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The Journal of Immunology, 2007, 178: 4072-4079.
Copyright © 2007 by The American Association of Immunologists, Inc.

Natural and Synthetic TLR7 Ligands Inhibit CpG-A- and CpG-C-Oligodeoxynucleotide-Induced IFN-{alpha} Production1

Beate Berghöfer, Gabriela Haley, Ture Frommer, Gregor Bein and Holger Hackstein2

Justus-Liebig University, Institute for Clinical Immunology and Transfusion Medicine, Giessen, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Plasmacytoid dendritic cells (pDCs) are unique with respect to their capacity to produce unsurpassed amounts of IFN-{alpha} and coexpress TLR7 and TLR9, mediating IFN-{alpha} production. Although TLRs are critical receptors of innate immunity, little is known about the immunological effects of TLR7/TLR9 costimulation. We have analyzed the effects of TLR7/TLR9 costimulation on IFN-{alpha} production by leukocytes and pDCs. Our experiments revealed that both synthetic (resiquimod and loxoribine) and natural (ssRNA40) TLR7 ligands abrogate CpG-A- and CpG-C-oligodeoxynucleotide (ODN)-induced IFN-{alpha} production by human leukocytes. Because TLR7 ligands themselves represent important IFN-{alpha} inducers, we demonstrated that substimulatory TLR7 ligand concentrations significantly inhibited CpG-A-induced IFN-{alpha}. Delayed addition of TLR7 ligands still resulted in complete suppression of CpG-A-ODN-induced IFN-{alpha} production, suggesting that the inhibition is unlikely to be caused by a kinetic uptake advantage. Unlike for CpG-A and CpG-C, TLR7 ligands did not inhibit CpG-B-ODN-induced IFN-{alpha} production. Experiments with purified human pDCs demonstrated that the inhibitory effects of TLR7/TLR9 costimulation were mediated directly by pDCs. Suppression of IFN-{alpha} production was not related to increased cell death and was also detectable in enriched mouse pDCs. Analyses of pDCs suggested that the TLR7 signal regulates the outcome of TLR7 ligand/CpG-A-ODN costimulation and can either inhibit (IFN-{alpha}) or promote (IL-8/CD40) cytokine and surface marker expression. Our data reveal for the first time a strong inhibitory effect of TLR7 stimulation on IFN-{alpha} production induced by CpG-A- and CpG-C-ODNs. These findings provide novel insight into the effects of TLR7/TLR9 costimulation and may support the development of novel TLR9 inhibitors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Human plasmacytoid dendritic cells (pDCs)3 represent unique IFN-{alpha}-producing, APCs in the immune system (1). In contrast to myeloid DCs, pDCs selectively express TLR7 and TLR9 and have the ability to produce exceptional amounts of IFN-{alpha}. Numerous studies have demonstrated the critical role of IFN-{alpha} in antiviral immunity and its function to bridge innate and adaptive immunity (2, 3). IFN-{alpha} has been demonstrated to prime the maturation and activation of DCs and NK cells and to promote the viability and killing activity of T cells (2, 4). IFN-{alpha} belongs to the group of few cytokines that have been established in routine medicine, being the most widely used cytokine for clinical therapy of chronic viral hepatitis and different malignancies (5).

Recently, independent groups have identified TLR7 and TLR9 as the principle pattern-recognition receptors mediating the IFN-{alpha} production capacity of pDCs after stimulation with their corresponding TLR ligand viral RNA and bacterial DNA (6, 7, 8, 9, 10, 11). The TLR7/TLR9-mediated type I IFN induction in pDCs is dependent on the adaptor protein MyD88 (12, 13). Subsequent to receptor ligation MyD88, IL-1R-associated kinase (IRAK) 1, IRAK4, and TNFR-associated factor 6 assemble a signaling complex (12, 14, 15), essentially required for downstream activation of IFN regulatory factor (IRF) 7 by phosphorylation (15). IRF7, the crucial transcription factor regulating IFN-{alpha} production, is constitutively expressed in high amounts, particularly in pDCs, and is enhanced by CpG-A-oligodeoxynucleotide (ODN) treatment (not by CpG-B-ODN) (16, 17, 18, 19). It is dimerized upon activation, translocated to the nucleus, and initiates high IFN-{alpha} production (16). The positive feedback loop for IFN-{alpha} production via IFNR in pDCs has been reported to be CpG-ODN class dependent (CpG-A, but not CpG-B-ODN) (20). IRF8 has been implicated in TLR9-induced cytokine synthesis (21) and human IRF5 was suggested to participate in TLR7-mediated type I IFN induction (22).

TLR9-induced IFN-{alpha} production can be inhibited in vitro by antagonists, for instance, ODN TTAGGGG, a sequence found in mammalian telomeres (23), or by inhibitors like chloroquine, which prevents endosomal acidification (24) that is required for activation of the signaling cascade (25). Wortmannin blocks sensitive members of the PI3K family that play a critical role in trafficking of CpG-ODN to TLR9 (26, 27). Furthermore, it was found that IL-10 inhibits CpG-ODN-induced pDC activation and IFN-{alpha} synthesis, whereas maturation of pDCs is not affected (28). Several intracellular signaling molecules regulate directly the TLR9-induced pathways, for instance, suppressor of cytokine signaling protein 1 (suppresses IRAK), MyD88s (MyD88 antagonist) or IRAKM (inhibits phosphorylation of IRAK1) (29).

The discovery and development of synthetic TLR7 and TLR9 ligands as novel immune adjuvants have greatly stimulated basic research and clinical study into novel and effective vaccination protocols as well as new anticancer and antiviral treatment options. Topical imiquimod, represents a synthetic TLR7 ligand that has been demonstrated in clinical trials to be safe and effective against superficial basal cell carcinoma and viral skin infections with human papillomavirus and other viruses that cause condylomata and cutaneous warts (30, 31, 32, 33, 34, 35).

The therapeutic potential of synthetic TLR9 ligands has been demonstrated in experimental animal models and human clinical trials with respect to protection from viral, bacterial, fungal and parasitic infections, vaccination, and substantial improvement of cancer therapy (36, 37, 38, 39). Interestingly, experimental asthma and allergy models revealed that the immunostimulatory capacity of synthetic TLR9 ligands can be used to effectively suppress pathogenic TH2 immune responses. Phase I–III clinical trials are underway to investigate the safety and efficacy of this new therapeutic option in humans (36).

In contrast to the fundamental importance of TLR7 and TLR9 for activation of innate and adaptive immunity through DCs and the critical role of IFN-{alpha}-producing pDCs, very little is known about the immunological effects of TLR7/TLR9 costimulation. Given the fact that most viruses and bacteria are unlikely to stimulate single TLRs exclusively, it is reasonable to analyze the immunobiological effects of TLR7/TLR9 costimulation. Currently, ongoing clinical trials use either TLR7 or TLR9 synthetic ligands, but we expect that experimental animal models and/or clinical trials will be planned that use the combination of TLR7 and TLR9 ligands to further augment immune adjuvant activity.

Therefore, in this study, we have, for the first time, systematically analyzed the impact of TLR7/TLR9 costimulation on IFN-{alpha} production of leukocytes and purified pDCs. We have focused on IFN-{alpha} and pDCs because many of the beneficial antiviral and antiproliferative effects of synthetic TLR7 and TLR9 ligands are believed to be mediated through the release of IFN-{alpha} by pDCs (30, 36). Unexpectedly, we found that TLR7/TLR9 costimulation with synthetic and natural TLR7 ligands is able to dramatically suppress CpG-A- and CpG-C-ODN-induced IFN-{alpha} production by human leukocytes. Our findings provide novel insight into the immunobiology of TLR7/TLR9 costimulation and may have important implications with respect to the clinical use of synthetic TLR7/TLR9 ligands for immune modulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Subjects

Blood samples and buffy coats were obtained from randomized, healthy, voluntary blood donors after giving their informed consent. BALB/c splenocytes and pDCs were provided by C. Steinschulte (Institute of Clinical Immunology and Transfusion Medicine, Justus-Liebig-University, Giessen, Germany). This study has been reviewed and approved by an appropriate institutional review committee.

Preparation and stimulation of human PBL and pDCs

PBL were isolated from EDTA-anticoagulated whole blood samples by ammonium chloride lysis (Puregene RBC Lysis solution; Gentra Systems). PBMC were isolated from buffy coats by Ficoll-Paque (Pharmacia) density gradient centrifugation. pDCs were purified from PBMC using the blood dendritic cell Ag 4 (BDCA-4) dendritic cell isolation kit (Miltenyi Biotec) according to the manufacturer’s instructions. Briefly, the pDCs were labeled with anti-BDCA-4 Ab coupled to colloidal paramagnetic MicroBeads and passed twice through a magnetic separation column using the magnetic cell sorter autoMACS (Miltenyi Biotec). Two x 106 PBL, 1 x 106 PBMC, or 2 x 105 pDCs were cultured and stimulated in 96-well round-bottom plates in 200 µl of medium, comprising RPMI 1640 with L-glutamine, penicillin/streptomycin, 10% heat-inactivated FCS Gold (PAA Laboratories), nonessential amino acids (Sigma-Aldrich), sodium pyruvate, and HEPES (Invitrogen Life Technologies). Stimulation of the cells was performed for 20 h with the following, in endotoxin-free water solved stimuli: 1 µg/ml resiquimod (R848), 3.4 mg/ml (1 mmol/ml) loxoribine (Loxo), 3 µg/ml CpG-ODN 2216/2006/M362 (InvivoGen), and 10 µg/ml ssRNA40 (5'-GCCCGUCUGUUGUGUGACUC-3' (11); MWG Biotec), supplemented with 50 µg/ml DOTAP Liposomal Transfection Reagent (Roche Diagnostics), added simultaneously (cocomplexation), unless otherwise noted. Trypan blue (Sigma-Aldrich) staining was performed for quantification of pDCs. Culture supernatants were collected after 20 h and frozen (–70°C) once.

Flow cytometry

For determination of viability and purity, a specimen of purified human pDCs was stained with anti-BDCA-2-PE (Miltenyi Biotec) and the vital dye 7-aminoactinomycin D (7-AAD; BD Pharmingen) and then analyzed by flow cytometry using a BD Biosciences FACSCalibur flow cytometer. Purity of human pDCs was 93% on average. CD40 expression was measured after CD40-allophycocyanin (BD Pharmingen) staining, in comparison with isotype-allophycocyanin staining. Murine splenocytes and pDCs were stained with anti-CD11c-allophycocyanin (BD Pharmingen) and anti-mouse PDCA-1-PE (Miltenyi Biotec). Purity of enriched murine pDCs was 67.2% on average (between 60.7 and 76.7%). The viability of murine cells was determined by trypan blue staining. The viability of human pDCs was determined after staining with annexin V-PE and 7-AAD in Annexin V Binding Buffer (Apoptosis Kit I; BD Pharmingen) according to the manufacturer’s instructions.

Preparation and stimulation of in vivo-expanded murine pDCs

Twelve-week-old BALB/c mice were purchased from Charles River. They were housed in the specific pathogen-free animal facility of the Justus-Liebig-University (Giessen, Germany). Mice were injected with 10 µg/mouse per day recombinant human Fms-like tyrosine kinase 3 ligand (Amgen) in aqua ad injectabila over a period of 10 days for in vivo expansion of DC. On day 11, animals were sacrificed and spleens were removed, chopped with fine scissors, and the resulting cell suspension was passed through a nylon cell strainer (70 µm, BD Falcon Cell Strainers) to obtain a single-cell suspension. Erythrocytes were lysed by ammonium chloride solution (Puregene RBC Lysis solution; Gentra Systems) for 1.5 min. Cells were washed once with RPMI 1640 (PAA Laboratories) and the residues of connective tissue were removed. Before staining, cells were passed through a preseparation filter (30-µm diameter; Miltenyi Biotec).

Murine pDCs were enriched with the Plasmacytoid Dendritic Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instructions. Briefly, splenocytes were labeled by biotin-conjugated Abs against CD3, CD19, CD11b (Mac-1) and CD49b (DX5), and anti-biotin MicroBeads to deplete T cells, B cells, NK cells, and myeloid DCs. The negative fraction represented the pre-enriched pDC fraction. pDCs subsequently were positively selected by labeling with CD45R (B220) MicroBeads. One x 105 enriched pDCs or 1 x 106 splenocytes were stimulated with TLR ligands in 96-well round-bottom plates in 200 µl of RPMI 1640 as described above.

Cytokine detection

Human and mouse IFN-{alpha} was measured by ELISA (PBL Biomedical Laboratories). IL-8 was measured using the BD OptEIA ELISA set (BD Pharmingen).

Statistics

Statistical analyses were performed using the Mann-Whitney U test. All tests were performed two-tailed. A p of < 0.05 was considered to be significant. All analyses were performed using the SPSS software version 12.0 (SPSS).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Suppression of TLR9-induced IFN-{alpha} production by TLR7 costimulation

pDCs are unique cell types with respect to their ability to produce huge amounts of IFN-{alpha} in response to TLR7 or TLR9 stimulation. However, when investigated directly, the TLR9 ligand CpG-ODN 2216 has been revealed to induce ~10 times more IFN-{alpha} in pDCs compared with the TLR7 ligands resiquimod (R848) and Loxo (8).

With respect to the paucity of information concerning the effects of TLR costimulation on IFN-{alpha} production and to identify possible novel ways to further augment IFN-{alpha} production, we analyzed the effects of TLR7/TLR9 costimulation on IFN-{alpha} in PBL and pDCs. At first, PBL of healthy blood donors were stimulated with TLR7 and TLR9 ligands alone or with a combination of both TLR ligands to examine the effects of TLR7/TLR9 costimulation on IFN-{alpha} production.

Optimal amounts of the TLR9 ligand CpG-ODN 2216 induced significantly higher amounts of IFN-{alpha} (~10 times) than maximal stimulation with the TLR7/8 ligand R848 (p < 0.000002, Mann-Whitney U test, Fig. 1A). Unexpectedly, these experiments revealed that costimulation with TLR7/TLR9 ligands resulted in massive inhibition of IFN-{alpha} production when compared with TLR9 monostimulation (Fig. 1A; n = 20; p < 0.000002). Interestingly, TLR7/TLR9 costimulation reduced IFN-{alpha} production to levels comparable to TLR7 monostimulation (Fig. 1). It is important to note, that in all experiments, the optimal stimulatory concentrations for each TLR ligand were determined after titration (data not shown). To further confirm the relevance of these findings, we repeated the experiments with Loxo, an exclusive TLR7 ligand, and with ssRNA40, a natural TLR7 ligand. Again, TLR7/TLR9 costimulation with either Loxo (Fig. 1B, n = 20; p < 0.0008) or ssRNA40 (Fig. 1C, n = 16; p = 0.01) profoundly decreased IFN-{alpha} levels when compared with TLR9 monostimulation. TLR7/TLR9 costimulation induced significant suppression of TLR9-induced IFN-{alpha} production by all subjects, irrespective of the individual’s capacity to produce IFN-{alpha} and occurred with either synthetic or natural TLR7 ligands.


Figure 1
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  		FIGURE 1. Abrogation of TLR9-induced IFN-{alpha} production by costimulation with synthetic (R848, Loxo) and natural (ssRNA40) TLR7 ligands. A and B, PBL were stimulated with 3 µg/ml CpG-ODN 2216 (CpG), 1 µg/ml R848 or 3.4 µg/ml Loxo alone, and in combination as described in Materials and Methods. C, PBMC were stimulated with 10 µg/ml ssRNA40 (RNA40), supplemented with 10 µl of DOTAP, alone, or in combination with CpG-ODN 2216, added simultaneously. IFN-{alpha} production was measured by ELISA. Costimulation with either synthetic (A and B) or natural (C) TLR7 ligands profoundly suppressed TLR9-induced IFN-{alpha} production. A, ***R848 vs CpG, p < 0.000002; ***CpG vs R848 + CpG, p < 0.000002; B, ***Loxo vs CpG, p < 0.000006; ***CpG vs Loxo + CpG, p < 0.0008; and C, ***RNA40 vs CpG, p < 0.0005; *RNA40 + CpG vs CpG, p = 0.01 (two-tailed Mann-Whitney U test). A–C, Results show mean IFN-{alpha} levels ± SE. A and B, n = 20 independent experiments; C, n = 16 independent experiments.

 
Suppression of TLR9-induced IFN-{alpha} production by TLR7 costimulation is CpG-ODN class dependent

To provide additional insight into the potential mechanism, we have used additional CpG-ODN classes (CpG-B- and CpG-C-ODN) that have been reported to be sublocalized in different endosomal compartments (40). The use of different CpG-ODN classes allowed us to analyze whether endosomal localization of CpG-ODNs interferes with the regulatory activity of TLR7 ligands. CpG-A-ODNs are known to be localized in transferrin receptor (TfR)-positive endosomes, CpG-B-ODNs in lysosome-associated membrane protein 1-positve endosomes, and CpG-C-ODNs were demonstrated to be localized in both types of endosomes (40). Moreover, the CpG-C-ODN M362 was used to examine whether the inhibitory effect of TLR7 ligands on TLR9-induced IFN-{alpha} production is due to the interaction of the TLR7 ligands with the tertiary structure of the CpG-A-ODN 2216.

Our experiments revealed that TLR7 ligands inhibit only CpG-A- and CpG-C-ODN-induced IFN-{alpha} production (Figs. 1 and 2, A and B). TLR7/TLR9 costimulation with CpG-B-ODN 2006 did not significantly enhance IFN-{alpha} production (Fig. 2C).


Figure 2
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  		FIGURE 2. Suppression of TLR9-induced IFN-{alpha} production by TLR7 costimulation is CpG-ODN class dependent. A and B, PBL were stimulated with 3 µg/ml CpG-ODN M362 (M362), 1 µg/ml R848, or 10 µg/ml ssRNA40 (RNA40), supplemented simultaneously with 10 µl of DOTAP, alone and in combination. C–E, PBMC were stimulated with R848, Loxo, or ssRNA40 (RNA40) supplemented with 10 µl of DOTAP were indicated, alone or in combination with CpG-ODN 2006 (2006), added simultaneously. IFN-{alpha} production was measured by ELISA. Costimulation with either synthetic or natural TLR7 ligands suppressed CpG-C-ODN M362-induced IFN-{alpha} production. CpG-B-ODN 2006 did not suppress TLR7 ligand induced IFN-{alpha} production. A, *R848 vs M362, p < 0.016; *M362 vs R848 + M362, p < 0.036; B, *RNA40 vs M362, p < 0.021; *M362 vs RNA40 + M362, p < 0.021; C, ***R848 vs 2006, p < 0.00016; ***2006 vs R848 + 2006, p < 0.00075; R848 vs R848 + 2006, n.s.; D, RNA40 vs 2006, p < 0.00076; ***2006 vs RNA40 + 2006, p < 0.00075; RNA40 vs RNA40 + 2006, p = 0.059; ***2006 vs 2006 + DOTAP, p < 0.0032; ***RNA40 + 2006 vs 2006 + DOTAP, n.s.; and E, ***Loxo vs 2006, p < 0.00078; Loxo vs Loxo + DOTAP, n.s.; ***2006 vs 2006 + DOTAP, p < 0.00078; ***Loxo + DOTAP vs Loxo + DOTAP + 2006, p < 0.00078; Loxo + DOTAP + 2006 vs 2006 + DOTAP, n.s., p = 0.674 (two-tailed Mann-Whitney U test). A–E, Results show mean IFN-{alpha} levels ± SE of n = 8 independent experiments, n.s., not significant.

 
Two groups have demonstrated recently that encapsulation of CpG-B-ODNs results in their redistribution into the TfR-positive endosomes and subsequent high IFN-{alpha} induction (40, 41). To analyze whether encapsulation of CpG-B-ODN by a liposomal transfection reagent can restore the inhibitory effects of TLR7 ligands on TLR9-induced IFN-{alpha}, we performed experiments with CpG-B-ODN, encapsulated by DOTAP.

These experiments revealed that IFN-{alpha} induction by encapsulated CpG-B-ODNs is not inhibited by costimulation with encapsulated ssRNA40 or Loxo (Fig. 2, D and E). Furthermore, there was a trend (p = 0.057) of encapsulated CpG-B-ODN 2006 to increase ssRNA40-induced IFN-{alpha} production, whereas Loxo, a nucleotide which is unlikely to be encapsulated by DOTAP, did not significantly change the IFN-{alpha} production of encapsulated CpG-B-ODN 2006 (p = 0.67).

Substimulatory TLR7 ligand concentrations that induce minimal IFN-{alpha} still inhibit TLR9-induced IFN-{alpha} production

Because TLR7 by itself represents an important IFN-{alpha} inducer, we questioned whether substimulatory TLR7 ligand concentrations (i.e., TLR7 ligand concentrations that induce only very low levels of IFN-{alpha}) were able to interfere with TLR9-induced IFN-{alpha} production. The TLR7 ligands R848, Loxo, and ssRNA40 each were titrated in the absence and presence of a constant CpG-ODN 2216 concentration in PBL cultures.

These experiments revealed that even the smallest amounts of TLR7 ligands were sufficient to profoundly inhibit TLR9-induced IFN-{alpha} production (Fig. 3). R848 (0.01–0.05 µg/ml), representing 1–5% of the maximal stimulatory concentration, were sufficient to inhibit 50–80% of TLR9-induced IFN-{alpha} production (Fig. 3A). TLR7 ligands Loxo and ssRNA40 similarly inhibited CpG-A-ODN-induced IFN-{alpha} production (Fig. 3, B and C). These results suggest that TLR7 ligands very efficiently inhibit CpG-A-ODN 2216-induced IFN-{alpha} production even at concentrations that induce near basal-level IFN{alpha} production.


Figure 3
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  		FIGURE 3. Substimulatory TLR7 ligand concentrations inhibit TLR9-induced IFN-{alpha} production. PBL (A and B) and PBMC (C) were stimulated with the indicated increasing concentrations of synthetic (A, R848; B, Loxo) and natural (C, ssRNA40) TLR7 ligands in the presence (filled symbols) or absence (open symbols) of 3 µg/ml CpG-ODN 2216 as described in Materials and Methods. Synthetic (A and B) and natural (C) TLR7 ligands suppress IFN-{alpha} production at concentrations that do induce very low IFN-{alpha}. Results show mean IFN-{alpha} levels. A, n = 6, B, n = 3, and C, n = 5 independent experiments, respectively.

 
Suppression of IFN-{alpha} production after TLR7/TLR9 costimulation is not caused by a kinetic uptake advantage of the TLR7 ligand

TLR7 and TLR9 receptors are located intracellularly in the endosomal compartment (42, 43). The TLR7 ligand R848 with its molecular mass of 340 g/mol is considerably smaller than the TLR9 ligand CpG-ODN 2216 (molecular mass, 6432 g/mol). Therefore, it could be considered that R848 has an uptake and transport-velocity benefit, reaching its receptor in the endosomal compartment earlier than the TLR9 ligand and therefore occupying the IFN-{alpha} signaling pathway and consequently inhibiting signaling through TLR9. CpG-ODN was detected intracellularly by Ahmad-Nejad et al. (43) within 15 min of exposure, and Latz et al. (42) found the ligand to interact with TLR9 5–30 min after uptake in a subcellular compartment. Based on these data, we designed the following experiments: we added CpG-ODN 2216 up to 2 h before the TLR7 ligand and analyzed IFN-{alpha} production by PBL. The results demonstrated that CpG-A-ODN-induced IFN-{alpha} production still was completely suppressed, even when the TLR7 ligand was added 2 h after the TLR9 ligand. These data suggest that the observed inhibitory effects of TLR7 ligands on TLR9-induced IFN-{alpha} production are independent of a kinetic uptake advantage of TLR7 ligands. Interestingly, the suppressive effects of R848 were even augmented when the TLR7 ligand was added 2 h after the TLR9 ligand (p = 0.01, Mann-Whitney U test, Fig. 4).


Figure 4
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  		FIGURE 4. Abrogation of IFN-{alpha} production after TLR7/TLR9 costimulation is not caused by a kinetic uptake advantage of TLR7 ligands. PBL were stimulated with 1 µg/ml R848 or 3 µg/ml CpG-ODN 2216 and with a combination of R848 + CpG (TLR7/TLR9 costimulation). In the TLR7/TLR9 costimulation experiments, R848 addition was delayed 0 min to 2 h after addition of CpG-ODN 2216. IFN-{alpha} levels were measured by ELISA. TLR9-induced IFN-{alpha} production was suppressed to TLR7-induced levels, even when TLR7 ligand was added up to 2 h later after CpG-ODN. **, 0 min vs 2 h, p = 0.01 (two-tailed Mann-Whitney U test). Bars, Mean values ± SE of n = 6 independent experiments.

 
Inhibitory effects of TLR7/TLR9 costimulation are mediated via direct effects on pDCs

Although pDCs specifically express TLR7 and TLR9 and represent the principle IFN-{alpha}-producing cell in the immune system, our previous experiments did not exclude the possibility that an indirect impact of the TLR7 ligands on other cells, such as B lymphocytes, could be responsible for the observed inhibitory effects. To exclude that the inhibition of TLR9-induced IFN-{alpha} production by TLR7 ligands was related to indirect effects, we purified pDCs from healthy blood donors by magnetic bead sorting (average purity, 93%). Isolated pDCs were stimulated with increasing concentrations of R848 or Loxo in the absence or presence of CpG-ODN 2216. Similar to the results with PBL, CpG-ODN 2216 induced up to 10 times more IFN-{alpha} in the pDC cultures compared with R848 or Loxo. Again, costimulation with TLR7/TLR9 ligands massively inhibited CpG-A-ODN-induced IFN-{alpha} production of purified pDCs. (Fig. 5).


Figure 5
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  		FIGURE 5. Abrogation of TLR9-induced IFN-{alpha} production by TLR7/TLR9 costimulation is mediated via direct effects on pDCs. pDCs were stimulated with the indicated concentrations of R848 (A) and Loxo (B) in the absence (open symbols) or presence (filled symbols) of 3 µg/ml CpG-ODN 2216. IFN-{alpha} was measured by ELISA. Costimulation of TLR7 and TLR9 suppressed the TLR9-induced IFN-{alpha} production of pDCs, indicating that the effects of TLR7/TLR9 costimulation are mediated via direct effects on pDCs. Mean values of IFN-{alpha}: A, n = 6 and B, n = 8 independent experiments, respectively.

 
Suppression of IFN-{alpha} production by purified pDCs after TLR7/TLR9 costimulation is not caused by increased apoptosis

To analyze the hypothesis that decreased levels of IFN-{alpha} in TLR7/TLR9-costimulated cultures were due to a higher rate of cell death, we examined the viability of purified pDCs after 20 h of stimulation. The frequencies of apoptotic and dead pDCs were determined after annexin V and 7-AAD staining by flow cytometry and compared with unstimulated cultured cells. There were no significant differences in the rate of dead or apoptotic cells between the different stimulatory conditions, indicating that selective enhancement of apoptosis is unlikely to account for the inhibition of IFN-{alpha} production after TLR7/TLR9 costimulation. Notably, pDCs stimulated with a combination of TLR7 and TLR9 ligands even exhibited a trend toward improved viability when compared with monostimulated or unstimulated pDC cultures (Fig. 6).


Figure 6
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  		FIGURE 6. Suppressed IFN-{alpha} production in TLR7/TLR9 costimulation is not caused by increased apoptosis. Purified pDCs were monostimulated with 1 µg/ml R848 or in combination with 3 µg/ml CpG-ODN 2216. Viable, apoptotic, and dead cells were determined by flow cytometry after staining with 7-AAD and annexin V-PE. The rate of dead and apoptotic cells did not differ significantly between the different stimulatory conditions. Control, unstimulated pDCs; {blacksquare}, dead cells; {square}, apoptotic cells; n.s., not significant. Bars, Mean values of n = 5 independent experiments.

 
Antagonistic effect of TLR7 costimulation on TLR9-induced IFN-{alpha} production is not species specific

Because significant species differences with respect to TLR expression and functionality have been reported, e.g., nonfunctional murine TLR8 (44), we questioned whether the abrogation of TLR9-induced IFN-{alpha} production by TLR7/TLR9 costimulation was also detectable in mice. Mice were injected with Fms-like tyrosine kinase 3 ligand to expand mouse pDCs in vivo. Subsequently, total murine splenocytes, as well as enriched pDCs, were stimulated with TLR7 (R848 and Loxo) and TLR9 (CpG-ODN 2216) ligands in mono- and costimulation combinations, and mouse IFN-{alpha} was determined in culture supernatants. The mouse experiments confirmed our results with human PBL and pDCs. Stimulation with optimal levels of R848 or Loxo induced moderate amounts of mouse IFN-{alpha} in mouse splenocytes and pDCs, and CpG-ODN 2216 induced 10–20 times higher levels of IFN-{alpha}. Interestingly, TLR7/TLR9 costimulation of mouse cells significantly suppressed the TLR9-induced IFN-{alpha} production (Fig. 7).


Figure 7
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  		FIGURE 7. Abrogation of TLR9-induced IFN-{alpha} production by TLR7/TLR9 costimulation is species spanning. Splenocytes (A) and enriched pDCs (B) of BALB/c mice were monostimulated with 1 µg/ml R848 or 3.4 µg/ml Loxo and in combination with 3 µg/ml CpG-ODN 2216. Mouse IFN-{alpha} levels were measured by ELISA. TLR7/TLR9 costimulation suppressed the TLR9-induced IFN-{alpha} production in mouse splenocytes and pDCs. A and B, **R848 vs CpG, **R848 + CpG vs CpG, **Loxo vs CpG, **Loxo + CpG vs CpG, (p = 0.004 each, two-tailed Mann-Whitney U test). {blacksquare}, Mean value of IFN-{alpha} ± SE of n = 6 individual animals.

 
Analysis of IL-8 production and CD40 surface expression by purified human pDCs after TLR7 ligand/CpG-A ODN costimulation suggests dominance of TLR7 signaling

Our results with human and mouse pDCs revealed a profound interference of TLR7- and TLR9-induced pathways by TLR7 costimulation, leading to complete abrogation of CpG-ODN 2216-induced IFN-{alpha} production. To investigate whether TLR7 ligand/CpG-A-ODN costimulation generally promotes an inhibitory signal to pDCs, we investigated IL-8 production and CD40 surface expression by purified human TLR7/TLR9-stimulated pDCs. We selected IL-8 as a functional readout, because our experiments indicated that CpG-A-ODNs are weaker IL-8 inducers when directly compared with TLR7 ligands R848 and Loxo (Fig. 8). CD40 surface expression was analyzed because it represents an important pDC maturation marker and is not expressed on immature pDC precursors (Fig. 9). In confirmation with the previous experiments, TLR7/TLR9 costimulation resulted in IL-8 (Fig. 8) and CD40 (Fig. 9) expression almost identical to those obtained after R848 or Loxo monostimulation. These results suggested that the TLR7 signal dominates the outcome of TLR7 ligand/CpG-A-ODN costimulation and can either strongly suppress (IFN-{alpha}) or promote pDCs’ activation (IL-8, CD40) depending on the intrinsic stimulatory capacity of TLR7 monostimulation.


Figure 8
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  		FIGURE 8. IL-8 production of pDCs indicates dominance of TLR7 signaling in TLR7/TLR9 costimulation. A and B, Purified pDCs were monostimulated with 1 µg/ml R848 (A) or 3.4 µg/ml Loxo (B) and in combination with 3 µg/ml CpG-ODN 2216. IL-8 levels were measured by ELISA. Equal levels of IL-8 were induced by TLR7 monostimulation and TLR7/TLR9 costimulation. A, *R848 vs CpG, p = 0.035; *CpG vs R848 + CpG, p = 0.035; B, *Loxo vs CpG, p = 0.009; *CpG vs Loxo + CpG, p = 0.006 (two-tailed Mann-Whitney U test); n.s., not significant. Results show mean IL-8 levels ± SE of n = 7 independent experiments.

 

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  		FIGURE 9. Analysis of CD40 surface expression by purified human pDCs after TLR7/TLR9 costimulation indicates dominance of TLR7 signaling. Purified pDCs were monostimulated with 1 µg/ml R848 or 3 µg/ml CpG-ODN 2216 and with a combination of 1 µg/ml R848 and 3 µg/ml CpG-ODN 2216. CD40 expression was analyzed on BDCA-2-positive, 7-AAD-negative pDCs by flow cytometry (A). The gating strategy with representative dot plot diagrams (A) and a summary (B) of the CD40 expression levels in the different groups are shown. TLR7 monostimulation induced significantly higher de novo CD40 expression as compared with TLR9 monostimulation (B). TLR7/TLR9 costimulation resulted in almost identical CD40 expression as compared with TLR7 monostimulation (A and B). B, Results of n = 7 independent experiments (±SE). ***R848 vs CpG, p = 0.004; *CpG vs R848 + CpG, p = 0.026 (two-tailed Mann-Whitney U test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Despite a large number of studies analyzing the results of TLR7 or TLR9 monostimulation, almost nothing is known with respect to the effects of TLR7/TLR9 costimulation. Synthetic TLR7 and 9 ligands are promising and powerful adjuvants that stimulate innate and adaptive immune responses. pDCs represent major targets of these adjuvants in the immune system. They express both TLR7 and TLR9, which mediate their capacity to produce eminently more IFN-{alpha} than any other cell in the immune system. IFN-{alpha} is of critical importance for antiviral immunity and for B, T, and NK cell activation (3). With respect to the central role of pDCs and IFN-{alpha}, in this study, we have analyzed the immunological effects of TLR7/TLR9 costimulation on IFN-{alpha} production by leukocytes and pDCs. The results reveal for the first time a strong inhibitory effect of synthetic and natural TLR7 ligands on CpG-A- and CpG-C-ODN-induced IFN-{alpha} production by human and mouse pDCs.

In contrast and with respect to other TLR ligands, other groups have reported synergistic effects of TLR ligation on cytokine release. Napolitani et al. (45) have analyzed the consequences of the combination of TLR8, TLR3, and TLR4 ligation and have reported synergistic IL-12 and IL-23 induction, which cooperate in a TH1-polarizing capacity. Likewise, Roelofs et al. (46) recently published data that showed a synergistic effect on DC activation and TNF-{alpha}, IL-12, and IL-10 release upon stimulation with combinations of two or more TLR3, TLR4, and TLR7/8 agonists. To our knowledge, only a single report so far has investigated immunological aspects of TLR7/TLR9 costimulation in mice, whereas no studies have been performed with native human cells. Interestingly, Weeratna et al. (47) reported that the combination of R848 and CpG-ODN 2006 could not enhance Ab responses in vivo above the levels seen with CpG-ODN alone. The lack of significant additive effects were unexpected, because each agonist acts via different TLRs and has been previously described as a potent vaccine adjuvant (36, 48, 49). In agreement, neither did our study indicate significantly enhanced IFN-{alpha} production after costimulation with CpG-ODN 2006 and R848. pDCs have recently been reported to control TLR7 sensitivity of B cells through type I IFNs (IFN-{alpha} and IFN-beta), indicating an important B cell regulatory loop mediated by IFN-producing pDCs (50).

In addition to the clinical implications, our study may contribute to the development of potentially immunosuppressive/immunoregulatory TLR7 ligands. Our experiments demonstrated for the first time that substimulatory TLR7 ligand concentrations, i.e., TLR7 ligand concentrations that induce very low levels of IFN-{alpha} production, can suppress dose dependently the IFN-{alpha} production by pDCs, indicating a strong interference of TLR9 signaling pathways by TLR7 ligation. Based on these results, we envisage the development of synthetic TLR7 ligands with less intrinsic IFN-{alpha}-inducing capacity and the initiation of further studies investigating the immunoregulatory effects of TLR7/TLR9 costimulation.

The question remains about the molecular mechanism that directs the suppressive effect of TLR7/8 ligands on TLR9 ligand-induced IFN-{alpha} production. Both TLRs belong to the same subfamily and activate DCs via MyD88-dependent signaling pathways (14, 51, 52, 53, 54). Because major molecular differences are currently unknown with respect to TLR7 and TLR9 signaling, our pDC model may facilitate the identification of differential signaling components.

It was suggested that the endosomal localization of different classes of CpG-ODNs is a key factor regulating IFN-{alpha} production. CpG-A-ODNs are located in early TfR-positive endosomes, whereas CpG-B-ODNs are located in lysosome-associated membrane protein 1-positive endosomes in pDCs (40). Their different localization is associated with different functions: CpG-A-ODNs induce high amounts of IFN-{alpha}, whereas CpG-B-ODNs induce maturation of pDCs. CpG-C-ODNs reside in both types of endosomes and induce both IFN-{alpha} and the maturation of pDCs. We hypothesized that TLR7-dependent inhibition of TLR9-induced IFN-{alpha} production may occur by direct interaction of TLR7 with TLR9 ligands and performed experiments with CpG-A, -B, and -C-ODNs. The results indicated that TLR7 ligands inhibit only CpG-A and –C-ODN-induced IFN-{alpha} production. Costimulation with CpG-B-ODN 2006 and TLR7 ligands with or without complexation did not show inhibitory activity of TLR7 ligands. Cocomplexed ssRNA40 and CpG-B-ODN showed a trend to enhance IFN-{alpha} production, whereas IFN-{alpha} induction by complexed CpG-B-ODN was not influenced significantly by costimulation with Loxo (which is unlikely to be complexed by DOTAP). These experiments indicated that TLR7 does not generally dominate TLR9-induced IFN-{alpha} production. Additionally, CpG-ODN class-dependent effects that regulate the interaction of TLR7 and TLR9 signaling are likely. Furthermore, differential uptake or release of TLR7/TLR9 ligands, modulation of TLR7/TLR9 ligand trafficking, and TLR7-induced signaling events that inhibit IRF-7 could account for the observed effects.

In addition to TLR7/TLR9-dependent factors, one might also discuss TLR7-independent molecular effects. Schön et al. (55) recently discovered that the synthetic TLR7/8 ligand imiquimod interacts with the adenosine receptor in a TLR7/8-independent fashion.

In summary, our study reveals for the first time a potent inhibitory effect of TLR7 ligands CpG-A/CpG-C-ODN-induced IFN-{alpha} production by pDCs. The massive inhibition of IFN-{alpha} production could be demonstrated for human and mouse pDCs, and our experiments showed that both natural and synthetic TLR7/8 ligands are able to suppress TLR9-induced IFN-{alpha}. We showed that these effects are mediated via direct interaction of TLR7 ligands with human and mouse pDCs and are unlikely to be related to a kinetic uptake advantage or proapoptotic effect of TLR7 ligands. We are confident these results will significantly stimulate further research of differential TLR7 and TLR9 signaling components, ligand interactions, and may have a direct impact on clinical studies aiming to combine synthetic TLR7 and TLR9 ligands.


   Acknowledgments
 
We thank Christoph Steinschulte (Institute for Clinical Immunology and Transfusion Medicine, Giessen, Germany) for providing the mouse splenocytes.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Bundesministerium für Bildung und Forschung (Germany), Nationales Genomforschungsnetz (NGFN1 IE-S08T03, NGFN2 NIE-S14T30) and by Grant GE-S13T01 (to G.B. and H.H.). Back

2 Address correspondence and reprint requests to Dr. Holger Hackstein, Institute for Clinical Immunology and Transfusion Medicine, Justus-Liebig University, Langhansstrasse 7, Giessen, Germany. E-mail address: Holger.Hackstein{at}immunologie.med.uni-giessen.de Back

3 Abbreviations used in this paper: pDC, plasmacytoid dendritic cell; IRAK, IL-1R-associated kinase; IRF, IFN regulatory factor; ODN, oligodeoxynucleotide; BDCA, blood dendritic cell Ag; DOTAP, N-[1-(2,3-dioeoyloxy)propyl]-N,N,N-trimethylammonium chloride; 7-AAD, 7-aminoactinomycin D; TfR, transferrin receptor; Loxo, loxoribine. Back

Received for publication July 12, 2006. Accepted for publication January 12, 2007.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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