Blockade of IL-33 release and suppression of type 2 innate lymphoid cell responses by helminth secreted products in airway allergy

Abstract

Helminth parasites such as the nematode Heligmosomoides polygyrus strongly inhibit T helper type 2 (Th2) allergy, as well as colitis and autoimmunity. Here, we show that the soluble excretory/secretory products of H. polygyrus (HES) potently suppress inflammation induced by allergens from the common fungus Alternaria alternata. Alternaria extract, when administered to mice intranasally with ovalbumin (OVA) protein, induces a rapid (1-48 h) innate response while also priming an OVA-specific Th2 response that can be evoked 14 days later by intranasal administration of OVA alone. In this model, HES coadministration with Alternaria/OVA suppressed early IL-33 release, innate lymphoid cell (ILC) production of IL-4, IL-5, and IL-13, and localized eosinophilia. Upon OVA challenge, type 2 ILC (ILC2)/Th2 cytokine production and eosinophilia were diminished in HES-treated mice. HES administration 6 h before Alternaria blocked the allergic response, and its suppressive activity was abolished by heat treatment. Administration of recombinant IL-33 at sensitization with Alternaria/OVA/HES abrogated HES suppression of OVA-specific responses at challenge, indicating that suppression of early Alternaria-induced IL-33 release could be central to the anti-allergic effects of HES. Thus, this helminth parasite targets IL-33 production as part of its armory of suppressive effects, forestalling the development of the type 2 immune response to infection and allergic sensitization.

Figure 1

Alternaria-induced allergic inflammation to ovalbumin (OVA) challenge is suppressed by H. polygyrus excretory-secretory (HES) products. (a) Schematic of the Alternaria model: OVA protein (20 μg) was administered to BALB/c mice intranasally, with or without 50 μg Alternaria extract and 10 μg HES. Two weeks later, mice were challenged by intranasal (IN) administration of 20 μg OVA or phosphate-buffered saline (PBS) for 3 days, and samples taken at day 17. (b) Numbers of SiglecF⁺CD11c^– eosinophils in bronchoalveolar lavage (BAL) fluid determined by flow cytometry. (c, d) Lung cell suspensions were analyzed by flow cytometry for eosinophils (as in panel b) and neutrophils (GR1^hiCD11b^hiSiglecf⁻CD11c⁻). (e) Formalin-fixed lungs were sectioned and stained by Hemotoxylin and Eosin (H&E) or Periodic Acid Schiff (PAS). Representative sections are presented, scale bar=100 μm. (f) HES was heat treated at 95 °C for 20 min (HT-HES) before administration, and numbers of BAL SiglecF⁺CD11c^– eosinophils counted. (g) BAL eosinophils from mice treated with Alternaria, OVA, and HES as described in panel a, or with 4 ng rTGF-β (TGF), or 10 μg Bovine Serum Albumin (BSA) as indicated. Data in panels b–d are representative of five repeat experiments, 3–5 per mice group, in panel f are pooled from two repeat experiments, total n=7 per group, and in panel g are representative of two repeat experiments with four mice per group. ***P<0.001, **P<0.01, *P<0.05, and NS, not significant.

Figure 2

Suppression of innate and adaptive type 2 responsiveness by HES. Following Alternaria-OVA sensitization and challenge, BAL fluids, lungs, and draining lymph nodes were collected; in some experiments (g–i), 1 × 10⁶ DO11.10 OVA-specific T cells were transferred to mice before sensitization. Fluids were assayed for soluble cytokines and markers, and cells stained for cytokines and Foxp3. (a) Cytokines in cell-free BAL supernatant measured by cytometric bead array. (b) Type 2 myeloid cell response markers in BAL measured by ELISA. (c–f) Intracellular cytokine-positive proportions (c, d) and absolute numbers (e, f) of lung CD4⁺TCRβ⁺ T cells (c, e) and ICOS⁺Lineage⁻ innate lymphoid cells (d, f). Lung tissue cells were cultured in Phorbol Myristate Acetate (PMA)/Ionomycin for 4 h in the presence of Brefeldin A, before staining for surface markers and intracellular cytokines. (g) Intracellular IL-13 expression by ICOS⁺Lineage⁻ lung cells. (h) Intracellular IL-4 within DO11.10 OVA-specific T cells from the draining lymph nodes co-staining with the clonotypic marker KJ126. (i) Intracellular Foxp3 expression in the draining lymph node CD4⁺KJ126⁺ T-cell population. Results are representative or pooled from at least two repeat experiments with 3–5 mice per group. Unless otherwise indicated differences are not significant. ***P<0.01, **P<0.01, and *P<0.05. ^#indicates significance when comparing Alt-OVA:PBS and Alt-OVA:OVA groups, * indicates significance when comparing Alt-OVA:OVA and Alt-OVA-HES:OVA groups in panels a–c, or as indicated by bars in panels d–j. BAL, bronchoalveolar lavage; HES, H. polygyrus excretory-secretory; OVA, ovalbumin; PBS, phosphate-buffered saline.

Figure 3

Alternaria-induced allergic reactivity is dependent on both innate and adaptive immune compartments in an IL-25-independent manner. The response to Alternaria-OVA administration was tested in mice deficient in recombinase-activating gene (RAG)-2 (a, b), IL-17RB (c, d), IL-17RBxST2 (e, f), MyD88xTRIF (g, h), or TLR4 (i, j) and their congenic wild-type strains; bronchoalveolar lavage (BAL) fluids were assayed for SiglecF⁺CD11c^– eosinophils (a, c, e, g, and i) and lung cell populations for intracellular IL-13 staining within the ICOS⁺Lineage⁻ subset (b, d, f, h, and j). Unless otherwise indicated differences are not significant. ***P<0.01, **P<0.01, and *P<0.05. OVA, ovalbumin.

Figure 4

Alternaria induces a rapid innate eosinophilia and type 2 innate lymphoid cell (ILC2) response which is suppressed by HES. Alternaria, without OVA, was administered intranasally to BALB/c mice (a, b, d–h, and l), IL-13-GFP reporter mice (c), or recombinase-activating gene (RAG)-2-deficient mice (j, k) in the presence or absence of HES, and samples harvested 24 or 48 h later. (a, b) Bronchoalveolar lavage (BAL) (a) and lung (b) eosinophils measured 24 and 48 h after administration of Alternaria with or without HES. (c) Expression of IL-13-GFP in reporter mice 48 h following administration of Alternaria with or without HES, measured in lung ICOS⁺Lineage^– cells. (d–f) Lung ICOS⁺Lineage^– cell expression of IL-4 (d), IL-5 (e), or IL-13 (f), measured by intracellular staining. (g, h) BAL eosinophils (g) and intracellular IL-13⁺ proportion of ICOS⁺Lineage^– cells (h) measured at 24 and 48 h following administration of Alternaria with or without HES or heat-treated HES (HT-HES). Significance shown are compared with the Alternaria+HT-HES group (i) BALB/c mice were treated with Alternaria with or without HES or 4 ng recombinant mammalian TGF-β, and BAL eosinophils measured by flow cytometry. (j, k) BAL eosinophils (j) and IL-13 expression by innate lymphoid cell (ILCs) (k) were measured 48 h after administration of Alternaria with or without HES to RAG-deficient mice. (l) BAL eosinophils 48 h after administration of HES with, or 6 h before, Alternaria exposure. Results are representative or pooled from at least two repeat experiments, 3–5 mice per group. Unless otherwise indicated differences are not significant. ***P<0.01, **P<0.01, *P<0.05. HES, H. polygyrus excretory-secretory; OVA, ovalbumin.

Figure 5

HES strongly suppresses early IL-33 production. (a–d) At 1, 24, and 48 h after Alternaria administration with or without HES, cell-free bronchoalveolar lavage (BAL) fluid was tested for levels of IL-5 (a), RELM-α (b), Ym1 (c), and IL-33 (d). Significance compares Alternaria level to Alternaria+HES level. (e) Alternaria, HES, and heat-treated HES (HT-HES) were administered to BALB/c mice and 1 h later levels of IL-33 in cell-free BAL fluid were measured. (f) Mean fluorescence intensity (MFI) of IL-33-Citrine within CD326⁺CD45.2^– epithelial cells 12 h after Alternaria±HES administration. (g) MFI of IL-33-Citrine within CD326⁺CD45.2^– epithelial cells 12 h after the final OVA administration in the Alternaria/OVA model shown in Figure 1a. Results are pooled from two repeat experiments, 3–5 mice per group. Unless otherwise indicated differences are not significant. ***P<0.01, **P<0.01, *P<0.05. HES, H. polygyrus excretory-secretory; OVA, ovalbumin.

Figure 6

Exogenous IL-33 can circumvent HES-mediated suppression in vivo. (a–c) Responses in mice given recombinant IL-33 in the presence or absence of HES. Recombinase-activating gene (RAG)-2 deficient mice received 200 ng per day IL-33±5 μg per day HES for 3 days, and samples harvested on day 4. Numbers of bronchoalveolar lavage (BAL) (a) or lung tissue (b) eosinophils were assessed, together with lung ICOS⁺Lineage^– intracellular IL-13 staining (c). (d–g) HES suppression is overridden by exogenous IL-33. Mice receiving 200 ng per mouse IL-33, Alternaria extract, OVA, and/or HES as shown in (d), and SiglecF⁺CD11c^– BAL eosinophils (e), IL-4 expression within lung CD4⁺ cells (f), and intracellular IL-13 expression among lung ICOS⁺Lineage^– cells (g) were assessed. Unless otherwise indicated differences are not significant. ***P<0.01, **P<0.01, *P<0.05, NS=not significant. Results are pooled from two repeat experiments, 3–5 mice per group. Unless otherwise indicated differences are not significant. ***P<0.01, **P<0.01, *P<0.05. HES, H. polygyrus excretory-secretory; OVA, ovalbumin.