Abstract
Purpose
Airway inflammation and remodeling are pivotal in asthma pathogenesis. Interleukin-17D (IL-17D), a recently characterized cytokine within the IL-17 family, is found to be expressed at higher levels in lung tissue; however, its role in asthma remains unclear. This study aims to elucidate the function and mechanisms of IL-17D in asthma, providing new insights for therapeutic strategies.
Methods
We analyzed IL-17D messenger RNA expression levels using datasets from the Gene Expression Omnibus (GEO). Bronchoscopic biopsy tissues and serum samples from asthmatics, along with lung tissues from chronic asthmatic mice, were collected to validate IL-17D expression. Lentivirus-mediated IL-17D knockdown was performed in a chronic asthma mouse model induced by ovalbumin (OVA). Recombinant murine IL-17D protein was intranasally administered to further investigate its impact. In vitro, human bronchial epithelial cells (16HBE) were treated with recombinant human IL-17D or CD93-targeting small interfering RNA to explore signaling mechanisms.
Results
GEO data and experimental findings revealed that IL-17D expression was reduced in the airway epithelia of asthmatics and chronic asthmatic mice. In vivo, knockdown of IL-17D exacerbated peri-airway inflammation and promoted epithelial-mesenchymal transition (EMT) in OVA-induced asthmatic mice. Conversely, recombinant IL-17D protein administration significantly attenuated airway inflammation, extracellular matrix (ECM) deposition, and EMT progression. In vitro, IL-17D mitigated transforming growth factor-β1-induced fibrotic remodeling in 16HBE cells, with CD93 receptor silencing partially reversing these effects.
Conclusions
Our findings suggest that IL-17D could play a protective role in asthmatic airway inflammation and remodeling, partially through the CD93 receptors. These results highlight IL-17D as a potential therapeutic target for asthma management.
Keywords: Asthma, inflammation, airway remodeling, IL-17D, epithelial-mesenchymal transition
INTRODUCTION
Asthma is a prevalent chronic non-communicable disease globally, characterized by variable airflow obstruction, leading to symptoms such as dyspnea and wheezing.1 Despite the availability of specific medications designed to manage asthma symptoms, the use of inhaled corticosteroids (ICS) or the addition of supplementary controller medications often fails to provide adequate relief for patients with severe asthma.2 The introduction of biologic therapies has proven beneficial for individuals with asthma, as these treatments can effectively prevent exacerbations, improve symptoms, and reduce the need for ICS and oral corticosteroids in patients with corticosteroid-dependent asthma.3
In fact, asthma is a heterogeneous disease, characterized by three main features: airway inflammation, airway hyperresponsiveness (AHR), and airway remodeling. Among these, airway remodeling, which includes extensive epithelial damage, airway smooth muscle hypertrophy and hyperplasia, collagen deposition, and subepithelial basement membrane thickening and fibrosis, is a major factor contributing to poor outcomes in asthma patients.4 Research involving biopsies of young children has indicated that airway remodeling may occur early in the pathogenesis of asthma, potentially predisposing these individuals to the condition by influencing lung development.5,6,7 Consequently, concentrating on the mechanisms underlying airway remodeling may significantly contribute to advances in future therapeutic approaches for asthma.
The interleukin (IL)-17 family consists of six members, including IL-17A through IL-17F. IL-17D is a relatively under-researched cytokine. In humans, IL-17D is situated on chromosome 13p11 and is characterized as a glycoprotein composed of 202 amino acids, rendering it the largest member of the IL-17 family.8 Studies have shown that IL-17D is preferentially expressed in various tissues, including the skeletal muscle, brain, lung, adipose tissue, heart, and pancreas. Recent studies have progressively elucidated the regulatory role of IL-17D in various diseases. Regarding tumor microenvironment modulation, multiple studies have demonstrated the significant anti-tumor activity of IL-17D. For instance, researchers found that local injection of recombinant IL-17D protein effectively suppressed tumor growth rate in a B16-ovalbumin (OVA) melanoma mouse model.9 In autoimmune diseases, IL-17D exhibits tissue-specific expression variations. Clinical sample analysis revealed the presence of IL-17D expression in rheumatoid nodules,10,11 while IL-17D messenger RNA (mRNA) expression was significantly reduced in psoriatic lesions.12 Notably, in IBD models, intestinal epithelium-derived IL-17D interacts with type 3 innate lymphoid cells (ILC3) to maintain intestinal mucosal barrier function,13 suggesting a tissue-protective role. In respiratory diseases, IL-17D expression is reduced in lung cancer and correlates with improved patient prognosis.14 Dong et al.15 discovered that IL-17D has the potential to mitigate septic lung injury resulting from heme oxygenase-1 deficiency. These studies indicate that IL-17D plays a crucial role in various diseases, often exhibiting a protective function in inflammatory responses and epithelial injury. Nevertheless, the function of IL-17D in the context of asthma is not well understood. Consequently, the objective of this study was to examine the role of IL-17D in asthma, focusing particularly on airway inflammation and remodeling.
MATERIALS AND METHODS
Bioinformatics analysis
Human gene expression profiles from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/), specifically datasets GEO Series (GSE) 179165, GSE63142, GSE143303, and GSE67472, were utilized to assess the expression of IL-17D in airway epithelial brush cells from both healthy individuals and asthmatic patients. Additionally, the GSE40374 dataset was employed to investigate IL-17D expression in M-BE cells, both with and without transforming growth factor (TGF)-β1 stimulation. The analyses were conducted using the Gene Expression Omnibus 2R (GEO2R) tool, and the results were visualized using GraphPad Prism 9.5 (GraphPad Software Inc., San Diego, CA, USA). Detailed information on each dataset is provided in Supplementary Table S1.
Patients and specimen sources
The diagnostic criteria for asthma are established in accordance with the Global Initiative for Asthma guidelines, which were updated in 2024. Serum samples were obtained from 32 asthma patients and 22 healthy controls at the First Affiliated Hospital of Shandong First Medical University. Peripheral blood (4 mL/subject) was collected in serum separator tubes, allowed to clot at room temperature for 30 minutes, and centrifuged at 3,000 rpm for 10 minutes. The supernatant serum was aliquoted, flash-frozen in liquid nitrogen, and stored at −80°C. Lung tissues from healthy controls were obtained from patients with benign lung nodules, all of whom demonstrated normal pulmonary function and were free from other respiratory conditions. Asthmatic lung tissues were collected via bronchoscopic biopsies, facilitated by the Department of Respiratory Medicine at Haihe Hospital, Tianjin University. This study received approval from the Ethics Committee of Shandong First Medical University (ethics approval No. 2021-S923). All participants in the study provided written informed consent. The fundamental clinical information for all subjects is presented in Supplementary Table S2.
Lung histology
Human lung biopsy tissues and murine lung tissues were preserved in 4% paraformaldehyde for a duration of 48 hours at room temperature, followed by paraffin embedding. Following sectioning at a thickness of 5 μm, the tissues were deparaffinised and subsequently employed in the processes of haematoxylin and eosin (H&E), Periodic Acid-Schiff (PAS) and Masson trichrome staining.
Mice
Female C57BL/6 mice, aged between 6 and 8 weeks and weighing approximately 20 g, were acquired from PengYue Laboratory Animal Breeding Co., Ltd. (Jinan, China). The construction of a mouse model of asthma commenced with the sensitization of the subjects via intraperitoneal injection of an OVA solution (20 µg in 200 µL phosphate-buffered saline [PBS]; Sigma-Aldrich, St. Louis, MO, USA) mixed with AL(OH)3 (2 mg) at Days 0, 7 and 14. Subsequently, the mice were given a 3% OVA aerosol every 2 days starting from Day 21. Mice were injected with lentiviral (LV) encoded IL-17D short hairpin RNA (shRNA) or negative control short hairpin RNA (shNC) in 3 separate doses via the tail vein between Days 21 and 58. Accordingly, the mice were divided into 4 groups (6 mice in each group): 1) LV2-shNC group; 2) LV2-shIL-17D group; 3) OVA+LV2-shNC group; 4) OVA+LV2-shIL-17D group. Furthermore, 3 experimental groups were established: 1) control group; 2) OVA group; 3) OVA+recombinant mouse IL-17D protein (rmIL-17D) group, in which mice received exogenous IL-17D administration. In the OVA+rmIL-17Dgroup, the experimental group of mice was administered intranasal drops of rmIL-17D on a weekly basis (Days 22, 29, 36, 43, 50 and 57) commencing after the initial OVA provocation test on Day 21. The administration of 0.5 µg of rmIL-17D was conducted on a per mouse. The airway resistance of mice was evaluated within 24 hours following the final challenge, using assessment techniques that our research team has previously detailed. All mice were subjected to euthanasia 24 hours following the final exposure.
Enzyme-linked immunosorbent assay (ELISA)
Levels of IL-4, IL-5, IL-13, immunoglobulin (Ig) E and IL-17D in serum or bronchoalveolar lavage fluid (BALF) were measured using commercial ELISA kits. The ELISA kits used were as follows: Human IL-17D ELISA Kit (EK5044; Sabbiotech, Nanjing, China); Mouse IL-17D ELISA Kit (EK0792; BOSTER, Wuhan, China); Mouse IL-4 ELISA Kit (70-EK204/2-96; MultiSciences, Hangzhou, China), Mouse IL-5 ELISA Kit (70-EK105-96; MultiSciences), Mouse IL-13 ELISA Kit (70-EK204/2-96, 70-EK213/2-96; MultiSciences). All samples were subjected to duplicate measurement.
Western blotting
Cellular or lung tissue proteins were subjected to lysis using a radioimmunoprecipitation assay buffer that contained phenylmethanesulfonyl fluoride (Servicebio, Beijing, China). Protein concentrations were determined utilizing the BCA Protein Quantification Kit (Beyotime, Shanghai, China). Protein samples were separated utilizing sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by transfer to polyvinylidene difluoride membranes with pore sizes of 0.45 or 0.22 μm. The membranes were subjected to blocking in tris-buffered saline containing 0.1% Tween 20 and 5% bovine serum albumin (BSA) at room temperature for a duration of 1.5 hours. Subsequently, the membranes were incubated with primary antibodies at 4°C overnight. The primary antibodies were used: anti-mouse IL-17D (1:1,000; Thermo Fisher Scientific, Waltham, MA, USA), anti-Vimentin (1:1,000; Cell Signaling Technology, Danvers, MA, USA), anti-alpha-smooth muscle actin (α-SMA) (1:1,000; HUABIO, Hangzhou, China), anti-glyceraldehyde-3-phosphate dehydrogenase (1:5,000; Abcam, Cambridge, MA, USA), anti-Collagen I (1:1,000; Abcam), anti-Fibronectin (1:1,000; HUABIO), anti-N-cadherin (1:1,000; Cell Signaling Technology), anti-E-cadherin (1:1,000; Cell Signaling Technology). On the second day, the bands were incubated with goat anti-mouse or goat anti-rabbit IgG (secondary antibodies) for a duration of 1 hour at room temperature. Subsequently, the bands were visualized through chemiluminescence utilizing an enhanced chemiluminescent reagent.
Immunohistochemistry (IHC) staining
Following deparaffinization and hydration with xylene and a gradual reduction in ethanol concentration, the sections were incubated in a citric acid buffer at pH 6.0 at 95°C for 15 minutes. Subsequently, the sections were washed in water for a period of 5 minutes, after which they were treated with a solution of 3% hydrogen peroxide (H2O2) for a further 10 minutes, with the objective of removing any endogenous peroxides. Afterwards the sections were blocked in a wet chamber containing goat serum for 1 hour. The samples were incubated with primary antibodies (anti-human IL-17D [1:200, Proteintech, Wuhan, China], anti-α-SMA [1:200; HUABIO], anti-zonula occludens-1 [ZO-1] [1:200; Abcam], anti-N-cadherin [1:200; Cell Signaling Technology]) at 4°C for a period of 12–16 hours. Following this, the samples were rewarmed for 30 minutes and then incubated in horseradish peroxidase-labelled secondary antibody for a further 30 minutes.
Immunofluorescence (IF) staining
The experimental protocol for IF staining closely resembled that of IHC staining, up until the point at which the sections were exposed to the primary antibody. Primary antibodies used for IF staining are as follows: anti-mouse IL-17D (1:200; Thermo Fisher Scientific), anti-α-SMA (1:200; HUABIO), anti-Collagen I (1:200; Abcam). In the subsequent day, the sections were incubated with the corresponding secondary antibodies for 1 hour at room temperature in the absence of light. Subsequently, the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (1:200) for 10 minutes. Images were obtained using an upright fluorescence microscope (Leica, Berlin, Germany).
RNA-sequencing analysis
The left lungs of mice in the OVA+LV2-shNC group and the OVA+LV2-shIL-17D group were promptly isolated, collected, and stored at −80°C to facilitate subsequent RNA extraction. RNA sequencing was performed by Biomarker Technologies Co., LTD. (Beijing, China).
Cell culture and treatment
The human bronchial epithelial cell line (16HBE) was acquired from FuHeng Biology (Shanghai, China) and subsequently cultured in Keratinocyte Medium (ScienCell, Carlsbad, CA, USA) enriched with 1% Keratinocyte Growth Supplement (ScienCell). The cells were maintained in a humidified incubator under conditions of 5% CO2 at 37°C. The human IL-17D protein (HY-P70587; MedChemExpress, Shanghai, China) was used to stimulate 16HBE cells at different concentrations and time points. The 16HBE cells were stimulated by 10 ng/mL TGF-β1 (Abbkine Scientific Co., Ltd., Wuhan, China) for 48 hours. To explore the possible receptors for IL-17D, we constructed small interfering RNA (siRNA) that knocked down CD93. The siRNAs sequences targeted CD93 were as follows:
siRNA 1:5′-CGAAGAGGGAGGAGAAGAATTUUCUUCUCCUCCCUCUUCGTT-3′
siRNA 2:5′-GGAGGACAAAGGAGAGAAATTUUUCUCUCCUUUGUCCUCCTT-3′
siRNA 3:5′-CAAUGUAGCCUGUGGGGAATTUUCCCCACAGGCUACAUUGTT-3′
16HBE cells were transfected with small interfering RNA negative control (siNC) or small interfering RNA targeting CD93 (siCD93) using the EndoFectin™ Max Transfection Reagent (GeneCopoeia, Guangzhou, China) in accordance with the manufacturer’s instructions.
Quantitative real-time-polymerase chain reaction (qRT-PCR)
Total RNA was isolated from 16HBE cells utilizing the RNA Fast 200 RNA Extraction kit (Fastagen Biotech, Shanghai, China), in accordance with the guidelines provided by the manufacturer. The Merinton SMA4000 UV-VIS spectrophotometer was employed for the measurement of RNA purity and concentration. Subsequently, cDNA was synthesized using reverse transcription reagents (Vazyme, Nanjing, China). The quantification of mRNA was conducted utilizing a Bio-Rad CFX96 Real-Time system in conjunction with the ChamQ Universal SYBR qPCR Master Mix (Vazyme). Primers sequences were available in Supplementary Table S3.
Cell IF staining
16HBE cells were cultured on cell culture slides within a 24-well plate. Subsequent to the specified treatments, the cell climbing slices were subjected to washing with PBS, followed by fixation using 4% paraformaldehyde. Thereafter, the slices were incubated with 0.5% Triton X-100 to facilitate permeabilization. Then, the cells were incubated with 1% BSA in PBS for one hour at room temperature. A primary antibody was administered to the slices and incubated overnight at a temperature of 4°C. Primary antibodies used for Cell IF staining are as follows: anti-Collagen I (1:200; Abcam), anti-E-cadherin (1:200; Cell Signaling Technology), anti-Fibronectin (1:200; HUABIO). The following day, the primary antibodies were removed from the sections using fresh PBS. After three washes with PBS, the sections were incubated with the appropriate secondary antibodies for a duration of one hour, protected from light exposure. The nuclei were counterstained with DAPI (1:200) for 10 minutes. Subsequent to the final washing with PBS, images were obtained using an upright fluorescence microscope (Leica).
Statistical analysis
Statistical analyses were conducted utilizing GraphPad Prism 9.5 software (GraphPad Software Inc.) and SPSS software (version 25.0; IBM Corp., Armonk, NY, USA). Normal distributed data was reported as the mean ± standard deviation, otherwise it was presented as medians accompanied by interquartile ranges. The Student’s t-test (normal distributed parameters) and the Mann-Whitney test (non-normal distributed parameters) were employed to ascertain significant differences between the 2 groups. A one-way analysis of variance was conducted, followed by Dunnett’s Test and Tukey’s post hoc test, to assess significant differences among multiple groups. A 2-tailed P value of < 0.05 was considered statistically significant. All experiments were conducted with a minimum of three replications.
RESULTS
IL-17D expression is reduced in the airway epithelium of asthma patients and chronic asthmatic mice induced by OVA
At present, the expression level of IL-17D in asthma has yet to be established. To address this, we initially analyzed the expression of IL-17D mRNA in the airway epithelium of asthmatics and healthy controls from the GEO database. In the GSE179156, GSE63142, and GSE143303 datasets, individuals were divided into an asthma group and a healthy control group. These datasets indicated that IL-17D mRNA expression was downregulated in asthmatic patients compared with healthy controls (Fig. 1A-C). In the GSE67472 dataset, the asthma group was categorized into high T2 asthma and low T2 asthma. Even so, the IL-17D mRNA expression was lower than that of healthy controls in both T2 high and T2 low asthma (Fig. 1D). We postulated that IL-17D is downregulated in the asthmatic epithelium.
Fig. 1. Lower IL-17D expression in the airway epithelium of asthmatics and OVA-induced chronic asthma mouse model. Box plots of IL-17D gene expression in airway epithelial cells from individuals in (A) GSE179156, (B) GSE63142, (C) GSE143303 and (D) GSE67472. (E) H&E staining showed infiltration of inflammatory cells around the airways, in healthy controls versus asthmatics (scale bar = 50 µm). (F) IHC analysis of IL-17D expression in airway epithelium from healthy individuals and asthmatics (scale bar = 50 µm). (G) IF analysis of IL-17D expression levels in the airway epithelium of control and asthmatic mice (scale bar = 50 µm). (H) ELISA analysis of IL-17D serum levels in healthy individuals and asthmatics. (I) ELISA was used to quantify IL-17D levels in the serum of control and asthmatic mice. (J) IL-17D levels in BALF from control and asthmatic mice were quantified by ELISA. Student’s t-test, Mann-Whitney test and analysis of variance analysis were used. Data were shown as the mean ± standard deviation.
IL, interleukin; OVA, ovalbumin; H&E, hematoxylin and eosin; IHC, immunohistochemistry; IF, immunofluorescence; ELISA, enzyme-linked immunosorbent assay; BALF, bronchoalveolar lavage fluid; ns, not significant.
*P < 0.05, **P < 0.01.
Furthermore, we obtained tissue samples and conducted a series of experimental validations to support our hypothesis. We obtained airway epithelial biopsy specimens from patients with asthma (Fig. 1E) and constructed a chronic asthma mouse model induced by OVA. The results showed that IL-17D expression was predominantly distributed in the airway epithelium. Notably, a reduction in IL-17D expression in the airway epithelium was observed in both asthma patients (Fig. 1F) and OVA-induced asthma mice (Fig. 1G). Concurrently, we assessed the serum concentrations of IL-17D in both healthy individuals and patients with asthma. The findings from the ELISA analysis indicated that the serum levels of IL-17D were significantly lower in asthmatic patients compared to healthy controls (Fig. 1H). Furthermore, we obtained serum and BALF from OVA-challenged chronic asthmatic mice and conducted an ELISA analysis on these samples. The results consistently demonstrated that the expression of IL-17D was reduced in the asthma group relative to the control group (Fig. 1I and J).
IL-17D deficiency exacerbates airway inflammation in OVA-induced asthma mice
To further elucidate the role of IL-17D in asthma, a lentivirus-mediated IL-17D knockdown animal model was established. This involved the intravenous injection of a lentiviral shRNA vector specifically targeting IL-17D into mice via the tail vein. Fig. 2A illustrates the methodology employed to construct an asthma mouse model and the timing of lentiviral interference. In addition, we extracted mouse lung tissue proteins to verify the efficacy of lentiviral interference using the experimental method of western blotting (Fig. 2B). The knockdown effect of shIL-17D in mouse airway epithelium was further verified by tissue IF staining (Supplementary Fig. S1). Analysis of lung function in mice revealed that administration of IL-17D shRNA further exacerbated airway resistance in OVA-induced asthmatic mice, demonstrating a more pronounced AHR (Fig. 2C). Concurrently, knockdown of IL-17D significantly increased the total cell count, neutrophil, eosinophil, and macrophage numbers in the BALF of asthmatic mice (Fig. 2D and E). Meanwhile, histological analysis using H&E and PAS staining demonstrated that IL-17D knockdown enhanced peribronchial inflammatory cell infiltration and airway mucus production in asthmatic mice (Fig. 2F and G). The findings indicate that IL-17D exerts a regulatory effect on airway inflammation in asthma.
Fig. 2. IL-17D deficiency increases OVA-induced airway inflammation in asthmatic mice. (A) Mouse models of chronic asthma experimental design and grouping. Part of the figure was generated with BioRender. (B) The effects of IL-17D knockdown with lentiviral shRNA was assessed by western blotting. (C) Airway resistance responses to increased doses of inhaled methacholine treatment in different groups. (D) Total cells number in BALF were counted. (E) The numbers of neutrophils, eosinophils and macrophages in the mouse BALF were detected. (F) Representative images of lung sections stained with H&E (scale bar = 50 µm). Peri-airway inflammation scores were measured. (G) PAS staining was used to assess the secretion levels of airway mucus in mice from each group (scale bar = 50 µm). One-way analysis of variance followed by Tukey’s post hoc test. Data are shown as the means ± standard deviation of 3 independent experiments.
IL, interleukin; OVA, ovalbumin; shRNA, short hairpin RNA; BALF, bronchoalveolar lavage fluid; H&E, hematoxylin and eosin; PAS, Periodic Acid-Schiff; PBS, phosphate-buffered saline; LV, lentivirus virus; NC, negative control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IgE, immunoglobulin E.
*P < 0.05, **P < 0.01.
Knockdown of IL-17D aggravates airway remodeling in the OVA-induced asthmatic mice
To investigate whether IL-17D affects airway remodeling processes in asthma, we performed Masson staining of mouse lung tissue, which showed increased collagen deposition around the airway epithelium in asthmatic mice after IL-17D knockdown (Fig. 3A). Concurrently with the histopathological analysis, lung tissue from all 4 groups of mice was homogenized, and protein was extracted for western blotting analysis to assess remodeling. Western blotting analysis revealed that the expression levels of collagen I, N-cadherin, and vimentin in the lung tissue of the OVA+LV2-shIL-17D group were significantly increased compared to the OVA+LV2-shNC group (Fig. 3B). This finding was further validated by IF staining of lung tissue (Fig. 3C and D). Furthermore, the disruption of intercellular adhesion and tight junctions plays a significant role in the remodeling of the airway epithelium. IL-17D knockdown in the asthmatic mouse model resulted in a marked decrease in the expression of the tight junction proteins ZO-1 and occludin (Supplementary Fig. S2).
Fig. 3. Impact of IL-17D deficiency on EMT in OVA-induced asthmatic mice. (A) Collagen deposition around the airways of mice belonging to different groups was determined through Masson staining (scale bar = 50 µm). Quantification of the collagen volume fraction was undertaken. (B) The protein expression levels and quantitative analysis of collagen I, N-cadherin and vimentin were assessed in various groups of mice. IF staining revealed the expression levels of (C) collagen I and (D) N-cadherin around the airway epithelium in mouse models (scale bar = 10 µm). One-way analysis of variance followed by Tukey’s post hoc test. Data are shown as the means ± standard deviation of 3 independent experiments.
IL, interleukin; EMT, epithelial-mesenchymal transition; OVA, ovalbumin; LV, lentivirus virus; shRNA, short hairpin RNA; NC, negative control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
*P < 0.05, **P < 0.01.
Based on these experimental results, we conducted an RNA-sequencing analysis of the OVA+LV2-shNC and OVA+LV2-shIL-17D groups (n = 3 mice/group). According to the filter criteria (|log2FC| ≥ 1.5, P < 0.05), 1,024 differentially expressed genes (DEGs) were identified in OVA+LV2-shIL-17D group vs OVA+LV2-shNC group, comprising 584 up-regulated genes and 440 down-regulated genes (Fig. 4A). For Gene Ontology enrichment, DEGs were enriched into 3 parts containing biological process, cellular component and molecular function. With specific regard to the cellular component, the DEGs were predominantly concentrated within the extracellular space, collagen-containing extracellular matrix (ECM) and extracellular region (Fig. 4B). Gene Set Enrichment Analysis indicated that the pathways identified as positively enriched in the OVA+LV2-shIL-17D group encompassed the extracellular space and extracellular region (Fig. 4C and D). The findings from the transcriptome analysis align with our experimental results, indicating that the deficiency of IL-17D may significantly contribute to airway remodeling via the ECM.
Fig. 4. The results of RNA-sequencing between OVA+LV2-shIL-17D group and OVA+LV2-shNC group. (A) Volcano plots of DEGs in OVA+LV2-shIL-17D group versus OVA+LV2-shNC group. Red dots represent up-regulated genes and blue dots represent down-regulated genes filtered based on FC ≥ 1.5 and P < 0.05. (B) Analyzing the cellular component showed an increased number of ECM genes in the OVA+LV2-shIL-17D group compared to the OVA+LV2-shNC group. GSEA indicated that the pathways identified as positively enriched in the OVA+LV2-shIL-17D group encompassed the (C) extracellular space and (D) extracellular region.
OVA, ovalbumin; LV, lentivirus virus; shRNA, short hairpin RNA; IL, interleukin; NC, negative control; DEG, differentially expressed gene; FC, fold change; ECM, extracellular matrix; GSEA, Gene Set Enrichment Analysis; NES, normalized enrichment score.
Exogenous IL-17D administration attenuates airway inflammation and remodeling in asthmatic mice
In order to ascertain the function of IL-17D in asthmatic mice, we administered rmIL-17D via intranasal injection to mice with asthma. Following the third challenge experiment, rescue experiments were initiated, in which rmIL-17D (0.5 µg) was administered intranasally to asthmatic mice (Fig. 5A). Firstly, we ruled out the effect of rmIL-17D alone on airway inflammation in mice (Supplementary Fig. S3). Analysis of pulmonary function in three groups of mice demonstrated that treatment with rmIL-17D significantly alleviated OVA-induced AHR (Fig. 5B). In parallel, H&E and PAS staining demonstrated a marked decrease in peribronchial inflammatory cell infiltration and mucus production in the rmIL-17D+OVA group relative to the OVA group (Fig. 5C and D). Furthermore, administration of rmIL-17D significantly reduced total cell counts and inflammatory cell counts in BALF compared to the OVA group (Fig. 5E and F). The expression of IgE in the serum and the concentration of certain inflammatory factors (IL-4/IL-5/IL-13) in the BALF (Fig. 5G) of asthmatic mice were also significantly decreased following the administration of rmIL-17D.
Fig. 5. Effect of intranasal delivery of rmIL-17D on airway inflammation in OVA-challenged mice. (A) Timeline of interventions in animal models. (B) Airway resistance responses to increased doses of inhaled methacholine treatment in different groups. (C) Representative H&E-stained lung sections showing inflammatory cell infiltration around the airways in different experimental groups (scale bar = 50 µm). (D) PAS staining was used to quantify airway mucus secretion levels in each group of mice (scale bar = 50 µm). (E) BALF was analyzed for total cell number. (F) Differential cell analysis of BALF was performed to quantify neutrophil, eosinophil, and macrophage populations. (G) ELISA was used to measure the levels of IgE in serum and the expression of IL-4, IL-5, and IL-13 in BALF. One-way analysis of variance followed by Tukey’s post hoc test. Data are shown as the means ± standard deviation of three independent experiments.
rmIL-17D, recombinant mouse interleukin-17D protein; OVA, ovalbumin; ; H&E, hematoxylin and eosin; PAS, Periodic Acid-Schiff stain; BALF, bronchoalveolar lavage fluid; ELISA, enzyme-linked immunosorbent assay; IgE, immunoglobulin E; IL, interleukin; PBS, phosphate-buffered saline.
*P < 0.05, **P < 0.01.
At the same time, we observed a reduction in collagen deposition around the airway of asthmatic mice following the administration of rmIL-17D, as indicated by Masson staining (Fig. 6A and B). Western blotting analysis demonstrated a notable decline in the expression levels of vimentin, collagen I and N-cadherin in the rmIL-17D+OVA group, compared to the OVA group (Fig. 6C and D). In addition, the results from IHC staining for N-cadherin (Fig. 6E and F) and IF staining for collagen I (Fig. 6G and H) were consistent with those obtained from western blotting analysis. This suggests that IL-17D may play a negative regulatory role in airway inflammation and remodeling in asthma.
Fig. 6. Role of IL-17D in airway remodeling in OVA-challenged mice. (A, B) Masson staining illustrated the degree of collagen deposition around the airways in different groups of mice (scale bar = 50 µm). (C, D) The expression and quantification of vimentin, collagen I and N-cadherin following treatment were assessed. (E, F) IHC analysis and measurement of N-cadherin protein levels in the airway epithelium of mice (scale bar = 50 µm). (G, H) Collagen I protein expression was detected by IF and quantitatively analyzed (scale bar = 50 µm). One-way analysis of variance followed by Tukey’s post hoc test. Data are shown as the means ± standard deviation of three independent experiments.
IL, interleukin; OVA, ovalbumin; IHC, immunohistochemistry; rmIL-17D, recombinant mouse interleukin-17D; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
*P < 0.05, **P < 0.01.
IL-17D plays an important role in epithelial-mesenchymal transition (EMT) and remodeling in bronchial epithelial cells
The results of the aforementioned experiments conducted with human and murine specimens indicate that the majority of IL-17D is localized within the airway epithelium. Consequently, we hypothesize that the airway epithelium serves as a critical site for the functional activity of IL-17D. Initially, we cultured 16HBE cells and subsequently introduced human recombinant IL-17D protein to the cultures. We established varying concentrations (0, 10, 50, and 100 ng/mL) and time intervals (0, 6, 24, and 48 hours) for the experimental conditions. Western blotting analysis indicate that IL-17D, when acting independently, possesses the capability to reduce the EMT and remodeling indicators of airway epithelium. This effect becomes more pronounced with higher concentration and longer duration (Supplementary Fig. S4).
An increasing body of research indicates that TGF-β1 is a key inducer of EMT and airway remodeling.16,17 In order to gain insight into the function of IL-17D in TGF-β1-induced EMT and remodeling models, an analysis of data from the GEO database was conducted. We selected the GSE40374 dataset, in which M-BE cells were divided into 2 groups, namely those with and without TGF-β1 stimulation. An analysis conducted utilizing the GEO2R tool indicated a reduction in IL-17D expression following stimulation with TGF-β1 (Fig. 7A). To investigate the role of IL-17D in conjunction with TGF-β1 in EMT and airway remodeling, this experiment was divided into 4 groups: 1) control group; 2) IL-17D group; 3) TGF-β1 group; 4) TGF-β1+IL-17D group. The addition of TGF-β1 stimulation induced EMT and remodeling in 16HBE cells, as evidenced by a decrease in E-cadherin and an increase in N-cadherin, vimentin, fibronectin and collagen I markers. Nevertheless, the combination of IL-17D and TGF-β1 resulted in a reversal of the aforementioned outcomes (Fig. 7C and D). Furthermore, the results of qRT-PCR (Fig. 7B) and IF staining (Fig. 7E) were found to be consistent with those obtained from western blotting. The results demonstrate that IL-17D inhibits the capacity of TGF-β1 to induce airway epithelium remodeling.
Fig. 7. IL-17D inhibits TGF-β1-induced fibrosis in 16HBE cells. (A) The GSE40374 dataset was analyzed using the GEO2R tool and volcano plots were produced. The red point represents up-regulated mRNAs, and the blue points indicates down-regulated mRNAs with significance (the arrow refers to IL-17D). (B) The mRNA expression levels of N-cadherin, vimentin, fibronectin and collagen I were assessed after different treatment. (C, D) The protein expression levels of E-cadherin, N-cadherin, vimentin, fibronectin and collagen I were assessed after different treatment. (E) The expression levels of E-cadherin, fibronectin and collagen I in 16HBE cells were examined through IF following the administration of various stimuli (scale bar = 10 µm). One-way analysis of variance followed by Tukey’s post hoc test. Data are shown as the means ± standard deviation of 3 independent experiments.
IL, interleukin; TGF, transforming growth factor; GEO2R, Gene Expression Omnibus 2R; mRNA, messenger RNA; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
*P < 0.05, **P < 0.01.
Potential mechanisms of IL-17D in the pathogenesis of bronchial asthma
CD93 has emerged as a significant functional receptor for IL-17D in recent years and has been linked to various immune-related conditions, including atopic dermatitis.13 In order to ascertain whether IL-17D exerts its effects through CD93 in the context of bronchial asthma, we constructed small interference RNAs that target CD93 and verified the efficacy of this knockdown (Fig. 8A). The inclusion or absence of IL-17D and small interference RNAs was grouped in a TGF-β1 induced airway fibrosis model. Through western blotting, we investigated the expression levels of EMT proteins (E-cadherin and vimentin) and discovered that the knockdown of CD93 attenuated the inhibitory regulatory effect exerted by IL-17D (Fig. 8B-D).
Fig. 8. Proposed mechanisms of IL-17D in bronchial asthma. (A) qRT-PCR to verify the knockdown effect of Si-CD93. (B-D) Protein expression levels of E-cadherin and vimentin after different treatments. One-way analysis of variance followed by Tukey’s post hoc test. Data are shown as the means ± standard deviation of 3 independent experiments.
IL, interleukin; qRT-PCR, quantitative real-time-polymerase chain reaction; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; TGF, transforming growth factor; si-NC, small interfering negative control; si-CD93, small interfering CD93.
**P < 0.01.
Next, we investigated the expression levels of p-Smad3, a protein associated with the TGF-β1 signaling pathway. The results indicated that IL-17D attenuated the TGF-β1-induced increase in p-Smad3 expression (Supplementary Fig. S5C). However, silencing CD93 diminished the inhibitory effect of IL-17D on TGF-β1, leading to a relative increase in p-Smad3 expression compared to the TGF-β1+IL-17D+Si-NC group (Supplementary Fig. S5D). Concurrently, in a murine model, we observed that intranasal administration of rmIL-17D significantly reduced the expression levels of TGF-β1 and p-Smad3 in the airway epithelium (Supplementary Fig. S5A and B). These findings suggest that IL-17D may negatively regulate the TGF-β1/Smad3 signaling pathway via the CD93 receptor, thereby exerting a protective effect in asthma. Furthermore, our study demonstrates that the administration of IL-17D led to a decrease in reactive oxygen species (ROS) produced by TGF-β1. This observation implies that IL-17D also may play an important role in the pathogenesis of bronchial asthma through the modulation of oxidative stress (Supplementary Fig. S6).
DISCUSSION
In this study, we found that IL-17D was present in the airway epithelium of asthmatics and OVA-induced asthma mice with reduced expression. In comparison to the knockdown of IL-17D, which intensified airway inflammation and associated remodeling indicators in chronic asthmatic mice, the administration of recombinant IL-17D protein mitigated the pathophysiological characteristics in OVA-induced asthma. This research establishes, for the first time, a correlation between IL-17D and chronic asthma induced by OVA, indicating that IL-17D could serve as a potential target for therapeutic intervention in the treatment of asthma.
The IL-17 family comprises a group of cytokines that hold significant immunological relevance. IL-17F exhibits the highest degree of homology, ranging from 40% to 55%, with IL-17A, and both cytokines are predominantly secreted by T helper (Th) 17 cells.8,18 IL-17A and IL-17F play significant roles in mediating inflammation and enhancing host defense mechanisms against infections by promoting the expression of pro-inflammatory cytokines, chemokines, and antimicrobial peptides in epithelial, fibroblast, and endothelial cells.19,20 Kouri et al.21 identified the production of IL-17B in the synovial tissues of patients with rheumatoid arthritis. This production facilitates the synthesis of various factors, including IL-6 and CCL20, in response to tumor necrosis factor (TNF)-α, thereby promoting the infiltration of neutrophils.21 IL-17C is an epithelial-derived molecule.22,23 Recent studies have demonstrated that IL-17C promotes the inflammatory response in neutrophilic asthma through pulmonary macrophage M1 polarization.24 IL-17E (also known as IL-25) stimulates the production of IgE and eosinophils, thereby augmenting type 2 immune responses.25,26 The findings of these studies indicate that these factors exhibit distinct functional roles across various disease contexts although there is some degree of homology among the members of the IL-17 family. It is important to note that IL-17D plays a significant role in inflammatory diseases. Research indicates that IL-17D impedes the phagocytic activity of abdominal macrophages towards Escherichia coli, thereby worsening the condition of sepsis.27 Concurrently, IL-17D suppresses the expression of DDX5 in keratinocytes, thereby exacerbating skin inflammation mediated by IL-36R.28 However, the expression of IL-17D was found to be diminished in both serum and BALF of individuals suffering from acute lung injury.15 Furthermore, Wang et al.29 demonstrated that the addition of recombinant human IL-17D protein into NCM460 cells subjected to hyperoxic conditions resulted in an upregulation of ZO-1 and occludin expression. This finding indicates that IL-17D may play a protective role in maintaining the integrity of the intestinal epithelial barrier in hyperoxic environments, thereby mitigating intestinal inflammation. Dong et al.15 found that intestinal epithelium-derived IL-17D stimulates IL-22 production in experimental IBD models, thereby promoting repair of the intestinal epithelial barrier.13 These findings align with our experimental results. We conducted an analysis of peripheral blood serum and airway mucosal epithelium from patients with asthma and observed a reduction in IL-17D expression when compared to healthy control subjects. Furthermore, our study revealed that chronic asthmatic mice lacking IL-17D exhibited heightened inflammation, characterized by the upregulation of both Th2-type inflammatory mediators (IL-4, IL-5, and IL-13) and non-Th2 inflammatory cytokines (IL-6, IL-1β, and TNF-α) (Supplementary Fig. S7A). Additionally, there was an increased infiltration of eosinophils in the vicinity of the airways, along with a reduction in the expression of the tight junction protein ZO-1 and occludin. These findings suggest that IL-17D plays a protective role in maintaining the integrity of the airway epithelial barrier in asthmatic mice.
Research has indicated that epithelial cells represent a primary cell type implicated in the pathogenesis of asthma. Furthermore, these cells may participate in EMT, a process that holds significant consequences for airway remodeling.30 The inhibition of EMT reduces airway hyperresponsiveness and mitigates fibrotic processes.31 TGF-β1 is a recognized cytokine that plays a significant role in EMT and the deposition of collagen.16,32 In our in vitro studies, we treated 16HBE cells with recombinant human IL-17D and observed that it significantly mitigated the TGF-β1-induced reduction in E-cadherin expression and the concomitant increases in vimentin and collagen I levels. These findings are consistent with our in vivo experimental results, suggesting a potential role for IL-17D in regulating EMT processes.
Additionally, we investigated the potential mechanisms by which IL-17D exerts its effects in bronchial asthma. The receptor for IL-17D will not be elucidated until the year 2021. Dong et al.15 employed surface plasmon resonance to demonstrate that IL-17D, secreted by intestinal epithelial cells, specifically interacts with CD93 on ILC3. This interaction supports the maintenance of intestinal homeostasis by modulating ILC3 function.13 Furthermore, Dong et al.15 demonstrated that IL-17D has the capacity to influence skin inflammation in murine models through the activation of the CD93-p38 MAPK-AKT-SMAD2/3 signaling pathway.28 CD93, referred to as C1qR1 or C1qRp, is a transmembrane glycoprotein composed of 652 amino acids. It is expressed across a diverse range of cell types, including endothelial cells, lymphocytes, and stem cells.33,34 Park et al.35 have identified the expression of CD93 in bronchial epithelial cells. We found that CD93 co-localized with IL-17D in the airway epithelium using a fluorescent double staining method (Supplementary Fig. S8). In order to examine the potential role of IL-17D in relation to CD93 in the context of asthma, we developed small interfering RNAs designed to downregulate CD93 expression. The results showed that knockdown of CD93 in vitro partly attenuated the inhibitory effect of IL-17D on TGF-β1-induced airway fibrosis and upregulated the expression of p-Smad3. This observation implies a possible interaction between IL-17D-CD93-TGF-β1/p-Smad3 in the pathophysiology of asthma. Future studies using in vivo CD93 knockout mice to further investigate the roles of CD93 and IL-17D in asthma would be highly valuable.
Moreover, research has shown that the expression of IL-17D is modulated by the transcription factor nuclear factor erythroid-derived 2 (Nrf2).15,36 Nrf2 is a transcription factor that is instrumental in the regulation of cellular homeostasis and the maintenance of redox equilibrium.37 Nrf2 agonists have the potential to mitigate ozone-induced inflammation in a mouse model of acute exacerbation asthma through the inhibition of ROS production.38 Our findings indicate that the incorporation of recombinant human IL-17D protein leads to a decrease in ROS production in response to TGF-β1 stimulation. In the future, the induction of IL-17D production through the administration of Nrf2 agonists may present a potential avenue for the treatment of asthma.
Although our study has yielded significant results, certain limitations remain, which warrant further investigation. While our study primarily examined the effects of IL-17D on the airway epithelium, we acknowledge that other immune cells (ILC2s, antigen-presenting cells) may also be involved. Consistent with this, Huang et al.13 have reported that IL-17D regulates the intestinal epithelium via modulation of ILC3s, suggesting that IL-17D may act on the airway epithelium in conjunction with immune cells. The observed lack of tissue specificity with lentivirus-mediated IL-17D knockdown necessitates further refinement. Future studies should focus on precisely quantifying the targeting efficiency within the lung tissue and rigorously comparing the knockdown efficiency in the lung to that in other relevant organs. This will be critical for minimizing off-target effects and ensuring the validity of conclusions drawn regarding IL-17D’s role in the lung.
In conclusion, our research indicates that IL-17D has the potential to mitigate airway inflammation and remodeling associated with chronic asthma induced by OVA, possibly through its interaction with the CD93 receptor or by reducing ROS production. This finding presents a novel target for therapeutic intervention in asthma management.
ACKNOWLEDGMENTS
This work was supported by the National Natural Science Foundation of China (grant No. 82270032, 82470028), the Key Research and Development Program of Shandong Province (grant No. 2021SFGC0504), Shandong Provincial Natural Science Foundation (ZR2021LSW015), Noncommunicable Chronic Diseases-National Science and Technology Major Project (2024ZD0529700; 2024ZD0529704), Natural Science Foundation of Shandong Province (ZR2024MH153), and Jinan Clinical Medicine Research Program for Respiratory Disease (grant No. 202132002), China International Medical Foundation (Z-2014-08-2309-1, Z-2017-24-2301).
Footnotes
Disclosure: There are no financial or other issues that might lead to conflict of interest.
SUPPLEMENTARY MATERIALS
Summary of the design of GEO datasets
Characteristics of the study subjects
Primer sequences
Validation of lentivirus knockdown of IL-17D in the airway epithelium. (A) Representative IF images of IL-17D in airway epithelium from LV2-shNC and LV2-shIL-17D groups. (B) Quantification of mean fluorescence intensity of IL-17D in the airway epithelium (scale bar = 10 µm). One-way analysis of variance followed by Tukey’s post hoc test. Data are shown as the means ± standard deviation of 3 independent experiments.
Expression of mouse airway epithelial tight junction proteins after lentiviral injection. (A-D) IHC analysis showed differential expression of ZO-1 and occludin protein in the airway epithelium of mice across the experimental groups (scale bar = 50 µm). One-way analysis of variance followed by Tukey’s post hoc test. Data are shown as the means ± standard deviation of 3 independent experiments.
Effect of rmIL-17D given alone on airway inflammation in mice. (A, B) H&E and PAS staining to assess the degree of inflammatory infiltration and mucus secretion levels in the PBS group and the PBS+rmIL-17D group of mice, followed by quantitative analysis (scale bar = 50 µm). (C, D) Expression levels of type 2 and non-type 2 inflammatory factors in the BALF of mice in the PBS group and PBS+rmIL-17D group were measured by ELISA. One-way analysis of variance followed by Tukey’s post hoc test. Data are shown as the means ± standard deviation of more than 3 independent experiments.
Effect of recombinant human IL-17D on 16HBE cells at different concentration and time gradients. (A, B) The protein expression levels of fibronectin, collagen I and vimentin, along with a quantitative analysis, were assessed following 48 hours of exposure to varying concentrations of IL-17D. (C, D) The protein expression levels of N-cadherin and Vimentin, along with a quantitative analysis, were assessed following various durations of IL-17D treatment at a concentration of 100 ng/mL. One-way analysis of variance followed by Tukey’s post hoc test. Data are shown as the means ± standard deviation of 3 independent experiments.
IL-17D administration reduces the expression of p-Smad3 in in vivo and in vitro experiments. (A, B) Representative IF images showing TGF-β1 (green) and p-Smad3 (red) expression in the airway epithelium of mice (scale bar = 10 µm). (C) Western blotting analysis of p-Smad3 expression in 16HBE cells following treatment with IL-17D and TGF-β1. (D) Western blotting analysis of p-Smad3 expression in 16HBE cells following transfection with siRNA. One-way analysis of variance followed by Tukey’s post hoc test. Data are shown as the means ± standard deviation of 3 independent experiments.
IL-17D reduces TGF-β1-induced ROS production. (A, B) Fluorescence assay showing ROS levels in 16HBE cells treated with IL-17D and TGF-β1. Data were quantitatively analyzed (scale bar = 50 µm). One-way analysis of variance followed by Tukey’s post hoc test. Data are shown as the means ± standard deviation of 3 independent experiments.
Determination of non-type 2 inflammatory factors in each group of mice. (A) ELISA quantification of IL-6, IL-1β, and TNF-α levels in BALF from lentivirus-shRNA transduced mice. (B) ELISA quantification of IL-6, IL-1β, and TNF-α levels in BALF from mice treated with exogenous IL-17D. One-way analysis of variance followed by Tukey’s post hoc test. Data are shown as the means ± standard deviation of 3 independent experiments.
Expression localization of IL-17D and CD93 in WT mice. Coimmunostaining of CD93 (red) and IL-17D (green) in WT mice (scale bar = 10 µm).
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Summary of the design of GEO datasets
Characteristics of the study subjects
Primer sequences
Validation of lentivirus knockdown of IL-17D in the airway epithelium. (A) Representative IF images of IL-17D in airway epithelium from LV2-shNC and LV2-shIL-17D groups. (B) Quantification of mean fluorescence intensity of IL-17D in the airway epithelium (scale bar = 10 µm). One-way analysis of variance followed by Tukey’s post hoc test. Data are shown as the means ± standard deviation of 3 independent experiments.
Expression of mouse airway epithelial tight junction proteins after lentiviral injection. (A-D) IHC analysis showed differential expression of ZO-1 and occludin protein in the airway epithelium of mice across the experimental groups (scale bar = 50 µm). One-way analysis of variance followed by Tukey’s post hoc test. Data are shown as the means ± standard deviation of 3 independent experiments.
Effect of rmIL-17D given alone on airway inflammation in mice. (A, B) H&E and PAS staining to assess the degree of inflammatory infiltration and mucus secretion levels in the PBS group and the PBS+rmIL-17D group of mice, followed by quantitative analysis (scale bar = 50 µm). (C, D) Expression levels of type 2 and non-type 2 inflammatory factors in the BALF of mice in the PBS group and PBS+rmIL-17D group were measured by ELISA. One-way analysis of variance followed by Tukey’s post hoc test. Data are shown as the means ± standard deviation of more than 3 independent experiments.
Effect of recombinant human IL-17D on 16HBE cells at different concentration and time gradients. (A, B) The protein expression levels of fibronectin, collagen I and vimentin, along with a quantitative analysis, were assessed following 48 hours of exposure to varying concentrations of IL-17D. (C, D) The protein expression levels of N-cadherin and Vimentin, along with a quantitative analysis, were assessed following various durations of IL-17D treatment at a concentration of 100 ng/mL. One-way analysis of variance followed by Tukey’s post hoc test. Data are shown as the means ± standard deviation of 3 independent experiments.
IL-17D administration reduces the expression of p-Smad3 in in vivo and in vitro experiments. (A, B) Representative IF images showing TGF-β1 (green) and p-Smad3 (red) expression in the airway epithelium of mice (scale bar = 10 µm). (C) Western blotting analysis of p-Smad3 expression in 16HBE cells following treatment with IL-17D and TGF-β1. (D) Western blotting analysis of p-Smad3 expression in 16HBE cells following transfection with siRNA. One-way analysis of variance followed by Tukey’s post hoc test. Data are shown as the means ± standard deviation of 3 independent experiments.
IL-17D reduces TGF-β1-induced ROS production. (A, B) Fluorescence assay showing ROS levels in 16HBE cells treated with IL-17D and TGF-β1. Data were quantitatively analyzed (scale bar = 50 µm). One-way analysis of variance followed by Tukey’s post hoc test. Data are shown as the means ± standard deviation of 3 independent experiments.
Determination of non-type 2 inflammatory factors in each group of mice. (A) ELISA quantification of IL-6, IL-1β, and TNF-α levels in BALF from lentivirus-shRNA transduced mice. (B) ELISA quantification of IL-6, IL-1β, and TNF-α levels in BALF from mice treated with exogenous IL-17D. One-way analysis of variance followed by Tukey’s post hoc test. Data are shown as the means ± standard deviation of 3 independent experiments.
Expression localization of IL-17D and CD93 in WT mice. Coimmunostaining of CD93 (red) and IL-17D (green) in WT mice (scale bar = 10 µm).