression reduced T_H17 and IL-21 levels and protected mice against EAE induction (Raveney et al. 2013). Moreover, it has been demonstrated that the frequencies of CNS-infiltrating T_H1, T_H17, IFNγ⁺IL-17A⁺ and IL-21⁺IL-17A⁺ CD4⁺ T-cells were significantly increased in the brain and spinal cord of B-lymphocyte-induced maturation protein-1 (BLIMP-1)-deficient NOD mice (Lin et al. 2014).

Contrary to the studies previously mentioned, there is evidence to indicate the IL-21/IL-21R axis has no effect on development of T_H17 cells and that IL-21/IL-21R-deficient mice are also susceptible to EAE progression (Coquet et al. 2008; Sonderegger et al. 2008). Moreover, increased infiltration of pro-inflammatory cells into the CNS has also been seen when IL-21 was suppressed (Piao et al. 2008). Another study showed the frequency of T_reg cells and expression of FoxP3 were significantly decreased in both blocked IL-21 and IL-21R deficient mice with EAE (Liu et al. 2008; Piao et al. 2008). Interestingly, it was seen that IL-21R-deficient mice developed EAE faster and in a more severe form when compared to control mice. This faster initiation was also associated with defective T_reg and FoxP3 expression and intact T_H17 development. Moreover, recovery among IL-21R-deficient mice was associated with expansion of T_reg cell levels and organ-specific redistribution of NK cells (Liu et al. 2008).

Recent studies have shown that the new sub-sets of B-cells that produce IL-10 can negatively regulate immune responses; therefore, these cells are known as regulatory B-cells (B10 or B_reg cells) (Yanaba et al. 2008; Yoshizaki et al. 2012). Generation of in vivo effector B10 cells depends on the presences of IL-21 and its receptor (Tedder and Leonard 2014). Therefore, B10 cells regulate immune responses by IL-10 production in neuro-inflammatory processes such as MS (Tedder and Leonard 2014). A study on purified spleen CD19⁺ B-cells showed that IL-21 significantly increased the production of IL-10 without the need of phorbol esters and ionomycin for stimulation. The effector function of B10 cells in EAE was dependent to the existence of IL-21R, MHC-II and CD40 (Yanaba et al. 2009; Yoshizaki et al. 2012). Moreover, initiation of EAE was significantly reduced in mice treated by adoptive transfer of antigen-specific (MOG-sensitized) B10 cells; it seems this function was related to CD4⁺ T-cell IL-21 production. Thus, it seems B10 cells could naturally regulate acute immune responses in EAE in an IL-21-dependent manner.

As noted earlier, there are conflicts between different studies regarding the role of IL-21/IL-21R in the immunopathogenesis of EAE (Table 2). However, these discrepancies may be due to genetic backgrounds of mice, different EAE-inducing protocols (using various doses of antigen for EAE induction [i.e. 100 μg (Bauquet et al. 2009), 150 μg (Coquet et al. 2008) or 200 μg (Vollmer et al. 2005)]) and the fact IL-21 is highly pleiotropic – affecting many immune and non-immune components of the body which have not been fully addressed until recently (Spolski and Leonard 2014; Croce et al. 2015). Further investigations with the same disease induction protocols (in large scale) and optimal doses of antigens are required to clarify the precise roles for the IL-21/IL-21R axis in EAE.

Role of IL-21/IL-21R in immunopathogenesis of MS

Since T_H17 cells play an important role in the neuro-inflammatory process of MS and IL-21 significantly promotes the development of T_H17, it seems that IL-21 may enhance MS progression. It has been reported that simvastatin reduces neuro-inflammatory CNS lesion formation in RR-MS patients (Giovannoni et al. 2014). Zhang et al. (2011) reported that the mechanism by which simvastatin exerted anti-inflammatory effects on RR-MS patients was due in part to inhibition of T_H17 and its secreted cytokines, particularly IL-21. Those authors demonstrated that simvastatin blocked differentiation of T_H17 cells and generation of T_H17-derived cytokines (IL-17A, IL-17F, IL-21 and IL-22) in in vitro-differentiated naive T_H cells.

IL-21 not only inhibits the development of T_reg cells (Deenick and Tangye 2007; Petrelli et al. 2011), but also converts T_reg to T_H17 (Yang et al. 2008) and increases the resistance of conventional T-cells against the suppressive function of T_reg cells (Peluso et al. 2007). Therefore, it seems that IL-21 enhances the development of MS, in part, through the suppression of T_reg cells. It has been reported that treatment of MS patients with UVB phototherapy was associated with down-regulation of IL-21 and up-regulation of T_reg cells and tolerogenic DC, resulting in disease attenuation (Breuer et al. 2014). It was also demonstrated that the frequency of T_FH cells was significantly increased in the peripheral blood and cerebrospinal fluid of RR-MS and SP-MS patients. Moreover, increased expression of IL-21, IL-21R and ICOS was noted in CD4⁺ T-cells obtained from progressive MS patients. Treatment of such patients with mitoxantrone was associated with the down-regulation of T_FH and IL-21, outcomes that led to disease alleviation (Romme Christensen et al. 2013). Further, accumulated populations of IL-21R⁺ and IL-21⁺CD4⁺ T-cells were observed in both active and chronic MS lesions (Tzartos et al. 2011; Romme Christensen et al. 2013). Although most studies have shown that IL-21 indirectly plays an important role in the pathogenesis of MS (Table 3

Table 3. Studies related to the role of IL-21/IL-21R in MS.

Main claim	Reference
MS
Simvastatin exerts its anti-inflammatory effects in RR-MS patients, in part through inhibition of TH17 and IL-21	Zhang et al. (2011)
UVB phototherapy down-regulates IL-21 and up-regulates Treg and tolerogenic DC cell levels in MS patients	Peluso et al. (2007)
Increased expression of IL-21, IL-21R and ICOS was observed in CD4^+ T-cells from progressive MS patients. Mitoxantrone decreased TFH and IL-21 in SP-MS patients	Romme Christensen et al. (2013)
Increased IL-21R^+ and IL-21^+CD4^+ T-cells have been observed in the both active and chronic MS lesions	Tzartos et al. (2011), Romme Christensen et al. (2013)
Treatment of MS patients with alemtuzumab led to decreased IL-21 levels	Zhang et al. (2013)
The secondary autoimmunity following treatment with alemtuzumab can be predicted via high baseline levels of IL-21 in MS sera	Costelloe et al. (2012), Jones et al. (2009)

), it is still necessary to conduct studies to clarify the precise function of IL-21 in the pathogenesis of the disease.

Alemtuzumab is a humanized monoclonal antibody targeting the campath-1 antigen (CD52) that is broadly expressed on immune cells. This antibody quickly eliminates CD52-expressing immune cells from the circulation. It has been suggested this antibody could also exert neuroprotective effects, presumably by inducing production of neurotrophic factors in autoreactive T-cells (Klotz et al. 2012; Fernandez 2014). Alemtuzmab can significantly reduce the rate of relapse in RR-MS patients (Brown and Coles 2013). However, treatment of MS patients with alemtuzumab may also be associated with new secondary autoimmune disorders, such as auto-immune thyroiditis or idiopathic thrombocytopaenic purpura (Jones et al. 2009; Klotz et al. 2012; Fernandez 2014).

Treatment of MS patients with alemtuzumab also led to decreased IL-21 levels (Zhang et al. 2013). Secondary autoimmunity following treatment with alemtuzumab usually occurs and can be predicted by the high baseline levels of IL-21 in the MS sera (Costelloe et al. 2012). Evaluation of IL-21 in the serum of MS patients prior to treatment with alemtuzumab showed that groups of patients who exhibited secondary autoimmunity had higher levels of IL-21 compared to non-autoimmune hosts (Jones et al. 2009; Costelloe et al. 2012). IFNβ has usually been used as a first-line treatment for RR-MS patients and can decrease production of IL-21 from CD4⁺ T-cells (Tao et al. 2014). Thus, it would seem that documented therapeutic effects of IFNβ are due in part to a down-regulation of IL-21.

As discussed earlier, little is known regarding the immunopathologic potential of IL-21/IL-21R in MS patients. Thus, it is difficult to reach a conclusive result based on previous studies about the precise role of IL-21/IL-21R in the immune status of MS patients. With that in mind, it appears IL-21 contributes to MS progression partly through up-regulation of T_H17 and down-regulation of T_reg cells. T_H17 cells, which are the main pathogenic cells in MS, produce IL-21 that can lead to the expansion of the same and other T_H17 cells in a positive autocrine loop. Moreover, there is no comprehensive data regarding precise effects of IL-21 on other immune cells involved in MS such as B-cells, macrophages/microglia and DC. Moreover, since IL-21 can affect several immune and non-immune components of the body, it is difficult to make a direct correlation between the therapeutic mechanisms of some agents and IL-21.

As discussed, there were several therapeutic agents that could affect the production of different cytokines (particularly IL-21) in the MS patients and EAE animal models. It is not rational to assume, just because IL-21 is included in affected cytokines, the major effect must come from it rather than from a combination of cytokines. Therefore, further comprehensive studies are needed to discriminate the precise role of IL-21 in the immunopathogenesis of MS between all affected cytokines.

Conclusion

IL-21 affects the development and function of various immune cells, including T_H17, T_reg, T_FH, NK and B-cells. Since these cells play an important role in the immune responses, IL-21 may be involved in the immunopathogenesis of several diseases, such as MS (Figure 1). Moreover, there is evidence supporting the theory that IL-21 is in fact involved in the development of MS. However, reports regarding the role of IL-21/IL-21R in EAE animal models remain controversial. One item that needs to be clarified is the role of IL-21 in the development and function of B10 cells in EAE and MS. This issue will determine whether IL-21 can be considered as having a protective role in MS or not. On the other hand, little is known regarding the precise role of the cytokine in the immunopathogenesis of MS. As IL-21 promotes development of effector cells involved in the neuro-inflammatory process associated with MS such as T_H17 and directly inhibits induction of T_reg cells, therefore inhibition of IL-21 may be considered a worthy target for MS therapy. However, it should be noted that T_reg and T_H17 cells are in the reciprocal regulation and development of each cell is associated with expansion of another one. Further investigations regarding the immunobiology of IL-21 are required to enable the designing of novel therapeutics based on IL-21 targeting.

Figure 1. IL-21 can affect various immune cells that are effective in the immunopathogenesis of MS.

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.