Targeting Toll-like receptors on dendritic cells modifies the T(H)2 response to peanut allergens in vitro

Abstract

Background: Delivery of allergens with bacterial adjuvants has been shown to be a successful immunotherapeutic strategy for food allergy treatment in animal models. How microbial signals, acting through the innate immune system, reshape ongoing allergic responses is poorly understood.

Objective: To investigate the contribution of Toll-like receptors (TLRs) in the response to bacterial adjuvants, we designed an in vitro system to characterize the effect of heat-killed Escherichia coli vector (HKE) on peanut-induced responses of dendritic cells (DCs) and T cells.

Methods: Wild-type or TLR signaling-deficient bone marrow-derived DCs were pulsed with crude peanut extract (CPE) alone (50 microg/mL) in the presence of HKE (10(6)/mL). DC maturation was analyzed by means of flow cytometry. Treated DCs were cocultured with carboxyfluorescein succinimidyl ester (CFSE)-labeled CD4(+) T cells from sensitized mice. Cytokine production from DCs and T cells was measured by using Bioplex assays.

Results: Peanut-pulsed DCs induced the production of IL-4, IL-5, and IL-13, as well as IL-17 and IFN-gamma, from primed T cells. Adding HKE to CPE-pulsed DCs resulted in a significant decrease in T(H)2 cytokine production associated with an increase in IFN-gamma levels and profound attenuation of T-cell proliferation. These effects were linked to HKE-induced TLR-dependent changes in DC reactivity to CPE, especially the production of polarizing cytokines, such as IL-12.

Conclusions: TLR signals modulate peanut-induced DC maturation in vitro, leading to changes in the T-cell response to peanut. These TLR effects must be confirmed in vivo and might constitute another alternative for allergen immunotherapies.

Figure 1. DC treatment with heat-killed E.coli alters T cell immune responses to peanut

CD4+ T cells were cocultured (ratio 10:1) with CPE-pulsed BMDC stimulated with CPE (50µg/ml), HKE (1 bacteria per BMDC) or both for 72hrs. [A] Concentrations in IL-4, IL-5, IL-13 and IFN-γ were determined. Concentrations represent mean ± SEM (n=5). (*=P<0.05). [B] After 5 days, proliferation of CD3+ cells was analyzed by flow cytometry. One representative experiment out of five is shown.

Figure 2. Heat-killed E.coli alters peanut response by triggering TLR signaling in DCs

WT CD4+ T cells were cocultured (ratio 10:1) with CPE-pulsed BMDC [WT vs. MyD88/TRIF-KO] stimulated with CPE (50µg/ml), HKE (1 bacteria per BMDC) or both for 72hrs. Concentrations in IL-4, IL-13 and IFN-γ were determined. Concentrations represent mean ± SEM (n=5). After 5 days, proliferation of CD3+ cells was analyzed by flow cytometry. Percentage of proliferative cells is indicated by mean ± SEM (n=5). Statistical differences are indicated as **=P<0.01.

Figure 3. Heat-killed E.coli has a long-lasting effect on peanut response

CD4+ T cells were cocultured (ratio 10:1) with BMDC stimulated with CPE (50µg/ml), HKE (1 bacteria per BMDC) or both. After 7 days, they were washed, counted and cocultured with CPE-pulsed BMDC for 72hrs. [A] Supernatants were analyzed for the presence of IL-5, IL-13, IL-17, IFN-γ (*=P<0.05, **=P<0.01). [B] Proliferation of CD3+ cells was determined by flow cytometry. One representative experiment out of five is shown.

Figure 4. Heat-killed E.coli changes peanut-induced cytokine production by BMDC

BMDC were stimulated either with CPE (50µg/ml) or HKE (ratio one bacteria per BMDC) or both stimuli for 24hrs. Unstimulated BMDC were used as control. Supernatants were analyzed by ELISA for production of IL-10, IL-12, IL-6, TNFα. Concentrations represent mean ± SEM (n=15) (*=P<0.05, **=P<0.01).

Figure 5. Adding TLR4 and TLR9 ligands to CPE stimulation synergizes IL-10 production whereas only TLR9 leads to IL-12 synergy by BMDC

BMDC (WT vs. TLR4- or TLR9-KO) were either stimulated with CPE (50µg/ml), ultrapure LPS (500 ng/ml), ultrapure CpG (500ng/ml) or combination of both stimuli for 24hrs. Supernatants were analyzed by ELISA for production of IL-10 and IL-12. Concentrations represent mean ± SEM values (n=4). (**=P≤0.01).

Figure 6. E. coli partially inhibits the peanut-induced immune response via IL-12 production

CD4+ T cells were cocultured (ratio 10:1) with CPE-pulsed BMDC stimulated with CPE (50µg/ml), HKE (1 bacteria per BMDC) or both for 72hrs. BMDC were preincubated with either an anti-IL-12 antibody or isotype control at 20µg/ml. The anti-IFN-γ antibody 20µg/ml was added with T cells. Antibody concentrations were maintained during the whole time of the coculture. Concentrations in IL-4, IL-5, IL-13 were determined as well as IFN-γ, and IL-10. Concentrations represent mean ± SEM (**=P≤0.01). After 5 days, proliferation of CD3+ cells was analyzed by flow cytometry. 4 combined experiments [involving at least 5 mice] are shown.