Increased Primary Cilia Contribute to Airway Epithelium Remodeling in Asthma

The primary cilium is an antenna-like organelle, serving as a sensor for extracellular signals and playing a critical role in cell proliferation and differentiation 1. It has been described that primary cilia appear in the epithelium during repair after airway injury, and increased primary cilia are associated with bronchial epithelium remodeling in patients with chronic obstructive pulmonary disease, suggesting a role for primary cilia in airway homeostasis and disease 2, 3. Epithelial hyperplasia and goblet cell metaplasia are key features of airway epithelium remodeling in asthma, which are more prominent in severe or fatal asthma 4. We hypothesized that epithelial primary cilia are increased and contribute to airway epithelium remodeling in asthma. We observed the presence and localization of primary cilia in the airway epithelium of subjects with mild-to-moderate asthma (n = 30) and control subjects (n = 15). Subject characteristics are summarized in Table S1. The study was approved by the medical ethics committee of Tongji Hospital, Huazhong University of Science and Technology (TJ-IRB-20210117). Immunofluorescent staining for ARL13B, a marker for cilium axoneme, in bronchial biopsies showed that primary cilia were mainly localized to the cells in the basal part of the epithelium, and the number of primary cilia is significantly greater in asthma patients compared to control subjects (79.5 (50.6–143.9) versus 14.4 (12.7–19.0); p < 0.001) (Figure 1A,B). Immunostaining for the basal cell marker KRT5 and the multiciliated cell marker FOXJ1 in bronchial biopsies revealed that primary cilia were localized in epithelial basal cells of asthma patients (Figure 1C, Figure S1A). We next examined whether the primary cilium is linked to goblet cell metaplasia and basement membrane thickening using Periodic acid-Schiff (PAS) and Masson's Trichrome staining, respectively. The numbers of PAS staining-positive goblet cells and basement membrane thickness were increased in asthma patients compared to control subjects (Figure 1D,E), and were strongly correlated with the number of primary cilia in asthma patients (Figure 1F,G). This suggests that an increased number of primary cilia may play a role in airway epithelium remodeling. We further observed that the number of primary cilia was correlated with sputum eosinophil percentages and fractional exhaled nitric oxide, the markers of type 2 inflammation, in asthma patients (Figure 1H,I). When stratifying our asthma patients as eosinophilic (sputum eosinophil ≥ 3%) and non-eosinophilic asthma (< 3%), the number of primary cilia was increased only in eosinophilic asthma (Figure S1B). We used IL-13, a type 2 cytokine, to stimulate normal human bronchial epithelial cells (HBECs) cultured at the air–liquid interface (Figure 2A). After 7 days of IL-13 (10 ng/mL) stimulation, the number of primary cilia was significantly increased in HBECs from 6 normal control donors (44.7 ± 6.2 vs. 8.8 ± 2.8/mm, p < 0.001; Figure 2B,C). IL-13 stimulation also markedly increased the number of PAS staining-positive goblet cells. However, chloral hydrate (4 mM), a primary cilia-ablating agent, significantly suppressed IL-13-induced goblet cell metaplasia (Figure 2D,E). Moreover, knockdown of IFT88, a gene driving primary ciliogenesis, also inhibited IL-13-induced goblet cell metaplasia in HBECs (Figure S2A,B). Our findings suggest that the type 2 milieu facilitates epithelial primary ciliogenesis, and the primary cilium contributes to goblet cell metaplasia. To our knowledge, this is the first report that primary cilia are increased in epithelial basal cells and contribute to goblet cell metaplasia in asthma. Basal cells are progenitor cells that can differentiate to secretory cells 5. The primary cilium drives cell differentiation through Hedgehog signaling 6. Elimination of primary cilia diminishes Notch signaling and inhibits epidermal basal cell differentiation 1. Our preliminary data showed that the elimination of primary cilia inhibits Hedgehog and Notch signaling and suppresses the Hedgehog and Notch agonists-augmented IL-13-induced goblet cell metaplasia in vitro (Figure S3A–H). These studies and ours suggest that increased primary cilia and upregulated ciliary signaling may promote the differentiation of basal cells to goblet cells in asthmatic airways. Our study has limitations including the inclusion of only mild-to-moderate asthma patients and a small sample size. Future studies focusing on the primary cilium as potential therapeutic targets for asthmatic airway remodeling are warranted. Study conceptualization: G.Z.; study design: G.C. and G.Z.; acquisition of data: G.C., H.J., C.H., Z.W., W.G., T.X., Y.L., Y.F., L.Y., and G.Z.; analysis and data interpretation: G.C., H.J., Z.X., and G.Z.; manuscript writing and revision: G.C. and G.Z. We sincerely thank all the participants in this study. This work is supported by the National Natural Science Foundation of China (grants 82570050, 82170036), Key Research and Development Program of Hubei Province (grant 2024BCB042), Noncommunicable Chronic Disease–National Science and Technology Major Project (grant 2024ZD0529900), Innovation and Translation Project of Tongji Hospital (grant 2023CXZH006). The authors declare no conflicts of interest. The data that support the findings of this study are available from the corresponding author upon reasonable request. Figure S1: Characterization of primary and motile cilia in asthmatic airway epithelium and IL-13-treated HBECs. (A) Immunofluorescent staining of ARL13B and FOXJ1 in bronchial biopsies from asthma patients. (B) Quantification of primary cilia in the epithelium of non-eosinophilic asthma patients (n = 6), eosinophilic asthma patients (n = 24), and control subjects (n = 15). Values indicate the number of primary cilia-positive cells per millimeter. (C) Representative images for immunofluorescent staining of FOXJ1 in normal human bronchial epithelial cells (HBECs) cultured at the air–liquid interface (ALI) under control conditions, IL-13 treatment, and IL-13 plus chloral hydrate (CH) treatment. (D) Quantification of FOXJ1 fluorescence intensity in HBECs. (E) The protein levels of FOXJ1 in control, IL-13, and IL-13 plus CH-treated HBECs cultured at ALI. **p < 0.01, ***p < 0.001. Figure S2: Supplemental analysis of epithelial barrier integrity following IL-13 treatment and intervention. (A) Representative images of PAS staining of NC siRNA plus IL-13, and IFT88 siRNA plus IL-13-treated normal human bronchial epithelial cells (HBECs) cultured at the air–liquid interface (ALI). (B) Quantification of PAS-positive cells in NC siRNA plus IL-13, and IFT88 siRNA plus IL-13-treated HBECs. (C) Representative transmission electron microscopy (TEM) images of HBECs cultured at ALI under control conditions, IL-13 treatment, and IL-13 plus chloral hydrate (CH) treatment. (D) Representative images for immunofluorescent staining of E-cadherin and ZO-1 in control, IL-13, CH, and IL-13 plus CH-treated HBECs at ALI. (E-F) Quantification of ZO-1 and E-cadherin positive area in HBECs. *p < 0.05, **p < 0.01, ***p < 0.001. Figure S3: IL-13 upregulates Hedgehog and Notch signaling, elimination of primary cilia suppresses the IL-13-induced as well as Hedgehog and Notch agonists-augmented IL-13-induced goblet cell metaplasia in vitro. (A) Representative images for immunofluorescent staining of NOTCH3 in normal human bronchial epithelial cells (HBECs) cultured at air–liquid interface (ALI) under control conditions, IL-13 treatment, and IL-13 plus chloral hydrate (CH) treatment. (B) Quantification of NOTCH3 fluorescence intensity in HBECs. (C) Representative images for immunofluorescent staining of SMO of HBECs cultured at ALI under control conditions, IL-13 treatment, and IL-13 plus chloral hydrate (CH) treatment. (D) Quantification of SMO fluorescence intensity in HBECs. (E) The protein levels of NICD3 and SMO in control, IL-13, and IL-13 plus CH-treated HBECs. (F) Representative images of PAS staining and MUC5AC immunostaining of HBECs cultured at ALI following treatment with IL-13, CH, Hedgehog signaling agonist SAG (Smoothened Agonist), and Notch signaling agonist Jagged-1. (G) Quantitative analysis of the percentage of PAS-positive area relative to the total area of HBECs. (H) Quantitative analysis of the percentage of MUC5AC-positive area relative to the total area of HBECs. *p < 0.05, **p < 0.01, ***p < 0.001. Table S1: all70309-sup-0003-Supinfo.docx. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.