Deconstructing Tick Saliva

Abstract

Dendritic cells (DCs) are powerful initiators of innate and adaptive immune responses. Ticks are blood-sucking ectoparasite arthropods that suppress host immunity by secreting immunomodulatory molecules in their saliva. Here, compounds present in Rhipicephalus sanguineus tick saliva with immunomodulatory effects on DC differentiation, cytokine production, and costimulatory molecule expression were identified. R. sanguineus tick saliva inhibited IL-12p40 and TNF-α while potentiating IL-10 cytokine production by bone marrow-derived DCs stimulated by Toll-like receptor-2, -4, and -9 agonists. To identify the molecules responsible for these effects, we fractionated the saliva through microcon filtration and reversed-phase HPLC and tested each fraction for DC maturation. Fractions with proven effects were analyzed by micro-HPLC tandem mass spectrometry or competition ELISA. Thus, we identified for the first time in tick saliva the purine nucleoside adenosine (concentration of ∼110 pmol/μl) as a potent anti-inflammatory salivary inhibitor of DC cytokine production. We also found prostaglandin E₂ (PGE₂ ∼100 nm) with comparable effects in modulating cytokine production by DCs. Both Ado and PGE₂ inhibited cytokine production by inducing cAMP-PKA signaling in DCs. Additionally, both Ado and PGE₂ were able to inhibit expression of CD40 in mature DCs. Finally, flow cytometry analysis revealed that PGE₂, but not Ado, is the differentiation inhibitor of bone marrow-derived DCs. The presence of non-protein molecules adenosine and PGE₂ in tick saliva indicates an important evolutionary mechanism used by ticks to subvert host immune cells and allow them to successfully complete their blood meal and life cycle.

Introduction

Ticks are phylogenetically distant from their hosts, but in general, these ectoparasites have developed, through their evolution, measures for adapting to host defense strategies. The most well studied approaches that ticks employ to evade host responses are the refined mixtures of proteic and non-protein molecules present in their saliva with anticlotting, anti-inflammatory, or immunomodulatory activities. As a result, a number of authors have demonstrated the suppression of cell-mediated and humorally mediated immune responses upon in vitro assays and following experimental models of tick infestations or naturally infested hosts (1–8).

In the last 4 decades, many of these proteic and non-protein molecules have been characterized and their specific functions identified. To date, a diversity of proteic (e.g. chitinases, mucins, ixostatins, cystatins, defensins, hyaluronidases, Kunitz, lectins, and lipocalins) and non-protein molecules (e.g. prostaglandins and endocannabinoids) have been characterized (9–14). Regarding anti-tick immunity, molecules present in tick saliva have been associated with modulation of various steps of host immune responses. For example, sialostatin L and PGE₂² inhibit maturation of dendritic cells (DCs) and prevent antigen presentation (13, 15); DAP-36 and SALP15 inhibit T cell proliferation and activation (16, 17); ISL 929 and ISL 1373 reduce recruitment of neutrophils (18); IgG-binding proteins theoretically decrease antibody functions (3, 19); ISAC, SALP-20, and OmCI inhibit alternative and/or classical pathways of the complement system (20, 21); and EVASIN-1, -3, and -4 bind chemokines and hamper cell migration (22, 23).

Despite the characterization of these molecules, most compounds in tick saliva thought to interfere in numerous other immunologic events described in the literature remain to be identified. In previous works, we have demonstrated the modulatory effects of saliva from Rhipicephalus sanguineus ticks on differentiation, migration, and maturation of DCs (24–26). Despite our wealth of knowledge, the molecules responsible for such effects have not yet been elucidated. In this study, we have identified for the first time the purine nucleoside adenosine (Ado), isolated directly from tick saliva, as an inhibitor of production of pro-inflammatory IL-12p40 and TNF-α cytokines and stimulator of production of anti-inflammatory IL-10 by murine DCs activated with Toll-like receptor (TLR) agonists. We have also identified PGE₂ in R. sanguineus tick saliva, which presented similar effects of Ado on cytokine production by DCs and additionally suppressed the differentiation of DCs from blood cell precursors. Our results also demonstrate that both Ado and PGE₂ exert their modulatory effects on cytokine production by inducing a common cAMP-PKA signaling pathway. Furthermore, both Ado and PGE₂ were able to inhibit expression of CD40 in mature DCs and presented additive effects when administered together. Thus, this study demonstrates the central involvement of tick salivary Ado and PGE₂ in modulation of the host inflammatory/immune responses. Moreover, the data presented here provide important insight to the evolution of host-tick interaction and provide a foundation for future pharmaceutical interventions that target non-protein molecules used by ticks to permit their blood feeding.

EXPERIMENTAL PROCEDURES

Experimental Animals

Female C57BL/6 mice (6–10 weeks old) were purchased from Taconic Farms (Germantown, NY). Mice were bred and maintained at an Association for Assessment and Accreditation of Laboratory Animal Care International accredited facility, NIAID, National Institutes of Health. All experiments with mice were evaluated and approved by the Animal Care and Use Committee of the NIAID, National Institutes of Health. The experiments with dogs were evaluated and approved by the Ethics Committee on Animal Use of the School of Medicine of Ribeirão Preto (USP)/Brazil and are in line with the Guidelines for Animal Users as issued by the National Institute of Health.

Saliva Collection

R. sanguineus ticks were laboratory reared, as described previously by Ferreira and Silva (27). To obtain engorged ticks for saliva collection, dogs (n = 10) were infested with 70 pairs of adult R. sanguineus ticks restricted by plastic feeding chambers fixed to their backs. The saliva collection procedure was performed in partially engorged female ticks (after 5–7 days of feeding) by inoculation of 10 μl of a 0.2% (v/v) solution of dopamine in phosphate-buffered saline, pH 7.4, into the hemocoel using a 12.7 × 0.33-mm needle (BD Biosciences). Saliva was harvested using a micropipette, kept on ice, pooled, filtered through a 0.22-μm pore filter (Costar-Corning Inc., Cambridge, MA), and stored at −70 °C for further use. Saliva protein concentration (∼970 μg/ml) was determined by molecular sieving using absorbance at 280 nm.

Separation of Tick Saliva Fractions by Microcon Filtration

To determine the approximate mass of the active component(s) of R. sanguineus tick saliva, samples were initially fractionated by microcon centrifugal filters (Millipore, Bedford, MA) following the manufacturer's instructions to provide fractions with apparent molecular masses of <5 and 5–100 kDa.

Reagents and Chemicals

Ultrapure Escherichia coli 0111:B4 LPS, Staphylococcus aureus peptidoglycan (PGN), and oligonucleotide CpG-1826 (CpG ODN-1826) were purchased from InvivoGen (San Diego). Ado, Ado deaminase (ADA), H-89 (B1427; PKA inhibitor), PGE₂, and recombinant murine GM-CSF were obtained from Sigma. Doses of each TLR ligand or reagent were determined based on the manufacturer's recommendations and/or our own concentration-response studies (data not shown). Cytokines were determined by OptEIA^TM ELISA sets from BD Biosciences. Antibodies for flow cytometry were also purchased from BD Biosciences.

Generation of Bone Marrow (BM)-derived DCs

DCs were generated according to the method of Inaba et al. (28), with modifications (13). Briefly, BM cells from femurs of C57BL/6 mice were cultured in complete medium (RPMI 1640 medium with 10% heat-inactivated FBS, 2 mm l-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 0.05 mm 2-mercaptoethanol) and 20 ng/ml GM-CSF. At day 0, cells were seeded at 10⁶ per 100-mm Petri dish (Falcon 1029 plates; Discovery Labware) in 10 ml of medium. At days 3 and 6, another 10 ml of complete medium containing 20 ng/ml GM-CSF was added to the plates. The differentiated cells were harvested on days 6–7 of culture for flow cytometry analysis and cytokine production assays.

Flow Cytometry

For experiments on the effect of saliva on DC differentiation, BM-derived DCs were cultured as described above in the presence of the indicated concentrations of tick saliva or saliva fractions and incubated with fluorochrome labeled antibodies. The percentage of CD11c⁺ cells (DC-restricted marker for mice) and CD11b⁺ cells (a myeloid cell marker) was evaluated.

In another set of experiments described below, DCs were also collected to evaluate the expression of costimulatory and stimulatory molecule expression. To that end, cells were stained with fluorochrome-labeled antibodies against CD11c, CD40, CD86, and MHC class II (I-A/I-E) molecules. Data were acquired using a FACSCalibur (BD Immunocytometry Systems) with CellQuest (BD Biosciences) and analyzed with FlowJo software (Tree Star Inc., Ashland, OR).

Cytokine Production Assay

Six-7-day cultured BM-derived DCs were gently collected, washed twice, and resuspended at 10⁶ cells/ml in complete medium. Cells were then cultured at 10⁵ cells/well in round-bottom 96-well cluster plates (Costar, Cambridge, MA) and incubated, depending on the experiments, with medium, saliva, saliva filtrates, or saliva fractions for 30 min before addition of TLR-2 (PGN, 10 μg/ml), TLR-4 (LPS, 100 ng/ml), or TLR-9 (CpG ODN-1826, 0.15 μm) ligands. Following overnight incubation (18 h) at 37 °C and 5% CO₂, cell-free supernatants were collected and levels of IL-12p40, TNF-α, and IL-10 determined.

HPLC Procedures

R. sanguineus saliva (1.5 ml) was centrifuged through a YM-5 microcon device (Millipore, Bedford, MA). The filtered <5-kDa fraction was submitted to a reversed-phase HPLC on a C18 column (4.3 × 150 mm; ThermoSeparation Products, Riviera Beach, FL) perfused at 0.5 ml/min using a CM-4100 pump (Thermo Separation Products, Riviera Beach, FL). The eluent was monitored at 220–500 nm using a diode array detector (model SPD M10AV, Shimadzu, Columbia, MD) A gradient of 80 min duration from 5 to 80% acetonitrile in water, containing 0.1% trifluoroacetic acid, was imposed after injection of the sample. Aliquots of these fractions were dried in 96-well plates and tested for cytokine inhibitory activity and costimulatory and stimulatory molecule expression on DCs at dilutions compatible for those employed for crude saliva.

Mass Spectrometry

Selected fractions from the reversed-phase HPLC experiment were analyzed by mass spectrometry by direct injection (5 μl) of the fractions into a Thermo-Finnigan LCQ Deca XP ion trap mass spectrometer, through an ion-spray interface supplied with a continuous flow of 50% methanol in water containing 0.1% acetic acid. Fragmentation of selected ions was performed with 30% maximum intensity. Calibration curves using commercial adenosine were performed to estimate salivary adenosine concentration.

PGE₂ Concentration in Saliva, Filtrate, and Fractions

The amount of PGE₂ in saliva samples as well as its concentration in the filtrate and fractions used for cell culture were determined by competition ELISA kit (R&D Systems, Minneapolis, MN), according to manufacturer's instructions. The detection limit for the assay was (∼42 pm).

Ado Determination in <5-kDa Filtered Saliva Fractions

The amount of Ado in saliva fractions used for cell culture was determined by mass spectrometry of the molecular ions present in fraction 11 (F11). To further confirm functional activity of Ado in the saliva, we also carried out experiments incubating the saliva or saliva fractions with ADA (3.0 units for 1 h at 25 °C) and testing each one for maturation of DCs.

Measurement of cAMP in DCs

DCs exposed to saliva or saliva fractions were used to measure production of intracellular levels of cAMP. Cells (10⁶) were seeded in 96-well round-bottom tissue culture plates and treated with saliva or saliva fractions for 15 min. Cells were then lysed with 0.1 m HCl, 0.1% Triton X-100, and intracellular cAMP was measured by commercial enzyme immunoassay kits (Cayman Chemicals, Ann Arbor, MI) according to the manufacturer's instructions.

Data Analysis

Data are shown as mean ± S.E. Statistical differences were analyzed by analysis of variance followed by Tukey-Kramer post-hoc analysis test (INSTAT software; GraphPad, San Diego). A p value of 0.05 or less was considered statistically significant.

RESULTS

R. sanguineus Tick Saliva Inhibits Production of IL-12p40 and TNF-α while Increasing IL-10 Cytokine Production by DCs Triggered by Different TLR Agonists

We first tested whether R. sanguineus tick saliva could modulate cytokine production by DCs matured with PGN, LPS, and CpG (TLR-2, -4, and -9 agonists, respectively). As expected, treatment of DCs for 18 h with these TLR agonists increased the levels of IL-12p40 and TNF-α (Fig. 1, A and B); however, when cell cultures were preincubated with whole saliva (dilution 1:20 v/v) for 30 min, significant inhibition (p < 0.05) of PGN-, LPS-, or CpG-induced IL-12p40 and TNF-α production by DCs was seen (Fig. 1, A and B). Conversely, preincubation with saliva (1:20 v/v) enhanced PGN-, LPS-, or CpG-induced IL-10 production by these cells (Fig. 1C) (p < 0.05). Levels of IL-12p40, TNF-α, and IL-10 were not changed in cultures incubated with saliva alone, suggesting that saliva lacks either contaminants or TLR ligands (Fig. 1).

FIGURE 1.

Tick saliva modulates cytokine production induced by diverse Toll-like ligands by use of molecules with molecular mass of <5 kDa. DCs from C57BL/6 mice were produced from BM cells cultured with GM-CSF (20 ng/ml) for 6–7 days. Next, cells were washed and preincubated with medium (−), saliva (Sal) (dilution 1:20) (A–C), or saliva (1:20) and saliva filtrates (Filt) (<5 or 5–100 kDa (1:20)) (D–F). After 30 min, cells were stimulated overnight with TLR-2 (PGN; 10 μg/ml), TLR-4 (LPS, 100 ng/ml), and TLR-9 (CpG, 150 nm) ligands. After 18 h of incubation, cytokine levels in culture supernatants were measured by ELISA. The results are expressed as the means ± S.E. obtained from one of three independent experiments performed in triplicate (n = 3). *, p < 0.05 versus respective LPS, CpG, and PGN groups without saliva or saliva filtrates.

To determine the closest molecular weight of the component(s) in the saliva responsible for modulation of cytokine production in DCs, 500 μl of saliva were sequentially centrifuged through microcon devices. The filtrate (equivalent volume to saliva diluted 1:20) with a molecular mass lower than 5 kDa or between 5 and 100 kDa was tested in the LPS-induced cytokine production assay (Fig. 1, D–F). The data indicate that when LPS-treated DCs were preincubated with the fraction of saliva containing molecules with a molecular mass lower than 5 kDa, their competence to produce IL-12p40 and TNF-α cytokines was repressed (p < 0.05). The inhibition was similar to that seen with nonfractionated saliva (Fig. 1, D and E). Saliva fractions containing molecules with molecular mass lower than 5 kDa also additively up-regulated production of IL-10 by LPS-treated DCs (Fig. 1F). Fractions containing molecules with molecular mass between 5 and 100 kDa did not modify production of these cytokines (Fig. 1, D–F).

Analysis of Tick Saliva Fractionated by Reversed-phase HPLC Indicates That It Contains Two Active Components Lower than 5 kDa

To identify the component(s) responsible for the effects of tick saliva on DCs, we further fractionated 1.5 ml of YM-5 filtrate by reversed-phase HPLC and tested the fractions for production of the cytokines in LPS-treated DCs. Fig. 2A depicts an HPLC fingerprint of the 80 fractions <5-kDa saliva filtrate at the absorbance of 220 nm. The 80 fractions were then assembled into 10 pools (containing eight fractions each) that were tested independently for production of IL-12p40 and TNF-α on DCs stimulated with LPS. When DCs were preincubated for 30 min with pools 2 (containing fractions 9–16) and 7 (containing fractions 49–56), but not the remaining pools, LPS-induced IL-12p40 and TNF-α production was diminished (Fig. 2, B and C). Next, we tested individually each of the fractions of pools 2 and 7 in a similar assay. As shown in Fig. 2 (D–G, respectively), only fractions 11 (F11, from pool 2) and 51 (F51, from pool 7) presented a positive inhibitory effect on production of IL-12p40 and TNF-α by DCs.

FIGURE 2.

Tick saliva contains two fractions with molecular mass of <5 kDa that inhibit IL-12p40 and TNF-α. DCs from C57BL/6 mice were produced from BM cells cultured with GM-CSF (20 ng/ml) for 6–7 days. To isolate the molecules from the 5-kDa saliva filtrate related to DC modulation, saliva was filtered using a YM-5 (cutoff 5,000 Da) membrane, and the filtrate was fractionated in 80 fractions by reversed-phase HPLC using the conditions described under “Experimental Procedures.” An HPLC of the 5-kDa filtrate at 220 nm is demonstrated in A. B and C demonstrate by ELISA the production of IL-12p40 and TNF-α from the supernatant of DCs that were preincubated with different pools (pools containing eight fractions each, according to the eluting time, successively) from the separated filtrate and 30 min later stimulated for 18 h with LPS (100 ng/ml). D–G show production of IL-12p40 and TNF-α from the supernatant of DCs preincubated with fractions 9–16 (D and E) and 49–56 (F and G), for 30 min and subsequently stimulated for 18 h with LPS (100 ng/ml). Arrows indicate the pools and the isolated fractions with strongest inhibitory effect for each assay. Results are expressed as the mean ± S.E. obtained from one of two independent experiments performed in triplicate (n = 3).

Ado (F11) and PGE₂ (F51) Are the Main Modulators of IL-12p40, TNF-α, and IL-10 Cytokine Production in R. sanguineus Saliva

To evaluate the chemical composition of the molecules present in the two subfractions (F11 and F51) with positive inhibitory effects on DCs, we employed different assays. For F11, we used a combination of two different apparatus as follows: molecule absorbance analysis and reversed-phase HPLC MS/MS. Absorbance measurement of F11, but not F10 or F12, showed a high concentration of molecules with 258 nm of absorbance, suggestive of the presence of adenosine nucleosides (Fig. 3A). The mass spectra of the molecular ions present in F11 were informative and analogous to Ado (Fig. 3B). Fragmentation of the 268 mass ion leads to production of an ion having m/z 136, as expected for adenine (Fig. 3C) (55). Finally, MS showed the presence of ∼110 pmol/μl of Ado in F11 (Fig. 3D).

FIGURE 3.

Ado (F11) and PGE₂ (F51) are the major modulators of R. sanguineus tick saliva. UV spectrum of fraction F11 as well as the adjacent fractions (F10 and F12) is demonstrated (A). Mass spectrogram of F11 (B). Mass spectrogram derived from the fragmentation of the m/z ion 268 with 30% maximum collision intensity (C). The concentration of the molecule present in F11 was also determined by MS (D). The concentration of the molecule present on F51 was measured using a standard commercial ELISA kit and compared with the concentration obtained with a given dilution of saliva (Sal), YM-5 saliva filtrate (Filt 0–5 kDa), and fractions 10–12 and the 51 adjacent fractions (50 and 52) (E). ND, not detected.

It has been demonstrated by Sá-Nunes et al. (13) that PGE₂ was present at the end of the gradient of a micro-HPLC from Ixodes scapularis saliva. Because this eicosanoid had already been described in the saliva of R. sanguineus ticks (28), as well as in the saliva of many other tick species (29–31), we asked whether F51 from R. sanguineus tick saliva, also found at the end of a micro-HPLC gradient, could be PGE₂. Using a PGE₂-specific competition ELISA, we found that saliva, <5-kDa saliva filtrate, and F51 indeed contained PGE₂ and that the concentration of both samples was ∼100 nm (Fig. 3E).

F11 Loses Activity on DCs When Exposed to the Enzyme ADA

To evaluate whether the Ado identified in F11 would have similar activity to synthetic Ado, we treated saliva or F11 with ADA, which degrades Ado into inosine, and tested it on DCs. As shown previously, R. sanguineus saliva (dilution 1:20) presented a potent inhibitory effect on LPS-induced production of IL-12p40 and TNF-α by DCs. When this saliva was treated with ADA, its effects were partially impaired (p < 0.05) (Fig. 4, A and B). Furthermore, when F11 was treated with ADA, this fraction entirely lost its cytokine modulating outcome (p < 0.05) (Fig. 4, A and B). Intriguingly, although F11 treated with ADA also lost its ability to enhance LPS-induced production of IL-10 (p < 0.05), this did not happen to ADA-treated saliva (Fig. 4C). The rationale for this is unknown; however, as saliva also contains PGE₂, possibly this component alone is enough to sustain production of IL-10 independently of Ado. Indeed, when DCs were precultured with F51 (which contains PGE₂), a similar enhancement of IL-10 production induced by LPS was seen in comparison with DCs precultured with total saliva (Fig. 4C). As expected, ADA treatment did not alter the effect of F51 (which contains PGE₂, not Ado) on production of cytokines by LPS-stimulated DCs (Fig. 4, A and B).

FIGURE 4.

Molecules from F11 lose activity when exposed to the enzyme ADA. DCs from C57BL/6 mice were produced from BM cells cultured with GM-CSF (20 ng/ml) for 6–7 days. Saliva (Sal), F11, or F51 (all diluted 1:20) were exposed or not with ADA (3.0 units) for 1 h and subsequently added to the DC culture. Thirty minutes later, cells were stimulated with LPS (100 ng/ml). After 18 h of incubation with LPS, cytokine levels in culture supernatants were measured by specific ELISA for IL-12p40 (A), TNF-α (B), and IL-10 (C) according to manufacturer's instructions. *, p < 0.05 versus LPS group without saliva or saliva fractions; &, p < 0.05 versus LPS + saliva; #, p < 0.05 versus LPS + F11.

It has been shown that inosine is able to modulate the effect of IL-12 and TNF-α of murine macrophages and splenic cells (32). To prove that inosine generated in the above experiment by the action of Ado degradation does not exert modulatory effects on murine DCs, we pretreated DCs with inosine and stimulated them overnight with LPS. The results showed that even at a concentration of 100 μm, standard inosine was not able to modulate production of IL-12 and TNF-α by murine LPS-matured DCs (data not shown).

F11 and F51 from R. sanguineus Saliva Cooperate in the Amplification of cAMP-PKA Signaling

Because initiation of the signaling pathway used by Ado and PGE₂ to modulate cytokine is via production of cAMP (33, 34), we evaluated whether F11 and F51 from R. sanguineus tick saliva increases production of cAMP in DCs. As depicted in Fig. 5A, when F11 or F51 was added separately, it induced a significant (p < 0.05) production of cAMP. Furthermore, when added together, they showed an additive effect, comparable with whole saliva, on the amplification of cAMP (Fig. 5A). As a positive control, DCs were also incubated with forskolin, a drug known to induce cAMP production in different cell types, and massive production (p < 0.05) of cAMP was seen (Fig. 5A).

FIGURE 5.

F11 and F51 have complementary immunosuppressive effects on DCs by amplification of the production of cAMP and activation of the enzyme PKA. DCs were harvested on day 6–7 of culture, washed with phosphate-buffered saline, and placed in RPMI 1640 fresh culture medium. For evaluation of cAMP production, DCs were incubated for 15 min with medium (−), F11, F51, F11 + F51, saliva (all diluted 1:20), or forskolin (positive control), and the cAMP levels were measured by competition ELISA according to manufacturers' instructions (A). *, p < 0.05 versus medium alone; &, p < 0.05 versus F11 or F51; #, p < 0.05 versus F11, F51, F11 + F51, or saliva. To evaluate the effect of F11 and F51 on activation of the enzyme PKA, DCs were exposed to H-89 (inhibitor of PKA; 3 μm) for 45 min and subsequently incubated with F11, F51, or F11 + F51 for 30 min, after which DCs were stimulated with medium (−) or LPS (100 ng/ml). After 18 h of incubation with LPS, cytokine levels in culture supernatants were measured by specific ELISA for IL-12p40 (B), TNF-α (C), and IL-10 (D) according to the manufacturer's instructions. *, p < 0.05 versus LPS group without F11, F51, or F11 + F51; &, p < 0.05 versus LPS + F11, LPS + F51, and LPS + F11 + F51. The results are expressed as the mean ± S.E. obtained from one of two independent experiments performed in triplicate (n = 3).

A downstream intracellular molecule activated by cAMP is protein kinase A (PKA), an essential enzyme for cell signaling, that among other effects plays an important role in modulation of cytokines induced by both Ado and PGE₂ (33–35). Having shown that F11 and F51 induce cAMP production, we further evaluated whether these fractions had their immunosuppressive effects blocked by H-89, a PKA inhibitor. To answer this question, DCs were treated with H-89 (3 μm), and the ability of F11 and F51 to modulate LPS-induced IL-12p40, TNF-α, and IL-10 cytokine production was tested. Our results demonstrate that H-89 reversed significantly (p < 0.05) but not completely the inhibitory effect of F11 and F51 (Fig. 5, B–D). When DCs were preincubated with H-89, the competence of F11 or F51 alone or F11 plus F51 to inhibit IL-12p40 and TNF-α production stimulated by LPS was significantly reversed (Fig. 5, B and C). Furthermore, production of IL-10 induced by F11 or F51 in the presence of LPS was completely restored after H-89 treatment, whereas IL-10 production induced by F11 plus F51 was restored only partially (Fig. 5D). No difference in cytokine production was seen when H-89 was added alone to DCs stimulated by LPS (data not shown). These results suggest F11 and F51 modulate only partially LPS-induced cytokine production in DCs by cooperating in amplification of cAMP-PKA signaling.

F11 and F51 Inhibit LPS-induced CD40 Expression in DCs

Previous results published by our group demonstrated that saliva from R. sanguineus ticks inhibits expression of CD40 and CD86 molecules on the surface of murine BM-derived DCs (24). To evaluate whether PGE₂ and/or Ado are responsible for these effects, BM-derived DCs were stimulated with LPS in the presence of medium, whole saliva (1:20), <5- and 5–100-kDa filtrates, F11, F51, or F11 plus F51, and the percentage and medium intensity of expression of CD40 and CD86 in CD11c⁺/I-A/I-E⁺ DCs were analyzed by flow cytometry. Following treatment with purified TLR agonists PGN (TLR2 ligand), LPS (TLR4 ligand), or CpG 1826 (TLR9 ligand), CD11c⁺/I-A/I-E⁺ DCs increased the intensity of expression of CD40 and CD86 (Fig. 6, A and B). When DC cultures were preincubated with whole saliva for 30 min at 1:20 dilution, significant inhibition (p < 0.05) of the percentage of PGN-, LPS-, or CpG-induced CD40 (Fig. 6A) and CD86 (Fig. 6B) expression was observed. Preincubation with the filtrate of 0–5 kDa but not 5–100 kDa also inhibited LPS-induced expression of CD40 and CD86 (Fig. 6, C and D).

FIGURE 6.

F11 and F51 cooperate in inhibition of CD40 expression in LPS-matured DCs. DCs from C57BL/6 mice were produced from BM cells cultured with GM-CSF (20 ng/ml) for 6–7 days. Next, cells were washed and preincubated with medium (−), saliva (Sal), saliva filtrates (Filt) (filtrate 0–5 kDa and filtrate 5–100 kDa), F11, F51, or F11 + F51 (all diluted 1:20). After 30 min, cells were stimulated by 18 h with TLR-2 (PGN; 10 μg/ml), TLR-4 (LPS; 100 ng/ml), or TLR-9 (CpG; 150 nm) agonists, depending on the experiment. CD11c⁺/I-A/I-E⁺ cells were gated for expression of CD40 and CD86 on their surface. It was demonstrated that saliva inhibits expression of CD40 and CD86 in PGN-, LPS-, and CpG-stimulated DCs (A and B). C and D show that inhibition of CD40 and CD86 in CD11c⁺/I-A/I-E⁺ DCs is mediated by molecule(s) presents on the filtrate lower than 5 kDa. Results are expressed as the mean ± S.E. obtained from one of two independent experiments performed in triplicate (n = 3 per group). Representative dot plots of each treatment (medium, LPS, LPS + F11, LPS + F51, or LPS + F11 + F51) are also shown (E). *, p < 0.05 versus LPS, CpG, and PGN groups without saliva or saliva filtrates.

To further investigate whether the effects of saliva or filtrate are mediated by F11, F51, or both, DCs were preincubated with F11, F51, or F11 plus F51 for 30 min and subsequently stimulated with LPS, and expression of CD40 and CD86 molecules was evaluated 18 h later. As shown in Fig. 6E, preincubation with F11 or F51 inhibited (p < 0.05) LPS-induced expression of CD40. Moreover, F11 and F51 demonstrated an additive inhibitory effect (Fig. 6E). However, F11, F51, or F11 plus F51 did not affect expression of CD86 molecules (data not shown). We attempted to analyze the different fractions from the filtrate of 0–5 kDa on CD86 expression, but following F11 and F51, no fractions were found with those properties (data not shown). These findings suggest that molecules present in saliva or the 0–5-kDa filtrate only suppress LPS-induced CD86 expression when these compounds act together on DCs. Further studies must be done to characterize which molecules can account for inhibition of expression of CD86 by saliva.

F51 Impairs Differentiation of BM-derived DCs

A previous work demonstrated that saliva from R. sanguineus ticks inhibits differentiation of DCs (24). To determine whether saliva-impaired differentiation of DCs was caused by the presence of saliva Ado, PGE₂, or both, BM-derived DCs were developed with GM-CSF in the presence of saliva, F11, F51, or both. Within 6–7 days of differentiation, the percentage of CD11c⁺ CD11b⁺ was determined by flow cytometry. Fig. 7, A and B, show that R. sanguineus saliva significantly inhibited differentiation of BM precursors into CD11c⁺/CD11b⁺ DCs (p < 0.05). Similarly to saliva, when F51 was added to cultures there was a significant inhibition of DC differentiation (p < 0.05). F11 alone or together to F51 did not impair DC differentiation (Fig. 7, A and B). More important, even when tested in a high concentration (100 μm), F11 did not hamper DC differentiation (data not shown).

FIGURE 7.

F51 from tick saliva inhibits differentiation of BM-derived DCs. BM-derived cells from C57BL/6 mice were cultured with GM-CSF (20 ng/ml) in the presence of tick saliva, F11, F51, or F11 + F51 as indicated. Cells were harvested on day 6–7, labeled with the designated monoclonal antibodies, and analyzed by flow cytometry. A, representative dot plots for CD11c and CD11b markers, in 7-day differentiated cells, are demonstrated. B shows the mean percentage ± S.E. of CD11c⁺ CD11b⁺ cells 7 days post-differentiation. The data are representative of two independent experiments. *, p < 0.05 compared with cells cultured with medium only.

DISCUSSION

The discovery of new molecules produced by parasites has become an important venue of many researchers who are trying to comprehend host-parasite interactions. This tendency is growing because information about the biological activities, structure, ligand-binding affinities, and concentration of these molecules opens to a greater extent the array of possibilities for finding out how these organisms evade hemostatic, inflammatory, and immunologic host responses.

Our group has worked to identify the molecules present in the saliva of the dog tick R. sanguineus with modulatory effects on diverse immune cells. Here, we identify for the first time in tick saliva the nucleoside Ado as a potent immunosuppressor of DCs. This study also demonstrates PGE₂ from saliva of R. sanguineus ticks with comparable modulatory effects. Our data demonstrate clearly that both Ado and PGE₂ cooperate in modulating the production of IL-12p40, TNF-α, and IL-10 cytokines and inhibiting expression of the stimulatory molecule CD40 on DCs activated with TLR agonists. The additive effect induced by Ado and PGE₂ probably results from the capacity of both molecules to cooperate in induction of cAMP-PKA signaling in DCs.

We demonstrated that salivary molecules from R. sanguineus ticks with a molecular mass below 5 kDa down-regulates production of IL-12p40 and TNF-α and up-regulates production of IL-10 by DCs stimulated with TLR-2, -4, and -9 agonists. These findings are of great interest, as IL-12p40 and TNF-α can induce functional maturation of DCs and could selectively stimulate their ability to induce Th1-type responses against ticks and tick-borne pathogens, although IL-10 is well known as an immunosuppressive cytokine (36–38).

Using microcon filtration and reversed-phase HPLC MS/MS, we found the nucleoside Ado in the saliva of R. sanguineus ticks. The saliva fraction that contained Ado presented a relevant effect on the biology of DCs, which may have important implications for our understanding of tick-host interactions. It is known that Ado is an endogenous purine nucleoside that modulates a wide variety of immunologic functions in antigen-presenting cells (39–42). It impairs TNF-α- and IL-12p40-mediated responses to LPS in human and murine DCs while enhancing production of IL-10 (43, 44). Ado also suppresses production of IL-12 and TNF-α in monocytes and macrophages (33, 45–46). In agreement with our results, others have shown that Ado-induced inhibition of TNF-α production by macrophages is not restricted to TLR-4-mediated activation, because Ado also down-modulates TNF-α production when cells are activated by TLR-2, -3, -7, and -9 agonists (47).

Besides blocking LPS-induced TNF-α production, Ado markedly induces heme oxygenase 1 (HO-1) in inflamed tissues (48). This is noteworthy because ticks are blood feeders and abundant quantities of heme, a potent pro-oxidant and pro-inflammatory agent, are probably released at the tick feeding site. HO-1 removes heme and, furthermore, generates three metabolites, carbon monoxide (CO), ferrous iron, and biliverdin, all of which have an immune protective effect (49). A positive feedback loop exists among Ado, HO-1, and CO and resolves the inflammatory response (48). Blocking of HO-1 by RNA interference abrogates the effects of adenosine on TNF-α and HO-1 in macrophages. Another venue in which tick salivary Ado may disrupt host defenses is the migration of phagocytic cells to the site of tissue damage caused by tick feeding. Signaling by means of purinergic receptors has been recently shown to be essential for chemotaxis of macrophages in a gradient of C5a (50). This requires sequential hydrolysis of nucleotides ending in Ado, and an excess of Ado provided by tick saliva may disrupt the sequence of events resulting in correct chemotactic navigation.

Components from tick saliva and salivary glands, including Ado, inhibit the biology of not only DCs but that of many other immune cells including neutrophils, mast cells, NK cells, and T and B cells (42, 51–55). This can be particularly relevant, as the tick Ado identified in this work could be responsible for the effects described above. Further studies must be performed to test this hypothesis. Furthermore, Ado possibly has an important role in evolution as an evasion mechanism for blood-sucking parasites. Indeed, salivary glands of other classes of hematophagous arthropods, notably sand flies Phlebotomus argentipes and Phlebotomus papatasi (55, 56), also produce Ado; however, it has not yet been tested for its immunomodulatory properties.

We found another fraction of R. sanguineus tick saliva that had a modulatory effect on cytokine production by DCs. Using a competition ELISA, we ascertained that this fraction contained PGE₂ in a considerable amount. Similarly to data described here, saliva of many tick species has been shown to contain PGE₂; moreover, it has been associated with the saliva effect on suppression of host immune responses (13, 57–58). As we reported with PGE₂ of the saliva, synthetic PGE₂ inhibits the ability of monocytes, macrophages, and DCs to secrete cytokines such as IL-12 and TNF-α (59–63) and may shift the balance in favor of a Th2 immune response. PGE₂ can also act directly on T cells by impairing production of IL-2 and IFN-γ (64), which also could induce a Th2-type response. In support of our findings, PGE₂ isolated from I. scapularis saliva is the major inhibitor of IL-12 and TNF-α induced by LPS-stimulated DCs (13). However, PGE₂ concentrations in I. scapularis saliva were 10–100-fold higher than that found in our study.³ This might explain why R. sanguineus saliva needs an additional molecule, Ado, to reach modulatory activities in pharmacological levels.

Related to Ado- and PGE₂-induced intracellular cascade, we observed as expected that both molecules in the saliva fractions were able to induce production of cAMP by DCs; furthermore, when combined, they showed an additive effect. Interestingly, Ado or PGE₂ used individually or in combination lose their modulatory activity when DCs are preincubated with H-89, a PKA inhibitor. This result is important because we demonstrate that both molecules affect production of cytokines by DCs by a common signal transduction pathway, linking increased production of cAMP and PKA activation. At the same line, it was demonstrated that Ado and PGE₂ produced by human regulatory T cells have additive effects in down-regulating functions of immune cells through intracellular cAMP-PKA signaling (65). It is essential to mention that the treatment of DCs with the PKA inhibitor did not restore completely the production of IL-12p40 and TNF-α, suggesting that Ado and PGE₂ may modulate DCs via signaling pathways other than cAMP-PKA. Our data are in accordance with those of Vassiliou et al. (60), who reported that inhibition of LPS-stimulated TNF-α production in murine DCs by PGE₂ was only partially prevented by the PKA inhibitor H-89. In support of this possibility, a recent work has revealed that alternative cAMP-binding targets, such as the guanine nucleotide exchange protein (Epac-1), can be also responsible for inhibition of TNF-α cytokine production in DCs (66).

Enhancement of costimulatory and stimulatory molecules on the surface of DCs is fundamental to induce a proper T cell response. Here, we demonstrate that tick saliva suppressed expression of CD40 and CD86 in DCs. Blocking binding of CD40 with its ligand CD40L (CD154) prevents T cell-dependent antibody production and reduces CD4⁺ T cell priming and expansion, which can impair the development of a protective host response to parasitic infections (67–70). Indeed, it was shown that the blockage of CD40-CD154 and CD86-CD28 interactions could result in T cell anergy and high IL-10 production (71). This aspect is relevant because the modulatory effect on cytokine production, together with reduction in expression of CD40 and CD86, could be an important “double-escape” mechanism used by ticks to induce regulatory T cells, T cell anergy, or to inhibit antigen presentation. These host conditions could probably help blood-feeding arthropods to complete their meal and, moreover, could facilitate transmission of vector-borne pathogens during the parasitic stage. Interestingly, only CD40 but not CD86 expression was down-regulated by saliva fractions containing Ado and/or PGE₂. The explanation for why crude tick saliva and saliva filtrate impair CD86 expression but saliva fractions individually do not is unknown. Possibly a combination of saliva fractions is required. Additional studies must be done to identify these molecules.

Finally, we showed that PGE₂ from saliva dramatically inhibits differentiation of BM-derived DCs. These results are in accordance with previous results showing that PGE₂ inhibits differentiation of murine and human DCs (72, 73). The effect of R. sanguineus saliva in inhibiting differentiation of DCs was previously demonstrated by Cavassani et al. (24); however, only now has the molecule responsible for this effect been identified. Saliva fractions that contain Ado showed no effect on DC differentiation, possibly explained by work demonstrating that Ado is related to macrophage inhibition and DC differentiation in human (74, 75) but not in murine (76) cells. Modulation of BM cells by PGE₂ during differentiation to DCs is vitally important because in addition to inhibiting of the number of DCs differentiated, the DCs differentiated in the presence of PGE₂ produce high levels of anti-inflammatory cytokines and induce a Th2-type immune response (76), a phenotype associated with immune suppression and tolerance to ticks.

In conclusion, we describe for the first time Ado as one of the most important immunomodulatory molecules identified to date in a tick saliva. This molecule, produced and secreted in high concentration, is able to modulate almost all cells of the immune system (42), in addition to being vasodilatory and inhibiting platelet aggregation (77, 78). Moreover, we also found PGE₂ in R. sanguineus saliva, which combined with Ado may augment anti-inflammatory and immunologic responses of their hosts. It is important to highlight that both salivary Ado and PGE₂ are non-protein endogenous small host molecules, and accordingly no host immune responses can be directed against them. Taken together, our results support the concept that the biological activities of Ado and PGE₂ counteract most of the host defenses and should help the tick to feed and reproduce.