Deconstructing tick saliva: non-protein molecules with potent immunomodulatory properties

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 E(2) (PGE(2) ∼100 nM) with comparable effects in modulating cytokine production by DCs. Both Ado and PGE(2) inhibited cytokine production by inducing cAMP-PKA signaling in DCs. Additionally, both Ado and PGE(2) were able to inhibit expression of CD40 in mature DCs. Finally, flow cytometry analysis revealed that PGE(2), but not Ado, is the differentiation inhibitor of bone marrow-derived DCs. The presence of non-protein molecules adenosine and PGE(2) 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.

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.

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).

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.

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.

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).

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.

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.