Tick saliva inhibits differentiation, maturation and function of murine bone-marrow-derived dendritic cells

Abstract

Haematophagous arthropod vectors such as mosquitoes, tsetse flies, sandflies and ticks have evolved salivary immunomodulatory factors that prevent the vertebrate host from rejecting them meanwhile enhancing pathogen transmission. As dendritic cells (DC) play a major role in host immune responses, we studied the effects of Rhipicephalus sanguineus tick saliva on DC differentiation and maturation. Flow cytometry analysis revealed that the addition of saliva to bone marrow cells inhibits the differentiation of DC and decreased the population of differentiated immature DC, increasing the levels of major histocompatibility complex (MHC) class II while not altering the expression of costimulatory (CD40, CD80 and CD86) and adhesion (CD54) molecules. Furthermore, maturation of DC stimulated by lipopolysaccharide (LPS) in the presence of saliva resulted in a lower expression of costimulatory molecules, but did not alter the up-regulation of MHC class II and CD54. The lipopolysaccharide (LPS)-matured DC cultured with saliva also presented reduced production of interleukin-12, whereas interleukin-10 production was unaltered. Assessment of the function of DC cultured with tick saliva revealed them to be poor stimulators of cytokine production by antigen-specific T cells. Our data indicate a novel modulatory role for the saliva of arthropod vectors at an initial step of the immune response through the inhibition of differentiation and maturation of DC into functional antigen-presenting cells.

Figure 1

Tick saliva inhibits differentiation of bone marrow (BM)-derived dendritic cells (DC). BM-derived cells from C57BL/6 mice were cultured in 24-well plates (2·5 × 10⁶ cells per well) with granulocyte–macrophage colony-stimulating factor (GM-CSF) (50 ng/ml) and interleukin-4 (IL-4) (10 ng/ml) in the presence or absence of tick saliva, as indicated. (a) Cell culture was performed in the presence of different concentrations of tick saliva protein, cells were harvested on day 9, labelled with the designated monoclonal antibodies (mAbs) and analysed by two-colour flow cytometry. Results are shown as the mean percentage ± standard error of the mean (SEM) of CD11c⁺ CD11b⁺ cells from duplicate wells. *P < 0·05 and **P < 0·01 compared to cells cultured only with GM-CSF + IL-4 (control group). Data shown are representative of two experiments. (b) The cell culture employed 64 µg/ml of saliva and cells were harvested on the days indicated. Results are shown as dot-plots and are representative of four independent experiments.

Figure 2

Tick saliva reduces the differentiation of bone marrow (BM)-derived dendritic cells (DC) that express costimulatory molecules CD86 and CD80. BM-derived cells from C57BL/6 mice were cultured in 24-well plates (2·5 × 10⁶ cells per well) with granulocyte–macrophage colony-stimulating factor (GM-CSF) (50 ng/ml) and interleukin-4 (IL-4) (10 ng/ml) in the absence (control group) or presence of tick saliva (64 µg/ml) (saliva group), as described in the Materials and methods. On days 3, 6 and 9 of culture the cells were harvested, labelled with the designated monoclonal antibodies (mAbs) and analysed by two-colour flow cytometry. Cells were gated on CD11c⁺ cells. Data are presented as the mean percentage of CD11c⁺ cells expressing CD86, CD80, CD40 or major histocompatibility complex (MHC) class II ± standard error of the mean (SEM) (a, b, c and d, respectively) from four independent experiments. *P < 0·05 compared with cells cultured with GM-CSF and IL-4 alone (control group).

Figure 3

Differentiated dendritic cells (DC) are also affected by tick saliva. Bone marrow (BM)-derived cells from C57BL/6 mice were cultured in 24-well plates (2·5 × 10⁶ cells per well) with granulocyte–macrophage colony-stimulating factor (GM-CSF) (50 ng/ml) and interleukin-4 (IL-4) (10 ng/ml) for 9 days in order to obtain immature differentiated DC (CD11c⁺ CD11b⁺). On day 9, when ≈ 70–80% of the cells were CD11c⁺, tick saliva (64 µg/ml) was added to the indicated groups. After 48 hr the cells were stained with designated antibodies or annexin V, and then analysed by two-colour flow cytometry. Data shown as dot-plots are representative of five experiments (a). Data in bar graphs are presented as the mean ± standard error of the mean (SEM) of the percentage of cells expressing the CD11c⁺ CD11b⁺, CD11c⁻ CD11b⁺ and CD11c⁻ CD11b⁻ phenotypes from five independent experiments (b) or as the mean ± SEM of the percentage of cells CD11c⁺ annexin V⁺ from two independent experiments (c). **P < 0·01 and *P < 0·05 compared with control cells.

Figure 4

Tick saliva inhibits dendritic cell (DC) maturation. Bone maarow (BM)-derived cells (2·5 × 10⁶/well) from C57BL/6 mice were differentiated for 9 days in the presence of granulocyte–macrophage colony-stimulating factor (GM-CSF) (50 ng/ml) and interleukin-4 (IL-4) (10 ng/ml). On day 9, when ≈ 80% of the cells were CD11c⁺, DC maturation was induced with the addition of lipopolysaccharide (LPS) (1 µg/ml) for 48 hr in the presence or absence of saliva (64 µg/ml) [rows (d) and (c) respectively]. In addition, these cells were cultured only with saliva (64 µg/ml) or with GM-CSF + IL-4 only [rows (e) and (b) respectively]. Cells were then harvested, labelled with the designated monoclonal antibodies (mAbs) and analysed by two-colour flow cytometry for the expression of surface molecules gated on CD11c. Histograms in row (a) represent the controls of DC labelled with isotype-matched, irrelevant mAbs. Results are expressed as the mean fluorescence intensity (MFI) of CD11c⁺ cells expressing the molecule of interest, which is indicated within each histogram. Data are representative of four independent experiments.

Figure 5

Lipopolysaccharide (LPS)-induced dendritic cell (DC) interleukin-12 (IL-12)p40 and p70 production is impaired in the presence of tick saliva. Bone marrow (BM)-derived cells (2·5 × 10⁶/well) from C57BL/6 mice were differentiated for 9 days in the presence of granulocyte–macrophage colony-stimulating factor (GM-CSF) (50 ng/ml) and IL-4 (10 ng/ml). On day 9, when ≈ 80% of the cells were CD11c⁺, maturation was induced by the addition of LPS (1 µg/ml) in the presence (black bar) or absence (white bar) of saliva (64 µg/ml). In addition, these cells were cultured with saliva alone (64 µg/ml) (hatched bar) or with GM-CSF + IL-4 alone (grey bar). After 48 hr of incubation, the supernatants were collected and assayed by specific enzyme-linked immunosorbent assay (ELISA) for IL-12p40 and p70. **P < 0·01 compared to cells stimulated with LPS, or ⁺⁺⁺P < 0·001 and ⁺P < 0·05 compared to cells cultured with GM-CSF and IL-4 alone. Results are presented as the mean of duplicate cultures ± standard deviation (SD). The figure is representative of three independent experiments.

Figure 6

Interferon-γ (IFN-γ) and interleukin (IL)-10 production is decreased when keyhole limpet haemocyanin (KLH)-specific T cells are stimulated with KLH-pulsed dendritic cells (DC) in the presence of saliva. Nine-day cultured murine bone marrow (BM)-derived DC were incubated with or without saliva for 24 hr and subsequently pulsed with KLH (10 µg/ml) for 2 hr, washed and injected into the footpads of mice. Six days later, the mice were killed and their popliteal lymph node cells were cultured with or without KLH (1, 5 and 10 µg/ml) or unrelated antigen [ovalbumin (OVA), 10 µg/ml]. After 48 hr of incubation, the supernatants were collected and assayed by specific enzyme-linked immunosorbent assay (ELISA) for IFN-γ (a and b) and IL-10 (c and d) or assayed for cell proliferation by methyl-[³H]thymidine incorporation [counts per minute (c.p.m.)] (inset box; the legends for bars are equivalent to the main figure). Results are presented as the mean of duplicate (cytokine production) or triplicate (cell proliferation) cultures ± standard deviation (SD). *P < 0·05, **P < 0·01 and ***P < 0·001 compared to cells cultured with medium only (M). The figure is representative of three independent experiments.