Ovalbumin (ova)

Le^X (lacto-N-fucopentose III; Dextra Labs, UK) carbohydrate structures were conjugated to OVA (Calbiochem) as previously described (Singh et al., 2009a (link)). In short; the bifunctional cross-linker (4-N-maleimidophenyl) butyric acid hydrazide (MPBH; Pierce) was cOVAlently linked to the reducing end of the Le^X and the maleimide moiety of the linker was later used for coupling the Le^X to OVA. Neo-glycoconjugates were separated from reaction by-products using PD-10 desalting columns (Pierce). Additionally, a Dylight 549-N-hydroxysuccimide (NHS) label (Thermo Scientific) was cOVAlently coupled with OVA or OVA-Le^X (Dylight-549-OVA). Free label was removed using a PD-10 column (Pierce).
The presence of Le^X and CLR binding to OVA was measured by ELISA. In brief, OVA-conjugates were coated directly on ELISA plates (NUNC) and binding of MR-Fc, MGL1-Fc, anti-Le^X and anti-OVA antibodies to OVA was determined as described (Singh et al., 2009a (link); Hawiger et al., 2001 (link)). The presence of endotoxin was measured using a LAL assay (Lonza) following manufacturer’s protocol.

BALB/c mice were divided into four treatment groups of 10 mice each as follows: (1) control group mice, sensitized and challenged with saline (group A), (2) OVA group mice, sensitized and challenged with OVA (group B), (3) isotype group mice, treated with isotype Abs for anti-IL-9 (group C), and (4) anti-IL-9 group mice, treated with an anti-IL-9 Ab (group D).
The OVA group mice were sensitized on days 0, 2, 4, 6, 8, 10, 12, and 14 by intraperitoneal (i.p.) injection with 1 mg/mL OVA (Sigma-Aldrich, St. Louis, MO, USA) and 20 mg/mL aluminum hydroxide (Sigma-Aldrich) in saline at a dose of 100 μL/mouse.
After 2 weeks, the animals were challenged by daily nasal instillation of 100 μg OVA in 20 μL saline per mouse, by means of a micropipette, from day 15 to day 25.
Mice from the isotype group and the anti-IL-9 group were given intranasal instillations of hamster IgG (isotype Ab for anti-IL-9, eBioscience, San Diego, CA, USA) and anti-IL-9 Abs (eBioscience), respectively, 30 min prior to the OVA challenge, at a dose of 10 μg in 20 μL saline per mouse. Control group mice were sensitized and challenged with saline instead of OVA at all stages.

The mice were divided into four treatment groups of ten mice each, as follows: (1) a control group, sensitized and challenged with saline; (2) an OVA group, sensitized and challenged with OVA ; (3) an isotype group treated with isotype Abs for anti-IL-17; and (4) an anti-IL-17 group treated with anti-IL-17 Abs.
The OVA group mice were sensitized on days 0, 2, 4, 6, 8, 10, 12, and 14 via an intraperitoneal injection of 1 mg/mL of OVA (Sigma-Aldrich, St. Louis, MO, USA) and 20 mg/mL of aluminum hydroxide (Sigma-Aldrich) in saline (100 μL/mouse). After 2 weeks, all animals were challenged by the daily nasal instillation of 100 μg of OVA in 20 μL of saline using a micropipette to day 25.
The isotype and anti-IL-17 Ab-treated mice were subjected to the intranasal instillation of mouse IgG (the isotype antibody control for anti-IL-17; eBioscience, San Diego, CA, USA) and anti-IL-17 Abs (eBioscience), respectively, 30 min prior to each OVA challenge; each mouse received 10 μg of antibodies in 20 μL of saline. The control mice were sensitized and challenged with saline instead of OVA.

Mouse models were established as previously described [8 (link)]. In brief, on Days 0, 7, and 14, the AS, EB, and DXM groups were administered with OVA (Sigma Company, USA, A5503-10G) sensitization solution at 200 μL/animal (containing 10 µg of OVA + 1.3 mg of aluminum hydroxide adjuvant (Thermo Fisher Scientific, USA, 77161)) by intraperitoneal injections and challenged with 200, 10, and 10 µg OVA on Days 21–23, respectively. Additionally, the DXM group was treated with 5 mg/kg of DXM (Guangzhou Baiyunshan Tianxin Pharmaceutical Co., Ltd., China, H44022091) by intraperitoneal injection 1 hr before stimulation and measurement of airway reactivity. The NS group was administered equal amount of NS for intraperitoneal sensitization and intranasal stimulation.
In the Sema3E treatment protocol (Figure 1(a)), the AS mouse model was established as described above while challenged i.n. with OVA (200 µg in 25 µL NS), mice were exposed intranasally to Sema3E (5 µg in 25 µL NS) (R&D Systems, Minneapolis, MN., USA, 3239-S3B-025), or NS as a control 1 hr before challenge. Twenty-four hours after the last administration, mice were anesthetized for invasive airway resistance detection and then sacrificed for analysis of airway inflammation, mucus production, and collagen deposition.

To measure Ig responses, 3-month-old mice were injected i.p. with 100 μg OVAlbumin (OVA, Sigma) or 30 μg Cholera Toxin B subtype (CTB, Enzo Life Science) emulsified in 100 μl of Imject® Alum solution (Thermofisher) on day 0, and 100 μg OVA or 30 μg CTB in PBS on day 9. Mice were bled on day 16 to measure OVA- or CTB- specific Igs in the serum. As indicated in the figures and their legends, some mice received 400 µg of anti-GARP:TGF-β1 i.p. on day -1 and 6. To measure T_H cell responses, 3-month-old mice were injected sub-cutaneously (s.c.) with 100 μg OVA emulsified in 100 μl of Complete Freund’s Adjuvant (CFA, Thermofisher) on day 0, then sacrificed to collect spleens on day 14.

Protocol of schema is shown in Figure S1A. Briefly, mice were sensitized through intraperitoneal injection of 50 μg OVA (Sigma-Aldrich, Missouri, USA.) or phosphate buffered saline (PBS) with 1 mg of aluminum hydroxide (Thermo Fisher Scientific, Massachusetts, USA) on Days 0 and 14 and daily challenged with OVA (nebulization of 1% OVA for 30 min) during Days 21–25. On Day 26, micro-CT analysis was performed 24 h after the last challenge. Then, OVA-induced AR mouse model was prepared (see histology in Figure S1B). In some experiments, mice were pretreated with an intraperitoneal injection of dexamethasone (10 mg/kg, Sigma-Aldrich) or sesame oil with 4% dimethyl sulfoxide as vehicle control 1 h before each OVA challenge on Days 21, 23 and 25.