Dendritic cells from C57BL/6 mice undergo activation and induce Th1-effector cell responses against Campylobacter jejuni

Abstract

Food-borne Campylobacter jejuni (Cj) is an important cause of enteritis. We showed that C57BL/6 and congenic interleukin (IL)-10(-/-) mice serve as models of Cj colonization and enteritis, respectively. Thus, C57BL/6 mice are resistant to Cj induced disease. Because dendritic cells (DCs) are central to regulating adaptive immune responses, we investigated the interaction of Cj with murine bone marrow-derived DCs (BM-DCs) to assess bacterial killing, DC activation, and the ability of Cj-infected BM-DCs to stimulate Campylobacter-specific T cell responses in vitro. BM-DCs challenged with Cj efficiently internalized and killed Cj 11168 and significantly upregulated surface MHC-II, CD40, CD80 and CD86 demonstrating a mature phenotype. Infected BM-DCs secreted significant amounts of tumor necrosis factor-alpha (TNF-alpha), IL-6 and IL-12p70. Formalin-killed Cj also induced maturation of BM-DCs with similar cytokine production but at a significantly lower magnitude than live bacteria. Maximal activation of murine BM-DCs required internalization of Cj; attachment alone was not sufficient to elicit significant responses. Also, various strains of Cj elicited different magnitudes of cytokine production from BM-DCs. Finally, in a coculture system, Cj-infected BM-DCs induced high level interferon-gamma (INF-gamma) production from CD4+T cells indicating Th1 polarization. Thus, DCs from resistant C57BL/6 mice initiate T cell responses against Cj.

Fig. 1

(A) Internalization and killing of Cj by BM-DCs. BM-DCs (2 × 10⁵) in a 24-well plate were infected with Cj at MOIs of 10:1 (2.1 × 10⁶ CFU; △) and 100:1 (1.91 × 10⁷ CFU; ▲). After 1 h of infection, non-phagocytosed bacteria were removed or killed by washing followed by gentamicin treatment. Subsequently BM-DCs were lysed at 2, 4, and 8 h p.i. and lysates cultured on BA plates to enumerate viable intracellular bacteria. (B) Viability of Cj in R10.2 medium without BM-DCs. R10.2 medium without BM-DCs (as a control) was inoculated with 2.1 × 10⁶ CFU or 1.91 × 10⁷ CFU of Cj and the bacterial viability was assessed at 2, 4, and 8 h post-inoculation. Data are from three-wells in an experiment and are expressed as mean ± SEM. One experiment representative of three independent experiments is shown. (C) Inhibition of BM-DC internalization of Cj by cytochalasin D (CCD) and nocodazole (NCD). BM-DCs, left untreated or preincubated with 1 μg/ml CCD, 20 μM NCD, 1 μg/ml CCD and 20 μM NCD or DMSO (0.1%, vehicle control) for 1 h, were inoculated with Cj (MOI of 100:1). The internalized bacteria were determined at 2 h p.i. by using gentamicin killing assay as described above. Internalized bacteria in each treatment group were presented as the % of that observed in DC + Cj group (without inhibitors). Data are from two-wells in an experiment and are expressed as mean ± SEM. One experiment representative of two independent experiments is shown.

Fig. 2

Maturation and cytokine production by BM-DCs infected with Cj. Data are from three-wells in an experiment and are expressed as mean ± SEM. One experiment representative of two independent experiments is shown. (A) Maturation: BM-DCs (2 × 10⁵ cells/ml) seeded in six-well plates (BD Falcon) were infected with Cj at a MOI of 100:1, treated with LPS (0.1 μg/ml) or R10.2 medium alone. After 24 h, cells were double-stained with R-PE-conjugated anti-CD11c and FITC-conjugated anti-MHC-II (I-A^b), anti-CD40, anti-CD80 or anti-CD86 antibodies or appropriate isotype controls and subjected to flow cytometric analysis. The mean fluorescence intensities of MHC-II, CD40, CD80, and CD86 markers on CD11c⁺ cells and the percentage of CD11c⁺ cells expressing these maturation markers are shown. Asterisks indicate P < 0.05 for 1) Cj-infected DCs or LPS-treated DCs vs. medium alone-treated DCs. (B) Cytokine production: BM-DCs (2 × 10⁵ cells per well of a 24-well plate) in R10 medium without antibiotics were treated with Cj at MOI of 10:1 and 100:1 or LPS (0.1 μg/ml) or R10.2 medium alone. The levels of IL-12, IL-6 and TNF-α cytokines in the culture supernatants at 4, 24 and 48 h p.i. were measured by cytokine-specific sandwich ELISA. Data from LPS treatment group was included in the statistical analysis but the significance levels are not shown in figure.

Fig. 3

Effect of viability of Cj on activation of DCs. BM-DCs were treated with approximately equal numbers of live or formalin-killed Cj (MOI of 100:1), LPS (0.1 μg/ml), or R10.2 medium alone. After 24 h, the expression of cell surface markers was analyzed by flow cytometry. (A) Mean fluorescence intensities of MHC-II, CD40, CD80, and CD86 markers on CD11c⁺ cells and the percentage of CD11c⁺ cells expressing these maturation markers are shown. (B) The levels of IL-12, IL-6 and TNF-α cytokines in the culture supernatants at 4, 24 and 48 h p.i. were measured by cytokine-specific sandwich ELISA. Asterisks indicate significance P < 0.05. Data are from three-wells in an experiment and are expressed as mean ± SEM. One experiment representative of two independent experiments is shown.

Fig. 4

Requirement for contact and internalization of Cj for activation of DCs. BM-DCs, preincubated with 1 μg/ml CCD and 20 μM NCD or DMSO (0.1% vehicle control) for 1 h, were treated with Cj (MOI of 100:1), LPS (0.1 μg/ml) or R10.2 medium alone. In experiments with transwells, BM-DCs were added to wells of cell culture plates and 0.2 μM Anopore membrane cell culture inserts were placed in wells. Then, Cj at an MOI of 100:1, R10.2 medium alone or 0.1 μg/ ml of S. typhimurium LPS was added to the top chamber. After 24 h of incubation, maturation and cytokine responses of BM-DCs were analyzed as described before. (A–D) The mean fluorescence intensities of MHC-II, CD40, CD80, and CD86 markers on CD11c⁺ cells and the percentage of CD11c⁺ cells expressing these maturation markers are shown. For the experiment with actin and microtubule inhibitors, MFI and percentage of CD11c cells positive for markers in the medium alone group were considered basal values and the increase in these parameters in the other two treatment groups was expressed as fold increase over basal value. (E) The levels of cytokines secreted at 24 h p.i. Data are from three-wells in an experiment and are expressed as mean ± SEM. One experiment representative of two independent experiments is shown.

Fig. 5

Cytokine responses of BM-DCs to various strains of Cj. BM-DCs were infected with the one of following strains of Cj: 11168, D2600, D0835, NW, D0121 and 33560 or treated with LPS (0.1 μg/ml) or R10.2 medium alone. At 24 and 48 h p.i., the concentrations of IL-12 (A), TNF-α (B) and IL-6 (C) in the culture supernatants were measured by sandwich ELISA. Data are from three-wells in an experiment and are expressed as mean ± SEM. One experiment representative of two independent experiments is shown. DCs treated with all Cj strains or LPS were significantly different (P < 0.05) from medium alone-treated DCs within each time point.

Fig. 6

Polarization of CD4⁺T cells by Cj-infected BM-DCs. BM-DCs treated with Cj (MOI of 100:1) or LPS (0.1 μg/ml) or R10.2 medium alone or zymosan (10 μg/ml) were cocultured with CD4⁺T cells isolated from spleen for 72 h. The levels of IFN-γ (A) and IL-4 (B) in the supernatants were determined by a sandwich ELISA. Asterisks indicate P < 0.05: (A) T + DC + LPS was significantly different from all the negative control groups; (B) T + DC + zymosan was significantly different from all other treatments. Data are from three-wells in an experiment and are expressed as mean ± SEM. One experiment representative of two independent experiments is shown. CM (conditioned medium) is 0.22 μM filtered supernatant from DC + Cj culture.