Journal: bioRxiv

Article Title: Interleukin 31 receptor alpha induces airway hyperresponsiveness in asthma

doi: 10.1101/2022.12.15.520615

Figure Lengend Snippet: (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

Article Snippet: HEK293 cells were transiently transfected with overexpressing plasmids for IL-31RA (Origene, RC218212L1) and CHRM3 (Origene, SC119736) or control empty plasmids (pLJM1-EGFP, Addgene 19319 and pLenti-C-Myc-DDK-P2A-Puro, Origene PS100092) using Lipofectamine 3000 and cultured for 48 h. Then, cells were fixed for 5 min in ice cold methanol at 4 0 C and incubated with Duolink blocking solution (Sigma) for 1 hour at room temperature followed by overnight incubation with primary antibodies for IL-31RA (15 µg/ml, R&D Systems AF2769) and CHRM3(1:500, Abcam ab126168).

Techniques: Two Tailed Test

Journal: bioRxiv

Article Title: Interleukin 31 receptor alpha induces airway hyperresponsiveness in asthma

doi: 10.1101/2022.12.15.520615

Figure Lengend Snippet: (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.

Article Snippet: HEK293 cells were transiently transfected with overexpressing plasmids for IL-31RA (Origene, RC218212L1) and CHRM3 (Origene, SC119736) or control empty plasmids (pLJM1-EGFP, Addgene 19319 and pLenti-C-Myc-DDK-P2A-Puro, Origene PS100092) using Lipofectamine 3000 and cultured for 48 h. Then, cells were fixed for 5 min in ice cold methanol at 4 0 C and incubated with Duolink blocking solution (Sigma) for 1 hour at room temperature followed by overnight incubation with primary antibodies for IL-31RA (15 µg/ml, R&D Systems AF2769) and CHRM3(1:500, Abcam ab126168).

Techniques: Staining, Cell Counting

Journal: bioRxiv

Article Title: Interleukin 31 receptor alpha induces airway hyperresponsiveness in asthma

doi: 10.1101/2022.12.15.520615

Figure Lengend Snippet: (A) Quantification of Th2 cytokine transcripts including IL-4, IL5, and IL-13 in the total lungs 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 total 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 total 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.

Article Snippet: HEK293 cells were transiently transfected with overexpressing plasmids for IL-31RA (Origene, RC218212L1) and CHRM3 (Origene, SC119736) or control empty plasmids (pLJM1-EGFP, Addgene 19319 and pLenti-C-Myc-DDK-P2A-Puro, Origene PS100092) using Lipofectamine 3000 and cultured for 48 h. Then, cells were fixed for 5 min in ice cold methanol at 4 0 C and incubated with Duolink blocking solution (Sigma) for 1 hour at room temperature followed by overnight incubation with primary antibodies for IL-31RA (15 µg/ml, R&D Systems AF2769) and CHRM3(1:500, Abcam ab126168).

Techniques: Reverse Transcription Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Interleukin 31 receptor alpha induces airway hyperresponsiveness in asthma

doi: 10.1101/2022.12.15.520615

Figure Lengend Snippet: (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.

Article Snippet: HEK293 cells were transiently transfected with overexpressing plasmids for IL-31RA (Origene, RC218212L1) and CHRM3 (Origene, SC119736) or control empty plasmids (pLJM1-EGFP, Addgene 19319 and pLenti-C-Myc-DDK-P2A-Puro, Origene PS100092) using Lipofectamine 3000 and cultured for 48 h. Then, cells were fixed for 5 min in ice cold methanol at 4 0 C and incubated with Duolink blocking solution (Sigma) for 1 hour at room temperature followed by overnight incubation with primary antibodies for IL-31RA (15 µg/ml, R&D Systems AF2769) and CHRM3(1:500, Abcam ab126168).

Techniques: Staining

Journal: bioRxiv

Article Title: Interleukin 31 receptor alpha induces airway hyperresponsiveness in asthma

doi: 10.1101/2022.12.15.520615

Figure Lengend Snippet: (A) Quantification of IFN-γ, IL-4, IL-5, and IL-13 in the total 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 total 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 total 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 total 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.

Article Snippet: HEK293 cells were transiently transfected with overexpressing plasmids for IL-31RA (Origene, RC218212L1) and CHRM3 (Origene, SC119736) or control empty plasmids (pLJM1-EGFP, Addgene 19319 and pLenti-C-Myc-DDK-P2A-Puro, Origene PS100092) using Lipofectamine 3000 and cultured for 48 h. Then, cells were fixed for 5 min in ice cold methanol at 4 0 C and incubated with Duolink blocking solution (Sigma) for 1 hour at room temperature followed by overnight incubation with primary antibodies for IL-31RA (15 µg/ml, R&D Systems AF2769) and CHRM3(1:500, Abcam ab126168).

Techniques: Reverse Transcription Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Interleukin 31 receptor alpha induces airway hyperresponsiveness in asthma

doi: 10.1101/2022.12.15.520615

Figure Lengend Snippet: (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 including ARG1, CHl3L3, and FIZZ1 in the total 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.

Article Snippet: HEK293 cells were transiently transfected with overexpressing plasmids for IL-31RA (Origene, RC218212L1) and CHRM3 (Origene, SC119736) or control empty plasmids (pLJM1-EGFP, Addgene 19319 and pLenti-C-Myc-DDK-P2A-Puro, Origene PS100092) using Lipofectamine 3000 and cultured for 48 h. Then, cells were fixed for 5 min in ice cold methanol at 4 0 C and incubated with Duolink blocking solution (Sigma) for 1 hour at room temperature followed by overnight incubation with primary antibodies for IL-31RA (15 µg/ml, R&D Systems AF2769) and CHRM3(1:500, Abcam ab126168).

Techniques: Isolation, Two Tailed Test, Staining

Journal: bioRxiv

Article Title: Interleukin 31 receptor alpha induces airway hyperresponsiveness in asthma

doi: 10.1101/2022.12.15.520615

Figure Lengend Snippet: (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 resistance to 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.

Article Snippet: HEK293 cells were transiently transfected with overexpressing plasmids for IL-31RA (Origene, RC218212L1) and CHRM3 (Origene, SC119736) or control empty plasmids (pLJM1-EGFP, Addgene 19319 and pLenti-C-Myc-DDK-P2A-Puro, Origene PS100092) using Lipofectamine 3000 and cultured for 48 h. Then, cells were fixed for 5 min in ice cold methanol at 4 0 C and incubated with Duolink blocking solution (Sigma) for 1 hour at room temperature followed by overnight incubation with primary antibodies for IL-31RA (15 µg/ml, R&D Systems AF2769) and CHRM3(1:500, Abcam ab126168).

Techniques: Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Interleukin 31 receptor alpha induces airway hyperresponsiveness in asthma

doi: 10.1101/2022.12.15.520615

Figure Lengend Snippet: (A) Wild-type and IL-31RA -/- mice were treated intratracheally with IFN-γ 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 = 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 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.

Article Snippet: HEK293 cells were transiently transfected with overexpressing plasmids for IL-31RA (Origene, RC218212L1) and CHRM3 (Origene, SC119736) or control empty plasmids (pLJM1-EGFP, Addgene 19319 and pLenti-C-Myc-DDK-P2A-Puro, Origene PS100092) using Lipofectamine 3000 and cultured for 48 h. Then, cells were fixed for 5 min in ice cold methanol at 4 0 C and incubated with Duolink blocking solution (Sigma) for 1 hour at room temperature followed by overnight incubation with primary antibodies for IL-31RA (15 µg/ml, R&D Systems AF2769) and CHRM3(1:500, Abcam ab126168).

Techniques: Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Interleukin 31 receptor alpha induces airway hyperresponsiveness in asthma

doi: 10.1101/2022.12.15.520615

Figure Lengend Snippet: (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 wildtype 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) HEK293 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) Increases in intracellular calcium levels were measured in HEK 293 cells transiently transfected with overexpressing plasmids for CHRM3 and IL-31RA or empty control plasmids for 72 h and treated with carbachol (10µM). (G) HEK 293 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. (H) 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

Article Snippet: HEK293 cells were transiently transfected with overexpressing plasmids for IL-31RA (Origene, RC218212L1) and CHRM3 (Origene, SC119736) or control empty plasmids (pLJM1-EGFP, Addgene 19319 and pLenti-C-Myc-DDK-P2A-Puro, Origene PS100092) using Lipofectamine 3000 and cultured for 48 h. Then, cells were fixed for 5 min in ice cold methanol at 4 0 C and incubated with Duolink blocking solution (Sigma) for 1 hour at room temperature followed by overnight incubation with primary antibodies for IL-31RA (15 µg/ml, R&D Systems AF2769) and CHRM3(1:500, Abcam ab126168).

Techniques: Isolation, Transfection, Hybridization, Labeling, Staining