Papain

Papain (0.5 units/g, Wako Pure Chemical Industries, Ltd., Osaka, Japan) was used for limited proteolysis of PspXyn10 by incubating 1 mL of 0.1 mg/mL PspXyn10 containing 0.3 mg of Papain powder in 50 mM sodium phosphate buffer (pH 6.5) at 35 °C for 2 h. The Papain-digested PspXyn10 was separated from unreacted PspXyn10 by affinity chromatography using microcrystalline cellulose. This separation step was performed at room temperature. Microcrystalline cellulose (300 mg) was loaded in an empty Micro Bio-Spin chromatography column (Bio-Rad), and the column was washed with 1 mL of 20 mM sodium acetate buffer (pH 5.0) and centrifuged at 1000×g for 1 min. The proteolysis reaction mixture (500 μL) was loaded, and the flow-through was collected as a non-cellulose-binding fraction by centrifugation at 1000×g for 1 min. The column was washed four times by adding 500 μL of 20 mM sodium acetate buffer (pH 5.0) containing 1 M NaCl and centrifuging at 1000×g for 1 min. Then, the cellulose-binding fraction was eluted three times with 0.5 mL of Milli-Q water by centrifugation at 1000×g for 1 min. To confirm that the nonbinding and binding fractions were PspXyn10ΔCBM and PspXyn10, respectively, the fractions were subjected to SDS-PAGE, the xylanase activity assay and the cellulose adsorption assay.

Negatively‐charged soft contact lenses (CLs) 2 mm in diameter were soaked with 25 mg/ml papain (Wako, Tokyo, Japan) for 24 h (papain‐CLs; Supplementary Fig. S1). Phosphate‐buffered saline (PBS) was used to dissolve the papain powder. After intraperitoneal anesthesia using pentobarbital, a papain‐CL was installed in the conjunctival sac of the right eye (at day 0), and the eyeid were sutured with 8–0 nylon (Mani, Tochigi, Japan). Two days after the surgery (day 2), the papain‐CL was removed and another newly prepared papain‐CL was inserted in the same conjunctival‐sac and the eyelids were resutured. For negative control experiments, heat‐inactivated papain‐CLs or PBS‐soaked CL were used instead of papain‐CLs. Three days after the second surgery (day 5), the papain‐CL was removed and the eye and eyelid were enucleated and fixed using 4% paraformaldehyde‐PBS. Five IL‐33 KO, IL‐25 KO and TSLPR KO mice and five wild‐type mice were used for histological analysis. Another three IL‐33 KO mice, IL‐25 KO and TSLPR KO mice and wild‐type mice were used for real‐time PCR analysis. These experiments are repeated three times and the representative data was shown. Control experiments using Rag2 KO mice, and heat‐inactivated papain‐CL models were carried out separately using five mice for each group.

To prepare primary neurons, embryonic brain cortices at E18-E19 were removed and transferred to new conical tubes and gently washed three times with PBS. The washed cortices were incubated in papain solution containing 3.5 mg/ml papain (0.5 units/g; Wako, VA, USA), 0.5 mg/ml EDTA-disodium salt (Wako, VA, USA), and deoxyribonuclease I (DNase I, 5 units/mL; Takara, Shiga, Japan) for 15 min. The digested cortices were dissociated by pipetting 12 times with a glass Pasteur pipette and filtered with a cell strainer (40-mm mesh; BD Biosciences, NJ, USA). The harvested neuronal cells were cultured in MEM containing 20% FBS and N2 supplement (Thermo Fisher Scientific, MA, USA).