Altered innate immune response of plasmacytoid dendritic cells in multiple sclerosis

Authors

  • A. Bayas,

    Corresponding author
    1. Department of Neurology, University of Würzburg, Würzburg,
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    • Present address: Department of Neurology, Klinikum Augsburg, Stenglinstr. 2, 86156 Augsburg, Germany.

    • These authors contributed equally to the work.

  • M. Stasiolek,

    1. Department of Neurology, Medical University of Lodz, Lodz, Poland
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    • Present address: Department of Neurology, Ruhr-University Bochum, 44791 Bochum, Germany.

    • These authors contributed equally to the work.

  • N. Kruse,

    1. Institute for MS-Research, Medical Faculty of the University and Gemeinnuetzige Hertie-Stiftung, Waldweg, Goettingen, Germany, and
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    • These authors contributed equally to the work.

  • K. V. Toyka,

    1. Department of Neurology, University of Würzburg, Würzburg,
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  • K. Selmaj,

    1. Department of Neurology, Medical University of Lodz, Lodz, Poland
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    • These senior authors contributed equally to the work.

  • R. Gold

    1. Institute for MS-Research, Medical Faculty of the University and Gemeinnuetzige Hertie-Stiftung, Waldweg, Goettingen, Germany, and
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    • Present address: Department of Neurology, Ruhr-University Bochum, 44791 Bochum, Germany.

    • These senior authors contributed equally to the work.

    • Senior author's e-mail: ralf.gold@ruhr-uni-bochum.de


Summary

Plasmacytoid dendritic cells (pDCs) are of crucial importance in immune regulation and response to microbial factors. In multiple sclerosis (MS), pDCs from peripheral blood showed an immature phenotype, but its role in susceptibility to MS is not determined. Because infectious diseases are established triggers of exacerbations in MS, in this study we have characterized the expression of Toll-like receptors (TLR) and the maturation and functional properties of peripheral blood pDCs from clinically stable, untreated MS patients in response to signals of innate immunity. After stimulation of TLR-9, interferon (IFN)-α production by pDCs was significantly lower in MS (n = 12) compared to healthy controls (n = 9). In an allogenic two-step co-culture assay we found an impaired effect of TLR-9 stimulation on IFN-γ expression of autologous naive T cells in MS patients (n = 4). In peripheral blood mononuclear cells, TLR-9 stimulation with type A CpG ODN resulted in a higher expression of TLR-1, -2, -4, -5 and -8 in MS patients (n = 7) compared with healthy controls (n = 11). These findings suggest an altered innate immune response to microbial stimuli in MS patients and may help understanding of why common infectious agents trigger MS attacks.

Introduction

Multiple sclerosis (MS) is a chronic and putatively immune-mediated inflammatory disease of the central nervous system (CNS). Autoreactive T cells, which are normally kept in check by tolerance mechanisms, are assumed to initiate and perpetuate an autoimmune reaction that may result eventually in severe CNS damage [1]. Several lines of evidence suggest that microbial factors might activate autoreactive T cells and contribute to development of autoimmune reactions [2–4].

There are at least two main subsets of peripheral blood dendritic cells (DCs) in man: myeloid (mDCs) and plasmacytoid (pDCs) [5,6]. pDCs exert functions as a part of the innate immune response by releasing large amounts of type I interferons (IFN). They are also important in the adaptive arm of the immune system by differentiation into DCs that can present antigens to T cells [7]. In the adaptive arm of the immune response, pDCs possess a pronounced ability to prime both T helper type 1 (Th1) [8] and Th2 cells [9]. Most importantly, pDCs were also shown to induce regulatory T cells [10–12].

Recently we have shown that in MS pDCs have an impaired maturation and a potentially relevant alteration in regulatory function when compared to healthy controls. The ex vivo expression of CD86 and 4-1BBL was significantly lower on pDCs from MS patients than from controls. When stimulated with interleukin (IL-3) and CD40L, pDCs of MS patients showed inefficient maturation, as demonstrated by a significantly lower or at least delayed up-regulation of CD86, 4-1BBL, CD40 and CD83 expression. In addition, pDCs in controls, but not in MS patients, facilitated the generation of forkhead box P3 (FoxP3)-positive regulatory T cells. Moreover, in MS but not in controls, pDCs failed to up-regulate proliferative responses and IFN-γ secretion of autologous peripheral blood mononuclear cells (PBMC) in a co-culture system [13].

Ligation of the ‘pattern recognition receptors’ has a particularly strong influence on the maturation and function of DCs [14,15]. Therefore, the family of Toll-like receptors (TLRs) has been shown to play the crucial role in developing and directing the response of DCs to microbial invasion [16]. Human pDCs express only TLR-7, -8 and -9. Recognition of microbial factors by pDCs is mediated primarily by TLR-7 and TLR-9. TLR-9 is of particular interest, because it is expressed exclusively on pDCs and B cells and its ligands, such as unmethylated cytosine–phosphate–guanosine oligodeoxynucleotides (CpG ODN), act as very effective immediate activators of these cells [17]. Three types of CpG ODN have been described [18]. Type A CpG ODN is a strong IFN-α inductor, type B CpG ODN leads to pDC differentiation and type C CpG ODN results in IFN-α induction and pDC maturation. Recently it has been shown that the endosomal CpG ODN location is the primary determinant of TLR-9 signalling [19]. In their experiments CpG type B, depending on its localization, influenced maturation or IFN-α expression by pDCs.

In the animal model of experimental autoimmune encephalomyelitis, disease expression is associated with a profound and sustained transcriptional activation of the gene encoding Toll-like receptor 2 in the mouse CNS [20]. In the study by Herrmann et al.[21], infection with Streptococcus pneumoniae increased the severity of autoimmune encephalomyelitis. Disease aggravation was mediated by engagement of TLR-2 in vivo, as the absence of TLR-2 in TLR-2-deficient mice prevented disease aggravation by S. pneumoniae infection, indicating the critical involvement of TLR-2. Kerfoot et al.[22] showed that pertussis toxin (PTX)-induced leucocyte recruitment is dependent upon TLR-4 and that the disease-inducing mechanisms initiated by PTX are also dependent, at least partly, on TLR-4. Thus the expression of different TLRs seems to be involved in autoimmunity.

As a first hint to a differential role of pDCs in MS and healthy individuals we have demonstrated that, in MS, stimulation of TLR-9 resulted in a significantly lower IFN-α secretion than in controls, indicating an altered response to signals of innate immunity [13].

In the following experiments we extended our previous study by specific investigation of the expression of TLRs and the response of their stimulation on pDCs in MS patients.

Materials and methods

Patients and controls

In our study we aimed at determining the immune state of MS patients without the influence of clinically overt acute exacerbations and of immunomodulatory treatments. Therefore, only patients with a clinically stable disease course and with relapsing–remitting MS were eligible. Patients had to be free of immunomodulatory treatment during the study period and for at least the preceding 6 months. Patients having suffered from a relapse and/or receiving glucocorticosteroid treatment for at least 90 days before cell collection were excluded from the study. This shorter washout interval compared to the exclusion criteria for immunomodulatory treatment was chosen for the assumed shorter immunological effects of glucocorticosteroids compared to immunomodulatory treatment. For the series of PBMC experiments, seven patients (six female, one male) with a mean age of 39 [± 4 standard deviation (s.d.)] years and a median expanded disability status scale (EDSS) score of 2·0 (range 1–2·5) were included, and 11 healthy subjects (eight female, three male) with a mean age of 32 (± 6 s.d.) years, without any kind of permanent treatment, served as a control group (all donors from Germany). For the series of experiments using material from leukaphereses, 12 patients (seven male, five female patients) with a mean age of 37 (± 7 s.d.) years and a median EDSS score of 2·25 (range 0–7) were included, and nine healthy subjects (two male, seven female) with a mean age of 32 (± 6 s.d.) years served as control. Most samples were derived from German donors; three MS patients and four controls came from Poland. Blood withdrawal and leukapheresis were approved by the local Ethics Committees at both institutions (Würzburg, Lodz), and all patients gave written informed consent. The allogenic proliferation assays were performed on four MS patients [mean age 35 ± 9 s.d. years; mean EDSS 1·75 (range 0–3·5); two female and two male] and three healthy controls [mean age 31 ± 7 (s.d.) years; two female, one male] at the department in Würzburg.

In all experiments patients and controls were of Caucasian origin.

Leukapheresis

For pDC sorting experiments large numbers of PBMC had to be obtained, which could be achieved only by performing leukapheresis. All leukaphereses were performed by use of cell separators, as described [13], at the Department for Transfusion Medicine of the University Hospital in Würzburg, and at the Blood Donation Center, Lodz. PBMC suspensions were used immediately for the DC isolation procedures and the culture experiments described below. None of the patients and controls suffered from major adverse events.

Fluorescence-activated cell sorting (FACS) analysis

Immune cells (pDCs, T cells) from MS patients and healthy controls were assessed by three- or two-colour flow cytometry using a FACSCalibur® cytometer and CELLQuest® software (BD Biosciences, San Jose, CA, USA). Monoclonal antibodies (mAb) specific for BDCA antigens were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). All remaining mAbs and appropriate isotype controls were purchased from BD Biosciences Pharmingen (San Jose, CA, USA). The BDCA2+ plasmacytoid DC subtype was recognized by staining with fluorescein isothiocyanate (FITC)-conjugated antibodies specific for BDCA2 [AC144, mouse immunoglobulin (IgG)1]. pDC were counterstained with phycoerythrin (PE)-conjugated mAbs: anti-BDCA4 (Ad 5-17F6, mouse IgG1), anti-human leucocyte antigen D-related (HLA-DR) [46-6(L243), mouse IgG2a], anti-CD11c (B-ly6, mouse IgG1), anti-CD40 (5C3, mouse IgG1), anti-CD80 (L307·4, mouse IgG1), anti-CD83 (HB15e, mouse IgG1), anti-CD86 [2331(FUN-1), mouse IgG1], anti-CD123 (9F5, mouse IgG1) and anti-CD137L (C65-485, mouse IgG1). T cell populations were analysed with the following mAbs: anti-CD3-PE (UCHT1, mouse IgG1), anti-CD4-FITC, anti-CD4-PE (RPA T4, mouse IgG1), anti-CD45RA-FITC (HI100, mouse IgG2b) and anti-CD25-PE (M-A251, mouse IgG1).

Isolation and culture of plasmacytoid DC and naive T cells

pDCs were isolated from the PBMC suspensions obtained by leukapheresis of MS patients and healthy subjects. PBMCs were isolated by Histopaque 1077 gradient centrifugation (30 min, 300 g, 20°C) (Sigma-Aldrich, St Louis, MO, USA); 2 × 109 cells were then sorted with a BDCA4 isolation kit (Miltenyi Biotec) in the magnetic field of a MidiMACS® sorter (Miltenyi Biotec). The pDC containing fractions had, routinely, more than 95% of BDCA2+ CD123+ pDCs.

For maturation and IFN-α secretion analysis, isolated pDCs were plated for 72 h on 48-well culture plates (Nunc, Roskilde, Denmark) at a concentration of 5 × 105/ml in a culture medium containing RPMI-1640, streptomycin 100 µg/ml, penicillin 100 U/ml, 2 mM L-glutamine (Gibco, Life Technologies, Vienna, Austria) and 10% heat-inactivated fetal calf serum (FCS; Boehringer Mannheim, Mannhein, Germany). pDCs were supplemented with 0·5 µM of type A unmethylated CpG ODN (CpG 2216, 20-mer), type B CpG 2006 (24-mer; both from Tib Molbiol, Berlin, Germany) as a stimulator of TLR-9 or 0·5 mM loxoribine (7-allyl-7,8-dihydro-8-oxo-guanosine; Sigma-Aldrich) as TLR-7 stimulator. After 24 and 72 h of culture, pDCs were harvested for flow cytometry. Culture supernatants were collected for measurement of IFN-α.

Untouched naive CD4+ CD45RA+ T cells were isolated by negative sorting using a CD4+ T cell isolation kit II (indirect magnetic labelling, Miltenyi Biotec) and subsequent depletion of CD45RO+ T cells by CD45RO MicroBeads (Miltenyi Biotec). The content of pDCs in the depleted fraction was routinely less than 0·1%, the purity of naive CD4+ CD45RA+ T cells was routinely > 98%.

Allogenic proliferation assay

Naive CD4+ T cells (2·5 × 105/well) were preincubated with freshly isolated autologous pDCs (2·5 × 104/well) on 48-well plates in culture medium supplemented with 1 µM CpG ODN 2006. After 7 days, culture supernatants were collected and T cells were harvested, washed twice, counted and co-cultured (104/well) on 96-well plates for 5 days with autologous naive CD4+ T cells (105/well) as responders stimulated with 105 allogenic irradiated PBMCs, always derived from the same healthy donor.

CpG ODN stimulation of PBMCs

PBMCs were isolated from peripheral venous blood samples by centrifugation on a discontinuous density gradient (Histopaque 1077; Sigma-Aldrich).

From MS patients and healthy controls, 2 × 106 PBMC/ml were cultured on round-bottomed 96-well culture plates for 72 h in culture medium or culture medium supplemented with 0·5 or 1 µM of CpG 2216. After 24, 48 and 72 h cells were collected for mRNA isolation, and supernatants were collected and stored at −20°C.

Isolation of RNAs and reverse transcription

RNA was purified from cultured cells as described previously [23] using the Qiagen RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany), with slight modifications. Purified RNA was eluted with 50 µl RNase-free water. For reverse transcription, 12 µl RNA solution was incubated with 1 µl oligodeoxythymidylic acid [oligo(dT)] (500 µg/ml) (Amersham Biosciences, Freiburg, Germany) and 1 µl 10 mM deoxynucleotide triphosphate (dNTPs) (Invitrogen, Karlsruhe, Germany) at 65°C for 5 min and chilled on ice. After the addition of 1 µl RNase-free water, 4 µl of 5× reverse transcriptase buffer and 1 µl of Superscript II reverse transcriptase (200 U/µl) (both from Invitrogen), the samples were incubated for 50 min at 40°C. Reverse transcriptase was denatured by incubating samples for 15 min at 75°C.

Polymerase chain reaction (PCR)

All PCR reactions were performed on a 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA) after appropriate dilution of cDNAs. Oligonucleotides were purchased as TaqMan gene expression assays and used in combination with TaqMan universal PCR master mix (both from Applied Biosystems) or platinum PCR SuperMix (Invitrogen), according to the manufacturer's instructions. The following TaqMan gene expression assays were used: TLR-1, Hs00413978_m1; TLR-2, Hs00152932_m1; TLR-3, Hs00152933_m1; TLR-4, Hs00152939_m1; TLR-5, Hs00152825_m1; TLR-6: Hs00271977_s1; TLR-7: Hs00152971_m1; TLR-8, Hs00152972_m1; TLR-9, Hs00152973_m1; TLR-10, Hs00374069_q1; and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), Hs99999905_m1. Oligonucleotides for beta-actin and RNA polymerase II PCR were from Kruse et al.[24] and Radonic et al.[25], respectively.

Cytokine secretion

Cell culture supernatants were collected, aliquoted and stored at −20°C. Immediately before the measurement, aliquots were brought to room temperature and analysed for cytokine content using a proper sandwich enzyme-linked immunosorbent assay (ELISA) kit (human IFN-α, Bender MedSystems, Vienna, Austria; IFN-γ, R&D Systems, Minneapolis, MN, USA) according to the manufacturers'protocols.

Statistical analysis

Cumulative FACS-data analysis, reverse transcription–polymerase chain reaction (RT–PCR) and cytokine measurements were performed in a blinded manner. The complete result-panels were divided further into appropriate experimental groups and subjected to statistical analysis.

Statistical analyses were performed using statgraphics plus version 5·0 software. All comparisons of group data were performed with the non-parametric Mann–Whitney U-test, with P = 0·05 as the level of significance.

Results

IFN-α secretion of pDCs in response to TLR stimulation

IFN-α secretion by pDCs in response to TLR stimulation was investigated by the use of loxoribine and CpG ODNs mimicking microbial stimulation of TLR-7 and TLR-9, respectively [17]. IFN-α secretion by PBMCs in healthy controls after stimulation with CpG 2216, a multimeric type A CpG ODN which is known to activate IFN-α secretion selectively by pDCs, was shown earlier to be twice as high compared to MS patients [13]. Here we analysed the effects of CpG 2216 and another TLR-9 ligand CpG 2006, a type B CpG ODN known to activate pDCs and to trigger B cells to proliferate and secrete [18], on purified pDCs. Furthermore, we stimulated cells with loxoribine, a TLR-7 ligand known for its anti-viral and anti-tumour activity [26]. In MS patients (n = 12), IFN-α production by pDCs was lower after all TLR ligand application when compared to healthy controls (n = 9). The difference in IFN-α secretion between MS and controls reached significance for CpG-ODN 2006 for days 1 and 3 taken together (P = 0·02; Fig. 1).

Figure 1.

Interferon (IFN)-α production in plasmacytoid dendritic cells (pDCs) after Toll-like receptor (TLR)-9 and -7 stimulation. Effects of TLR stimulation by (a) type B cytosine–phosphate–guanosine oligodeoxynucleotides (CpG ODN) 2006, (b) loxoribine and (c) type A CpG ODN 2216 on IFN-α expression in pDC culture over 3 days (multiple sclerosis n = 12, healthy controls n = 9). Difference is significant for CpG-ODN 2006 when data are pooled for day 1 (d1) and 3 (d3) (P = 0·02). Shown are mean and standard error of the mean.

pDC maturation in response to TLR stimulation

In parallel experiments the effects of CpG ODN 2006, CpG ODN 2216 and loxoribine on pDC maturation were investigated. There was no significant effect on the expression of CD40, CD80, CD83, CD86 and 4-1BBL after 24 and 72 h of culture comparing MS patients and controls as measured by flow cytometry (Fig. 2).

Figure 2.

Figure 2.

Plasmacytoid dendritic cells (pDCs) maturation in response to Toll-like receptor (TLR) stimulation. Effects on the expression of CD40, CD80, CD83, CD86 and 4-1BBL after 24 and 72 h of culture comparing multiple sclerosis patients and controls as measured by flow cytometry after stimulation with (a) cytosine–phosphate–guanosine oligodeoxynucleotides (CpG ODN) 2006, (b) loxoribine and (c) CpG ODN 2216.

Figure 2.

Figure 2.

Plasmacytoid dendritic cells (pDCs) maturation in response to Toll-like receptor (TLR) stimulation. Effects on the expression of CD40, CD80, CD83, CD86 and 4-1BBL after 24 and 72 h of culture comparing multiple sclerosis patients and controls as measured by flow cytometry after stimulation with (a) cytosine–phosphate–guanosine oligodeoxynucleotides (CpG ODN) 2006, (b) loxoribine and (c) CpG ODN 2216.

Figure 2.

Figure 2.

Plasmacytoid dendritic cells (pDCs) maturation in response to Toll-like receptor (TLR) stimulation. Effects on the expression of CD40, CD80, CD83, CD86 and 4-1BBL after 24 and 72 h of culture comparing multiple sclerosis patients and controls as measured by flow cytometry after stimulation with (a) cytosine–phosphate–guanosine oligodeoxynucleotides (CpG ODN) 2006, (b) loxoribine and (c) CpG ODN 2216.

Altered regulatory function of pDCs stimulated by TLR-9

In order to assess the influence of TLR-9 signalling on pDC regulatory function in MS patients, we investigated the ability of pDCs stimulated by CpG ODN 2006 to prime naive CD4+ T cells to secrete IFN-γ. After 7 days of preincubation of naive T cells with CpG ODN and freshly isolated pDCs, T cells were used to modulate cytokine secretion of autologous CD4+ CD45RA+ naive T cells stimulated with irradiated allogenic PBMCs. After 7 days IFN-γ concentration in supernatants of co-cultures of pDCs and T cells from MS patients and controls was not significantly different (data not shown). In the subsequent allogenic co-culture at day 5, MS patients and controls showed a similar IFN-γ secretion without adding responder cells. However, in healthy controls naive T cells preincubated with autologous pDCs induced significantly higher IFN-γ secretion in the allogenic co-culture assay with responders. In contrast, in MS patients no significant difference was observed (Fig. 3). Thus, we have seen a clear difference between MS and control in relation to modulate allogenic responses by T cells pre-exposed to pDCs.

Figure 3.

Immunoregulatory effect of plasmacytoid dendritic cells (pDCs) in allogenic co-culture assay. In healthy controls (n = 3, black bars) pDCs co-cultured with autologous naive T cells and type B cytosine–phosphate–guanosine oligodeoxynucleotides (CpG ODN) 2006 induced significantly higher interferon-gamma expression in the subsequent allogenic co-culture assay with autologous naive T cells as responders than in those without responders. In multiple sclerosis patients (n = 4, grey bars) no significant difference could be observed. Shown are means and standard error of the mean (*P < 0·05).

CD4+ CD25+ cells induction by autologous pDCs

Our previous results [13] and the current finding on the decreased allogenic reaction might imply the role of regulatory T cells induced with pDCs. Therefore, we have assessed the induction of CD4+ CD25+ T cells in co-culture of naive CD4+ CD45RA+ T cells with autologous pDCs stimulated by type B CpG ODN. After 7 days of co-culture the percentage of CD4+ CD25+ regulatory T cells was determined by flow cytometry. The number of CD4+ CD25+ T cells increased in naive T-cells co-cultures with pDCs and CpG ODN 2006, derived from both MS patients and controls, compared to naive T cells alone, but no significant differences between MS patients and controls were observed (Fig. 4).

Figure 4.

CD4+ CD25+ T cells after co-culture of T cells with autologous plasmacytoid dendritic cells (pDCs) stimulated by type B cytosine–phosphate–guanosine oligodeoxynucleotides (CpG ODN). After 7 days of culture the number of CD4+ CD25+ T cells increased in naive T cells co-cultures with pDCs and CpG ODN 2006 derived both from multiple sclerosis (MS) patients and controls compared to naive T cells alone, but no significant differences between MS patients and controls were observed. Shown are means and standard error of the mean.

Increased TLR expression in MS after TLR-9 stimulation

Because the involvement of different TLRs is known in autoimmune processes, and TLR-4 has been shown to be higher-expressed in MS CNS tissues [27], we assessed the influence of TLR-9 stimulation on expression of TLR-1–10 by quantifying TLR expression in PBMCs over 3 days of culture with CpG ODN 2216 (0·5 and 1 µM) by RT–PCR. In PBMCs from MS patients stimulated with CpG-ODN 2216, TLR expression was significantly higher at day 1 for TLR-1, -2, -4, -5 and -8 after CpG-ODN 2216 stimulation than in controls (Fig. 5). At days 2 and 3 no significant differences could be observed.

Figure 5.

Toll-like receptor (TLR) expression after type A cytosine–phosphate–guanosine oligodeoxynucleotides (CpG ODN) stimulation. Stimulation of TLR-9 by type A CpG ODN 2216 in peripheral blood mononuclear cells resulted in a significantly higher mRNA expression of TLR-1, -2, -4, -5 and -8 in multiple sclerosis patients (n = 7) than in healthy controls (n = 11) after 24 h. Shown are means and standard error of the mean (*P < 0·05; **P < 0·01).

Discussion

The main finding of this study is that in MS pDCs have a clearly altered response to agents mimicking microbial infections, i.e. in innate immunity, when compared to healthy controls. This was observed at the level of IFN-α secretion by pDC after TLR-9 stimulation, priming of naive T cells for allogenic reactivity and expression of other TLRs.

The lower capacity of MS patients to secrete IFN-α upon TLR-9 stimulation strengthens our previous findings of functional impairment of these cells in MS [13]. It has been suggested that the secretion of IFN-α by pDCs shapes the immune response by influencing several cell types with regulatory properties, including mDCs and natural killer (NK) cells [28–30]. In turn, these cells and their products control at least partially the maturation process of pDCs and thus influence pDC function in adaptive immunity [31]. Type I interferons can be both inhibitors and effectors of autoimmune disease. Meyers et al.[32] investigated how pDC-derived type I IFN might regulate Th cells in systemic lupus erythematosus (SLE). The role of CpG-A/TLR9-induced type I IFN in regulating PBMC was determined by blocking with a virus-derived soluble type I IFN receptor. They observed that pretreatment with either recombinant human (rh)rhIFN-α/β or CpG-A inhibited PBMC secretion of superantigen-induced IFN-γ and IL-17, and CD40L induced IL-12p70 and IL-23. B18R, a virus-derived soluble type I IFN receptor, prevented these effects. It was concluded that CpG-A-induced type I IFN inhibits IL-12p70-dependent PBMC IFN-γ secretion by enhancing IL-10. Thus, as observed in this study, the lack of appropriate IFN-α secretion after TLR-9 stimulation might contribute to the inefficient inhibition of inflammatory response and might enhance the proinflammatory environment in the pathogenesis of MS.

CpG ODN 2006, CpG ODN 2216 and loxoribine had no significant effect on the expression of CD40, CD80, CD83, CD86 and 4-1BBL comparing MS patients and controls as measured by flow cytometry. As we have shown already in our previous study [13], pDCs of MS patients show inefficient maturation, as demonstrated by significantly lower or delayed up-regulation of CD86, 4-1BBL, CD40 and CD83 when stimulated with classical pDCs maturation factors: IL-3 and CD40L. Thus, dependent upon the type of stimulation, there seem to be differences in the expression of co-stimulatory molecules. These findings might indicate that the different expression of maturation markers might occur in the adaptive rather than in the innate arm of immunity.

Moseman et al.[12] observed that type B CpG ODN promoted pDCs to prime allogenic naive CD4+/– CD25 T cells to differentiate into CD4+/- CD25+/– regulatory T (Treg) cells. The CD4+/– CD25+/– T cells induced by CpG ODN-activated pDCs expressed FoxP3 and produced IL-10, transforming growth factor (TGF)-β, IFN-γ and IL-6, but low IL-2 and IL-4, a typical Treg cell type cytokine profile. In the allogenic co-culture assay used in our study we observed an altered function of pDCs stimulated with type B CpG ODN in MS with regard to their capacity to drive naive T cells to produce IFN-γ. However, we did not detect a difference in the occurrence of CD4+ CD25+ Treg cells in the first step of the co-culture assay. Thus, the reduced IFN-γ induction in the second step of the allogenic stimulation in MS patients might indicate the reduced capacity of other immunoregulatory circuits, possibly also affecting the maintenance of immunological tolerance.

Karni et al.[33] investigated the polarization effect of DCs from MS patients on cord blood-derived naive T cells in an allogenic mixed lymphocyte reaction (MLR) assay. In their study, CD11+ DCs from relapsing–remitting MS induced higher levels of Th1 (IFN-γ, TNF-α) and Th2 (IL-4, IL-13) cytokines than did controls. We have performed as yet unpublished experiments (A. B. and M. S.) with autologous naive T cells co-cultured with mDCs or pDCs with or without CD40L for 5 days, followed by 48 h stimulation with IL-2 and restimulation with CD3 and CD28 for 24 h. In co-cultures from MS patients with mDCs or pDCs, we observed a trend for a higher IFN-γ concentration than in healthy controls after the total culture period, with no statistical significance between the two groups (data not shown). The discrepancy between our data and the study by Karni et al.[33] might be explained by the autologous co-culture condition, in contrast to the allogenic assay used by these investigators.

In PBMCs, type A CpG ODN stimulation resulted in a significantly higher expression of TLR-1, -2, -4, -5 and -8 in MS patients than in controls.

The up-regulation of TLR expression upon stimulation with CpG ODN is consistent with reports describing cross-regulation among TLR family members, both with regard to functional properties as well as a level of expression [34,35]. The exact mechanisms underlying the mutual influence of various TLRs remain not fully understood. However, it has been shown recently that the stimulation of TLR-9 with CpG ODN results in a time-dependent (30 min–72 h) activation of multiple regulatory pathways leading in consequence to up- or down-regulation of numerous genes – among them cytokines and TLRs. Importantly, TLR-9 stimulation in those experiments induced a counter-regulatory mechanism involving a number of suppressors actively reversing the stimulatory effects of CpG ODNs [36]. Those results and other studies describing inhibitory circuits in TLR-9 signalling [37,38] may suggest the insufficient activity of suppressive factors as a possible mechanism of overexpression of TLRs in response to CpG ODN stimulation in MS patients. However, that concept needs to be investigated further. Because the secretion of IFN-α by pDCs in response to CpG ODN, at least under specific pathological conditions associated with human immunodeficiency virus (HIV) infection, seems to be regulated separately from proinflammatory factors as TNF-α or IL-6 [39], the impairment of IFN-α secretion after CpG ODN stimulation shown in our study in MS patients does not stand in contrast to the overexpression of TLR family members under similar experimental conditions.

TLR-2 and -4 have been shown to be involved in autoimmune disorders [20–22]. Bsibsi et al.[27] investigated in vivo expression of TLR-3 and TLR-4 of brain and spinal cord sections from both control and MS brains and found enhanced expression of either TLR in inflamed CNS tissues. In light of these results, the higher expression of TLRs as found in our study might contribute to a higher disease susceptibility, although we cannot ascribe this observation as yet to one of the currently proposed pathogenetic pathways [40,41].

Experiments providing insight in the biology of naturally occurring peripheral blood pDCs demand very high presorting numbers of PBMC used for pDCs purification via sorting. Such amounts of biological material could be derived only from leukaphereses, performed in all the individual cases. Due to this and the stringent inclusion criteria only few of numerous patients screened in our MS centres entered the study. In consequence, the small cohorts do not allow patients and controls to be matched confidently with regard to age and gender. The HLA genotype was not assessed in the study. Taking these limiting factors into consideration, we provide data showing that some important reactions in the innate immune system may be altered in patients with relapsing–remitting MS. TLR-9 stimulation in our study resulted in several types of responses in MS patients that are different from controls, and these new observations are in line with our previous findings of an impaired maturation and altered regulatory function of pDCs in MS [13].

The risk of developing MS is determined by both genetic and environmental factors. In particular, infections may play a role in triggering the onset of the disease or in inducing subsequent exacerbations. Resistance to infections is based primarily on the innate mechanisms of host defence and hence any alteration in the innate immune system might therefore provide clues to understanding the early steps of the pathogenetic process in MS. Because molecular tools have now become available which can modulate the innate response at various levels, a better insight into crucial steps of the disordered innate immune system in MS may evolve and this, in turn, may help eventually to develop new strategies for prevention and treatment of disease exacerbations.

Acknowledgements

This work was supported by the Polish–German Cooperation Grant in Neuroscience (PBZ-MIN-001/P05/26), a binational grant from the Bundesministerium für Bildung und Forschung, Germany (01GZ0303), the German National MS Society Research Fund (DMSG, Hannover, Germany), the University Research Fund at the University of Würzburg and a visiting scientist scholarship to M. S. from the SFB 581. We wish to thank Professor Hermann Wagner, Institute of Medical Microbiology, Immunology and Hygiene, Munich, Germany, for very helpful discussions on DC and TLR biology and for a gift of CpG ODN. We also thank Professor Heinz Wiendl for helpful discussion on the manuscript. The continuous support of Drs A. Opitz, Department for Transfusion Medicine of the University of Wuerzburg, Germany and E. Gauer, Blood Donation Center, Lodz, Poland, in performing leukapheresis is gratefully acknowledged. We thank our patients and the healthy volunteers for agreeing to participate in our study. The excellent technical assistance by A. Schmitt and A. Horn is gratefully acknowledged.

Disclosure

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