Impairment of dendritic cell function and induction of CD4⁺CD25⁺Foxp3⁺ T cells by excretory-secretory products: a potential mechanism of immune evasion adopted by Echinococcus granulosus

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention. The protocol was approved by the Laboratory Animal Welfare & Ethics Committee (LAWEC), National Institute of Parasitic Diseases, Chinese Center for Diseases Control and Prevention (Permit Number: IPD 2011-006). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.

BMDC were obtained according to a method adapted from Inaba et al. [13] and Lutz et al.[14]. Briefly, total bone marrow cells were collected from the femur of BALB/c mice and red blood cells were lysed using RBC Lysis Buffer (Beyotime, Shanghai, China). After washing with RPMI-10 (Hyclone, Utah, USA) three times, the bone marrow cells were resuspended in RPMI-10 containing 10 ng/ml granulocyte-macrophage colony stimulating factor (GM-CSF; Peprotech, London, UK) at 1 × 10⁶ cells/ml and cultured for 7 days at 37 °C in a humidified CO₂ incubator. Cells were fed on days 3 and 5. On day 7, DC clusters were harvested and subcultured overnight to remove adherent cells. Non-adherent cells were collected on day 8 by gentle pipetting and washed CD11c⁺ cells were then purified using a Magnetic-activated Cell Sorting System (Miltenyi Biotec, Auburn, CA) as immature BMDC for use in experiments.

BMDC were resuspended at 10⁶ cells/ml in complete medium containing 10 ng/ml GM-CSF and stimulated with PBS (Gibco, California, USA), lipopolysaccharide (LPS; 100 ng/ml; from E. coli strain 0111:B4; Sigma-Aldrich, Missouri, USA), ES (50 μg/ml) or AWA (50 μg/ml) for 24 h. Cells were harvested for flow cytometric analysis of surface marker expression and supernatants were collected for cytokine quantification using an enzyme-linked immunosorbent assay.

For cell surface staining, 10⁶ cells were washed and treated for 30 min on ice with Fc receptor-blocking reagent (FcγIII/IIR Ab; BD PharMingen, California, USA) in sorting buffer (PBS with 1 % fetal calf serum). Blocked cells were then incubated with PE-labeled anti-CD86 monoclonal antibody (mAb; clone GL-1), APC-labeled anti-CD80 mAb (clone 16-10A1), PeCY5-labeled anti-CD40 mAb (clone 3/23) and FITC-labeled anti-MHC class II mAb (A_βb, clone AF6-120.1) purchased from BD PharMingen. After washing, cells were resuspended in sorting buffer for flow cytometric analysis using a FACSCalibur (BD Biosciences, New Jersey, USA). At least 10,000 gated events were acquired per sample and data analysis was performed using CellQuest or FlowJo software.

BMDC were resuspended at 10⁶ cells/ml in complete medium and treated with ES (50 μg/ml) for 2 h before pulsing with 5 mg/ml OVA for 16 h at 37 °C in the presence or absence of 1 μM CpG during the last 12 h. After pulsing, cells were harvested for the flow cytometric analysis of surface marker expression.

For determination of the T cell-polarizing function of differentially activated BMDC, CD4⁺ T cells were enriched from spleens of DO11.10 transgenic mice by positive selection using anti-CD4 magnetic Beads (Miltenyi Biotec, Auburn, CA) according to the instructions provided by the manufacturer. These cells express a T cell receptor specific for an ovalbumin (OVA) peptide (323-ISQAVHAAHAEINEAGR-339) presented in the context of I-A^d MHC class II. Purified CD4⁺ T cells were washed three times and resuspended at 2 × 10⁶ cells/ml in complete medium. BMDC were resuspended at 4 × 10⁵ cells/ml in complete medium and were treated with ES (50 μg/ml), AWA (50 μg/ml) and LPS (100 ng/ml). Untreated DC were used as a control. BMDC were extensively washed and seeded into 48-well culture plates (250 μl/well) in complete medium containing 50 nM OVA323-339 peptide (AnaSpec, California, USA). CD4⁺ T cells were added (250 μl/well) to obtain a final T cell/BMDC ratio of 5:1. Co-cultures were incubated for 48 h at 37 °C before the medium was removed and fresh complete medium containing 10 ng/ml mouse rIL-2 (R&D Systems, Wisconsin, USA) was added. Following an additional incubation for 72 h at 37 °C, cells were harvested, washed and resuspended at 1 × 10⁶ cells/ml. For cytokine analysis by ELISA, 200 μl of this cell suspension were seeded into 96-well flat-bottomed culture plates coated with 5 μg/ml anti-mouse-CD3 mAb (Perprotech, London, UK) and incubated for 48 h at 37 °C prior to harvesting supernatants. For flow cytometric analysis of T cell phenotype, 500 μl of the cell suspension was seeded into 48-well flat-bottomed plates coated with anti-mouse-CD3, incubated for 12 h at 37 °C prior to addition of monensin (2 μl GolgiStop; BD PharMingen, California, USA) and incubated for a further 5 h. Cells were harvested and stained as described above.

Levels of IL-4, IL-10 and IFN-γ in culture supernatants were determined using an ELISA kit (Biolegend, California, USA) according to the instructions provided by the manufacturer.

The ability of ES and AWA to induce DC maturation and cytokine production was investigated by using LPS as a positive control and PBS as a negative control. Expression of co-stimulatory molecules (CD40, CD80, CD86 and MHC class II) was analyzed by flow cytometry flowing in vitro stimulation of CD11c⁺ BMDC with ES, AWA, LPS or PBS alone. Interestingly, ES and AWA showed different effects on DC maturation: DC stimulated with AWA expressed higher levels of CD40, CD80, MHC class II and CD86 compared with DC stimulated with PBS (Fig. 1, data are shown in Table 1), manifesting a conventional mature phenotype, similar to that obtained with LPS. In contrast, the expression of co-stimulatory molecules on ES-treated BMDCs was equivalent to that observed in PBS-treated cells.

Next, we detected the production of cytokines in BMDCs in response to different stimuli. AWA treatment induced the elevation of IL-6, IL-10, TNF-α, IL-12p40 and IL-12p70 levels in BMDCs (Fig. 2) compared to PBS (negative control), but these were lower than LPS-induced cytokine production levels. In contrast, ES treatment only induced marginal IL-6 and IL-10 production compared to negative controls. Thus, AWA rather than ES induced DC maturation and robust cytokine production.

To evaluate the effects of ES on DC maturation induced by the Toll-like receptor (TLR) ligand, BMDCs were stimulated with CpG in the presence of ES. As shown in Fig. 3 (data are shown in Table 2), BMDCs pulsed with OVA alone expressed higher levels of CD40 compared with the unpulsed control cells stimulated with PBS. The addition of CpG as an adjuvant during the final 12 h of the OVA pulsing procedure induced higher levels of CD40, CD80, CD86 and MHC class II expression compared with BMDCs pulsed with OVA alone. The ES treatment of DC before they were pulsed with OVA and CpG reduced the expression of the co-stimulatory molecule compared with the responses induced by DCs pulsed with OVA and CpG. These results indicated that ES suppressed DC maturation induced by the TLR ligand.

The effects of ES and AWA on the ability of BMDC to stimulate CD4⁺ T cell cytokine production was also examined using naïve CD4⁺ T cells purified from the spleens of DO11.10 OVA-specific T cell receptor transgenic mice. Preliminary studies demonstrated that stimulation of DO11.10 CD4⁺ T cells co-cultured with control BMDC and 50 nM OVA peptide induced high levels of expression of both IFN-γ and IL-4, and a modest level of IL-10. DCs alone or T cells co-cultured with BMDCs in the absence of OVA peptide produced negligible amounts of these cytokines (data not shown). As shown in Fig. 4, co-culture with LPS-pretreated BMDCs led to a significant increase in the production of IFN-γ and IL-10, and a reduction of IL-4 production in CD4⁺ T cells. Similarly, AWA-pretreated BMDC stimulated CD4⁺ T cells to secrete higher levels of IFN-γ compared with the control. In contrast, the ES pretreatment of BMDCs with ES did not modify their capacity to stimulate IFN-γ or IL-4 production by T cells.

Surface expression of CD4 and CD25 and intracellular expression of Foxp3 were also analyzed. ES-pretreated BMDC induced an increase in the percentage of CD4⁺ T cells expressing CD25 and Foxp3 (Fig. 5, data are shown in Table 3). These data suggest that BMDC exposed to ES induced a regulatory population of CD4⁺ T cells that express these two markers.