Bordetella pertussis-infected human monocyte-derived dendritic cells undergo maturation and induce Th1 polarization and interleukin-23 expression

Abstract

Bordetella pertussis, the causative agent of whooping cough, is internalized by several cell types, including epithelial cells, monocytes, and neutrophils. Although its ability to survive intracellularly is still debated, it has been proven that cell-mediated immunity (CMI) plays a pivotal role in protection. In this study we aimed to clarify the interaction of B. pertussis with human monocyte-derived dendritic cells (MDDC), evaluating the ability of the bacterium to enter MDDC, to survive intracellularly, to interfere with the maturation process and functional activities, and to influence the host immune responses. The results obtained showed that B. pertussis had a low capability to be internalized by-and to survive in-MDDC. Upon contact with the bacteria, immature MDDC were induced to undergo phenotypic maturation and acquired antigen-presenting-cell functions. Despite the high levels of interleukin-10 (IL-10) and the barely detectable levels of IL-12 induced by B. pertussis, the bacterium induced maturation of MDDC and T helper 1 (Th1) polarized effector cells. Gene expression analysis of the IL-12 cytokine family clearly demonstrated that B. pertussis induced high levels of the p40 and p19 subunits of IL-23 yet failed to induce the expression of the p35 subunit of IL-12. Overall our findings show that B. pertussis, even if it survives only briefly in MDDC, promotes the synthesis of IL-23, a newly discovered Th1 polarizing cytokine. A Th1-oriented immune response is thus allowed, relevant in the induction of an adequate CMI response, and typical of protection induced by natural infection or vaccination with whole-cell vaccines.

FIG. 1.

(A) Internalization of B. pertussis by monocytes and MDDC. Sytox green-stained bacteria were incubated with monocytes or MDDC from the same donor. The samples were analyzed by flow cytometry to assess bacterial internalization. Black bars, percentage of fluorescent phagocytic cells; open bars, results of control experiments with the phagocytosis inhibitor cytochalasin D. Results are means ± SEs from three independent experiments. (B) Intracellular survival of phagocytosed bacteria. Black squares, counts of viable intracellular bacteria recovered at the indicated time points after MDDC infection; open squares, results of control experiments with the phagocytosis inhibitor cytochalasin D. Results are means ± SEs from 13 independent experiments.

FIG. 2.

Cytokine production and allostimulatory activity of MDDC treated with B. pertussis. (A and B) IL-12 (A) and IL-10 (B) production by MDDC after incubation with live (Bp) or HI (hi Bp) bacteria. Values are expressed as means ± SEs and are reported as percentages of cytokine production levels by cells treated with E. coli LPS, taken as 100% (in response to LPS, IL-12 production was 286.9 ± 60.78 pg/ml [n = 15] and IL-10 production was 546.3 ± 147.6 pg/ml [n = 15]). (C) MDDC treated with Bp, hi Bp, or LPS, or left untreated (none), were cocultured, at different numbers, with allogeneic purified T cells. Proliferation was assessed by [³H]thymidine incorporation. Results are reported as mean counts per minute (CPM) ± SE from three independent experiments.

FIG. 3.

B. pertussis-treated MDDC drive Th1 polarization of naïve T lymphocytes but do not produce a bioactive IL-12 p70 dimer. (A) MDDC either treated with HI B. pertussis (hi Bp) or E. coli LPS or left untreated (none) were cocultured with purified CD45 RA⁺ T cells as described in Materials and Methods. On day 12, supernatants were collected and secreted cytokines were measured by a CBA assay. Results of one representative experiment of three performed are shown. (B) T cells were washed and restimulated with PMA-ionomycin for IFN-γ and IL-4 intracellular staining. Numbers in each quadrant indicate the percentage of positive cells. Results of one representative experiment of three performed are shown. (C) IL-12 production by MDDC either treated with hi Bp or LPS or left untreated, in the absence or presence of an anti-IL-10 MAb (10 μg/ml), as indicated. Results are means ± SEs from three independent experiments. (D) MDDC either treated with Bp, hi Bp, or LPS or left untreated were stained for intracellular IL-12. Numbers in each quadrant indicate the percentage of IL-12 p40/p70- and CD1a-positive cells. (E) IL-12 p70 release in cell culture supernatants from the experiment for which results are shown in panel D. Results of one representative experiment out of three performed for hi Bp and LPS and two performed for Bp are shown in panels D and E.

FIG. 4.

B. pertussis-treated MDDC expressed p40 and p19 but did not express p35 monomers: MDDC were cultured in the presence of B. pertussis (A), HI B. pertussis (B), or E. coli LPS (C). At the indicated time point, a Taqman real-time quantitative RT-PCR for p35, p40, and p19 gene expression was performed. mRNA transcript levels were expressed as fold increase over those in unstimulated MDDC at 2 h. Results of one representative experiment of four performed for HI B. pertussis and LPS and two performed for B. pertussis are shown.