Oesophagostomum dentatum extract modulates T cell-dependent immune responses to bystander antigens and prevents the development of allergy in mice

Abstract

One third of the human population is currently infected by one or more species of parasitic helminths. Certain helminths establish long-term chronic infections resulting in a modulation of the host's immune system with attenuated responsiveness to "bystander" antigens such as allergens or vaccines. In this study we investigated whether parasite-derived products suppress the development of allergic inflammation in a mouse model. We show that extract derived from adult male Oesophagostomum dentatum (eMOD) induced Th2 and regulatory responses in BALB/c mice. Stimulation of bone marrow-derived dendritic cells induced production of regulatory cytokines IL-10 and TGF-beta. In a mouse model of birch pollen allergy, co-administration of eMOD with sensitizing allergen Bet v 1 markedly reduced the production of allergen-specific antibodies in serum as well as IgE-dependent basophil degranulation. Furthermore, eMOD prevented the development of airway inflammation, as demonstrated by attenuation of bronchoalveolar lavages eosinophil influx, peribronchial inflammatory infiltrate, and mucus secretion in lungs and IL-4 and IL-5 levels in lung cell cultures. Reduced secretion of Th2-related cytokines by birch pollen-re-stimulated splenocytes and mesenteric lymph node cells was observed in eMOD-treated/sensitized and challenged mice in comparison to sensitized and challenged controls. The suppressive effects of eMOD were heat-stable. Immunization with model antigens in the presence of eMOD reduced production of antibodies to thymus-dependent but not to thymus-independent antigen, suggesting that suppression of the immune responses by eMOD was mediated by interference with antigen presenting cell or T helper cell function but did not directly suppress B cell function. In conclusion, we have shown that eMOD possesses immunomodulatory properties and that heat-stable factors in eMOD are responsible for the dramatic suppression of allergic responses in a mouse model of type I allergy. The identification and characterization of parasite-derived immune-modulating molecules might have potential for designing novel prophylactic/therapeutic strategies for immune-mediated diseases.

Figure 1. Antibody production in mice immunized with eMOD.

Mice were immunized intraperitoneally with 50 µg of eMOD-alum (dark bars) or sham treated with PBS-alum (white bars) on days 0 and 10. Serum was collected on day 21. The levels of eMOD-specific IgM (A), IgG (B), IgG1 (C), IgG2a (D), IgE (E), and IgA (F) were detected by ELISA. The dashed line indicates the background level of the assay. Results are representative of three repeat experiments each with four to six mice per group. Data are expressed as mean ±SEM. *p<0.05; **p<0.01.

Figure 2. Immunization with eMOD induces Th2 and regulatory responses.

Mice were immunized intraperitoneally with 50 µg of eMOD-alum on days 0 and 10. Spleen and MLN for cell preparation were collected on day 21. Cell cultures were stimulated with 100 µg of eMOD (dark bars; eMOD) or left untreated (white bars; Med) for 72 hours. Concentrations of IL-4 (A, E), IL-5 (B, F), IL-10 (C, G) and TGF-β (D, H) in the supernatants were measured by ELISA. Results represent three combined experiments each with four to six mice per group and are expressed as mean ±SEM. *p<0.05; **p<0.01; ***p<0.001.

Figure 3. Stimulation of BM-DC with eMOD results in the production of regulatory cytokines.

Bone marrow-derived dendritic cells (BM-DC) derived from naïve BALB/c mice were stimulated with 100 µg/ml of eMOD (dark bars; eMOD) or left untreated (white bars; Med) for 24 hours. Levels of IL-10 (A) and TGF-β (B) in the supernatants were measured by ELISA. Results are representative of three repeat experiments. Data are expressed as mean ±SEM.

Figure 4. Co-application of eMOD at sensitization suppresses Bet v 1-specific but not eMOD-specific humoral responses.

(A) A diagram of experimental setup. Mice were immunized intraperitoneally with 50 µg of eMOD admixed to 1 µg of Bet v 1-alum (black bars; eMOD/sens) on days 0, 14 and 28, then intranasally challenged with 100 µg of birch pollen extract (BP) on three consecutive days one week after the last sensitization (days 35–37) and sacrificed on day 40. Control mice (white bars; sens) were immunized with Bet v 1-alum admixed to PBS. Serum was collected at day 40. Levels of Bet v 1-specific and eMOD-specific IgG1 (B; E); IgG2a (C; F), and eMOD-specific IgE (G) were measured by ELISA. Functional IgE was measured by Bet v 1-mediated β-hexosaminidase release from rat basophil leukemia cells (D). The dashed line indicates the background level of the assay. Results represent three combined experiments each with five mice per group. Data are expressed as mean ±SEM. ***p<0.001.

Figure 5. Co-application of eMOD at sensitization reduces the development of airway inflammation.

Mice were immunized intraperitoneally with 50 µg of eMOD admixed to1 µg of Bet v 1-alum (black bars; eMOD/sens) on days 0, 14 and 28, then intranasally challenged with 100 µg of birch pollen extract (BP) on three consecutive days one week after the last sensitization (days 35–37) and sacrificed on day 40. Control mice (white bars; sens) were immunized with Bet v 1-alum admixed to PBS. Bronchoalveolar lavage fluid (BALF) and lung tissue for cell preparation and histology were collected at day 40. (A) The numbers of total and differential cells in BALF. Production of IL-4 (B), IL-5 (C) and IL-10 (D) in BP-re-stimulated lung cell cultures was assessed by ELISA. Results represent three combined experiments each with five mice per group. Data is expressed as mean ±SEM. *p<0.05; **p<0.01; Mac = macrophages; Eos = eosinophils, Neu = neutrophils; Lym = lymphocytes. (E) Representative haematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) staining of paraformaldehyde-fixed lung tissue sections at 40×magnification. Representative BALF cytospins were stained with H&E and are shown at 100×magnification.

Figure 6. Co-application of eMOD at sensitization reduces allergen-specific recall responses.

Mice were immunized intraperitoneally with 50 µg of eMOD admixed to 1 µg of Bet v 1 (black bars; eMOD/sens) on days 0, 14 and 28, then intranasally challenged with 100 µg of birch pollen extract (BP) on three consecutive days one week after the last sensitization (days 35–37). Control mice (white bars; sens) were immunized with Bet v 1 admixed to PBS only. On day 40, splenocytes and mesenteric lymph node (MLN) cells were stimulated with 50 µg/ml of BP extract for 72 hours. Levels of IL-4 (A, D), IL-5 (B, E) and IL-10 (C, F) were assessed by ELISA. Results are representative of at least two independent experiments each with five mice per group. Data is expressed as mean ±SEM. *p<0.05; **p<0.01.

Figure 7. The suppressive effects of eMOD are heat-stable.

Mice were immunized intraperitoneally with 1 µg of Bet v 1 admixed to 50 µg of eMOD (black bars; eMOD/sens) or heat-treated eMOD (striped bars; hi-eMOD/sens) on days 0, 14 and 28, then intranasally challenged with 100 µg of birch pollen extract (BP) on three consecutive days one week after the last sensitization (days 35–37). Control mice (white bars; sens) were immunized with Bet v 1 admixed to PBS. Serum, bronchoalveolar lavage fluid (BALF) and lung tissue for histology were collected at day 40. Functional IgE was measured by Bet v 1-mediated β-hexosaminidase release from rat basophil leukemia cells (A). The dashed line indicates the background level of the assay. (B) The numbers of total and differential cells in BALF. Results are representative of two experiments each with five mice per group. Data is expressed as mean ±SEM. **p<0.01; ns = not significant; Mac = macrophages; Eos = eosinophils, Neu = neutrophils; Lym = lymphocytes. (C) Representative haematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) staining of paraformaldehyde-fixed lung tissue sections at 40×magnification. Representative BALF cytospins were stained with H&E and are shown at 100×magnification.

Figure 8. Co-application of eMOD suppresses humoral responses to thymus-dependent diphtheria toxoid antigen.

Mice were immunized intraperitoneally with 50 µg of eMOD (dark bars; eMOD) or 10 µg of diphtheria toxoid-alum (white bars; DT) or 50 µg of eMOD admixed to 10 µg of diphtheria toxoid-alum (striped bars; eMOD/DT) on days 0 and 7. Sera were collected on day 21. Levels of DT-specific IgM (A), IgG1 (B), IgG2a (C), IgE (D), and IgA (E) were detected by ELISA. The dashed line indicates the background level of the assay. Results are representative of two experiments each with five mice per group and data are expressed as mean ±SEM. *p<0.05; **p<0.01; ns = not significant.

Figure 9. Co-application of eMOD does not interfere with humoral responses to thymus-independent NIP-Ficoll antigen.

Mice were immunized intraperitoneally with 50 µg of eMOD (dark bars; eMOD) or 200 µg of NIP-Ficoll (white bars; NIP) or 50 µg of eMOD admixed to 200 µg of NIP-Ficoll (striped bars; eMOD/NIP) on days 0 and 7. Sera were collected on day 21. Levels of NIP-specific IgM (A), IgG1 (B), IgG2a (C), IgE (D), and IgA (E) were detected by ELISA. The dashed line indicates the background level of the assay. Results are representative of two experiments each with five mice per group and data are expressed as mean ±SEM. **p<0.01; ns = not significant.