IL-33, IL-25 and TSLP contribute to development of fungal-associated protease-induced innate-type airway inflammation

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Fungal-associated proteases (FAP) induce asthma-like airway eosinophilia in mice without prior sensitization

Others observed infiltration of neutrophils rather than eosinophils in BAL fluids of naïve mice at 18 hours after one inhalation of FAP22,23. We previously reported that inhalation of a high dose of papain resulted in infiltration of neutrophils, whereas inhalation of the optimal dose of papain resulted in infiltration of eosinophils, into BAL fluids of naïve mice16. Therefore, we hypothesized that inhalation of FAP also may lead to infiltration of eosinophils into BAL fluids of naïve mice. Based on those reports, we attempted to establish a murine model of FAP inhalation-induced airway eosinophilia. C57BL/6 wild-type mice were intranasally treated with 800 μg/ml FAP once per day for 1 to 3 days (see the box in Fig. 1). Twenty-four hours after the last FAP inhalation, the cell profiles in BAL fluids were investigated. As shown in Fig. 1a, the numbers of eosinophils, neutrophils, macrophages and lymphocytes were slightly, but not significantly, increased after a single inhalation of FAP. Macrophages and lymphocytes, but not eosinophils or neutrophils, were significantly increased after two inhalations of FAP, while macrophages, lymphocytes and eosinophils were significantly increased after three inhalations of FAP (Fig. 1). Therefore, our model for studying the mechanisms of FAP-induced airway eosinophilia used mice treated intranasally with FAP once per day for 3 days.

Figure 1
Figure 1

Induction of airway eosinophilia in naïve mice after FAP inhalation. Naïve C57BL/6-wild-type mice were treated intranasally with or without 800 μg/ml FAP once per day for 1 to 3 days (see the box). Twenty-four h after the last FAP inhalation, BAL fluids were collected. The numbers of various leukocytes in each BAL fluid were determined. Data show the mean ± SEM (naïve mice, n = 8; FAP-treated mice, n = 10), and representative results that were obtained in 2 independent experiments. **p < 0.005 and ***p < 0.0005 vs. the corresponding values for naive mice, and ††p < 0.005 and †††p < 0.0005 vs. the corresponding values for FAP-treated mice (×1).

Next, C57BL/6 wild-type mice were treated intranasally with various concentrations of FAP (100–1600 μg/ml), heat-inactivated FAP (100–1600 μg/ml) or PBS once per day for 3 days. In our pilot studies, we found that the numbers of leukocytes (total, macrophages, lymphocytes, neutrophils and eosinophils) in the BAL fluids of mice treated with 100, 200, 400, 800, 1200 and 1600 μg/ml heat-inactivated FAP were comparable to those of mice treated with PBS (data not shown). Therefore, we used 800 μg/ml heat-inactivated FAP in the later experiments. The numbers of various leukocytes in the BAL fluids and the severity of lung inflammation determined by histological analysis were not dramatically increased at the lowest concentrations of FAP (100 and 200 μg/ml) compared with PBS (Fig. 2a–c). Although the numbers of leukocytes in the BAL fluids were not significantly increased at 400 μg/ml FAP, lung inflammation accompanied by epithelial hyperplasia and mucus secretion was observed (Fig. 2a–c). Consistent with Fig. 1, a significant increase in eosinophils but not neutrophils was observed in BAL fluids from mice that inhaled 800 μg/ml FAP, while both eosinophils and neutrophils were significantly increased after inhalation of the highest FAP doses (1200 and 1600 μg/ml) (Fig. 2a). Asthma-like inflammation was seen in the lungs of mice treated with 800 μg/ml FAP, while chronic obstructive pulmonary disease-like inflammation associated with tissue destruction was observed at 1200 and 1600 μg/ml FAP (Fig. 2b,c; and data not shown). Similar observations were reported in mice treated with a cysteine protease, papain16. In that study, ELISA hardly detected cytokines in the BAL fluids of mice after papain inhalation because they had been cleaved by the protease activity of papain16. Likewise, using ELISA, we hardly detected IFN-γ, IL-4, IL-13 or IL-17A in the BAL fluids of mice after FAP inhalation (data not shown). On the other hand, we found that the levels of IL-5 were significantly increased in the BAL fluids of mice after inhalation of 800 μg/ml FAP (Fig. 2d).

Figure 2
Figure 2

Importance of protease activity of FAP for induction of airway eosinophilia. Naïve C57BL/6-wild-type mice were treated intranasally with various concentrations of FAP (100–1600 μg/ml), heat-inactivated FAP (HI-FAP; 800 μg/ml) or PBS once per day for 3 days. Twenty-four h after the last inhalation, BAL fluids and lungs were collected. (a) Numbers of leukocytes in BAL fluids (PBS, n = 5; HI-FAP, n = 5; FAP, n = 4-5). (b) Lung sections stained with hematoxylin-eosin (×200). (c) Severity scores of lung inflammation (PBS, n = 6; HI-FAP, n = 9; FAP, n = 11–14). (d) The levels of IL-5 in BAL fluids by ELISA (PBS, n = 5; HI-FAP, n = 5; FAP, n = 5). Data show the mean ± SEM, and representative results that were obtained in 2 independent experiments. *p < 0.05, **p < 0.005 and ***p < 0.0005 vs. the corresponding values for PBS-treated mice.

Therefore, in our subsequent experiments, we used 800 μg/ml FAP to induce asthma-like airway eosinophilia in mice. In addition, inhalation of heat-inactivated FAP did not induce airway eosinophilia accompanied by eosinophilia or elevation of IL-5 in the BAL fluids (Fig. 2a–d). Those findings suggest that the protease activity of FAP is crucial for induction of asthma-like airway eosinophilia. In support of this, mice deficient in protease-activated receptor-2 (Par2−/− mice) showed reduced inflammatory cell counts in the BAL fluids during FAP-induced airway eosinophilia (Fig. 3a). In addition, the number of BAL cells was comparable between wild-type and Tlr2−/−Tlr4−/− mice during FAP-induced airway eosinophilia, suggesting that contamination by endotoxin does not influence the induction of FAP-induced airway eosinophilia (Fig. 3b).

Figure 3
Figure 3

Importance of Par2, but not TLR2 or TLR4, for development of FAP-induced airway eosinophilia. Mice were treated intranasally with 800 μg/ml FAP, 800 μg/ml heat-inactivated FAP (HI-FAP) or PBS once per day for 3 days. Twenty-four h after the last inhalation, BAL fluids were collected. (a) Numbers of leukocytes in BAL fluids from C57BL/6-wild-type mice (PBS, n = 12; HI-FAP, n = 14; FAP, n = 32) and –Par2−/− mice (PBS, n = 8; HI-FAP, n = 8; FAP, n = 20). (b) Numbers of leukocytes in BAL fluids from C57BL/6-wild-type mice (HI-FAP, n = 5; FAP, n = 17) and –Tlr2−/−Tlr4−/− mice (HI-FAP, n = 3; FAP, n = 12). Data show the mean ± SEM, which are pooled from 2 independent experiments. *p < 0.05, **p < 0.005 and ***p < 0.0005 vs. the corresponding values for PBS-treated mice, and ††p < 0.005 and †††p < 0.0005 vs. the corresponding values for FAP-treated wild-type mice.

IL-33, but not IL-25 or TSLP, is critical for FAP-induced airway eosinophilia, independently of acquired immune cells

Infiltration of neutrophils into BAL fluids was seen even in Rag1−/− mice at 18 hours after one inhalation of FAP, indicating that acquired immune cells such as T, B and NKT cells are not essential for that response22. In our model, as well, FAP-induced airway inflammation was observed in Rag2−/− mice (Fig. 4a). In particular, neutrophils, macrophages and lymphocytes, but not eosinophils, were increased in BAL fluids of Rag2−/− mice compared with wild-type mice (Fig. 4a), suggesting that acquired immune cells such as T, B and NKT cells were not essential for FAP-induced airway eosinophilia. On the other hand, FAP-induced airway eosinophilia was markedly impaired in Rag2−/−Il2rg−/− mice, which lack acquired immune cells, innate lymphoid cells (ILCs) and IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, compared with wild-type mice (Fig. 4b), suggesting that ILCs and/or IL-2Rγ-ligands may be important for FAP-induced airway eosinophilia. We found that the number of group 2 ILCs (ILC2s) was increased in the lungs of wild-type mice, but not Rag2−/−Il2rg−/− mice, after FAP inhalation (Fig. 4c).

Figure 4
Figure 4

Importance of innate-type, but not acquired, immune cells for development of FAP-induced airway eosinophilia. Mice were treated intranasally with 800 μg/ml FAP, 800 μg/ml heat-inactivated FAP (HI-FAP) or PBS once per day for 3 days. Twenty-four h after the last inhalation, BAL fluids were collected. (a) Numbers of leukocytes in BAL fluids from C57BL/6-wild-type mice (PBS, n = 6; HI-FAP, n = 9; FAP, n = 20) and –Rag2−/− mice (PBS, n = 5; HI-FAP, n = 8; FAP, n = 21). (b) Numbers of leukocytes in BAL fluids from C57BL/6-wild-type mice (PBS, n = 5; FAP, n = 9) and –Rag2−/−Il2rg−/− mice (PBS, n = 5; FAP, n = 13). (c) Profiles of CD25+ CD127+ or CD25+ ST2+ ILC2s among 7-aminoactinomycin D-negative, lineage marker-negative CD45+ cells in lungs from C57BL/6-wild-type mice and –Rag2−/−Il2rg−/− mice. Data show the mean + SEM, which are pooled from 2 independent experiments (a,b) and representative of similar results that were obtained in 3 independent experiments (c). *p < 0.05, **p < 0.005 and ***p < 0.0005 vs. the corresponding values for PBS-treated mice, and p < 0.05, ††p < 0.005 and †††p < 0.0005 vs. the corresponding values for FAP-treated wild-type mice.

IL-25, IL-33 and TSLP are known to be produced by epithelial cells in the lungs2,24 and involved in activation of ILC2s to produce type 2 cytokines, thereby contributing to induction of type 2 cytokine-dependent inflammation such as airway eosinophilia25,26. We found that the mRNA expression level of Il33, but not Il25 or Tslp, was significantly increased in the lungs of wild-type mice treated intranasally with 800 μg/ml FAP once per day for 1 to 3 days as shown in Fig. 1 (Fig. 5a), or with 0-1,200 μg/ml FAP once per day for 3 days as shown in Fig. 2 (Fig. 5b), in comparison with wild-type mice treated intranasally with PBS or heat-inactivated FAP. In association with this, the IL-33 protein levels were increased in the nuclei of alveolar epithelial cells from FAP-treated wild-type mice compared with heat-inactivated FAP-treated wild-type mice (Fig. 5c). To elucidate the contributions of IL-25, IL-33 and TSLP to induction of FAP-induced airway inflammation, Il25−/−, Il33−/− and TSLP receptor-deficient (Crlf2−/−) mice were treated intranasally with 800 μg/ml FAP, 800 μg/ml heat-inactivated FAP or PBS once per day for 3 days. As shown in Fig. 6a, eosinophils were significantly decreased in the BAL fluids of all mutant mice (Il25−/−, Il33−/− and Crlf2−/− mice) compared with wild-type mice after the last FAP inhalation, while the neutrophil counts in the BAL fluids were comparable in all groups. In comparison with FAP-treated wild-type mice, macrophages were significantly reduced in the BAL fluid of FAP-treated Il33−/−, but not Il25−/− or Crlf2−/−, mice (Fig. 6a). On the other hand, lymphocytes were significantly reduced in the BAL fluids of FAP-treated Il33−/− and Crlf2−/−, but not Il25−/−, mice (Fig. 6a). In addition, the histological score of lung inflammation was significantly lower in FAP-treated Il33−/−, but not Il25−/− or Crlf2−/−, mice than in FAP-treated wild-type mice (Fig. 6b,c). These observations suggest that IL-25, IL-33 and TSLP are crucial for FAP-induced airway eosinophilia, but IL-33 is more important than IL-25 and TSLP for FAP-induced airway inflammation accompanied by infiltration of macrophages and lymphocytes as well as eosinophils.

Figure 5
Figure 5

Increased expression of IL-33 in the lungs of FAP-treated mice. (a) Naïve C57BL/6-wild-type mice were treated intranasally with or without 800 μg/ml FAP once per day for 1 to 3 days as in Fig. 1. Twenty-four h after the last FAP inhalation, lungs were collected. mRNA expression levels of Il25, Il33 and Tslp in the lungs were determined by quantitative PCR (naïve, n = 10; FAP, n = 10). (b) Naïve C57BL/6-wild-type mice were treated intranasally with various concentrations of FAP (100–1600 μg/ml), heat-inactivated FAP (HI-FAP; 800 μg/ml) or PBS once per day for 3 days as in Fig. 2a. Twenty-four h after the last inhalation, lungs were collected. mRNA expression levels of Il25, Il33 and Tslp in the lungs were determined by quantitative PCR (PBS, n = 9–12; HI-FAP, n = 6–11; FAP, n = 6–10). (c) Mice were treated intranasally with 800 μg/ml FAP and heat-inactivated FAP (HI-FAP) once per day for 3 days. Twenty-four h after the last inhalation, lungs were collected. IL-33 expression in sections of the lungs was detected by immunohistochemistry. Blue = Mayer’s hematoxylin, and brown = anti-IL-33 Ab staining, respectively. ×10 and ×160 (inserted photos). Data show the mean ± SEM and representative results that were obtained in 2 independent experiments (a,b). *p < 0.05, **p < 0.005 and ***p < 0.0005 vs. the corresponding values for PBS-treated mice.

Figure 6
Figure 6

Involvement of IL-25, IL-33 and TSLP in development of FAP-induced airway eosinophilia. Mice were treated intranasally with 800 μg/ml FAP, 800 μg/ml heat-inactivated FAP (HI-FAP) or PBS once per day for 3 days. Twenty-four h after the last inhalation, BAL fluids and lungs were collected. (a) Numbers of leukocytes in BAL fluids from C57BL/6-wild-type mice (PBS, n = 8–12; HI-FAP, n = 10–14; FAP, n = 29–43), –Il25−/− mice (PBS, n = 8; HI-FAP, n = 10; FAP, n = 52), –Il33−/− mice (PBS, n = 8; HI-FAP, n = 11; FAP, n = 31) and –Crlf2−/− mice (PBS, n = 10; HI-FAP, n = 13; FAP, n = 33). (b) Lung sections stained with hematoxylin-eosin (×200). (c)Severity scores of lung inflammation in C57BL/6-wild-type mice (PBS, n = 9–10; FAP, n = 11–19), –Il25−/− mice (PBS, n = 8; FAP, n = 13), –Il33−/− mice (PBS, n = 11; FAP; n = 11) and –Crlf2−/− mice (PBS, n = 8; FAP, n = 10) Data show the mean + SEM, which are pooled from 2 or 3 independent experiments (a,c). *p < 0.05 and **p < 0.005 and ***p < 0.0005, the corresponding values for PBS-treated mice, and p < 0.05, ††p < 0.005 and †††p < 0.0005 vs. the corresponding values for FAP-treated wild-type mice.

Type 2 cytokines such as IL-5 and IL-13 were shown to be important for IL-33-induced airway eosinophilia13,27,28. Therefore, we investigated involvement of type 2 cytokines in airway eosinophilia after FAP inhalation. The numbers of eosinophils in the BAL fluids were similar in Il4−/− mice compared with wild-type mice after FAP inhalation (Fig. 7). On the other hand, eosinophils in the BAL fluids were greatly reduced in both Il5−/− mice and Il13−/− mice compared with wild-type mice (Fig. 7). These observations indicate that IL-5 and IL-13 are crucial for FAP-induced airway eosinophilia.

Figure 7
Figure 7

Importance of IL-5 and IL-13, but not IL-4, for development of FAP-induced airway eosinophilia. Mice were treated intranasally with 800 μg/ml FAP or heat-inactivated FAP (HI-FAP) once per day for 3 days. Twenty-four h after the last inhalation, BAL fluids were collected. Numbers of leukocytes in BAL fluids from C57BL/6-wild-type mice (HI-FAP, n = 9; FAP, n = 18), –Il4−/− mice (HI-FAP, n = 5; FAP, n = 8), –Il5−/− mice (HI-FAP, n = 3; FAP, n = 5) and –Il13−/− mice (HI-FAP, n = 4; FAP, n = 10). Data show the mean + SEM. *p < 0.05, **p < 0.005 and ***p < 0.0005 vs. the corresponding values for HI-FAP-treated mice, and p < 0.05, ††p < 0.005 and †††p < 0.0005 vs. the corresponding values for FAP-treated wild-type mice.



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