Epithelial Cell-derived IL-25, but not Th17 cell-derived IL-17 or IL-17F, is Crucial for Murine Asthma¹

Mice

C57BL/6 mice (CD45.1 and CD45.2) were purchased from Sankyo Lab (Tsukuba, Japan). IL-25^+/− mice were obtained by mating chimeric mice, which were generated by using il25-targeted 129 ES cells (OYC069) by Lexicon Pharmaceuticals, Inc. (The Woodlands, TX), with C57BL/6 female mice (N8). IL-25^−/− mice, IL-17A^−/− IL-17F^−/− mice (8), IL-23p19^−/− mice (17), ROR-γt^−/− mice (18), TNF^−/− mice (19) and Kit^W-sh/W-sh mice (20) on the C57BL/6 background were also used. Rag-2^−/− and OTII mice on the C57BL/6 background mice were obtained from Taconic (Hudson, NY). Sex- and age-matched mice were used (6–12 weeks old at the start of immunization). All animals were kept under specific-pathogen-free conditions in an environmentally controlled cleanroom at the National Center for Child Health and Development, The Institute of Medical Science, The University of Tokyo or Animal Research Center, Tokyo Medical University. All animal experiments were approved and conducted according to the ethical safety guidelines of those institutions.

Induction of allergic airway inflammation

Mice were sensitized with OVA, with and without (in selected experiments) alum, as described previously (21). Twenty-four hours after the last challenge, bronchoalveolar lavage fluids (BALFs), lungs and sera were collected. The analysis of BAL cells, lung histology, eosinophil peroxidase (EPO) assay and OVA-specific immunoglobulins in sera were performed as described elsewhere (21, 22). The OVA-specific cell proliferative responses were determined as described elsewhere (22).

Measurement of airway hyperreactivity (AHR)

Twenty-four hours after the last challenge, bronchial responses to aerosolized methacholine (Sigma-Aldrich, St. Louis, MO) were measured by an invasive approach using plethysmograph chambers with a ventilator (Elan Series Mouse RC Site; Buxco Electronics, Sharon, CT), as described elsewhere (6).

Histology and Immunohistochemistry

Twenty-four hours after the last challenge, lungs were harvested and fixed in Carnoy’s solution. The fixed tissues were embedded in paraffin and sliced into 5-μm sections, followed by hematoxylin-eosin staining or periodic acid-Schiff (PAS) staining. For immunohistochemistry, paraffin sections were incubated with 0.5-μg/ml anti-mouse IL-25 mAb (clone 207710, R&D Systems, Minneapolis, MN) or isotype-matched control Ab, and then IL-25 producing cells were detected by the immunoperoxidase method, as previously described (23).

DC migration

Mice were intranasally administered 100 μl of 10-mg/ml FITC-conjugated OVA (FITC-OVA) together with 5 μg of rmIL-25 (R&D Systems) or PBS alone. Submaxillary and thoracic LNs were collected separately at 12 or 24 hours after the inhalation. The proportion of FITC⁺ cells among 7-amino actinomycin D-negative I-A^bhi CD11c^hi cells in LN cells was determined with a FACSCalibur (BD Biosciences, San Jose, CA) using CellQuest software (BD Biosciences), as described elsewhere (21, 24).

T cell transfer

T cells were purified as described elsewhere (25). The purified T cells (1.0 × 10⁷ cells in 200 μl PBS) were intravenously injected into Rag-2^−/− mice. For OVA-specific Th2 cell transfer, spleen cells from OTII mice were cultured with 0.1 μM OVA peptides in the presence of 40-ng/ml rmIL-4 (PeproTech, Rocky Hill, NJ), 40-ng/ml rmTSLP (R&D systems) and 40-μg/ml anti-mouse IFN-γ mAb (XMG1.2; Biolegend, San Diego, CA) for 4 days (approx. 20% IL-4⁺ Th2 cells). Then, Th2 cells were enriched using a Mouse IL-4 Secretion Assay-Cell Enrichment and Detection Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) in accordance with the manufacturer’s instructions. The enriched OTII Th2 cells (5 × 10⁶ cells in 200 μl PBS, approx. 80% IL-4⁺ Th2 cells) were intravenously injected into Rag-2^−/− and IL-25^−/− Rag-2^−/− mice.

Mast cell transfer

Mouse bone-marrow-derived cultured mast cells (BMCMCs) were obtained and intravenously injected (1.0 × 10⁷ cells in 200 μl PBS) into Kit^W-sh/W-sh mice (3 weeks old), as described elsewhere (21). One month after this cell transfer, the mice were used for experiments. The cells were shown to be > 95% c-Kit^high FcɛRIα^high by flow cytometric analysis at the time of cell transfer (data not shown).

Bone marrow cell transfer

CD45.1-C57BL/6 or IL-25^−/− mice were irradiated with 8 Gy and then injected intravenously with bone marrow cells from CD45.2-C57BL/6 wild-type or IL-25^−/− mice (2 × 10⁷ cells). One month after this cell transfer, the mice were used for experiments (CD45.2⁺ cells > 95% in LN cells and spleen by FACS).

Epithelial cell culture

Tracheas from C57BL/6 wild-type mice were harvested and digested with 1.4-mg/ml pronase (Roche Applied Sciences, Mannheim, Germany) and 0.1-mg/ml deoxyribonuclease I (Sigma-Aldrich) in Ca²⁺/Mg²⁺-free MEM at 4°C overnight. Then the cells were suspended in DMEM/F12 supplemented with 120-mU/ml insulin and 1x non-essential amino acids and incubated at 37°C for 3 hours. Non-adherent cells were collected and cultured in serum-free PCT Airway Epithelial Medium Complete (Millipore #CnT-17, Temecula, CA) in collagen-coated plates (BD BioCoat Collagen I Cellware, BD Biosciences). The purity of the cultured epithelial cells was determined by immunohistochemical staining for cytokeratin (>99%). The isolated mouse lung epithelial cells and a mouse lung epithelial cell line (MLE12) (2 × 10⁵ cells per well in 24-well culture plates) were stimulated with various concentrations of rmIL-25, 100-ng/ml rmIL-4 or 100-ng/ml rmIL-13 (R&D Systems) in the presence and absence of PI3K inhibitor LY294002, p38MAPK inhibitor SB203580, MEK1/2 inhibitor U0126, MAPK/ERK1 (MEK1) inhibitor PD98059, NF-κB inhibitor Bay11-7082, or JNK inhibitor SP600125 (Cayman Chemical, Ann Arbor, MI) for 4 hours (for quantitative PCR) or 24 hours (for ELISA).

Mast cell and eosinophil culture

BMCMCs were sensitized with 2 μg/ml of mouse monoclonal anti-DNP IgE (SPE-7, Sigma-Aldrich) at 37°C overnight. IgE-sensitized BMCMCs and naïve BMCMCs were stimulated with DNP-conjugated human serum albumin (DNP-HSA) (Sigma-Aldrich) at 37°C for 6 hours. The culture supernatants were collected, and cell lysates were prepared.

Bone marrow cell-derived cultured eosinophils (92–95% by Wright-Giemsa stain) were generated by cultivation of bone marrow cells in the presence of 100-ng/ml rmflt3 ligand (PeproTech) and 100-ng/ml rmSCF (PeproTech), followed by addition of 10-ng/ml rmIL-5 (R&D Systems), as described elsewhere (26). The eosinophils were stimulated with the various concentrations of rmIL-25 or 100-ng/ml rmIL-5 at 37°C overnight.

Quantitative PCR

Total RNA was extracted from cells, and cDNA was prepared as described previously (27). The expression levels of CCL1 and CCL11 in MLE12 cells were determined by quantitative PCR using an Mx3000P QPCR System (Agilent Technologies, Santa Clara, CA). The primers and probes (CCL1: forward, 5’-GCTTACGGTCTCCAATAGCTGC-3’ and reverse, 5’-GCTTTCTCTACCTTTGTTCAGCC-3’, and CCL11: forward, 5’-TCCATCCCAACTTCCTGCTGCT-3’ and reverse, 5’-CTCTTTGCCCAACCTGGTCTTG-3’) were designed by Origene Technologies (Rockville, MD). Data were quantitated by the comparative Ct method after normalization with the value of endogenous GAPDH gene expression, as described elsewhere (27). Data are expressed as the RQ values to the expression without stimulation.

Measurement of cytokines

The levels of cytokines in BALFs, culture supernatants and cell lysates were determined using a Bio-Plex Mouse Cytokine Group I 23-Plex (Bio-Rad Laboratories) and LUMINEX 200 (Luminex Corporation, Austin, TX), or with ELISA kits, respectively, according to the manufacturers’ instructions. Mouse IFN-γ BD OptEIA ELISA set (BD Biosciences), Mouse IL-4, IL-6, IL-13 and IL-17 Ready-Set-Go ELISA sets (eBioscience) and Mouse IL-13, IL-17E, CCL5/RANTES and CXCL1/KC DuoSets (R&D Systems) were used.

Statistics

Unless otherwise specified, ANOVA (measurement of airway hyperresponsiveness) and the unpaired Student’s t-test, two-tailed, were used for statistical evaluation of the results. All results are shown as means + SEM.

IL-17A, IL-17F, IL-23 and ROR-γt are not required for the development of Th2-type allergic airway inflammation

Consistent with previous observations using IL-17A^−/−IL-17F^−/− mice on the BALB/c background (8), we found that the increased influx of eosinophils, neutrophils, macrophages and lymphocytes in BALFs was comparable in wild-type and IL-17A^−/−IL-17F^−/− mice on the C57BL/6 background during OVA/aluminum hydroxide (alum)-induced Th2-type airway inflammation (Fig. 1A). These observations suggest that Th17 cells may not be essential in this setting. In support of this, the numbers of eosinophils in BALFs from mice deficient in IL-23 and ROR-γt—which are a key cytokine and transcription factor, respectively, for Th17 cell commitment and development—were equivalent to those in BALFs from wild-type mice 24 hours after the last OVA challenge during OVA/alum-induced Th2-type airway inflammation (Fig. 1B, 1C). However, IL-23- or ROR-γt-deficiency influenced the numbers of macrophages, lymphocytes and neutrophils in BALFs (Fig. 1B, 1C). Consistent with the number of eosinophils in BALFs, as well as the baseline of AHR (data not shown), AHR to methacholine in those gene-deficient mice was similar to that in wild-type mice 24 hours after the last challenge (Fig. 1D). These findings suggest that IL-17A, IL-17F, IL-23 and ROR-γt are not essential for the development of OVA/alum-induced Th2-type/eosinophilic airway inflammation.

IL-25 is responsible for the development of Th2-type allergic airway inflammation

OVA/alum-induced Th2-type/eosinophilic allergic inflammation was attenuated in mice deficient in IL-17RA, which is a component of receptors for IL-17A and IL-17F (9), but it developed normally in IL-17A^−/−IL-17F^−/− mice (Fig. 1A) (8). This suggests that IL-25, which can bind to IL-17RA (10), rather than IL-17A and IL-17F, may contribute to the responses. Indeed, we found that the numbers of such inflammatory cells as eosinophils, neutrophils, macrophages and lymphocytes in BALFs were profoundly reduced in OVA-challenged IL-25^−/− mice in comparison with OVA-challenged wild-type mice during OVA/alum-induced airway inflammation (Fig. 2A). In accordance with this, pathological changes such as airway inflammation and mucin-deposition on the airway walls were strongly reduced in the lungs of IL-25^−/− mice compared with wild-type mice after the last OVA challenge (Fig. 2B, 2C). In addition, AHR was also significantly suppressed in IL-25^−/− mice compared with wild-type mice 24 hours after the last OVA challenge (Fig. 2D). Moreover, the levels of such Th2-cytokines as IL-4, IL-5, IL-9 and IL-13—but not the levels of Th1-cytokines such as IFN-γ, Th17-cytokines such as IL-17A, chemokines such as CXCL1/KC, CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β and CCL5/RANTES, or the activity of eosinophil peroxidase (EPO)—were significantly reduced in BALFs from OVA-challenged IL-25^−/− mice compared with OVA-challenged wild-type mice (Fig. 3A, 3B). The levels of OVA-specific IgE, but not IgG1 or IgG2a, were also significantly lower in sera from OVA-challenged IL-25^−/− mice than in sera from OVA-challenged wild-type mice (Fig. 3C), although the proportion of CD4⁺ PD-1⁺ CXCR5⁺ follicular helper T cells (28) in the spleen was similar in both groups (wild-type mice vs. IL-25^−/− mice = 0.20 ± 0.08 vs. 0.13 ± 0.02; each n=5). These observations clearly indicate that IL-25, rather than IL-17A and IL-17F, is responsible for the development of OVA/alum-induced Th2-type/eosinophilic airway inflammation.

IL-25 enhances, but is not essential for, lung DC migration

Dendritic cells (DCs) in the bronchi and lungs are considered to be the major antigen-presenting cells in allergic pulmonary disorders. After mice inhale FITC-conjugated OVA (FITC-OVA), lung DCs, which have captured FITC-OVA, are known to migrate into the draining LNs, with peak responses observed at 24 hours after the inhalation of FITC-OVA (29). Overexpression of IL-25 in mice resulted in elevation of serum IL-5, IL-13 and TNF, leading to increased blood eosinophils and neutrophils (30). We previously demonstrated that TNF is important for lung DC migration after inhalation of FITC-OVA (24), suggesting that IL-25 may be involved in that migration, at least by promoting TNF production. Indeed, the number of FITC-positive DCs in thoracic LNs, but not in submaxillary LNs, was significantly increased in wild-type mice treated with FITC-OVA plus rmIL-25 compared with those treated with FITC-OVA plus PBS, even at 12 hours after treatment (Fig. 4A). In addition, although this enhancement of DC migration by rmIL-25 treatment was observed even in TNF^−/− mice, it was impaired in TNF^−/− mice compared with wild-type mice (Fig. 4B). Therefore, exogenous IL-25 (or excessive IL-25 production) can enhance DC migration from the lungs to draining LNs, dependent at least in part on TNF.

We next investigated the role of intrinsic IL-25 in lung DC migration using IL-25^−/− mice. Twenty-four hours after FITC-OVA or PBS treatment, the numbers of FITC-positive DCs in thoracic LNs were comparable in wild-type and IL-25^−/− mice after FITC-OVA inhalation (Fig. 4C). These observations indicate that IL-25 can enhance, but is not essential for, DC migration from the lungs to draining LNs after allergen challenge.

IL-25 is not essential for expansion of antigen-specific memory T cells

IL-25 was reported to enhance the differentiation of Th2 cells from naïve T cells directly (31) and/or indirectly through modulation of DC function (32). This suggests that IL-25-deficiency may result in impaired induction of antigen-specific memory T cells, particularly Th2 cells, causing attenuation of OVA/alum-induced Th2-type/eosinophilic airway inflammation. To elucidate this, spleen cells and mesenteric LN (MLN) cells from OVA/alum-sensitized wild-type and IL-25^−/− mice were cultured in the presence and absence of OVA. However, the OVA-specific proliferative responses were comparable between spleen and MLN cells from wild-type and IL-25^−/− mice after OVA stimulation (Fig. 5A). The levels of IL-4 as well as IFN-γ and IL-17A in the culture supernatants of MLN cells from IL-25^−/− mice were also identical to those from wild-type mice (Fig. 5B). These observations indicate that IL-25 is not essential for OVA-specific T cell expansion, at least in the present setting. On the other hand, like intact IL-25^−/− mice, the number of eosinophils in BALFs and mucus secretion in the lungs from IL-25^−/−Rag-2^−/− mice injected with OVA-specific OTII mouse-derived Th2 cells was significantly decreased compared with Rag-2^−/− mice injected with OVA-specific OTII mouse-derived Th2 cells (Fig. 5C and data not shown). In association with this, AHR to methacholine in IL-25^−/−Rag-2^−/− mice injected with OVA-specific OTII mouse-derived Th2 cells was also reduced compared with Rag-2^−/− mice injected with OVA-specific OTII mouse-derived Th2 cells (Fig. 5D). Thus, these results suggest that IL-25 is not essential for induction of antigen-specific Th2 cells in the sensitization phase, but is crucial for activation of Th2 cells in the elicitation phase of Th2-mediated allergic airway inflammation.

Immune cell-derived IL-25 is not required for the development of Th2-type allergic airway inflammation

Although it was also shown that IL-25 is involved in the development of Th2-type/eosinophilic airway inflammation (15, 16), the source of IL-25 remains to be elucidated. As described above, IL-25-deficiency did not influence OVA-specific T-cell expansion in the sensitization phase of OVA/alum-induced Th2-type airway inflammation. On the other hand, since IL-25 mRNA is known to be expressed in Th2 cells (33, 34), T-cell-derived IL-25 may be important for activation of such target cells in local inflammatory sites, contributing to the development of allergic airway inflammation. Therefore, we next examined the role of T-cell-derived IL-25 in the pathogenesis of OVA/alum-induced Th2-type airway inflammation by performing adoptive transfer of T cells from wild-type or IL-25^−/− mice to Rag-2-deficient mice (wild-type T cells or IL-25^−/− T cells → Rag-2^−/− mice). The levels of IL-4, IL-17 and IFN-γ in the culture supernatants and proliferation of purified splenic CD3⁺ T cells in response to plate-coated anti-CD3 and anti-CD28 mAbs were similar in wild-type and IL-25^−/− mice (data not shown), suggesting that IL-25-deficiency did not influence the intrinsic functions of T cells before adoptive transfer. The number of inflammatory cells in BALFs, mucus secretion in the lungs and AHR to methacholine did not differ significantly between wild-type T cells → Rag-2^−/− mice and IL-25^−/− T cells → Rag-2^−/− mice after the last OVA challenge (Fig. 6A, B and data not shown). This indicates that T-cell-derived IL-25 is not crucial for induction of OVA/alum-induced Th2-type eosinophilic airway inflammation.

Mast cells (i.e., BMCMCs) were reported to express/produce IL-25 mRNA/proteins in response to PMA + ionophore and IgE + Ags (34). Although the levels of IL-25 proteins in the culture supernatants of BMCMCs after stimulation with PMA + ionophore and IgE + Ags were below the limit of detection by ELISA, the lysates of wild-type BMCMCs contained ELISA-detectable levels of IL-25 proteins, irrespective of stimulation, compared with lysates of IL-25^−/− BMCMCs as negative controls (Fig. 6C). These observations suggest that mast-cell-derived IL-25 may contribute to the development of OVA-induced Th2-type/eosinophilic airway inflammation. However, mast cells were shown not to be essential for the development of OVA-induced airway inflammation in mice sensitized with OVA emulsified with alum (hereinafter, mast-cell-independent airway inflammation) (5). Therefore, mast cells and mast-cell-derived IL-25 do not seem to be important in this setting.

On the other hand, mast cells are known to be responsible for the development of OVA-induced airway inflammation in mice repeatedly sensitized with OVA alone (without alum) (called mast-cell-dependent airway inflammation) (5). Since mast-cell-dependent airway inflammation (Fig. 6D) and also mast-cell-independent airway inflammation (Fig. 2A, 2B, 2C) were significantly impaired in IL-25^−/− mice, mast-cell-derived IL-25 may be involved in the pathogenesis of mast-cell-dependent airway inflammation. To elucidate the role of mast-cell-derived IL-25 in this setting, we used mast-cell-specific IL-25-deficient mice generated by injection of IL-25^−/− BMCMCs to mast cell-deficient Kit^W-sh/W-sh mice (IL-25^−/− BMCMCs → Kit^W-sh/W-sh mice), as described elsewhere (35). IL-25-deficiency did not exert any influence on the development of BMCMCs, expression of c-kit and FcɛRIα on the cell surface of BMCMCs, or β-hexosaminidase release and IL-6, IL-13 and TNF production by BMCMCs after stimulation with PMA + ionophore or IgE + Ags and after stimulation with IgE, LPS and IL-33, respectively (data not shown).

The number of eosinophils in BALFs was decreased in the mast cell-deficient Kit^W-sh/W-sh mice compared with wild-type mice during mast-cell-dependent OVA-induced airway inflammation (Fig. 6E). That decreased eosinophilia returned to the wild-type mouse level in WT BMCMCs → Kit^W-sh/W-sh mice after the last OVA challenge (Fig. 6E). Similar recovery was observed even in IL-25^−/− BMCMCs → Kit^W-sh/W-sh mice in the setting (Fig 6E) These observations indicate that mast-cell-derived IL-25 is dispensable for mast-cell-dependent OVA-induced airway inflammation. Moreover, in contrast to intact IL-25^−/− mice, the number of BAL cells was comparable between CD45.1-wild-type mice that had been reconstituted with CD45.2-wild-type bone marrow cells or IL-25^−/− bone marrow cells after irradiation (Fig. 7A) and between CD45.1-IL-25^−/− bone marrow mice that had been reconstituted with CD45.2-wild-type bone marrow cells or IL-25^−/− bone marrow cells after irradiation (Fig. 7A) 24 hours after OVA challenge during mast-cell-independent airway inflammation.

Thus, these observations indicate that IL-25, which is produced by immune cells derived from hematopoietic stem cells, is not essential for induction of Th2-type allergic airway inflammation. In support of this, our immunohistochemical studies showed that bronchial and alveolar epithelial cells in naïve and OVA-inhaled lungs from wild-type, but not IL-25^−/−, mice were IL-25-positive cells (Fig. 7B). Thus, taken together, our findings suggest that IL-25 produced by epithelial cells, but not immune cells, is crucial for induction of Th2-type allergic airway inflammation.

IL-25 is important for activation of epithelial cells and eosinophils

In allergic airway inflammation, NKT cells (36), lung macrophages (37), innate lymphoid cells (38) and epithelial cells (39) are considered to be target cells in the response of IL-25. In addition, we demonstrated that IL-25 is important for Th2 cell activation in the elicitation phase—but not Th2 cell differentiation in the sensitization phase—of Th2-type allergic airway inflammation (Fig. 5). In support of this, IL-25 enhanced mRNA expression for, or production of, CCL1/I-309, CCL11/eotaxin, CCL5/RANTES and/or CXCL1/KC, which are potential chemoattractants for Th2 cells and/or eosinophils (40, 41) by primary epithelial cells isolated from mouse trachea (Fig. 8) and by a mouse airway epithelial cell line, MLE-12 (Fig. 9A, B). Regarding the signal transduction of IL-25R, which consists of IL-17RA and IL-17RB, like IL-17RA (42, 43), Act1 is recruited to SEFIR of IL-17RB through interaction of SEFIR between IL-17RB and Act1 after IL-25 binding (35). In contrast to IL-17RA, IL-17RB—which has a TRAF6-binding motif in the cytoplasmic region—can directly associate with TRAF6 irrespective of IL-25 binding, resulting in activation of NF-κB, but not MAPK, after IL-25 binding (44). Production of CCL5/RANTES by MLE-12 cells in response to IL-25 was significantly reduced in the presence of inhibitors of PI3K or NF-κB, but not MEK1/2, MEK1, JNK or p38MAPK, while production of CXCL1/KC was suppressed in the presence of those inhibitors, except the p38MAPK inhibitor (Fig. 9C). Moreover, IL-25-mediated CCL5/RANTES, but not CXCL1/KC, production by MLE-12 cells was partially, but dose-dependently, suppressed by addition of dexamethasone (Fig. 9D). These observations suggest that IL-25-mediated CCL5/RANTES and CXCL1/KC production in epithelial cells is regulated by distinct signal cascades. In addition to epithelial cells, IL-25 was able to induce IL-13, but not IL-4, production by bone marrow cell-derived eosinophils from wild-type mice (Fig. 9E). Taken all together, these observations suggest that IL-25 plays important roles in the induction of local inflammation by activating airway structural cells such as epithelial cells and immune cells such as Th2 cells and eosinophils during Th2-type allergic airway inflammation.