Ovalbumin (ova)

A murine model of neutrophilic asthma that was characterized by Th17 cell responses was generated as described previously [18 ]. In brief, mice were intranasally sensitized with 75 μg LPS-depleted OVA (grade V; Sigma-Aldrich, St. Louis, MO, USA) plus 10 μg LPS (E. coli serotype 026 : B6; Sigma-Aldrich) on days 0, 1, 2, and 7 and then challenged with 50 μg OVA alone on days 14, 15, 21, and 22. One day after the final challenge, the mice were euthanized for further analyses.
In this study, the mice were divided randomly into four groups (n = 6 mice) as follows: (i) mice sensitized with phosphate-buffered saline (PBS) and challenged with OVA (control group); (ii) mice sensitized with OVA plus LPS and challenged with OVA (asthma group); (iii) mice treated with control IgG (R&D Systems) for half an hour before sensitization to OVA plus LPS and the same challenge with OVA later (control IgG group); (iv) mice treated with anti-HMGB1 IgG (R&D Systems) half an hour before sensitization to OVA plus LPS and the same challenge with OVA later (anti-HMGB1 group). Anti-HMGB1 IgG or control IgG was administered intranasally (200 μg/kg) on days 14, 15, 21, and 22 a half an hour before OVA challenge (Figure 1(a)). The dose of anti-HMGB1 IgG was predetermined using staining analysis of airway inflammation in mice receiving 100–400 μg/kg of anti-HMGB1 IgG.

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.

Eosinophilic asthma model was created by intranasally inoculating six‐week‐old C57BL/6 mice (female) with 20 μL OVA (0.5 mg·mL⁻¹) and recombinant mouse IL‐33 protein, carrier free (5 μg·mL⁻¹) (R&D systems, MN, USA) in PBS on 0 and 7 day. Mice were fasted for 48 h from day 0 to day 2 or from day 7 to day 9. Bronchoalveolar lavage fluid (BALF), serum, mediastinal lymph node (mLN), lung, spleen and bone marrow (BM) samples were collected on day 10. All animal experiments in this manuscript were carried out in accordance with both the ARRIVE Guideline for reporting in vivo experiments and the guidelines approved by the Ethics Committee for Animal Care and Use of Kumamoto University composed of a third party (ApprOVAl ID: A020‐030). All experimental animals were bred in a 12‐h light‐dark cycle environment with free access to food and water except during the experimental period. To minimise the pain or suffering, the animal experiments were performed under appropriate anaesthesia and analgesia to minimise pain.