Signals involved in the commitment of Th17 differentiation are of substantial interest for our understanding of antimicrobial defense mechanisms and autoimmune disorders. Various ways in which myeloid dendritic cells modulate Th17 differentiation have been identified. However, although plasmacytoid dendritic cells (PDCs) are regarded as important players in antiviral/antimicrobial host defense and autoimmune diseases, a putative modulatory role of PDCs in Th17 differentiation has not yet been elucidated in detail. We demonstrated that PDCs are capable of promoting Th17 differentiation in response to TLR7 stimulation. Further, both the differentiation of Th17 cells from naive T cells and the amplification of Th17 effector functions of memory T cells are promoted by PDCs after TLR7 activation. Our data are of strong clinical relevance because TLR7 activation in PDCs might represent one of the missing links between innate and adaptive immune mechanisms and contribute to the amplification of Th17-driven autoimmune disorders as well as viral host defense.
Plasmacytoid dendritic cells (PDCs) sense the environment for nucleic acids with the help of pattern-recognition receptors in the context of viral and microbial infections as well as autoimmune diseases and, thereby, link innate and adaptive immune responses (1). TLR7 is localized in the endosomal compartment of PDCs and recognizes single-stranded RNA (ssRNA), genomic RNA, or synthetic nucleoside analogs (2, 3). Activation of PDCs via TLR7 results in enhanced IFN-α production, which is supposed to be a crucial protective mechanism to establish and sustain an efficient viral defense (1). However, TLR7-mediated IFN-α production by PDCs also represents an important disease-promoting mechanism that is believed to determine the severity and activity of autoimmune disorders, such as lupus erythematosus (LE) (1, 4). As a supposed disease model of LE, viral DNA or RNA and immune complexes composed of autoantibodies specific to self-DNA or -RNA are suspected to act as ligands for TLR7/TLR9 and, thereby, might promote IFN-α release and impair the disease (4, 5). However, the exact mechanistic basis for the induction and amplification of immune responses leading to autoimmunity by PDCs remains to be elucidated. Based on current knowledge, TLR7 fulfills opposing tasks in the immune system, with the unwanted activation of TLR7-bearing cells leading to autoreactive immune responses as well as protective functions represented by antiviral and antimicrobial defense mechanisms mediated by TLR7 (e.g., after stimulation with influenza virus [FLU V] or HIV-1 ssRNA) (3, 6–8). In addition, TLR7-expressing cells are of particular importance as targets of therapeutic approaches because TLR7 ligands, such as the synthetic nucleoside analog imiquimod (R837), are used successfully as treatment for viral infections and precancerous skin and mucosal lesions (9–11). These divergent functions of TLR7 in PDCs illustrate the versatile character of TLR7 and the complex nature of immune responses mediated by this structure, which are far from being completely understood.
Within the last few years, the discovery of IL-17–secreting Th cells (Th17 cells) revealed them to be additional important players in microbial infections and autoimmune diseases and stimulated strong research activities in this field (12, 13). IL-17 is a proinflammatory cytokine that modulates tissue inflammation. The IL-17–induced release of TNF-α, IL-6, chemokines, and other soluble mediators, such as matrix metalloproteases, promotes tissue infiltration and destruction (14). Another task of IL-17 is to control the differentiation and chemotaxis of neutrophils (14). Consequently, a lack of Th17 cells results in recurrent or persistent infections (e.g., mucocutaneous candidiasis) or severe infections (e.g., those observable in patients with hyper IgE syndrome) (15–17). Th17 cells produce IL-17A, -17F, -22, and -26, IFN-γ, and CCL20 and, in lesser amounts, IL-6 and TNF-α (14, 18). Additionally, the retinoic acid-related orphan receptor (RORC), a transcription factor characteristic of the Th17 lineage, is upregulated during differentiation of Th17 cells (12). In humans, the impact of the soluble factors IL-1β, -6, -21, and -23, TGF-β, and prostaglandin E2 on the development of Th17 cells is controversially discussed (13, 19). IL-23, a member of the IL-6 family that consists of a unique p19 subunit linked to the p40 subunit of IL-12 (18, 19), shares, in addition to the p40 subunit, some signal transduction pathways with IL-12; however, IL-23, in contrast to IL-12, does not promote the expansion of Th1 cells (20). Macrophages and dendritic cells (DCs) are regarded as a cellular source of IL-23. IL-23 affects the functional properties of T cells via a heterodimeric receptor consisting of IL-23R and -12Rβ1 (20, 21). IL-23 and -17 are supposed to play a major role in chronic inflammatory diseases driven by autoimmune mechanisms, including rheumatoid arthritis, multiple sclerosis, and psoriasis (18, 22, 23). Further proof of the concept that IL-17 is involved in the augmentation of autoimmune disorders is provided by the observation that IL-17–deficient mice develop experimental autoimmune encephalitis with profoundly attenuated severity (24). Several mechanisms have been described that promote the differentiation of Th17 cells through myeloid DCs, such as the induction of NOTCH signaling in DCs (25), stimulation of the nucleotide oligomerization domain 2 with muramyldipeptide (26), activation of DCs with thymic stromal lymphopoietin and TLR3 ligands (27), stimulation of DCs with schistosome eggs (28), or incubation of DCs with TLR2, TLR4, and TLR7 agonists (29–31).
Considering the importance of TLR-transferred signals in PDCs in antimicrobial host defense and autoimmune responses, a TLR-mediated modulatory role of PDCs on Th17 cell differentiation and effector function would represent an intriguing concept that could introduce a new, vitally important way in which PDCs might link innate and adaptive immunity in health and disease. Therefore, we investigated the role of TLR stimulation in PDCs and its impact on the regulation of soluble factors required for the differentiation and function of Th17 cells. Our data demonstrate that PDCs are able to promote Th17 differentiation under certain circumstances. Interestingly, the functional relationship between PDCs and Th17 highlighted in this study is established via TLR7 activation in PDCs. Also, we showed that the differentiation of Th17 cells from naive T cells, as well as the amplification of Th17 effector functions of memory T cells, is promoted by PDCs in response to TLR7 activation. Our data are of strong clinical relevance because repetitive TLR7 activation in PDCs might lead to the amplification of Th17-driven inflammatory immune responses in autoimmune disorders and represents an important pathway in the context of viral immune defenses.
Materials and Methods
PBMCs were prepared by Ficoll gradient centrifugation (Lymphoprep, Axis-Shield, Dundee, Scotland), as described previously (32), from buffy coats of human blood from healthy donors provided by the blood bank of the University of Bonn. Purification of PDCs was accomplished by a combination of negative and positive selection with immunomagnetic beads. Briefly, PBMCs were depleted twice with the Diamond Plasmacytoid Dendritic Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer’s instructions, with the AutoMACS (Miltenyi Biotec) to enrich PDCs. In a second incubation step, the enriched PDCs were directly labeled by the BDCA-4 MicroBead Kit to positively select PDCs. The purity of BDCA2+CD123+ PDCs was >98%, as confirmed by flow cytometry.
Human memory CD4+ T cells.
Untouched CD4+CD45RO+CD45RA− T cells were obtained from PBMCs by the Memory CD4+ T cell Isolation Kit (Miltenyi Biotec), according to the manufacturer’s manual. The purity of memory CD4+ T cells was ∼96–99%, as confirmed by flow cytometric staining.
Human naive CD4+ T cells.
Naive CD4+CD45RA+CD45RO− T cells were isolated by depletion of CD4+CD45RO+ memory Th cells and non-Th cells with the naive CD4+ T Cells Isolation Kit II (Miltenyi Biotec) and the AutoMACS technique, according to the manufacturer’s instructions. The purity of untouched naive T cells was >96%, as measured by flow cytometry.
PDC stimulation and coculture with T cells
PDCs were seeded at 2 × 10533,34). Supernatants were collected after 24 h of stimulation with the TLR7 ligand imiquimod (R837, 10 μM; Invivogen, Toulouse, France) or LPS (100 ng/ml; Sigma-Aldrich, Taufkirchen, Germany).
Coculture of PDCs with T cells was done according to literature (18, 35). Briefly, naive CD4+CD45RA+18). After 5 d, PDCs were removed by magnetic depletion, and primed naive CD4+CD45RA+ T cells were washed and cultured in fresh medium supplemented with IL-2 (20 U/ml) for 7 d (35). Memory CD4+CD45RO+ T cells were cocultured with PDCs at a ratio of 1:10 (PDC/T cells) for 7 d in the absence of anti-CD3 mAb and IL-2.
PDC stimulation with FLU V
Influenza A virus, subtype H3N2 isolated from a patient, was prepared as described in the literature (7, 36). Briefly, virus was propagated in Madin-Darby canine kidney cells by using serum-free Eagle’s MEM. After 5 d, cells were collected by centrifugation. Cell pellets were resuspended in 700 μl PBS and frozen–thawed twice. The suspension was centrifuged at 3000 × g, and clarified supernatant was heated for 30 min at 56°C. Virus concentration was measured by real-time RT-PCR. To adjust 4 × 107 genome equivalents/ml, 30 μl (1.2 × 106 genome) of the supernatant was used for inoculation of 2 × 105 PDCs cultured in 200 μl growth medium with 10 ng/ml IL-3 for 24 h. Then PDCs were washed twice and cocultured with CD4+CD45RO+ memory Th cells.
PDC stimulation with HIV1-derived ssRNA
PDCs (1 × 105) cultured in 200 μl growth medium with 10 ng/ml IL-3 were stimulated with 15 μg/ml HIV-1 derived ssRNA: GagRNA1166 (5′-UUGUUAAGUGUUUCAAUUGU-3′), Gp160RNA2093 (5′-UUUUUGCUGUACUUUCUAUA-3′), VIFRNA327 (GUAUUACUUUGACUGUUUUU), or control ssRNA VIFRNA327A (GAAAAACAAAGACAGAAAAA) (Eurofins MWG, Ebersberg, Germany) and ssRNA A21 (5′-AAAAAAAAAAAAAAAAAAAAA-3′), complexed with 30 μg/ml DOTAP liposomal transfection reagent (Roche Diagnostics, Mannheim, Germany). After 24 h of stimulation, PDCs were washed twice and cocultured with CD4+CD45RO+ memory Th cells.
Intracellular cytokine staining
For intracellular staining of cytokines, PDCs were stimulated with TLR7 ligand or LPS for a total of 12 h; GolgiPlug and GolgiStop were added after 2 h of stimulation. Intracellular staining was performed as reported in detail elsewhere (32
Quantitative real-time PCR analysis
For extraction of mRNA from cocultured T cells, PDCs were removed by magnetic depletion, and T cells were restimulated for 5 h with PMA and ionomycin. Then purified T cells were lysed, and total RNA was isolated with the help of a NucleoSpin RNA XS kit (Macherey-Nagel, Dueren, Germany), including digestion of genomic DNA, and subjected to cDNA synthesis with TaqMan reverse transcription reagents with random hexamers, according to the manufacturer’s instructions (Applied Biosystems, Darmstadt, Germany). The prepared cDNA was amplified using TaqMan Gene Expression Master Mix and predesigned TaqMan Gene Expression Assays, according to the recommendations of the manufacturer, on an ABI Prism 7300 Sequence Detection System (all from Applied Biosystems). Primers, including probes, were as follows: IL1-β (Hs00174097_m1), IL-6 (Hs99999032_m1), IL-23A (Hs00372324_m1), TNF-α (Hs00174128_m1), IL-17A (Hs00936345_m1), IL-17F (Hs00369400_m1), IL-21 (Hs00222327_m1), IL-22 (Hs00220924_m1), IL-23R (Hs00332759_m1), IL-26 (Hs00218189_m1), CCL20 (Hs00171125_m), CCL22 (Hs00171080_m1), RORC (Hs01076112_m1), and endogenous control (18s) (Applied Biosystems). All assays were performed according to the manufacturer’s instructions. Relative quantification and calculation of the range of confidence was performed using the comparative CT method (37). All analyses were conducted in duplicate.
Analysis of cytokines produced by T cells
After 7 d of coculture with PDCs, memory T cells were restimulated for 7 h with PMA (100 ng/ml) and ionomycin (1 μg/ml), in the presence of GolgiStop and GolgiPlug (BD Bioscience) for the final 5 h. Then intracellular cytokine staining of T cells was performed.
After naive CD4+ T cells were cocultured with PDCs, stimulated with anti-CD3 mAb for 5 d, and cultured with IL-2 for 7 d, primed CD4+ T cells were restimulated with ionomycin and PMA as described above.
After restimulation, T cells were fixed and permeabilized with BD Bioscience Perm/wash buffer, according to the manufacturer’s instructions. T cells were further stained with PE-labeled mAb against IL-17A (eBio64DEC17) (eBioscience/NatuTec, Frankfurt, Germany), PE-labeled mAb against IL-4 (8D4-8), FITC-labeled mAb against IFN-γ (B27), PE-Cy5–labeled mAb against-CD4 (RPA-T4), and respective isotype controls (all from BD Biosciences). Flow cytometry was done using a FACSCanto flow cytometer (BD Biosciences), and data were analyzed by FACSDiva and FlowJo software.
To analyze the amount of cytokines in the cell culture supernatant, 5 × 105
Statistical analysis was performed with SPSS 17.0 for Windows (SPSS, Chicago, IL). Quantitative values were compared among the groups using the Mann–Whitney U test for data not distributed normally and the paired t test for normally distributed data. Results are shown as mean ± SEM. Any p values are two-sided and subject to a significance level of 5%.
TLR7 ligation of human PDCs induces increased IL-1β, -6, and -23p19 and TNF-α mRNA and protein expression
PDCs were shown to play important roles in the defense against microbial infections as well as autoimmune disorders (1, 2). Stimulation of PBMCs with TLR7/8 ligands and TLR ligation of human myeloid DCs promote the generation of Th17 cells (27, 31, 38, 39). Because signals involved in the commitment of soluble factors essential for Th17 differentiation and increase of their effector functions are of substantial interest, we aimed to determine whether the production of soluble mediators required for Th17 differentiation and their effector functions are affected by TLR7 stimulation in human PDCs. Therefore, we tested IL-1β, -6, and -23p19 and TNF-α mRNA expression of PDCs in response to TLR7 ligation by quantitative real-time PCR. We found upregulation of IL-1β, -6, and -23p19 and TNF-α mRNA expression of PDCs 2 h after TLR7 ligation (Fig. 1); mRNA expression for IL-6 and -23p19 was even greater after 18 h of stimulation (data not shown). Intracellular cytokine staining, ELISA, and Flex-Set assays of supernatants of TLR7- or LPS-stimulated PDCs were performed to compare the results on the mRNA level with the respective protein synthesis. TLR7 ligation profoundly upregulated intracellular amounts of IL-6 (p = 0.031; Fig. 2A) and TNF-α (p = 0.002; Fig. 2B) of PDCs, as well as their IL-6, TNF-α, and IFN-α protein release (data not shown), whereas stimulation with LPS did not induce any cytokine production (Fig. 2A, 2B). Furthermore, increased amounts of IL-1β (Fig. 2C) and IL-23p19 (Fig. 2D) were detectable in the supernatants of TLR7-stimulated PDCs. Similar to a recent report (31), we also observed high and low responders to TLR7 stimulation of PDCs in terms of IL-23p19 production (Fig. 2D). In line with previous reports (40, 41), neither IL-12 nor TGF-β production by PDCs was detectable (data not shown).
PDCs stimulated with the synthetic TLR7 ligand R837 augment Th17 effector functions
In mice, the differentiation of Th17 cells requires TGF-β, IL-6, and the transcription factor RORC. In contrast, Th17 differentiation in humans is promoted by IL-1β, -6, and -23p19 and TNF-α, whereas TGF-β and IL-12 were reported to prevent Th17 differentiation under specific circumstances (35). Therefore, we aimed to determine the capacity of PDCs to induce Th17 cell differentiation from human memory CD4+ T cells after TLR7 ligation with the synthetic TLR7 agonist R837. For this purpose, PDCs were incubated with R837 or LPS as a control for 24 h, washed twice, and cocultured with allogeneic memory CD4+ T cells. Upregulated expression of IL-17A, -17F, -22, and -26 and CCL22 mRNA was observable in the differentiating T cells cocultured with PDCs, which underwent TLR7 stimulation (Fig. 3). In contrast, no change in CCL20 and IL-21 and -23R mRNA expression was observed (data not shown). Furthermore, the intracellular amount of IL-17A increased significantly in T cells cocultured with PDCs after TLR7 stimulation (Fig. 4A).
Because pure Th17 clones and IFN-γ+/IL-17+ and IL-4+/IL-17+ double-positive T cell clones were described in previous studies (42, 43), we ascertained the proportion of IL-17/IFN-γ– and IFN-γ/IL-4–positive T cells.
Intracellular staining revealed that the percentage of memory T cells positive for IL-17A−/IFN-γ+ or for IL-4 did not change profoundly after coculture with TLR7-stimulated PDCs (Fig. 4A, 4B, 4E, 4F). In contrast, TLR7-stimulated PDCs induced significant upregulation of the percentage of IL-17A+/IFN-γ+ and IL-17A+/IFN-γ− memory T cells (Fig. 4A, 4C, 4D). In line with a previous report describing low and high responders in terms of induction of Th17 cells from naive T cells after TLR7/8 activation of human myeloid DCs and PDCs (31), we observed interindividual differences in the IL-17A production of memory T cells. Most interestingly, the capacity to induce intracellular IL-17A production of memory T cells correlated positively with the amount of IL-23p40 produced by PDCs after TLR7 ligation (Fig. 4G, 4H, Supplemental Fig. 1). Furthermore, significant upregulation of IL-17A protein was detected in the supernatants of memory T cells cocultured with TLR7-stimulated PDCs (Fig. 4I) but not in the supernatants of memory T cells cocultured with unstimulated or LPS-stimulated PDCs.
In accordance with a recent report (31), increased IL-17A and -17F mRNA levels were detectable in allogeneic naive T cells cocultured with TLR7-stimulated PDCs in our study (Fig. 4J). However, in naive T cells, a stronger induction of IL-22 mRNA was observed compared with IL-17A/F and -26 mRNA. The induction of IL-17A/F mRNA expression was as strong as the induction of IL-26 mRNA expression.
Stimulation of PDCs with FLU V or HIV-1 ssRNA promotes Th17 immune responses
Because small synthetic nucleoside analogs, such as R837, exhibit additional functions that strongly contribute to their clinical efficacy (44), we next evaluated whether similar effects are achievable by TLR7 stimulation with natural ligands. Therefore, we stimulated PDCs with inactivated FLU V and cocultured them with memory CD4+CD45RO+ T cells. We observed significantly increased induction of intracellular IL-17A+/IFN-γ+ and IL-17A+/IFN-γ− production (Fig. 5A, 5B), greater IL-17A protein release (Fig. 5C), and elevated mRNA expression of cytokines and chemokines associated with Th17 cells in cocultured T cells (Fig. 5D). The effects mediated by FLU V-stimulated PDCs were qualitatively and quantitatively comparable to the effects mediated by R837. As a next step, PDCs were stimulated with different HIV-1–derived ssRNA and respective ssRNA controls and cocultured with memory T cells. Significant upregulation of intracellular IL-17A production by memory T cells cultured with PDCs stimulated with GAGRNA1166, GP160RNA2093, and VIFRNA327 (Fig. 6A, 6B), as well as greater IL-17A protein release (Fig. 6C) and IL-17A and -17F mRNA expression, was observed compared with the respective control ssRNA (Fig. 6D). Interestingly, the induction of Th17 effector functions by HIV-1–derived ssRNA was not as strong as the induction achieved by R837-stimulated PDCs.
Induction of Th17 cells by TLR7-stimulated PDCs is mainly related to increased IL-1β and -23p19 production of PDCs
Having demonstrated that PDCs promote the differentiation of Th17 cells after TLR7 stimulation, we next evaluated whether the induction of Th17 differentiation resulted from the release of soluble mediators by PDCs. Furthermore, we investigated which of the soluble factors released by PDCs after TLR7 activation had the strongest effect on Th17 cell differentiation. Therefore, coculture experiments of PDCs and T cells were conducted in the presence of neutralizing Abs against IL-1β, -6, and IL-12/IL-23p40 subunit and TNF-α alone or in combination, as well as isotype control Abs. As a result, the induction of IL-17A and -17F mRNA in memory T cells was strongly abrogated by IL-1β neutralization and the combination of IL-1β and IL-6 neutralization, as well as IL-12/IL-23p40 neutralization alone or in combination with neutralization of IL-1β and/or IL-6. Neutralization of IL-6 and TNF-α alone did only slightly revert TLR7/PDC-driven Th17 differentiation in terms of IL-17A mRNA expression, but not IL-17F mRNA expression (Fig. 7A). Similar effects of IL-1β neutralization alone or in combination with IL-6 or IL-12/IL-23p40 neutralization were observed for intracellular IL-17A production (Fig. 7B) and protein release (Fig. 7C). Furthermore, neither culture of PDCs without IL-3 nor neutralization of IFN-α in the cell culture supernatant with recombinant B18R protein, a vaccinia virus-encoded type I IFNR, modified the capacity of PDCs to induce effector Th17 cell responses (data not shown). From these data, we conclude that the induction of Th17 cells by PDCs is mainly related to TLR7-triggered IL-1β and -23p19 production.
In this study, we demonstrated that PDCs are able to promote and modify Th17 cell differentiation and function after TLR7 stimulation by synthetic or natural ligands. This identifies a new way in which PDCs connect innate and adaptive immune responses via the TLR/Th17 axis. Furthermore, we showed that amplification of Th17 effector functions of memory T cells and the induction of characteristics of Th17 cells in naive T cells are promoted by PDCs in response to TLR7 activation. These data are in line with a recent publication that demonstrated that incubation of human PBMCs with TLR7/8 ligands promoted the differentiation of Th17 cells from naive T cells (38) and another study showing that the stimulation of TLR7 on human PDCs promoted Th17 differentiation from naive T cells (31). We showed that, in particular, the release of IL-1β and -23p19 by PDCs after TLR7 activation accounts for this effect, that synthetic and natural TLR7 ligands are capable of inducing this effect, and that enhancement of Th17 effector functions of memory T cells is stronger than priming of Th17 cells from naive T cells. These data expand our knowledge about both human DC types capable of inducing Th17 immune responses and the functional significance of TLR7 in PDCs.
Taken together, our findings could have important implications for autoimmune diseases, such as LE, in which the activation of TLR7 and TLR9 in PDCs has been shown to play a critical disease-promoting role (4, 45). In view of the data presented, repetitive TLR7 stimulation in PDCs by ssRNA/autoantibodies might increase the differentiation of Th17 cells and boost the effector functions of already differentiated Th17 cells as a disease-promoting and perpetuating factor in autoimmune disorders, such as LE. This hypothesis is in line with previous studies reporting increased IL-23 and -17 plasma levels in patients with LE (46, 47). In addition, the induction and amplification of Th17 effector functions in response to TLR7 stimulation by viral ssRNA might play roles in the immune activation related to viral immune responses, in particular, because it was recently shown that the number of peripheral Th17 cells increased during chronic virus infections (48, 49).
Moreover, the data presented herein might be of relevance during the treatment of neoplastic skin lesions with R837, because it was shown that increasing numbers of PDCs infiltrate the treated skin area (10). Furthermore, profound neutrophil recruitment (10) and the upregulation of IFN-α and IFN-γ genes were noted in the R837-treated tissue (50). Because the recruitment of neutrophils is a characteristic feature of Th17 cells (51), it is tempting to speculate that TLR7 activation on infiltrating PDCs by the TLR7 ligand R837 might contribute to the release of soluble factors that promote the recruitment of neutrophils as well as the differentiation of Th17 cells or amplification of Th17 effector functions. Together, this might support the therapeutic effect in terms of tissue inflammation and tissue destruction observable during the treatment of viral warts or cancerous skin lesions with the TLR7 ligand R837. Finally, these data underscore the strong role of PDCs as an important link between innate and adaptive immunity, coordinating signals mediated via pattern-recognition receptors by the induction of specific T cell responses.
Disclosures The authors have no financial conflicts of interest.
This work was supported by Deutsche Forschungsgemeinschaft Grants NO454/2-4, NO454/1-4, SFB704 TPA4, TPA14, TPA15, and TPA16 and a BONFOR grant from the University of Bonn. N.N. is supported by a Deutsche Forschungsgemeinschaft Heisenberg Professorship (NO454/5-2).
The online version of this article contains supplemental material.
Abbreviations used in this paper:
- dendritic cell
- FLU V
- influenza virus
- lupus erythematosus
- plasmacytoid dendritic cell
- retinoic acid-related orphan receptor
- single-stranded RNA.
- Received June 2, 2009.
- Accepted November 18, 2009.
- Copyright © 2010 by The American Association of Immunologists, Inc.