Eosinophil-derived neurotoxin acts as an alarmin to activate the TLR2-MyD88 signal pathway in dendritic cells and enhances Th2 immune responses

Abstract

Eosinophil-derived neurotoxin (EDN) is an eosinophil granule-derived secretory protein with ribonuclease and antiviral activity. We have previously shown that EDN can induce the migration and maturation of dendritic cells (DCs). Here, we report that EDN can activate myeloid DCs by triggering the Toll-like receptor (TLR)2-myeloid differentiation factor 88 signaling pathway, thus establishing EDN as an endogenous ligand of TLR2. EDN activates TLR2 independently of TLR1 or TLR6. When mice were immunized with ovalbumin (OVA) together with EDN or with EDN-treated OVA-loaded DCs, EDN enhanced OVA-specific T helper (Th)2-biased immune responses as indicated by predominant production of OVA-specific interleukin (IL)-5, IL-6, IL-10, and IL-13, as well as higher levels of immunoglobulin (Ig)G1 than IgG2a. Based on its ability to serve as a chemoattractant and activator of DCs, as well as the capacity to enhance antigen-specific immune responses, we consider EDN to have the properties of an endogenous alarmin that alerts the adaptive immune system for preferential enhancement of antigen-specific Th2 immune responses.

Figure 1.

EDN activation of DCs. (A and B) Measurement of EDN preparation by the Wako SLP Reagent Set or Cambrex QCL-1000 Chromogenic LAL Assay kit for potential contamination with PGN and LPS, respectively. EDN preparation at the concentration tested (1∼5 μg/ml) did not generate an optical density (O.D.) higher than that generated by the lowest concentration of standard PGN or LPS. (C) Flow cytometric analysis of the expression of DC surface markers. Monocyte-derived DCs were incubated in the absence (sham-treated) or presence of EDN at 1 μg/ml (EDN-treated) and LPS at 1 μg/ml (LPS-treated) for 48 h before immunostaining and flow cytometric analysis. The position of quadrants was determined by DCs stained with FITC- and PE-conjugated isotype-matched control antibodies. The density plots of the results of one of three representative experiments are shown. (D) Production of cytokines by DCs in response to EDN. Monocyte-derived iDCs (5 × 10⁵ cells/ml) were incubated in triplicate in the absence or presence of EDN and LPS at the specified concentrations for 48 h before the supernatant was harvested for the measurement of IL-6, IL-8, IL-12p70, and TNF-α levels by SearchLight microarray. The average (mean ± SD) of two experiments using independent donors is shown. *, P < 0.05; **, P < 0.001 when compared with the corresponding sham-treated DC supernatant (open bars) using the unpaired t test. (E) Migration of EDN-treated DCs in response to selected chemokines. Monocyte-derived DCs were cultured in the absence (sham) or presence of 1 μg/ml EDN or LPS for 24 h before the measurement of their migration to the indicated chemokines using chemotaxis assay. RANTES, SLC, and SDF-1α were all used at 100 ng/ml. The migration of DCs is depicted as the average number of DCs per high-powered field (mean ± SD) of triplicate wells. (F) The proliferative response of human peripheral blood T lymphocytes to EDN-treated allogeneic DCs. Monocyte-derived DCs were treated with or without (sham) 1 μg/ml EDN or LPS for 48 h at 37°C in humidified air with 5% CO₂ for 48 h. Subsequently, the treated DCs were added together with T cells (10⁵/well) at the indicated DC/T ratio into 96-well plates and incubated at 37°C in humidified air with 5% CO₂ for 6 d with the addition of [³H]-TdR (0.5 μCi/well) for the last 18 h of incubation. The cells were harvested and measured for the incorporation of [³H]-TdR, illustrated as the average CPM (mean ± SD) of triplicate wells. *, P < 0.05; **, P < 0.001 when compared with the corresponding sham group (unpaired t test).

Figure 2.

EDN induction of I-κBα degradation and phosphorylation of MAPKs in DCs. Serum-starved DCs (4 × 10⁶ cells/tube in serum-free RPMI 1640) were incubated at 37°C in the absence or presence of EDN or E. coli LPS (final concentration, 1 μg/ml) for 30 min. EDN or LPS was added into corresponding tubes at the beginning and 10, 20, 25, and 29 min of the incubation to ensure treatment for 30, 20, 10, 5, and 1 min, respectively. At the end of incubation, the cells were solubilized in SDS-PAGE sample buffer. Identical amounts of cell lysate (15 μl) were run on a gradient SDS-PAGE gel and transferred onto a piece of PVDF membrane and sequentially Western blotted for I-κBα, phosphorylated JNK (p-JNK), p-p38, and p-Erks as described in the Materials and methods. Similar results were obtained in three independent experiments.

Figure 3.

EDN-induced IL-6 production was dependent on MyD88. (A) Human monocyte–derived DCs were transfected with or without (Chariot II alone) gripNAhMyD88. After 24 h of culture, the DCs (4 × 10⁵ cells/ml) were incubated with EDN at the indicated concentrations for 40 h, and the concentration of IL-6 in the culture supernatant was measured by ELISA. Shown are the data of one experiment representative of two. (B) DCs generated from the bone marrow progenitors of WT (MyD88^+/+) and MyD88 knockout (MyD88^−/−) mice were incubated at 10⁶/ml in the presence of 1 μg/ml Pam3, EDN, or LPS for 48 h, and the production of IL-6 in the culture supernatants was measured by ELISA. The results of one experiment representative of two are presented as the average (mean ± SD) of triplicate wells.

Figure 4.

EDN activates TLR2. (A) HEK293 cells in a 96-well tissue culture plate were transfected with IgκB-luciferase and pSV–β-galactosidase in the presence of human TLR2, TLR3, or TLR4 plus MD2. After overnight culture, the cells were treated with of 20 μg/ml PGN, 10 μg/ml polyI:C, 1 μg/ml LPS, or 1 μg/ml EDN for 24 h. The relative luciferase activity was measure and illustrated as the average RLU (mean ± SD) of triplicate wells. (B) DCs generated from the bone marrow progenitors of WT (TLR^+/+), TLR2 knockout (TLR^−/−), C3H/HeN, and C3H/HeJ mice were incubated at 10⁶/ml in the presence of 1 μg/ml Pam3, EDN, or LPS for 48 h. The production of IL-6 in the culture supernatants was measured by ELISA and presented as the average (mean ± SD) of triplicate wells. The results of one experiment representative of three are shown.

Figure 5.

EDN induction of DC cytokine production does not depend on TLR1 or TLR6. DCs were generated from the bone marrow HPCs of WT, TLR1^−/−, TLR2^−/−, and TLR6^−/− C57BL/6 mice (10∼12 wk old) as described in Materials and methods. Subsequently, DCs were plated at 2 × 10⁵/ml and treated in the absence (sham) or presence of 300 ng/ml Pam3, 1 μg/ml FSL-1, 1 μg/ml EDN, or 100 ng/ml E. coli (K12) LPS for 48 h at 37°C in humidified air with 5% CO₂ before the supernatants were harvested and assayed for TNF-α (top) and IL-6 (bottom) by ELISA. Similar results were obtained in two separate experiments.

Figure 6.

Enhancement of OVA-specific immune responses by EDN. C57BL/6 mice (female, 8-wk old, n = 10) were immunized by i.p. injection of 50 μg OVA in the presence or absence of alum (3 mg/mouse), EDN, or hAng on day 1 and boosted by i.p. injection of 50 μg OVA alone on day 14. Serum samples were taken on days 10 (A and C) and 21 (B and D) from each mouse for the measurement of total (A and B) and subclass (C and D) anti-OVA IgG antibodies using ELISA. The average OVA-specific IgG antibody titers (mean ± SEM) of each group is shown. *, P < 0.001 compared with the group immunized with OVA alone (Mann-Whitney test). Similar results were obtained in three independent experiments.

Figure 7.

EDN enhancement of OVA-specific Th2-biased immune responses is ablated in TLR2 knockout (TLR2^−/−) mice. TLR2^−/− and littermate-matched TLR2^+/+ mice (female, 8-wk old, n = 4) were immunized by i.p. injection of 50 μg OVA in the presence or absence of EDN (1 μg/mouse) or LPS (1 μg/mouse) on day 1 and boosted by i.p. injection of 50 μg OVA alone on day 14. Serum samples were taken on days 10 and 20 for the measurement of anti-OVA IgG antibodies using ELISA. On day 20, mice were killed and their spleens removed for the determination of OVA-specific proliferation and cytokine production. (A) Pooled splenocytes (5 × 10⁵/0.2 ml/well) were stimulated with OVA at concentrations specified for 60 h with [³H]-TdR pulse (1 μCi/well) for the last 18 h. Splenocyte proliferation was shown as the average (mean ± SD) [³H]-TdR incorporation (CPM) of triplicate wells of one experiment representative of two. *, P < 0.001 by Student's t test. (B) Pooled splenocytes (5 × 10⁶/1 ml/well) of each group were stimulated with 100 μg/ml OVA for 48 h, and the cytokines in the supernatants were measured by multiplex ELISA. Shown are the averages (mean ± SD) of individual cytokines of two independent experiments. (C) The average (mean ± SEM) of OVA-specific IgG antibody titers (a and b) and subclass (c and d) of each group (n = 4) on days 10 (a and c) and 21 (b and d) are shown. *, P < 0.001 by Mann-Whitney test. Similar results were obtained in two independent experiments.

Figure 8.

DCs treated with EDN promote Th2 immune response. DCs generated from the bone marrow progenitors of WT or TLR2 knockout mice were incubated with 100 μg/ml OVA for 24 h, and then cultured at 2 × 10⁵/ml in the presence or absence of 1 μg/ml EDN or E. coli LPS for another 24 h. After harvesting supernatants for cytokine measurement, DCs were washed with PBS three times and suspended in PBS at 2.5 × 10⁶/ml. C57BL/6 mice (three mice/group) were injected i.p. with 0.2 ml DC suspension on day 0, immunized i.p. with OVA (50 μg/0.2 ml PBS/mouse) on day 4, and killed on day 11 for the removal of spleens. Single splenocyte suspensions in complete RPMI 1640 were prepared from pooled spleens of each group and stimulated in vitro with 100 μg/ml OVA for 48 h for the measurement of OVA-specific cytokine production by splenocytes. (A) The production of cytokines in the supernatants of differently treated DCs was shown as the average (mean ± SD) of two independent experiments. (B) The generation of OVA-specific cytokines by splenocytes of various groups of mice was shown as the average (mean ± SD) of two independent experiments. *, P < 0.05 compared with sham-treated group (Mann-Whitney test).