Adenylate cyclase toxin from Bordetella pertussis synergizes with lipopolysaccharide to promote innate interleukin-10 production and enhances the induction of Th2 and regulatory T cells

Abstract

Adenylate cyclase toxin (CyaA) from Bordetella pertussis can subvert host immune responses allowing bacterial colonization. Here we have examined its adjuvant and immunomodulatory properties and the possible contribution of lipopolysaccharide (LPS), known to be present in purified CyaA preparations. CyaA enhanced antigen-specific interleukin-5 (IL-5) and IL-10 production and immunoglobulin G1 antibodies to coadministered antigen in vivo. Antigen-specific CD4(+)-T-cell clones generated from mice immunized with antigen and CyaA had cytokine profiles characteristic of Th2 or type 1 regulatory T (Tr1) cells. Since innate immune cells direct the induction of T-cell subtypes, we examined the influence of CyaA on activation of dendritic cells (DC) and macrophages. CyaA significantly augmented LPS-induced IL-6 and IL-10 and inhibited LPS-driven tumor necrosis factor alpha and IL-12p70 production from bone marrow-derived DC and macrophages. CyaA also enhanced cell surface expression of CD80, CD86, and major histocompatibility class II on immature DC. The stimulatory activity of our CyaA preparation for IL-10 production and CD80, CD86, and major histocompatibility complex class II expression was attenuated following the addition of polymyxin B or with the use of DC from Toll-like receptor (TLR) 4-defective mice. However, treatment of DC with LPS alone at the concentration present in the CyaA preparation (0.2 ng/ml) failed to activate DC in vitro. Our findings demonstrate that activation of innate cells in vitro by CyaA is dependent on a second signal through a TLR and that CyaA can promote Th2/Tr1-cell responses by inhibiting IL-12 and promoting IL-10 production by DC and macrophages.

FIG. 1.

CyaA induces Th2/Tr1 responses to a coadministered antigen. BALB/c mice were immunized s.c. in the hind footpad with PBS, KLH (5 μg) alone, or KLH with CyaA (1 μg). After 7 days mice were sacrificed and popliteal lymph node cells were prepared and stimulated with KLH (2 to 50 μg/ml), PMA, and anti-CD3 or medium only. After 3 days supernatants were tested for IFN-γ, IL-4, IL-5, and IL-10 by ELISA. Proliferation was assayed on day 4 by [³H]thymidine incorporation. Results represent means (error bars, SD) of five mice per group and are representative of three experiments. NT, not tested. Symbols: *, P < 0.05; ***, P < 0.001 (KLH versus KLH plus CyaA).

FIG. 2.

KLH-specific Tr1- and Th2-cell lines and clones generated from mice immunized with KLH in the presence of CyaA. (A) CD4⁺-T-cell lines were generated from lymph nodes of 10 individual mice immunized with KLH and CyaA. (B) T-cell line 7.2 was cloned by limiting dilution. T-cell lines or clones were stimulated with KLH (50 μg/ml) in the presence of autologous APC and cytokine concentrations tested in the supernatants after 3 days. Results are cytokine profiles for lines or clones cultured in individual wells of 96- or 24-well plates and are representative of three different assays. IFN-γ was at background levels in each of the T-cell clones.

FIG. 3.

CyaA enhances IgG1 antibodies to coadministered antigens in vivo. Mice were immunized s.c. in the hind footpad with PBS, KLH (5 μg) alone or with CyaA (1 μg) and boosted 21 days later. Serum samples taken 7 days after one (A) or two (B) immunizations and KLH-specific IgG, IgG1, and IgG2a determined by ELISA. Results are mean titers (error bars, SD) for five mice per group and are representative of two experiments. ***, P < 0.001 KLH versus KLH plus CyaA.

FIG. 4.

CyaA enhances LPS-induced anti-inflammatory cytokines and suppresses proinflammatory cytokines by macrophages. J774 macrophages (10⁶/ml) were incubated with the indicated concentrations of LPS (0 to 1,000 ng/ml), CyaA (1 μg/ml), in the presence or absence of polymyxin B (PB) (10 μg/ml). Supernatants were collected at the indicated times and were tested for IL-10, IL-6, and TNF-α by immunoassay. Results are means (error bars, SD) of triplicate assays and are representative of three experiments. Symbols: *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus CyaA; ++, P < 0.01, +++, P < 0.001 versus LPS alone at the same concentration.

FIG. 5.

CyaA enhances LPS-induced anti-inflammatory cytokines and suppresses LPS-induced proinflammatory cytokines from DC. Murine bone marrow-derived immature DC (10⁶/ml) were incubated with the indicated concentrations of LPS (0 to 1,000 ng/ml), CyaA (1 μg/ml) in the presence or absence of polymyxin B (PB) (10 μg/ml). Supernatants were collected at the indicated times and tested for IL-10, IL-6, TNF-α, and IL-12p70 by immunoassay. Results are means (error bars, SD) of triplicate assays and are representative of three experiments. Symbols: *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus CyaA; +, P < 0.05; ++, P < 0.01; +++, P < 0.01 versus LPS alone at the same concentration.

FIG. 6.

CyaA enhances CD80, CD86, and MHC-II but inhibits CD40 and ICAM-1 expression on DC. DC were stimulated with CyaA (1 μg/ml) in the presence of polymyxin B (10 μg/ml), LPS (1 μg/ml), CyaA and LPS, or medium only. After 24 h of incubation, cells were washed and stained with antibodies specific for CD80, CD86, MHC-II, CD40, and ICAM-1 or with isotype matched controls. Results of immunofluorescence analysis are shown for treated (black line) compared to untreated (grey histogram) DC. The numbers on the right of each histogram refer to the mean fluorescence intensity of the treated cells for the relevant fluorescent antibody, the value for cells treated with medium only is shown on the left of the first histogram in each case. Profiles are shown for a single experiment and are representative of four experiments.

FIG. 7.

CyaA-induced cytokine and chemokine production is altered in TLR4-defective mice. Bone marrow derived DC from C3H/HeN or C3H/HeJ mice (10⁶/ml) were cultured with the indicated concentrations of LPS (0 to 10 ng/ml), CyaA (1 μg/ml), in the presence or absence of polymyxin B (PB) (10 μg/ml). Supernatants were tested by immunoassay for IL-10 and MIP-1α (4 h) and IL-12p70 and TNF-α (24 h). Results are means (error bars, SD) of triplicate assays and are representative of three experiments. Symbols: **, P < 0.01 versus CyaA; +++, P < 0.001 versus LPS alone at the same concentration.

FIG. 8.

CyaA-induced DC activation is altered in TLR4-defective mice. Bone marrow derived DC from C3H/HeN (A) or C3H/HeJ (B) mice (10⁶/ml) were cultured with CyaA (1 μg/ml) either alone or with polymyxin B (PB) (10 μg/ml) or LPS (10 ng/ml) or with medium. After 24 h of incubation, cells were washed and stained with antibodies specific for CD80, CD86, MHC-II, CD40, and ICAM-1 or with isotype matched control antibodies. Immunofluorescence analyses are shown for treated (black line) compared to untreated (grey histogram) DC. The numbers on the right of each histogram refer to the mean fluorescence intensity of the treated cells for the relevant fluorescent antibody, the value for cells treated with medium only is shown on the left of the first histogram in each case. Profiles are shown for a single experiment and are representative of three experiments.