Brieﬂy, femurs and tibias were ﬂushed with RPMI-1640(Gibco-BRL Life Technologies, Grand Island, NY) to releasetheBMcellsthatwereculturedin24-well-cultureplatesinRPMI supplemented with 10% heat-inactivated fetal calf serum (FCS), 100
g/mL of penicillin, 100
g/mL of streptomycin, 5
M 2-mercaptoethanol (all fromSigma) plus murine granulocyte-macrophage colony-stimulating factor (GM-CSF) (30ng/mL) and IL-4 (10ng/mL) (Peprotech, Rocky Hill, NJ). On days 3 and 6 thesupernatants were gently removed and replaced with thesamevolumeofsupplementedmedium.Onday9,thenon-adherent cells were removed and analyzed by ﬂowcytometry using DCs surface markers, given that morethan 80% of cells expressed CD11c.
2.5. Measurement of the expression of DC surface markers
To evaluate the effect of tick saliva on activation of antigen-presenting cells and TLR surface expression, DCswere exposed for 24h to tick saliva (1:20) in the presenceof TLR-2 and TLR-4 ligands (lipoteicoic acid (LTA) andlipopolysacaride (LPS), respectively). After incubation,culture media was carefully collected and stored at
C for subsequent cytokine measurements and theDCs were collected for ﬂow cytometric analysis. Afterincubating with anti-CD16/32 (Fc block) for 35min on ice,DCswerewashedwithPBSandculturedwiththefollowingantibodies: FITC-conjugated anti-CD11c or -CD40 mAb,PE-conjugated anti-MHC II, -CD80, -CD86, TLR-2 or -TLR-4antibodies for 45min. After washing twice in PBS,cytometric and ﬂuorescence data were acquired. Inaddition, the effect of tick saliva alone (1:20) on TLR-2and TLR-4 expression was evaluated in 16, 24 and 48hafter incubation.
2.6. Cytokine assays
Nine-day cultured BM-derived DCs were gently col-lected, washed twice, and resuspended at 10
cells/mL incomplete medium. Cells were seeded at 10
cells/well in24-well cluster plates (Costar, Corning Glass) and incu-bated with medium, saliva (1:20), TLR-4 (LPS, 1
g/mL),TLR-2 (LTA, 10
g/mL), TLR-3 (Poly I:C, 5
g/mL), TLR-5(ﬂagellin, 1.5
g/mL), TLR-9 (CPG-ODN 1826, 10
g/mL)ligandswithorwithoutticksaliva(1:20).Measurementsof IL-12p40, IL-12p70, TNF-
, IL-1, IL-10 and IL-6 wereperformed using speciﬁc solid-phase sandwich enzyme-linkedimmunosorbentassay(ELISA).BDOptEIAELISAsetswere used according to manufacturer’s instructions (BDBiosciences). None of the tested samples were thawedmore than once.
2.7. Determination of phosphorylated forms of ERK and p38MAPKs by Western blot analysis
Activated forms of ERK 1/2 (p42/44) and p38 MAPKsweredetectedbyWesternblotanalysisusingantibodiestothe phosphorylated forms of these kinases. Extracts fromuntreated cells (controls) and from cells cultured withsaliva, LPS or LPS plus saliva (15, 30 or 60min of incubation) were made by sonication in ice-cold RIPAbuffer (150mM NaCl, 0.1mM Tris–HCl, pH 7.2, 1% TritonX-100, 1% sodium deoxycholate, 0.1% SDS, 5mM EDTA)containing a mixture of protease inhibitors and thephosphatase inhibitor sodium orthovanadate (Sigma,1mM). For Western blot analysis, 30
g cell extractproteins were electrophoresed in a SDS polyacrylamidegel12%andelectroblottedontoanitrocelluloseﬁlter.Blotswere blocked overnight at 4
C in PBS containing 0.2%Tween 20 and 5% low fat milk. They were then incubatedovernight at 4
C with rabbit polyclonal antibodiesrecognizing either the phosphorylated or unphosphory-lated forms of ERK or p38. Blots were then washed ﬁvetimes and incubated for 1h at room temperature withhorseradish peroxidase-conjugated goat anti-rabbit IgG(JacksonImmunoResearchLaboratories,Inc.,WestGroove,PA). After further washing, the blots were developed withthe Enhanced Chemiluminescence’s Detection ECL-kit(Pierce, Thermo Fisher Scientiﬁc Inc., Rockford, IL). Bandsof ERK MAPK or p38 MAPK were visualized after exposingthe blots to a Kodak RX ﬁlm.
Signiﬁcant differences between saliva-treated andcontrol groups were determined by analysis of variance(ANOVA) followed by post hoc analysis with the Tukey–Kramer test (INSTAT software; GraphPad, San Diego, CA).
3.1. Saliva from R. sanguineus ticks induces production of IL-10 and inhibits production of IL-12p70 and TNF-
in DCsstimulated with LPS
In order to carry out their main role of initiating aprimary immune response, immature DCs must bestimulated for maturation and a widely used stimulusthat induces maturation of these cells is bacterial LPS. Toevaluate the effect of tick saliva on LPS-matured DCs, weexposed BM-derived DCs to LPS (1
g/mL) for 24h in thepresenceorabsenceofticksaliva(1:20),andmeasuredtheproduction of IL-12 p40 and p70, IL-10, IL-1, IL-6 and TNF-
by ELISA. As shown inFig. 1, saliva led to a decrease of 36.1, 63.6 and 26.2% in the production of IL-12 p40, IL-12p70andTNF-
0.01,respectively,comparedtoLPSonly).As previously published (Harizi et al., 2002), anti-inﬂammatory cytokines such as IL-10 are also producedin low quantities by DCs stimulated with LPS. However,whenticksalivawasadded,theeffectofLPSininducingIL-10 production was enhanced by 72.9% (
0.01) (Fig. 1D).Interestingly, tick saliva did not modulate the productionof the LPS-induced IL-1 and IL-6 cytokines (Fig. 1E and F).DCsculturedinthepresenceofsalivaalonedidnotpresenta signiﬁcant difference in the production of any cytokine,when compared to immature DCs cultured with mediumonly (Fig. 1). To eliminate the possibility that saliva wascontaminated with LPS, which could affect cytokineproduction, we quantiﬁed the level of LPS in saliva byusing the LAL (limulus amoebocyte lysate) assay (Sigma).The LAL assay demonstrated that the amount of tick saliva
C.J.F. Oliveira et al./Veterinary Parasitology 167 (2010) 288–297