C57BL/6 mice were purchased from Clea Japan, Tokyo, Japan. They were maintained under specific pathogen-free conditions with a temperature-controlled room (22°C) on a 12-h light-dark cycle at the Institute for Animal Experimentation, Hirosaki University Graduate School of Medicine. All animal experiments were carried out in accordance with the Institutional Animal Care and Use Committees/ethics Committee of Hirosaki University. The study was approved by the Ethics Committee of the Institute for Animal Experimentation, Hirosaki University Graduate School of Medicine (approval no. M08028).

Mice were sensitized and challenged with papain as described previously (12,13), with minor modifications. Briefly, papain (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was dissolved in phosphate-buffered saline (PBS). Mice (6 to 12-week old) were sedated by intraperitoneal injection with a mixture of anesthetic agents [0.075 mg/ml medetomidine (Zenoaq, Tokyo, Japan), 0.4 mg/ml midazolam (Sandoz, Tokyo, Japan) and 0.5 mg/ml butorphanol (Meiji Seika Pharma Co., Ltd., Tokyo, Japan)] at 100 µl/10 g body weight and allowed to inhale papain (1 µg in 40 µl) into both nostrils (20 µl each). Inhalation of PBS (40 µl) was performed in the control group. The sedated mice were reversed by intraperitoneal injection with 0.075 mg/ml of atipamezol hydrochloride (Antisedan; Zenoaq) at 100 µl/10 g body weight. The intranasal administration of papain was performed daily for 5 consecutive days. After 2 weeks, mice were boosted using the same regimen. A total of 2 weeks post-boost, mice were challenged by daily intranasal delivery of papain (100 µg in 40 µl) for 3 consecutive days. At 24 h after the final challenge, mice were euthanized. Serum and BALF were collected for further analysis. To accelerate acute allergic responses, mice were administered intranasally as described above with 10 µg papain (in 40 µl) on days 0 and 7. At 6, 12, 24 and 72 h after the second sensitization with papain, all mice were euthanized. Then, the serum and BALF were collected.

PG extracted from salmon nasal cartilage was purchased from Kakuhiro Co., Ltd., (Aomori, Japan). It was added into drinking distilled water (DW) at a concentration of 0.2 mg/ml and the mice drank ad libitum by starting on the first papain-sensitized day. DW only was used as a negative control. A mouse drank approximately 5 ml/day. Thus, the consumption of PG was estimated at 1 mg/day.

The titer of total IgE in the serum was determined by sandwich enzyme-linked immunosorbent assay (ELISA) using purified rat anti-mouse IgE monoclonal antibody (catalog number 1130-01; Southern Biotechnology Associates, Inc., Birmingham, AL, USA) as a capture reagent, whereas heat-inactivated papain was used to determine papain-specific IgE. For detection, goat anti-mouse IgE antibody conjugated with horseradish peroxidase (cat. no. 1110-05; Southern Biotechnology Associates, Inc.) was used. Mouse IgE isotype control (cat. no. 0114-01; Southern Biotechnology Associates, Inc.) was used to establish a standard curve for total IgE. On the other hand, a pool of sera with a high titer of papain-specific IgE was used as a standard for the specific IgE. The optical density obtained from 100 times-diluted standard serum was assigned the arbitrary unit of 1.0. The titer of papain-specific IgE in the samples was determined as a relative value to the standard IgE-positive serum.

After anesthetization of the mice, BALF was collected via an incision to the trachea (12). PBS (0.5 ml) was flushed into the lungs and recovered using 1 mm diameter polyethylene tubing and a 1 ml syringe. BALF cells were collected by centrifugation at 100 × g at 4°C for 10 min and suspended in 40 µl of PBS. BALF cells were spread on a glass slide and Giemsa staining was performed according to the standard protocol. The granulocytes were then counted under a light microscope (Olympus CX31; Olympus Corporation, Tokyo, Japan) and the proportion of eosinophils was evaluated from the number of granulocytes. The number of granulocytes per observed area was in the range of 100-148. BALF was collected and stored at −20°C until use.

A total of 72 h following the second sensitization of papain in the acute allergic model, mice were euthanized. Lung tissue was collected and fixed in 4% (w/v) paraformaldehyde buffer at 4°C overnight. Tissues were then embedded in paraffin and cut into 5-µm thick sections. Deparaffinized sections were stained with hematoxylin and eosin. The tissues were observed under a BZ-X700 microscope (Keyence, Tokyo, Japan). Infiltrated cells were randomly counted from four histological sections.

The titer of TSLP, IL-4, IL-13 and IL-5 in the BALF and serum was determined using ELISA kits, according to the manufacturer's protocol. Determination of TSLP and IL-13 production was achieved using a Mouse TSLP ELISA Ready-SET-Go!™ kit (cat. no. 5017406; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and Novex™ IL13 Mouse Antibody Pair (cat. no. 10180373; Thermo Fisher Scientific, Inc.), respectively. The production of IL-4 and IL-5 was determined using Mouse IL-4 Antibody Pair and Mouse IL-5 CytoSet™, respectively (cat. nos. CMC0043 and CMC0053; Invitrogen; Thermo Fisher Scientific, Inc.).

Data are expressed as the mean ± standard deviation. Statistical analyses were performed using Ystat 2018 software (Igaku Tosho Shuppan Co., Ltd., Tokyo, Japan) together with Microsoft Excel software (Microsoft Corporation, Redmond, WA, USA). The statistical significance between two samples was performed by a Mann-Whitney U-test, whereas ANOVA analysis with a post-hoc Tukey's test was used for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

To investigate the effect of PG on allergic responses in papain-sensitized mice, serum IgE levels were determined. As presented in Fig. 1, the IgE level of papain-sensitized mice with DW-drinking increased after challenging with papain. The serum IgE of PG-administered mice was significantly decreased compared with mice with DW-drinking. This result is promising for the further examination of the suppressive effect of PG in allergy mouse models.

To observe the effect of PG on eosinophil infiltration in the lung, the cells in the BALF were collected and counted. In the naive mice, the infiltration of eosinophils could not be detected. Conversely, there was increased infiltration of eosinophils into the BALF following papain sensitization and challenge for 3 consecutive days. This infiltration significantly decreased when mice were orally administered with PG (Fig. 2A and B). To accelerate the acute allergic response, mice were administered intranasally with 10 µg papain on days 0 and 7. In this acute model, the effect of PG on eosinophil infiltration was also confirmed. PG reduced eosinophil infiltration in the BALF from 12 h after the second sensitization, and the effect was retained for up to 3 days (Fig. 2C).

The histology of the lung tissue was then analyzed to investigate the effect of PG on lung tissue damage. Lung tissue damage was not observed in papain-sensitized mice. However, the papain-sensitized mice with DW-drinking had an increased number of inflammatory cells infiltrating around the alveolar septa, compared with the control mice. Conversely, lung inflammation was reduced by salmon PG. Almost no inflammatory cell infiltration was observed in the papain-sensitized mice with PG-administration (Fig. 3A). Fig. 3B shows the quantitative analysis of cell infiltration in the lung tissue.

Epithelium-derived cytokines have been known to activate T cells in an antigen-dependent manner and ILC2s in an antigen-independent manner. Thus, production of the epithelium-derived cytokine TSLP was examined in acute allergic-induced mice. At 12 h after the second sensitization, the titer of TSLP in the BALF and serum was determined by ELISA. Although the titer of TSLP of papain-sensitized mice in the BALF was low and PG had no effect, PG suppressed TSLP production in the serum (Fig. 4).

Th2 cells are known to drive asthma pathobiology, thus titers of Th2-related cytokines were evaluated. At 12 h after the second sensitization of acute allergic-induced mice, BALF and serum were collected and the titers of IL-4, IL-5 and IL-13 were determined by ELISA. The results showed that both IL-4 and IL-13 could not be detected in the serum (Fig. 5A and B). Although IL-4 production was detected at low levels in the BALF, PG had no significant effect (Fig. 5A). In contrast, the production of IL-13 in the BALF could be detected in papain-sensitized mice and PG suppressed this IL-13 production (Fig. 5B). The high titer of IL-5 was detected in both the serum and BALF of papain-sensitized mice. The production of IL-5 in PG-administered mice was significantly lower compared with DW-drinking control mice (Fig. 5C).