Interleukin 31 receptor α promotes smooth muscle cell contraction and airway hyperresponsiveness in asthma

Abstract

Asthma is a chronic inflammatory airway disease characterized by airway hyperresponsiveness (AHR), inflammation, and goblet cell hyperplasia. Multiple cytokines, including IFNγ, IL-4, and IL-13 are associated with asthma; however, the mechanisms underlying the effects of these cytokines remain unclear. Here, we report a significant increase in the expression of IL-31RA, but not its cognate ligand IL-31, in mouse models of allergic asthma. In support of this, IFNγ, IL-4, and IL-13 upregulated IL-31RA but not IL-31 in both human and mice primary airway smooth muscle cells (ASMC) isolated from the airways of murine and human lungs. Importantly, the loss of IL-31RA attenuated AHR but had no effect on inflammation and goblet cell hyperplasia in mice challenged with allergens or treated with IL-13 or IFNγ. We show that IL-31RA functions as a positive regulator of muscarinic acetylcholine receptor 3 expression, augmenting calcium levels and myosin light chain phosphorylation in human and murine ASMC. These findings identify a role for IL-31RA in AHR that is distinct from airway inflammation and goblet cell hyperplasia in asthma.

Fig. 1. Loss of IL-31RA attenuates house dust mite (HDM)-induced airway hyperresponsiveness.

a Schemata of HDM-induced allergic asthma model. Image was created with biorender.com. b Quantification of IL-31RA transcripts in the lungs of wildtype (n = 5) and IL-31RA^–/– (n = 5) mice treated with saline or wildtype (n = 6) and IL-31RA^–/– (n = 6) mice treated with HDM. Data shown as mean $\pm$ SEM. Two-way ANOVA test was used. c Quantification of IL-31 transcripts in the lungs of wildtype (n = 5) and IL-31RA (n = 5) mice treated with saline or wildtype (n = 5) and IL-31RA (n = 6) mice treated with HDM. Data shown as mean ± SEM. Two-way ANOVA test was used. d Representative images of immunohistochemical staining for IL-31RA on lung sections from wild-type mice treated with saline (n = 4) or HDM (n = 4). Scale bar, 50 µm. e Representative images of immunohistochemical staining for IL-31RA on lung sections from healthy controls (n = 8) and asthmatics (n = 8). Arrow heads highlight the IL-31RA staining (brown) in smooth muscle cells with spindle-shaped nuclei. Scale bar, 50 µm. f Measurement of resistance with increasing doses of methacholine (MCh) in wildtype (n = 7) and IL-31RA^–/– (n = 8) mice treated with saline or wildtype (n = 5) and IL-31RA^–/– (n = 8) mice treated with HDM using FlexiVent. Data are shown as mean $\pm$SEM. A two-way ANOVA test was used. g The percent of contraction of airways with increasing doses of MCh compared to the baseline area of airways between wild-type (n = 8) and IL-31RA^–/– (n = 6) mice. A two-way ANOVA test was used. Data shown as mean $\pm$SEM. h The percent contraction of collagen gels embedded with airway smooth muscle cells from wild-type (n = 3) and IL-31RA^–/– (n = 3) mice in culture media and treated with carbachol (10 μM) for 60 min. Data shown as mean ± SEM. Unpaired t test was used. At least two independent experiments produced similar results. Source data are provided as a Source Data file.

Fig. 2. Loss of IL31RA has no effect on house dust mite (HDM)-induced airway inflammation and goblet cell hyperplasia.

a Representative image of hematoxylin and eosin-stained lung sections from wildtype (n = 5) and IL-31RA^–/– (n = 5) mice treated with saline or HDM. Images were taken at ×20 magnification, scale bar 100 µm. b, c Total bronchoalveolar lavage (BAL) cell number and the differential cell count of BAL cells of wild-type and IL-31RA^-/- mice treated with saline (n = 5) or HDM (n = 5). Eos, eosinophils; Mac, macrophages; Lym, lymphocytes; and Neu, neutrophils. One-way ANOVA was used. d Representative images of Alcian blue periodic acid-Schiff (ABPAS) staining of lung sections from wildtype (n = 7) and IL-31RA^–/– (n = 7) mice treated with saline or HDM. Images were taken at ×20 magnification, scale bar 100 µm. e The percent of ABPAS-positive cells normalized to total cell in the airways of wild-type and IL-31RA^–/– mice treated with saline (n = 7) or HDM (n = 7). Data are shown as mean $\pm$SEM. One-way ANOVA was used. At least two independent experiments produced similar results. Source data are provided as a Source Data file.

Fig. 3. Loss of IL-31RA has no effect on Th2 responses and goblet cell hyperplasia during house dust mite (HDM)-induced allergic asthma.

a Quantification of Th2 cytokine transcripts including IL-4, IL5, and IL-13 in the whole lung tissue of wild-type and IL-31RA^-/- mice treated with saline (n = 6) or HDM (n = 6) using RT-PCR. Data are shown as mean $\pm$SEM. One-way ANOVA was used. b Quantification of chemokines and inflammatory cytokines including CCL11, CCL24, and IL-10 in the whole lungs of wild-type and IL-31RA^-/- mice treated with saline (n = 6) or HDM (n = 6) using RT-PCR. Data are shown as mean $\pm$SEM. One-way ANOVA was used. c Quantification of Th2 response-associated gene transcripts including CHI3L3, ARG1, and FIZZ1 in the total lungs of wild-type and IL-31RA^–/– mice treated with saline (n = 6) or HDM (n = 6) using RT-PCR. Data are shown as mean $\pm$SEM. One-way ANOVA was used. d Quantification of GOB5 and MUC5AC gene transcripts in the whole lungs of wild-type and IL-31RA^–/– mice treated with saline (n = 6) or HDM (n = 6) using RT-PCR. Data are shown as mean $\pm$SEM. One-way ANOVA was used. e Concentration of cytokines IL-4, IL-13, IL-5, IL-6, IFNγ, TNF-α and IL-10 in the BAL fluids of wild-type and IL-31RA^–/– mice treated with saline (n = 6) or HDM (n = 6) using the cytometric bead assay LEGENDplex. Data are shown as mean $\pm$SEM. One-way ANOVA was used. At least two independent experiments produced similar results. Source data are provided as a Source Data file.

Fig. 4. Loss of IL31RA attenuates AHR but not inflammation and goblet cell hyperplasia during SEA-induced allergic asthma.

a Schemata of SEA-induced allergic asthma model. Image was created with biorender.com. b Quantification of IL-31RA transcripts in the lungs of wildtype (n = 5) and IL-31RA^–/– (n = 6) mice treated with saline or wild-type (n = 6) and IL-31RA^–/– (n = 6) mice treated with SEA. Data shown as mean $\pm$SEM. Two-way ANOVA test was used. c Quantification of IL-31 transcripts in the lungs of wildtype (n = 5) and IL-31RA^–/– (n = 5) mice treated with saline or wild-type (n = 6) and IL-31RA^–/– (n = 5) mice treated with SEA. Data shown as mean $\pm$SEM. Two-way ANOVA test was used. d, e Measurement of resistance with increasing doses of methacholine (MCh) in wildtype (n = 6) and IL-31RA^–/– (n = 7) mice treated with saline or wildtype (n = 5) and IL-31RA^–/– (n = 4) mice treated with SEA using FlexiVent. Data are shown as mean $\pm$SEM. A two-way ANOVA test was used. f Representative images of hematoxylin and eosin-stained lung sections from wildtype (n = 5) and IL-31RA^–/– (n = 5) mice treated with saline or SEA. Images were taken at ×20 magnification. Scale bar, 100 µm. g Representative images of Alcian blue periodic acid-Schiff staining of lung sections from wildtype (n = 5) and IL-31RA^–/– (n = 5) mice treated with saline or SEA. Images were taken at ×20 magnification. Scale bar, 100 µm. At least two independent experiments produced similar results. Source data are provided as a Source Data file.

Fig. 5. IL-31 is dispensable for the induction of AHR, inflammation, and Th2 responses.

a Schemata showing intratracheal administration of IL-31 or saline in wild-type mice. Image was created with biorender.com. b Measurement of resistance with increasing doses of methacholine (MCh) in wild-type mice treated with saline (n = 5) or IL-31 (n = 5) using FlexiVent. Data are shown as mean $\pm$SEM.Two-way ANOVA was used. The data is representative of two independent experiments with no statistical significance between groups. c Representative images of hematoxylin and eosin-stained lung sections from wildtype mice treated with IL-31 (n = 5) or saline (n = 5). Images were captured at ×20 magnification. Scale bar, 100 µm. d Quantification of IL-31-induced SOCS3 gene expression in the whole lung tissue of wild-type mice treated with saline (n = 6) or IL-31 (n = 6). Unpaired t test was used. Data shown as mean $\pm$SEM. e, f Quantification of inflammation-associated gene transcripts IFNγ, TNFα, IL-6, and IL-17 and Th2-associated gene transcripts including IL-4, IL-13, ARG1, MUC4, and MUC5AC in whole lungs of wild-type mice treated with saline (n = 6) or IL-31 (n = 6). Unpaired t test was used and no significance found between groups. Data shown as mean $\pm$SEM. g Representative images of precision cut lung sections (PCLS) from wildtype mice treated with media or IL-31 (500 ng/mL) for 24 h. Airway contractility was measured in response to MCh (10^-4M) compared to baseline lumen area. The percent of airway lumen area contraction with increasing doses of MCh was calculated for media (n = 5) and IL-31-treated (n = 8) PCLS from wildtype mice. Two-way ANOVA test was used. Data shown as mean $\pm$SEM. h The percent contraction of collagen gels embedded with airway smooth muscle cells from wild-type mice that were treated with media, IL-13 (50 ng/mL) or IL-31 (500 ng/mL). The percent contraction was measured at different time points compared to baseline. Two-way ANOVA was used. n = 4/group. Data shown as mean $\pm$SEM. At least two independent experiments produced similar results. Source data are provided as a Source Data file.

Fig. 6. Th2 cytokines upregulate IL-31RA to induce AHR with no effect on inflammation and goblet cell hyperplasia.

a, b Quantification of IL-31RA transcripts in mouse ASMC treated with increasing doses of IL-4 (n = 3) and IL-13 (n = 4) for 16 h. Two-way ANOVA test was used. Data shown as mean $\pm$SEM. c Wildtype (n = 4) and IL-31RA^–/– (n = 4) mice were treated intratracheally with IL-13 on days 0 and 6, and resistance in the lungs was measured with increasing doses of methacholine (MCh) using FexiVent. Data are shown as mean $\pm$SEM. Two-way ANOVA test was used. d ASMC isolated from wildtype (n = 3) and IL-31RA^–/– (n = 3) mice were seeded into collagen gels and treated with IL-13 to measure the contraction of collagen gels after 48 h. Unpaired t test was used. Data shown as mean $\pm$SEM. e Representative images of hematoxylin and eosin -stained lung sections from wildtype (n = 4) and IL-31RA^–/– (n = 4) mice treated with IL-13. Images were captured at ×20 magnification. Scale bar, 100 µm. f, g, h Quantification of inflammatory chemokines (CCL11, CCL24 and IL-17), Th2 cytokines (IL-4 and IL-5), and Th2 response-associated genes (ARG1, CHl3L3, and FIZZ1) in the whole lungs of wildtype (n = 4) and IL-31RA^–/– (n = 4) mice treated with IL-13. Data are shown as mean $\pm$SEM. Unpaired t test was used, and no statistical significance observed between groups. i Representative images of Alcian blue periodic acid-Schiff-stained lung sections from wildtype (n = 4) and IL-31RA^–/– (n = 4) mice treated with IL-13. Images were captured at ×20 magnification. Scale bar, 100 µm. j Quantification of goblet cell hyperplasia associated genes including GOB5 and MUC5AC transcript levels in the total lungs of wildtype (n = 4) and IL-31RA^–/– (n = 4) mice treated with IL-13. Data are shown as means $\pm$SEM. Unpaired t test was used, and no statistical significance observed between groups. At least two independent experiments produced similar results. Source data are provided as a Source Data file.

Fig. 7. IFNγ is a positive regulator of IL31RA expression, along with AHR, inflammation, and goblet cell hyperplasia.

a Quantification of IL-31RA transcript levels in mouse airway smooth muscle cells (ASMC) treated with increasing doses of IFNγ (n = 3) or media (n = 3) for 16 h. Data are shown as the mean $\pm$SEM. Two-way ANOVA was used. b Quantification of IL-31RA transcript levels in human ASMCs treated with media, IL-31 (500 ng/mL) or IFNγ (50 ng/mL) for 16 h (n = 4). Data are shown as the mean $\pm$SEM. Two -way ANOVA was used. c Wildtype mice were intratracheally treated with saline (n = 4) or IFNγ (5 µg) (n = 4) on days 0 and 6, and airway resistance with increasing doses of methacholine was measured on day 7. Data are shown as mean $\pm$SEM. Two-way ANOVA was used. d Representative images of hematoxylin and eosin -stained lung sections from wildtype mice treated with saline (n = 4) or IFNγ (n = 4). Images were captured at ×20 magnification. Scale bar, 100 µm. e, f, g Quantification of inflammatory chemokines (CCL11 and IL-17), Th2 cytokines (IL-4, IL-5, and CCL24), and Th2 response-associated genes, including ARG1 and CHl3L3 in the lungs of wildtype mice treated with saline (n = 4) or IFNγ (n = 4). Data are shown as the mean $\pm$SEM. Unpaired t test was used. h Representative images of Alcian blue periodic acid-Schiff-stained lung sections from wild-type mice treated with saline (n = 4) or IFNγ (n = 4). Images were captured at ×20 magnification. Scale bar, 100 µm. i Quantification of mucus-associated gene transcripts, including GOB5 and MUC5AC, in the lungs of wildtype mice treated with saline (n = 4) or IFNγ (n = 4). Data are shown as the mean $\pm$SEM. Unpaired t test was used. At least two independent experiments produced similar results. Source data are provided as a Source Data file.

Fig. 8. Loss of IL-31RA is sufficient to attenuate IFNγ-induced AHR with no effect on inflammation and goblet cell hyperplasia.

a Wildtype (n = 9) and IL-31RA^–/– (n = 9) mice were treated intratracheally with IFNγ (5 μg) on days 0 and 6, and resistance was measured with increasing doses of MCh using Fexivent. Data are shown as mean $\pm$SEM. Two-way ANOVA was used. b Representative images of hematoxylin and eosin-stained lung sections from wildtype (n = 6) and IL-31RA^–/– (n = 6) mice treated with IFNγ. Images were captured at ×20 magnification. Scale bar, 100 µm. c, d, e Quantification of inflammatory cytokines (CCL11, CCL24 and IL-17), Th2 cytokines (IL-4 and IL-5), and Th2 response-associated genes including ARG1, and CHl3L3 in the lungs of wildtype (n = 6) and IL-31RA^–/– (n = 6) mice treated with IFNγ. Data are shown as mean $\pm$SEM. Unpaired t test was used, and no statistical signification observed between groups. f Representative images of Alcian blue periodic acid-Schiff-stained lung sections from wildtype (n = 6) and IL-31RA^–/– (n = 6) mice treated with IFNγ. Images were captured at ×20 magnification. Scale bar, 100 µm. g Quantification of mucus-associated genes including GOB5 and MUC5AC transcript levels in the lungs of wildtype (n = 6) and IL-31RA^–/– (n = 6) mice treated with IFNγ. Data are shown as means $\pm$SEM. Unpaired t test was used, and no statistical signification observed between groups. At least two independent experiments produced similar results. Source data are provided as a Source Data file.

Fig. 9. The post-transcriptional regulation of CHRM3 expression by IL-31RA.

a Quantification of CHRMs transcripts in the lungs of wildtype (n = 5) and IL-31RA^–/– (n = 5) mice using RT-PCR. Data shown as mean $\pm$SEM. Two-way ANOVA was used. b, c Quantification of CHRM3 transcripts in ASMC isolated from wildtype (n = 3) and IL-31RA^–/– (n = 3) mice and treated with media, IL-4 (10 ng/mL) or IL-31 (500 ng/mL) for 16 h. Data shown as mean $\pm$SEM. Two-way ANOVA was used. d, e The total lung lysates of HDM-treated wildtype (n = 5) and IL-31RA^–/– (n = 5) mice were immunoblotted with antibodies against CHRM3 and GAPDH. Data are shown as mean ± SEM. Unpaired t test was used. f ASMC isolated from wildtype (n = 3) and IL-31RA^–/– (n = 3) mice were lysed and immunoblotted with antibodies against CHRM3 and GAPDH. Data are shown as mean $\pm$SEM. Unpaired t test was used. g hTERT cells were transiently transfected with IL-31RA-specific (n = 3) or control siRNA (n = 3) for 72 h. Cell lysates were immunoblotted with antibodies against IL-31RA, CHRM3 and β-actin. Bar graph shows CHRM3 protein levels normalized to β-actin. Unpaired t test was used. Data are shown as means ± SEM. h Cell surface proteins were biotinylated, and affinity purified using neutravidin to measure cell surface levels of IL-31RA and CHRM3 in hTERT cells transfected with IL31RA-specific (n = 2) or control siRNA (n = 2) for 72 h. hTERT cells without biotinylation were used as a negative control. CHRM3 protein levels were normalized to ITGB1. i HEK293 cell were transiently transfected with overexpressing plasmids for CHRM3 and IL-31RA or empty control plasmids for 48 h. Cells were treated with antibodies against IL-31RA and CHRM3 and the IL-31RA-CHRM3 complex formation was visualized using hybridization probes at an excitation λex 594 nm (Red). The plasma membrane was stained with cholera toxin subunit b conjugated with Alexa Fluor 488 (Green) and the nuclei with DAPI (Blue). The white arrowheads highlight colocalization between the cholera toxin and puncta of the IL-31RA-CHRM3 complex. Images were captured at ×40 magnification. Scale bar, 50 µm. j hTERT cells were treated with antibodies against IL-31RA and CHRM3 or isotype IgG (negative PLA staining). The IL-31RA-CHRM3 complex formation was visualized as described in Fig. 9i. k HEK293T cells transiently transfected with a control plasmid or FLAG-tagged IL-31RA^OE plasmid for 72 h. Total cell lysates and eluted fractions were immunoblotted with anti-Flag, anti-IL31RA and anti-CHRM3 antibodies. At least two independent experiments produced similar results. Source data are provided as a Source Data file.

Fig. 10. The positive regulation of agonist-induced calcium elevation and MLC phosphorylation by the IL-31RA-CHRM3 axis.

a Intracellular calcium levels were measured using Fluo-4 AM dye in HEK293T cells transiently transfected with control or overexpressing plasmids for CHRM3 and/or IL-31RA plasmids for 72 h and treated with 10 µM carbachol (n = 4). b Intracellular calcium levels were measured using Fluo-4 AM dye in HEK293T cells transiently transfected with control or overexpressing plasmid for IL-31RA for 72 h and treated with 50 µM bradykinin (n = 3). c, d, e, f Intracellular calcium levels were measured using Fluo-4 AM dye in hTERT cells transiently transfected with control or overexpressing plasmid for IL-31RA for 72 h and cells treated with 10 µM carbachol (n = 8), 10 µM bradykinin (n = 8), 100 µM serotonin (n = 4) or 10 µM ionomycin (n = 4). g hTERT cell line 72 (CHRM3^LOW) was transiently transfected with control or IL-31RA overexpression plasmids for 72 h and intracellular calcium levels were measured in cells treated with 10 µM carbachol (n = 5) or 10 µM ionomycin (n = 3) using Fluo 4 AM dye. Data are shown as means ± SEM. h Intracellular calcium levels were measured using Fluo-4 AM dye in hTERT cells transiently transfected with IL-31RA-specific or control siRNA for 72 h and treated with 10 µM carbachol (n = 8). i hTERT cells were transiently transfected with IL-31RA overexpressing plasmid for 48 h and treated with IL-31 (500 ng/mL) or media for another 24 h. Media or IL-31 treated cells were stimulated with 10 µM carbachol (n = 4) and intracellular calcium levels were measured using Fluo-4 AM dye. Data are shown as means ± SEM. j hTERT cells were transiently transfected with IL-31RA-specific or control siRNA for 72 h and treated with 10 µM carbachol (n = 3) for 10 min. Cell lysates were immunoblotted with antibodies against phospho-MLC, and β-actin. Data are shown as means $\pm$SEM. Unpaired t test was used. k ASMC isolated from wildtype and IL-31RA^–/– mice were treated with 10 µM carbachol (n = 3) for 10 min and cell lysates were immunoblotted with antibodies against phospho-MLC, total-MLC and GAPDH. Data are shown as means $\pm$SEM. Unpaired t test was used. At least two independent experiments produced similar results. Source data are provided as a Source Data file.