Interleukin 31 receptor alpha augments muscarinic acetylcholine receptor 3-driven calcium signaling and airway hyperresponsiveness in asthma

Abstract

Asthma is a chronic inflammatory airway disease characterized by airway hyperresponsiveness (AHR), inflammation, and goblet cell hyperplasia. Both Th1 and Th2 cytokines, including IFN-γ, IL-4, and IL-13 have been shown to induce asthma; however, the underlying mechanisms remain unclear. We observed a significant increase in the expression of IL-31RA, but not its cognate ligand IL-31 during allergic asthma. In support of this, IFN-γ and Th2 cytokines, IL-4 and IL-13, upregulated IL-31RA but not IL-31 in airway smooth muscle cells (ASMC). Importantly, the loss of IL-31RA attenuated AHR but had no effects on inflammation and goblet cell hyperplasia in allergic asthma or mice treated with IL-13 or IFN-γ. Mechanistically, we demonstrate that IL-31RA functions as a positive regulator of muscarinic acetylcholine receptor 3 expression and calcium signaling in ASMC. Together, these results identified a novel role for IL-31RA in AHR distinct from airway inflammation and goblet cell hyperplasia in asthma.

Keywords: airway hyperresponsiveness; allergic asthma; calcium signaling; interleukin 31; lung.

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

(A) Schemata of HDM-induced allergic asthma model. (B and C) Quantification of IL-31RA and IL-31 transcripts in the lungs of wild-type and IL-31RA^−/− mice treated with HDM or saline. Data shown as mean ± SEM, n = 5–6/group, one-way ANOVA test, * p < 0.05. (D) Measurement of resistance with increasing doses of methacholine (MCh) in wild-type and IL-31RA^−/− mice treated with saline or HDM using FlexiVent. Data are shown as mean ± SEM, n = 7–8/group. A two-way ANOVA test, * p < 0.05. (E) Representative images of precision cut lung sections (PCLS) from the lungs of wild-type and IL-31RA^−/− mice treated with MCh (10⁻⁴ M) or prior to the treatment (baseline), scale bar 150 μm. (F) The percent of contraction of airways with increasing doses of MCh compared to the baseline area of airways between wild-type and IL-31RA^−/− mice. A two-way ANOVA test; n = 3–8 per group. * p < 0.05. (G) The percent contraction of collagen gels embedded with airway smooth muscle cells from wild-type and IL-31RA^−/− mice in culture media and treated with carbachol (10 μM) for 60 minutes. N = 3/group. Data shown as means mean ± SEM, two-tailed Student’s t-test, *p < 0.05

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

(A) Representative images of hematoxylin and eosin -stained lung sections from wild-type and IL-31RA^−/− mice treated with saline or HDM. Images were taken at 20x magnification, scale bar 100 μm. (B and 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 or HDM. Eos, eosinophils; Mac, macrophages; Lym, lymphocytes; and Neu, neutrophils. (D) Representative images of alcian blue periodic acid shiff(ABPAS) staining of lung sections from wild-type and IL-31RA^−/− mice treated with saline or HDM. Images were taken at 20x 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 or HDM. Data are shown as means ± SEM. One-way ANOVA was used, with n = 7–8/group. * p < 0.05.

Figure 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 or HDM using RT-PCR. (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 or HDM using RT-PCR. (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 or HDM using RT-PCR. (D) Quantification of GOB5 and MUC5AC gene transcripts in the whole lungs of wild-type and IL-31RA^−/− mice treated with saline or HDM using RT-PCR. Data are shown as mean ± SEM and representative of two independent experiments with n=5–6/group. One-way ANOVA was used. * p < 0.05.

Figure 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. (B and C) Quantification of IL-31RA and IL-31 transcripts in the lungs of wild-type and IL-31RA^−/− mice treated with house dust mite (HDM) or saline. Data shown as mean ± SEM, n = 5–6/group, one-way ANOVA test, *p < 0.05. (D) Measurement of resistance with increasing doses of methacholine (MCh) in wild-type and IL-31RA^−/− mice treated with saline or HDM using FlexiVent. Data are shown as mean ± SEM, n = 5–6/group. The above data is representative of two independent experiments with similar results. A two-way ANOVA test for multiple comparisons was used. * p<0.01. (E) Representative images of hematoxylin and eosin-stained lung sections from wild-type and IL-31RA^−/− mice treated with saline or SEA. Images were taken at 20x magnification, scale bar 100 μm. (F) Representative images of alcian blue periodic acid shiff staining of lung sections from wild-type and IL-31RA^−/− mice treated with saline or SEA. Images were taken at 20x magnification, scale bar 100 μm.

Figure 5.. Loss of IL31RA has no effect on Th2 responses and goblet cell hyperplasia during SEA-induced allergic asthma.

(A) Quantification of IFN-γ, IL-4, IL-5, and IL-13 in the whole lungs of wild-type and IL-31RA^−/− mice treated with saline or SEA using RT-PCR. (B) Quantification of CCL11, CCL24, and IL-10 in the whole lungs of wild-type and IL-31RA^−/− mice treated with saline or SEA using RT-PCR. (C) Quantification of Th2 response-associated genes including CHI3L3, ARG1, and FIZZ1 in the whole lungs of wild-type and IL-31RA^−/− mice treated with saline or SEA using RT-PCR. (D) Quantification of GOB5 and MUC5AC gene transcripts in the whole lungs of wild-type and IL-31RA^−/− mice treated with saline or SEA using RT-PCR. Data are shown as means ± SEM and representative of two independent experiments with n=5–6/group. One-way ANOVA was used. * p < 0.05.

Figure 6.. 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. (B) Measurement of resistance with increasing doses of methacholine (MCh) in wild-type mice treated with saline or IL-31 using FlexiVent. Data are shown as mean ± SEM, n = 6/group. 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 wild-type mice treated with IL-31 or saline. Images were captured at 20x 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 or IL-31. Unpaired two-tailed Student’s t-test was used. ***P < 0.001. (E and F) Quantification of inflammation-associated gene transcripts IFN-γ, 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 or IL-31. Unpaired two-tailed Student’s t-test was used and no significance found between groups, n=6/group. (G) Representative images of precision cut lung sections (PCLS) from wild-type mice treated with or without IL-31 (500 ng/ml) for 24 h. Airway contractility was measured in response to MCh (10⁻⁴ M) compared to baseline diameter. The percent of airway lumen area contraction with increasing doses of MCh was calculated for saline and IL-31-treated PCLS from wild-type mice. Two-way ANOVA test, n=5–8/group. (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.

Figure 7.. Th2 cytokines upregulate IL-31RA to induce AHR with no effect on inflammation and goblet cell hyperplasia.

(A and B) Quantification of IL-31RA transcripts in mouse airway smooth muscle cells (ASMC) treated with increasing doses of IL-4 and IL-13 for 16 h. One-way ANOVA test was used, n = 3/group; * p<0.05. (C) Wild-type and IL-31RA^−/− mice were treated intratracheally with IL-13 on days D0 and D6 and resistance in the lungs was measured with increasing doses of methacholine (MCh) using FexiVent. Data are shown as mean ± SEM, n=4/group. Two-way ANOVA test was used, *p<0.05. (D) ASMC isolated from wild-type and IL-31RA^−/− mice were seeded into collagen gels and treated with IL-13 to measure the contraction of collagen gels after 48 hours. Two-tailed Student’s t-test was used, n=3/group; * p<0.05. (E) Representative images of hematoxylin and eosin -stained lung sections from wild-type and IL-31RA^−/− mice treated with IL-13. Images were captured at 20x magnification, scale bar 100 μm. (F, G and 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 wild-type and IL-31RA^−/− mice treated with IL-13. Data are shown as mean ± SEM, n=4/group, Two-tailed Student’s t-test was used and no statistical significance observed between groups. (I) Representative images of alcian blue periodic acid shiff stained lung sections from wild-type and IL-31RA^−/− mice treated with IL-13. Images were captured at 20x magnification, scale bar 100 μm. (J) Quantification of goblet cell hyperplasia associated genes including GOB5 and MUC5AC transcript levels in the total lungs of wild-type and IL-31RA^−/− mice treated with IL-13. Data are shown as means ± SEM, n=4/group, Two-tailed Student’s t-test was used and no statistical significance observed between groups.

Figure 8.. IFN-γ 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 IFN-γ for 16 h. Data are shown as the mean ± SEM (n = 3/group). One-way ANOVA was used; *P < 0.05. (B) Quantification of IL-31RA transcript levels in human ASMCs treated with IL-31 (500 ng/ml) or IFN-γ (50 ng/ml) for 16 h. Data are shown as the mean ± SEM, n = 3–4/group. One-way ANOVA was used; *P < 0.05. (C) Wild-type mice were intratracheally treated with IFN-γ (5 μg) on days 0 and 6, and airway resistance with increasing doses of methacholine was measured on day 7. Data are shown as mean ± SEM, n = 4/group; two-way ANOVA was used, * P < 0.05. (D) Representative images of hematoxylin and eosin -stained lung sections from wild-type mice treated with saline or IFN-γ. Images were captured at 20x magnification, with a scale bar of 100 μm. (E, F, and G) Quantification of inflammatory chemokines (CCL11 and IL-17), Th2 cytokines (IL-4, IL-5, and CCL24), and Th2 response-associated genes, including ARG1, CHl3L3, and FIZZ1, in the lungs of wild-type mice treated with saline or IFN-γ. Data are shown as the mean ± SEM, n = 6/group. Two-tailed Student’s t-test was used; * P < 0.05. (H) Representative images of alcian blue periodic acid shiff -stained lung sections from wild-type mice treated with saline or IFN-γ. Images were captured at 20x magnification, scale bar = 100 μm. (I) Quantification of mucus-associated gene transcripts, including GOB5 and MUC5AC, in the lungs of wild-type mice treated with saline or IFN-γ. Data are shown as the mean ± SEM (n = 6/group). Two-tailed Student’s t-test was used; * P < 0.05.

Figure 9.. Loss of IL-31RA is sufficient to attenuate IFNγ-induced AHR with no effect on inflammation and goblet cell hyperplasia.

(A) Wild-type and IL-31RA^−/− mice were treated intratracheally with IFN-γ (5 ng) on days D0 and D6 and resistance was measured with increasing doses of methacholine (MCh) using FexiVent. Data are shown as mean ± SEM, n = 6/group. Two-way ANOVA was used, *p<0.05. (B) Representative images of hematoxylin and eosin -stained lung sections from wild-type and IL-31RA^−/− mice treated with IFN-γ. Images were captured at 20x magnification, scale bar 100 μm, n = 6/group. (C, D and E) Quantification of inflammatory cytokines (CCL11, CCL24 and IL-17), Th2 cytokines (IL-4 and IL-5), and Th2 response-associated genes including ARG1, CHl3L3, and FIZZ1 in the lungs of wild-type and IL-31RA^−/− mice treated with IFN-γ. Data are shown as mean ± SEM, n = 6/group. Two-tailed Student’s t-test was used and no statistical signification observed between groups. (F) Representative images of alcian blue periodic acid shiff-stained lung sections from wild-type and IL-31RA^−/− mice treated with IFN-γ. Images were captured at 20x magnification, scale bar 100μm, n = 6/group. (G) Quantification of mucus-associated genes including GOB5 and MUC5AC transcript levels in the lungs of wild-type and IL-31RA^−/− mice treated with IFN-γ. Data are shown as means ± SEM, n = 6/group. Two-tailed Student’s t-test was used, and no statistical signification observed between groups.

Figure 10.. IL-31RA augments CHRM3-driven calcium signaling and MLC phosphorylation in ASMC.

(A) Quantification of the transcripts of different isoforms of CHRMs in the lungs of wild-type and IL-31RA^−/− mice. (B) Quantification of the transcripts of CHRM3 in ASMC isolated from wild-type mice and treated with media, IL-4 (10 ng/ml) or IL-31 (500 ng/ml) for 16 h. (C) Quantification of the transcripts of CHRM3 in ASMC isolated from IL-31RA^−/− mice and treated with media, IL-4 (10 ng/ml) and IL-31 (500 ng/ml) for 16 h. (D) ASMC isolated from wild-type and IL-31RA^−/− mice were lysed and immunoblotted with antibodies against CHRM3 and GAPDH. CHRM3 protein levels were normalized to GAPDH and shown as fold induced using a bar graph (***P < 0.0005; n = 3, Student’s 2-tailed t test). (E) HEK293T cells were transiently co-transfected with overexpressing plasmids for CHRM3 and IL-31RA or empty control plasmids for 48 h. The IL-31RA-CHRM3 complex formation was visualized using hybridization probes labeled with Alexa 594 (Red). The nuclei were stained with DAPI (blue) and images were captured at 40X magnification. Scale bar, 100 μm. (F) Western blot analyses of CHRM3 in anti-FLAG or control IgG immunoprecipitants from cell lysates of HEK293T cells transiently transfected with a control plasmid or IL-31RA^OE plasmid for 72 hrs. Total cell lysates and eluted fractions were immunoblotted with anti-Flag, anti-IL31RA and anti-CHRM3 antibodies. The data presented is a representative of three independent experiments with similar results. (G) Increases in carbachol-induced intracellular calcium levels were measured in HEK293T cells transiently transfected with overexpressing plasmids for CHRM3 and IL-31RA or empty control plasmids for 72 h and treated with carbachol (10μM). (H) HEK293T cells were transiently co-transfected with overexpressing plasmids for CHRM3 and IL-31RA or empty control plasmids for 48 h and treated with carbachol (10μM) for 10 min. Cell lysates were immunoblotted with antibodies against phospho-MLC, total-MLC and GAPDH. Data are shown as means ± SEM, n = 3/group. (I) ASMC isolated from wild-type and IL-31RA^−/− mice were treated with carbachol (10μM) for 10 min and cell lysates were immunoblotted with antibodies against phospho-MLC, total-MLC and GAPDH. Data are shown as means ± SEM, n = 3/group