Oncostatin M Contributes to Airway Epithelial Cell Dysfunction in Chronic Rhinosinusitis with Nasal Polyps

Abstract

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a typical type-2 inflammation involving several cytokines and is associated with epithelial cell dysfunction. Oncostatin M (OSM) (belonging to the interleukin(IL)-6 family) could be a key driver of epithelial barrier dysfunction. Therefore, we investigated the presence of OSM and IL-6 and the expression pattern of tight junctions (TJs) in the nasal tissue of CRSwNP patients and controls using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and Western blotting. Then, their potential role in the epithelial barrier was evaluated in vitro in 27 different primary cultures of human nasal epithelial cells (HNECs) by measuring TJ expression and transepithelial electric resistance (TEER) with or without OSM or IL-6 (1, 10, and 100 ng/mL). The effect on ciliary beating efficiency was evaluated by high-speed videomicroscopy and on repair mechanisms with a wound healing model with or without OSM. OSM and IL-6 were both overexpressed, and TJ (ZO-1 and occludin) expression was decreased in the nasal polyps compared to the control mucosa. OSM (100 ng/mL) but not IL-6 induced a significant decrease in TJ expression, TEER, and ciliary beating efficiency in HNECs. After 24 h, the wound repair rate was significantly higher in OSM-stimulated HNECs at 100 ng/mL. These results suggest that OSM could become a new target for monoclonal antibodies.

Keywords: CRSwNP; IL-6; OSM; ciliary beating efficiency; epithelial electric resistance; nasal epithelium; primary culture; repair rate; tight junctions.

Figure 1

OSM and IL-6 were overexpressed, and epithelial TJs were altered compared to non-inflammatory nasal mucosa. The ex vivo expression of OSM and IL-6 were analyzed by RT-qPCR in nasal polyps and in non-inflammatory nasal mucosa (controls) (n = 9) (A) and expression of occludin and ZO-1 were analyzed by Western blotting (n = 5) (B), both using GAPDH as a housekeeping gene (HKG) to normalize gene and protein expression. OSM and IL-6 were overexpressed in nasal polyps, and occludin and ZO-1 were underexpressed. The type II OSM receptor and IL-6 receptor were expressed in nasal-polyp-derived HNECs (n = 5) (C). The y-axis represents the relative expression normalized to HKG. * p = 0.0159; ** p = 0.0079; **** p < 0.0001.

Figure 2

OSM but not IL-6 reduced the expression of occludin and ZO-1 in HNECs. Occludin (n = 7) and ZO-1 (n = 5) relative expression at the protein level were analyzed by Western blotting using GAPDH as HKG to normalize protein expression (A,B). The y-axis represents the relative expression normalized to HKG. Comparisons were made between the groups ** p = 0.0079; *** p = 0.0006.

Figure 3

OSM but not IL-6 reduced the expression of ZO-1 in HNECs. ALI cultures of HNECs were stimulated on day 21 with OSM or IL-6 1, 10, and 100 ng/mL for 48 h. ZO-1 (red) and actin (green) expression after immunolabeling and nuclear DAPI labeling (blue) with stimulation by OSM at 1 ng/mL (A), 10 ng/mL (B), and 100 ng/mL (C), and IL-6 at 1 ng/mL (D), IL-6 at 10 ng/mL (E), IL-6 at 100 ng/mL (F), or without any stimulation (G,H), for ZO-1 and actin, respectively, are shown. A negative control without a primary antibody or rhodamine–phalloidin was performed (I).

Figure 4

OSM but not IL-6 decreased TEER in HNECs. ALI cultures of HNECs were stimulated on day 21 with OSM or IL-6 1, 10, and 100 ng/mL for 48 h and compared to the unstimulated cultures. TEER measurements in unstimulated controls (blue), OSM 1 ng/mL (light green), OSM 10 ng/mL (green), and OSM 100 ng/mL (dark green), and IL-6 1 ng/mL (light orange), IL-6 10 ng/mL (orange), and IL-6 100 ng/mL (dark orange), were expressed in Ohms/cm². OSM at 100 ng/mL exposure demonstrated a significant decrease in TEER (A) whereas IL-6 had no effect (B). Data are presented as the change in TEER measurement (Ohm/cm²) between the baseline and after the 48 h cytokine exposure. * p = 0.049; “ns” indicates p > 0.05 between IL-6 stimulated wells and controls.

Figure 5

OSM decreased ciliary beating efficiency and increased the in vitro repair rate at 24 h. (A) The ciliary beating efficiency (mPa) was evaluated after 48 h of OSM stimulation at various concentrations (1 ng/mL, 10 ng/mL, and 100 ng/mL) and compared with the unstimulated controls (n = 7). Stimulation of HNECs with OSM at 10 and 100 ng/mL decreased ciliary beating efficiency. * p < 0.0001, and ** p < 0.0001. (B) The repair rate was evaluated as the proportion of the wounded area closed at each time point for OSM stimulated wells (either 1 ng/mL, 10 ng/mL, or 100 ng/mL) and compared to one of unstimulated control wells (n = 5). Stimulation of HNECs with OSM at 100 ng/mL increased the wound repair rate. * Indicates statistical significance between the control and OSM 100 ng/mL at 24 h, p = 0.016.