DOI 10.1515/hsz-2013-0213      Biol. Chem. 2014; 395(3): 335–346

Review Georgina Galicia and Jennifer L. Gommerman*

Plasmacytoid dendritic cells and autoimmune inflammation Abstract: Plasmacytoid dendritic cells (pDC) are a subpopulation of dendritic cells (DC) that produce large amounts of type I interferon (IFN) in response to nucleic acids that bind and activate toll-like-receptor (TLR)9 and TLR7. Type I IFN can regulate the function of B, T, DC, and natural killer (NK) cells and can also alter the residence time of leukocytes within lymph nodes. Activated pDC can also function as antigen presenting cells (APC) and have the potential to prime and differentiate T cells into regulatory or inflammatory effector cells, depending on the context. In this review we discuss pDC ontogeny, function, trafficking, and activation. We will also examine how pDC can potentially be involved in regulating immune responses in the periphery as well as within the central nervous system (CNS) during multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Keywords: dendritic cells (DC); experimental autoimmune encephalomyelitis (EAE); multiple sclerosis (MS); type I interferon (type I IFN). *Corresponding author: Jennifer L. Gommerman, Department of Immunology, University of Toronto, 1 King’s College Circle, Toronto, ON M5S 1A8, Canada, e-mail: [email protected] Georgina Galicia: Department of Immunology, University of Toronto, 1 King’s College Circle, Toronto, ON M5S 1A8, Canada

Introduction Dendritic cells (DC) are hematopoietically-derived cells that efficiently link the innate and the adaptive immune responses. DC function as sentinels within the body. They survey tissues to capture and present antigens in order to instruct and shape the adaptive immune response either to respond or to be tolerant against peripheral antigen. DC can be derived from common DC progenitors (CDPs) that give rise to different DC subsets characterized by the expression of lineage-specific transcription factors: (1) conventional DC (CD11b+ or CD8α+) (cDC) that are derived

from pre-cDC committed precursors and express the Zbtb46 or Batf3 transcription factors respectively (Satpathy et al., 2012a) and (2) plasmacytoid DC (pDC) that are derived directly from CDPs and express the E2-2 transcription factor (Cisse et al., 2008). In addition, some DC can be derived from blood-borne monocytes such as TNF/iNOS producing DC (TiP-DC) and monocyte-derived DC (Mo-DC) (Satpathy et al., 2012b). Unique functions of different DC subsets are beginning to be revealed by using tissue-specific knock-outs of the various transcription factors that dictate DC lineage specification. Broadly speaking, cDC are well-suited for Ag presentation to CD4+ and CD8+ T cells. cDC are found in most tissues of the body and are particularly abundant in locations that experience a heavy burden of Ag exposure such as the skin as well as the gastrointestinal, reproductive, and respiratory tracts. cDC capture and take up foreign and self antigens so that they may be processed into peptides that are loaded onto major histocompatibility complex (MHC) molecules to be presented on the cDC surface. cDC then migrate to local lymph nodes (LN) to present these antigen/MHC complexes to T cells. Under steady state conditions, cDC exhibit an immature phenotype, and antigen presentation in the absence of inflammation by immature cDC can result in tolerance (Kornete and Piccirillo, 2012). In contrast, in the context of inflammation, cDC detect microbial infection and tissue damage by various types of pattern recognition receptors (PRR), such as toll-like receptors (TLR), resulting in cDC activation. Activated cDC up-regulate co-stimulatory molecules and MHC class II, allowing them to potently activate CD4+ T cells, as well as cognate CD8+ T cells through MHC class I-antigen cross-presentation, thereby bridging the innate and adaptive immune response. Furthermore, cDC can secrete an array of cytokines such as IL-12, type I interferon (IFN), and IL-23 that contribute to the polarization of activated T cells into different T cell fates in the periphery (Banchereau et  al., 2000; Merad et al., 2013), and also contribute to promote the function of innate lymphoid cells in the gut (Wang et  al., 2010; Tumanov et al., 2011).

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336      G. Galicia and J.L. Gommerman: pDC and inflammation In contrast to cDC, pDC are specialized cells with the morphology of an antibody-secreting plasma cell. They circulate within the blood compartment and lymphoid organs, playing a critical role as part of a first line of defense against various infections, particularly viral infections. Notably, pDC express TLR7 and TLR9 within endosomal compartments that detect nucleic acids derived from viruses, bacteria, or dead cells. Engagement of TLR7 and 9 with their ligands (RNA or DNA) triggers a downstream signaling cascade that induces the secretion of type I IFN (Fonteneau et al., 2004; Gilliet et al., 2008). In addition to type I IFN production, after TLR activation pDC undergo morphological changes and upregulate the expression of co-stimulatory and MHC molecules that enable them to present antigen to T cells (Di Pucchio et  al., 2008; Merad et  al., 2013). It has been reported that pDC are able to activate CD8+ T cells (Di Pucchio et al., 2008; Mouries et al., 2008), however, pDC have low capacity to stimulate CD4+ T cells (Young et al., 2008; Kool et  al., 2011). Differences in antigen uptake and presentation by pDC compared to cDC may account for the poor capacity of pDC to activate CD4+ T cells (Villadangos and Young, 2008; Young et al., 2008). In fact, as will be described below, priming of CD4+ T cells by pDC can induce regulatory IL-10 producing CD4+ T cells (Ogata et al., 2013). As first-line responders to infection and inflammation, both cDC and pDC shape subsequent immune responses and, as such, they are appealing targets for immuno-modulatory therapeutics. In this review, we will focus on how pDC develop and traffic within the body followed by how they may be recruited to sites of autoimmune inflammation and exert immunomodulatory functions, particularly in the context of autoimmune diseases such as multiple sclerosis (MS).

Immunobiology of plasmacytoid dendritic cells Origin and development of pDC pDC were first described in 1958, and based on their plasma-like morphology, they were originally identified as plasmacytoid monocytes or plasmacytoid T cells (Facchetti et  al., 1988). Subsequent studies have shown that pDC can express MHC-II, and B220 (CD45R), and more importantly, they were found to produce large amounts of type I IFN upon microbial stimulation (Cella et  al.,

1999; Siegal et al., 1999). In mice, pDC can be identified by the expression of sialic acid-binding immunoglobulinlike lectin (Siglec-H) and bone marrow stromal antigen 2 (BST2). Human pDC express the blood DC antigens (BDCA-2 and BDCA-4), leukocyte immunoglobulin-like receptor subfamily A member 4 (LILRA4-ILT7), and CD123 (Crozat et al., 2010). pDC can be derived directly from a common DC progenitor (CDP) that can also give rise to cDC subsets. New evidence suggests that a more primitive lymphoid-primed multipotent progenitor (LMPP) can also give rise to pDC (Onai et al., 2013). Furthermore, it has also been proposed that a certain fraction of pDC can be derived from bone marrow-derived CLP that express the Flt3 receptor (Sathe et al., 2013). CLP stimulated with Flt3 ligand can give rise to pDC that are able to produce IFNα when stimulated with CpG, and such CLP-derived pDC show evidence of a priori RAG1 expression with D-J rearrangements of IgH genes (Sathe et  al., 2013). Thus, not only CDPs, but also CLP and LMPPs can serve as pDC precursors (Shigematsu et al., 2004; Onai et al., 2013; Sathe et al., 2013). Several cytokines and transcription factors have been described to play a central role in pDC development (Moore and Anderson, 2013). Although cDC and pDC development depends on Flt3L-Flt3 signaling, ablation of Flt3 signaling (via a non-functional mutation or a targeted deletion of the Flt3 receptor) causes a more profound diminishment of pDC compared to cDC in the periphery (Waskow et al., 2008; Eidenschenk et al., 2010). In addition, several transcription factors control pDC lineage commitment, including PU.1 (both pDC and cDC) (Carotta et al., 2010), Spi-B in mice (Sasaki et  al., 2012) and humans (Schotte et  al., 2004) and high levels of Ikaros are also needed for pDC differentiation to silence alternative lineage genes and to regulate the Flt3L response in pDC (Allman et al., 2006). However, the unique pDC-specific transcription factor that drives pDC development is the E protein E2-2 transcription factor, which is indispensable for pDC lineage commitment (Cisse et  al., 2008). The E2-2 transcription factor directly binds to the promoters of several pDC selective genes such as BDCA2, LILRA4, IRF7, IRF8, and SPIB (Moore and Anderson, 2013) (Figure 1).

Activation of pDC As mentioned, pDC characteristically express TLR7 and TLR9, the TLRs that specifically sense nucleic acids within endolysosomal compartments. The endolysosomal localization of TLR7 and TLR9 depends on the endoplasmic reticulum resident protein UNC93B1. UNC93B1 delivers

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G. Galicia and J.L. Gommerman: pDC and inflammation      337

Figure 1 Depiction of DC lineage specification. A combination of cytokines and transcription factors have been determined to participate in the pDC development. E2-2 is an indispensable transcription factor required for pDC development. Hematopoietic stem cells (HSC), lymphoid-primed multipotent progenitor (LMPP), common lymphoid progenitors (CLP), and common dendritic cell progenitor (CDP), common myeloid progenitor (CMP), macrophage and dendritic cell progenitor (MDP).

TLR7/9 from the endoplasmic reticulum to endolysosomes where the ectodomains of TLR7/9 are cleaved by cathepsins and asparagine endopeptidase to generate functional TLR for ligand recognition (Gilliet et  al., 2008). TLR7 is triggered by guanosine- or uridine-rich single strand RNA (ssRNA) and TLR9 responds to single strand DNA (ssDNA) that contain unmethylated CpG-containing motifs normally found in virus and bacteria. In addition, pDC can also sense host genomic material released by apoptotic cells (Heyder et  al., 2007; Gilliet et  al., 2008). Once TLR7/9 bind to their ligands, the myeloid differentiation primary response protein (MyD88) couples to the TLR receptor. MyD88 contains a C-terminal TIR domain and an N-terminal death domain, which is required for the recruitment and assembly of a multi-protein signal transduction complex that contains the IL-1 receptor-associated kinase 4 (IRAK4), Buton’s tyrosine kinase (BTK), IRF-7 and TNF receptor-associated factor 6 (TRAF6). IRF7 is further activated through TRAF3, IRAK1, IKKα, and osteopontin. IRF7 is ubiquitinylated and phosphorylated to enable translocation into the nucleus for type I IFN gene transcription (Gay et al., 2011; Bao and Liu, 2013). pDC express constitutively low levels of IRF7, thus facilitating rapid type I IFN expression following TLR activation (Dai et al., 2004) (Figure 2).

Immunoregulatory effects of Type I IFN production by pDC Type I IFNs constitute a large family of related proteins including IFNα, β, κ, ω, λ, and τ. All type I IFNs signal

through a common type I IFN receptor (IFNAR). IFNAR signaling leads to the expression of IFN-stimulated genes such as IFIT3, IFI44L, IFI35, IFI44, MX1, MX2, OAS1, OAS2, OAS3 or SIGLEC1, and collectively these genes comprise a ‘type I IFN signature’ that has been described in autoimmune diseases such as systemic lupus erythematosus (Siegal et al., 1999; Obermoser and Pascual, 2010). In the context of viral infection, type I IFN can directly interfere

Endosome RNA

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Figure 2 Activation of pDC by innate signals. TLR7 and 9 are activated by nucleic acids derived from pathogens and apoptotic cells. TLR7 and 9 are expressed in endosomes and signal through MyD88 adaptor molecules to induce the expression of type I IFN genes.

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338      G. Galicia and J.L. Gommerman: pDC and inflammation with viral replication, and modulate the immune response for the clearance of pathogens. IFNAR is ubiquitously expressed on all nucleated cells, and type I IFN can exert a potent inflammatory effect on various types of immune cells. IFNAR activation can trigger DC maturation, which leads to increased expression of co-stimulatory molecules, further promoting the generation of Th1 and cytotoxic T lymphocytes (Cella et al., 2000). For B cells, type I IFN has been shown to enhance antibody production and immunoglobulin class switching (Le Bon et  al., 2001, 2006; Jego et  al., 2003). Type I IFN produced by activated pDC is essential for clearance of rotavirus in the gut by virtue of B cell activation and antibody production (Deal et  al., 2013) and pDC can regulate steady state IgA production via a type I IFN-dependent mechanism (Tezuka et al., 2011). As type I IFNs have autocrine, paracrine, and pleiotropic effects, the amplitude of the type I IFN response by pDC is also tightly regulated to prevent a harmful immune response. For example, type I IFN production is negatively regulated by surface receptors such as BDCA-2, ILT7 (human), NKp44, and Siglec-H (mouse). BDCA-2 and immunoglobulin-like transcript 7 (ILT7) potently suppresses the ability of pDC to produce type I IFN in response to TLR ligands (Cao and Bover, 2010). Upon antibody crosslinking, NKp44 inhibits the production of IFNα induced by CpG ODN (Fuchs et al., 2005). Also, antibody crosslinking of Siglec-H reduces type I IFN production in vivo and in vitro (Lande and Gilliet, 2010). Interestingly, these pDC receptors, when targeted with antibodies coupled to antigen, mediate endocytosis, processing, and presentation of antigen whilst suppressing type I IFN expression (Loschko et al., 2011). However, little is known of the physiologic ligands for surface receptors such as NKp44 and Siglec-H.

Immunoregulatory effects of pDC independent of Type I IFN As mentioned, cDC are important APC both in the periphery and in the CNS during neuroinflammation (Bailey et  al., 2007; Ji et  al., 2013). After exposure to maturation stimuli such as TLR ligands and CD40L, pDC can prime CD8+ T cells and CD4+ T cells, although, they appear to be much less potent APC compared to cDC in stimulating T cells (Di Pucchio et al., 2008; Mouries et al., 2008; Young et al., 2008; Kool et al., 2011). It is now appreciated that pDC may not represent a homogenous population, but rather subpopulations with different functions (Dzionek et  al., 2000). For example, blood-derived pDC can be subdivided into two different subsets: pDC1 and pDC2. These subsets

differ in their phenotype and in their capacity to induce pro-inflammatory or regulatory T cell responses. pDC1 exhibit a more immature phenotype with low expression of MHCII and null expression of the co-stimulatory molecule CD86, while pDC2 have a more mature phenotype (MCHII+ and CD86+). Stimulation of naïve allogeneic T cells with pDC1 versus pDC2 results in the adoption of opposing T cell properties: pDC1 induce a regulatory T cell phenotype whereas pDC2 induce pro-inflammatory T cells. However, the pDC1/pDC2 spectrum exhibits a certain level of plasticity because stimulation of pDC1 with CpGB results in the acquisition of a pDC2 phenotype (Schwab et al., 2010). In addition to this functional plasticity, ligation of CD123 on pDC can indirectly polarize T cells towards a regulatory phenotype. CD123 is the receptor for IL-3, a cytokine that can induce up-regulation of ICOS-ligand in pDC, which in turn can mediate the differentiation of IL-10-producing regulatory T cells via ICOS ligation (Ogata et al., 2013). Furthermore, pDC have the capacity to regulate IL-10 production by B cells (Georg and Bekeredjian-Ding, 2012). Thus, taken together, pDC can have important effects in shaping the regulatory potential of lymphocytes.

Migration of pDC After development in the bone marrow, pDCs circulate through the body via the bloodstream, and enter secondary lymphoid tissues via high endothelial venules (HEV). Under steady state conditions, pDCs are found in the thymus, in secondary lymphoid tissues, and in rare numbers in peripheral tissues. After challenge via infection or inflammation, pDCs migrate and accumulate in inflamed tissues where they can secrete type I IFN and take up antigen. Activated pDC further migrate to LN for antigen presentation (Cella et al., 1999). pDC migration is facilitated through the expression of L-selectin and PSGL1, the ligands of E and P selectins expressed in HEV (Diacovo et al., 2005). pDC also express CXCR4 that is a receptor for CXCL12, a chemokine that is expressed by cells adjacent to HEVs and is displayed on the HEV lumen (Okada et al., 2002). Deficiency of L-selectin or the CXCR4 signaling molecule DOCK2 results in reduced numbers of pDC in secondary lymphoid organs (Sozzani et al., 2010). Additionally, CCR7, a receptor for CCL19 and CCL21 chemokines, is expressed on both resting and activated pDC and contributes to LN homing (Seth et al., 2011). Under reactive conditions, CCR5 also promotes pDC migration to LN (Diacovo et  al., 2005). Furthermore, pDC accumulation in peripheral tissue is observed in pathological situations such as autoimmunity, cancer, and allergy. This accumulation can

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be mediated by ChemR23/chemerin axis and the abovementioned chemokines (Sozzani et al., 2010). Upon viral infection or systemic exposure to recombinant IFNβ, pDC can accumulate within LN, and their residence time may be influenced by sphingosine-1-phosphate (S1P) receptor 4 signaling (Gao et al., 2009). Lastly, the chemokine receptor CCR9 is also expressed by pDC and facilitates migration within the small intestine (Wendland et al., 2007).

Plasmacytoid DC and multiple sclerosis Dendritic cells and neuroinflammation MS is an inflammatory demyelinating disease that affects the central nervous system (CNS). It is hypothesized that MS is initiated by DC-induced activation of myelin reactive T cells in the periphery. Activated CD4+ T cells subsequently migrate into the CNS, crossing the blood-brain barrier (BBB) and accumulating in the perivascular spaces where they are reactivated by resident APC such as DC. DC derived from inflammatory monocytes that secrete TNFα and iNOS can also cross-prime CD8+ T cells within the CNS (Ji et al., 2013). This second CNS-intrinsic triggering event endows perivascular-resident activated T cells with the ability to enter the CNS parenchyma and the capacity to secrete proinflammatory cytokines such as IFNγ and IL17 (Domingues et al., 2010). These pro-inflammatory cytokines encourage the recruitment of other leukocytes and also cause direct damage to neural tissue (Sospedra and Martin, 2005). Because DC are involved in both the primary activation and in the subsequent re-activation and/or polarization of encephalogenic T cells, DC are key players in the pathogenesis of MS. Indeed, accumulation of both cDC and pDC in the CNS and in the cerebrospinal fluid (CSF) of MS patients has been reported (Pashenkov et al., 2001; Serafini et al., 2006). Studies using MS patient samples, as well as the animal model of MS, experimental autoimmune encephalomyelitis (EAE), have revealed emerging functions (both regulatory and pro-inflammatory) for both cDC and pDC in neuroinflammation (Zozulya et al., 2010; Yogev et al., 2012).

pDC phenotype and function are altered in MS patients In MS patients, pDC accumulate in the CSF, in white matter lesions, and in the leptomeninges (Pashenkov

et al., 2001; Lande et al., 2008), and pDC are increased in the CSF during MS exacerbations (Longhini et al., 2011). Enumeration of circulating pDC in the blood of relapsingremitting (RR) MS patients has shown conflicting results: while Sanna et  al. (2008) have reported a reduced proportion of circulating pDC, others have found no change in the percentage or numbers in the circulating BDCA-2+ CD123+ pDC compared to healthy controls (Lopez et  al., 2006; Lande et al., 2008). Irrespective of their frequency in the periphery, it has been found that pDC in the blood of MS patients may exhibit a dysfunctional phenotype. For example, one report analyzed pDC derived from the blood of clinically stable, untreated RRMS patients and found that they express lower levels of co-stimulatory molecules CD86 and 4-1BBL compared to healthy controls. Upon in vitro stimulation with IL-3 and human soluble CD40 ligand (sCD40L), the low expression of CD86 and 4-1BBL on MS pDC persisted along with a reduced expression level of CD40 and CD83. In an in vitro assay of allogeneic stimulation, pDC from the MS patients described above exhibited a reduced capacity to induce proliferation and IFNγ secretion compared with healthy donors (Stasiolek et al., 2006). Others have examined the ratio of pDC1/pDC2 in peripheral blood of stable and untreated RRMS patients, and interestingly, pDC from MS patients predominantly exhibit a pDC2 phenotype (Schwab et  al., 2010). When pDC were isolated from MS patients and co-cultured with allogeneic naïve T cells, a Th17 differentiation bias was observed (Schwab et al., 2010). Taken together, the accumulation of pDC in the CNS and the phenotypic and functional abnormalities of circulating pDC of MS patients suggest that pDC can play an important role in the pathogenesis of MS, possibly by controlling the aberrant immune responses in MS patients.

How does Type I IFN modulate neuroinflammation via pDC-dependent effects? As mentioned, pDC can have both activating and tolerogenic properties, and some of these properties are related to their capacity to secrete high amounts of type I IFN upon activation. On one hand, independent studies have revealed a defective production of type I IFN by pDC in MS patients. RRMS patients had significantly lower serum type I IFN activity compared to healthy donors, which was reduced even further as disease progressed (Feng et al., 2012). Single cell analysis and quantitative assessment have also revealed that peripheral pDC from RRMS patients stimulated in vitro with a TLR7 or TLR9 agonist secreted lower levels of IFNα than healthy controls

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340      G. Galicia and J.L. Gommerman: pDC and inflammation (Stasiolek et al., 2006; Bayas et al., 2009; Chiurchiu et al., 2013). On the other hand, a wealth of data has shown that IFNβ treatment has a therapeutic benefit in MS concomitant with changes in the pDC compartment: First, treatment with IFNβ in RRMS patients can increase the expression of CD123 in pDC, which, as mentioned, may have an impact on the generation of T-regulatory (Treg) cells (Lopez et al., 2006). Secondly, IFNβ therapy can differentially regulate the activation of pDCs themselves, normalizing the altered pDC1/pDC2 distribution in MS patients towards a more regulatory pDC1 phenotype. Therefore, a positive feedback loop may exist where pDC secrete type I IFN that in turn promotes their regulatory pDC1 phenotype, suppressing chronic inflammation (Schwab et al., 2010). Third, IFNβ therapy has been shown to enhance the secretion of IFNα by pDC of MS patients upon TLR7 or TLR9 activation, and was shown to specifically up-regulate the expression of TLR7 on pDC from MS patients (Aung et al., 2010; Derkow et al., 2013). Although it is unclear what consequence this would have on chronic inflammation during MS, nevertheless one potential ‘sideeffect’ of IFNβ therapy could be an enhanced ability to detect and respond to viral infections, providing indirect patient benefit. Indeed, epidemiological data suggest that protracted viral infections that last longer than 5  days can trigger clinical exacerbation in patients with RRMS (Andersen et al., 1993). Thus enhanced activation of pDC

Human

How does Type I IFN modulate neuroinflammation via effects on other immune cell types? In addition to modulating pDC function, IFNβ is able to regulate the function of other immune cells involved in the pathogenesis of MS [reviewed in Limmroth et  al. (2011)] (Figure 3). For instance, in MS the suppressive capacity of Treg cells is diminished compared to healthy individuals (Viglietta et al., 2004; Venken et al., 2006), potentially because of a reduced thymic output of naïve Tregs (Haas et al., 2007). IFNβ treatment has been shown to increase overall Treg capacity in RRMS patients by helping to restore the homeostatic balance within the Treg cell compartment, increasing the numbers of naïve Treg cells to levels observed in healthy controls (Korporal et al., 2008). The effect of IFNβ in effector T cells has also been studied. The addition of recombinant IFNβ to in vitro cultured naïve CD4+ T cells that were polarized towards a Th1 cytokine profile resulted in the co-production of IL-10 compared to non-treated cells. In contrast, CD4+ T cells that were polarized towards a Th17 cytokine profile treated with IFNβ exhibited a reduction in IL-17 expression

Mouse Treg Siglec-H

pDC Signal I + Signal II

by IFNβ therapy may help to decrease the frequency of MS attacks associated with viral infections (Aung et al., 2010; Balashov et al., 2010).

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Figure 3 Putative roles for pDC during neuroinflammation in MS and EAE. pDC can exert a regulatory role in MS by polarizing CD4+ T cell towards a regulatory cell phenotype that produces IL-10. IL-10 is an antiinflammatory cytokine that restrains the activation of cDC. Similarly, production of IFNβ by pDC can dampen inflammation within the CNS. IFNβ also has autocrine and paracrine functions regulating the activation of pDC themselves and favoring the differentiation of regulatory T cells. In mice, delivery of myelin antigen to pDC via Siglec-H results in the suppression of CD4+ T cells. Lastly, IFNβ can regulate the exit of activated T cells from the LN and can also diminish the production of IL-17.

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(Axtell et al., 2010). Furthermore, it was found that RRMS patients that were unresponsive to IFNβ treatment had higher IL-17F serum levels compared to patients who responded to IFNβ therapy. Non-responder RRMS patients exhibited faster disability progression, higher number of relapses, and required steroid interventions before and after IFNβ treatment compared with responder patients (Axtell et al., 2010). IFNβ can also modulate NK cell subpopulations of RRMS patients, with an expansion of CD56bright NK cells (Saraste et al., 2007). Remarkably, CD56bright NK cells can have immunoregulatory functions as they can produce anti-inflammatory cytokines, such as IL-10 (Cooper et  al., 2001; Li et  al., 2005). Lastly, IFNβ may also serve to dampen MS by sequestering pro-inflammatory leukocytes within LN and away from the CNS itself, as it has been observed that IFN-β treatment results in a significant accumulation of lymphocytes in the LN concomitant with a decrease in circulating lymphocytes (Shiow et al., 2006; Gao et al., 2009). Collectively these studies suggest that the capacity of pDC from MS patients to secrete type I IFN is dysregulated, and this may shape a pro-inflammatory environment that drives MS pathology. Moreover, MS pDC may have an impaired ability to interact and instruct other immune cells, and in particular to generate Treg cells. Therefore pDC not only can be targets of immunomodulatory therapies such as IFNβ, but also could be utilized as a potential therapeutic strategy to control autoimmunity of the CNS.

Plasmacytoid DC and experimental autoimmune encephaloymyelitis (EAE) EAE as a model for studying the role of pDC in MS The function of pDC has also been studied in the most common and widely use animal model of MS, known as EAE. EAE can be induced by active immunization with myelin peptides or protein in CFA, or by adoptive transfer of myelin reactive T cells into susceptible animals (Baxter, 2007). Induction of EAE causes progressive paralysis, demyelination, and inflammation of the CNS. Studies in EAE have revealed that pDC accumulate in the CNS (spinal cord and brain) of EAE mice, suggesting that pDC may also contribute to EAE in a similar manner as observed for MS in humans (Bailey et al., 2007; Galicia-Rosas et al., 2012).

SJL mice immunized with proteolipid protein (PLP) develop a relapsing-remitting EAE pathology. Using this model, we previously identified the precise dynamics of pDC accumulation within the CNS during EAE. Specifically, we showed that 2 days following the entry of T cells into the CNS, pDC subsequently enter the CNS concordant with a reduction of pDC in the blood. Interestingly, if we treat mice with an S1P receptor 1 (S1P1R)-agonist (AUY954) at the time of pDC migration into the CNS, we found that AUY954 treatment reduces lymphocyte accumulation in the CNS and dampens EAE symptoms, but does not affect the accumulation of pDC at the site of inflammation [perhaps because pDC express more S1P4 receptor than S1P1 receptor, the target of AUY954 (Gao et  al., 2009)]. However, the therapeutic efficacy of AUY954 in reducing the clinical severity of EAE was reliant on the presence of pDC, and if pDC were pre-depleted, administration of AUY954 at a time point when we would normally observe pDC migration to the CNS was no longer effective at reducing EAE symptoms (Galicia-Rosas et al., 2012) (Figure 4). Indeed, depending on the stage of the disease course, other studies have also shown that pDC exert a regulatory role in the progression of EAE. Therefore, studies on when pDC exert an effect on EAE pathogenesis have been performed. Depletion of pDC during the effector phase of the disease results in exacerbated pathology of EAE (BaileyBucktrout et al., 2008). However, if pDC are depleted at the time of immunization, EAE is attenuated (Isaksson et al., 2009). Thus, depending on when pDC are removed, differing outcomes on EAE disease trajectory may be observed.

How do pDC reduce neuroinflammation during EAE? Different mechanisms may account for the anti-inflammatory function of pDC in EAE. We have found that in the absence of pDC, during the symptomatic phase of the disease, an increased accumulation of T cells that produce IL17 and IFNγ, either alone or in combination, is observed in the CNS (Galicia-Rosas et al., 2012). Others have linked pDC in the CNS with an accumulation of Treg cells and depletion of pDC results in impaired T-regulatory cell frequency (Bailey-Bucktrout et al., 2008). In addition, pDC may play an important role in tolerance induction (Goubier et  al., 2008): using mice that selectively lack MHCII expression in pDC, increased auto-reactive CD4+ T cell responses were observed. In this context, pDC were required for the expansion of thymus-derived Treg cells that are required to dampen CNS autoimmunity (Irla et  al., 2010). Siglec-H antibody-mediated delivery of the

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342      G. Galicia and J.L. Gommerman: pDC and inflammation Lymph node

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B cell

Figure 4 Effects of trafficking inhibitors on neuroinflammation. An S1P1R-agonist (AUY954) given at the time of pDC migration into the CNS reduces lymphocyte accumulation in the CNS but is permissive for pDC migration to the CNS concomitant with a reduction in EAE severity. The therapeutic efficacy of AUY954 was shown to depend on the presence of pDC.

CNS-derived antigen MOG to Siglec-H expressing pDC also reduces the severity of EAE induced by active immunization. This correlated with hypo-responsiveness of MOG specific CD4+ T cells and poor secretion of IFNγ and IL17 (Loschko et al., 2011). Whether via tolerance induction or through the actions of Treg cells, it remains unclear which pDC-specific properties contribute to their immunomodulatory potential in the context of chronic inflammation. pDCgenerated type I IFN may play a critical role in disease modulation, as mice deficient in IFNβ (Teige et al., 2003) or IFNAR exhibit more severe EAE (Prinz et al., 2008), and type I IFN can dramatically ameliorate the clinical outcomes of EAE (Kalinke and Prinz, 2012). Moreover, when myelin-specific T cells were treated with type I IFN and adoptively transferred into naïve recipients, they induced milder EAE compared with EAE mice that received an adoptive transfer of non-treated cells (Zhang et al., 2011). However, the therapeutic effect of IFNβ in EAE seems to depend on the cytokine signature of the encephalitogenic T cells that are used to induce EAE. IFNβ effectively reduced EAE severity induced by Th1 cells, but worsened symptoms in EAE induced by Th17 cells, and IFNβ treatment resulted in more severe EAE in INFγ deficient mice (Axtell et al., 2010; Naves et al., 2013). Tissue-specific type I IFN knock-outs or complex cell transfer experiments may resolve whether the capacity to produce type I IFN is related to the immunoregulatory effects of pDC in EAE.

Conclusions and perspectives pDC have a very relevant role in the pathogenesis of MS and an intervention to regulate their function may be desirable in order to restore tolerance against CNS antigens. For example, a novel proposed treatment of RRMS, Cladribine, seems to diminish the number of T and B lymphocytes but spares pDC numbers, allowing them to accumulate in blood, although Cladribine remains to be FDA approved pending safety and risk-benefit analysis. Whether these pDC accumulate in the CNS has not been determined (Mitosek-Szewczyk et al., 2013). In addition, a S1P1-specific functional agonist such as AUY-945 that may selectively effect lymphocyte trafficking will also be useful to study the effect of the accumulation of circulating pDC in MS pathology (Galicia-Rosas et  al., 2012). However, there are several questions that still need to be answered about the mechanism(s) that pDC use to negatively regulate autoimmune inflammation of the CNS (type I IFNdependent vs. independent), and the relevant timing of these anti-inflammatory effects with respect to a complex disease trajectory. As we are now in a position to dissect unique functions of different DC subsets using tissue-specific knock-outs of the various transcription factors that dictate DC lineage specification, a further understanding of which type of DC and/or pDC dampen or promote neuroinflammation is on the horizon. As such, discerning

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G. Galicia and J.L. Gommerman: pDC and inflammation      343

at what point during chronic disease these various DC subsets exert regulatory versus pro-inflammatory functions will inform treatment strategies.

post-doctoral fellowship from the MS Society of Canada to G.G. The authors would like to acknowledge the helpful comments from Dennis Ng.

Acknowledgments: This work was funded by an operating grant from the MS Society of Canada to J.L.G and a

Received June 27, 2013; accepted October 29, 2013; previously published online October 30, 2013

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Georgina Galicia completed her PhD at Catholic University of Leuven, Belgium where she studied experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Dr. Galicia then moved to Toronto to continue her studies in the Department of Immunology at the University of Toronto. Upon joining the Gommerman lab, Dr. Galicia won a post-doctroral fellowship from the MS Society of Canada. Currently Dr. Galicia investigates the immunopathology of EAE, with particular focus on cells of immune cells such as plasmacytoid dendritic cells (pDC) and B cells.

Jen Gommerman received her PhD in 1998 from the Department of Immunology at the University of Toronto. She went on to do a post-doctoral fellowship with Dr. Michael Carroll at Harvard Medical School and subsequently joined Biogen Idec as a staff scientist under the direction of Dr. Jeffrey Browning. Dr. Gommerman returned to Toronto in 2003 to establish her laboratory in the Department of Immunology at the University of Toronto. She has received funding from the Canadian Institutes of Health Research, the MS Society of Canada and the Kidney Foundation to study the immune system in the context of health and disease. Her funding from the MS Society has allowed her to examine the role of different immune cell types in the immunopathology of EAE.

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