Evidence that oncostatin M synergizes with IL-4 signaling to induce TSLP expression in chronic rhinosinusitis with nasal polyps

Abstract

Background: Oncostatin M (OSM) may promote type 2 inflammation in chronic rhinosinusitis with nasal polyps (CRSwNP) by inducing thymic stromal lymphopoietin (TSLP).

Objective: We sought to study the impact of OSM on TSLP synthesis and release from nasal epithelial cells (NECs).

Methods: OSM receptors, IL-4 receptors (IL-4R), and TSLP were evaluated in mucosal tissue and primary NECs from patients with CRSwNP by quantitative PCR and immunofluorescence. Air-liquid interface-cultured NECs were stimulated with cytokines, including OSM, and quantitative PCR, ELISA, Western blot, and flow cytometry were used to assess the expression of OSM receptors, IL-4R, and TSLP.

Results: Increased levels of OSM receptor β chain (OSMRβ), IL-4Rα, and TSLP were observed in nasal polyp tissues and primary epithelial cells from nasal polyps of patients with CRSwNP compared with control tissues or cells from control subjects. The level of expression of OSMRβ in tissue was correlated with levels of both IL-4Rα and TSLP. OSM stimulation of NECs increased the expression of OSMRβ and IL-4Rα. Stimulation with IL-4 plus OSM augmented the production of TSLP; the response was suppressed by a signal transducer and activator of transcription 6 inhibitor. Stimulation of NECs with IL-4 plus OSM increased the expression of proprotein convertase subtilisin/kexin 3, an enzyme that truncates and activates TSLP.

Conclusions: OSM increases the expression of IL-4Rα and synergizes with IL-4 to induce the synthesis and release of TSLP in NECs. Because the combination of IL-4 and OSM also augmented the expression of proprotein convertase subtilisin/kexin 3, these results suggest that OSM can induce both synthesis and posttranslational processing/activation of TSLP, promoting type 2 inflammation.

Keywords: IL-4Rα; OSM; OSMRβ; TSLP; chronic rhinosinusitis; nasal epithelial cells; nasal polyps; type 2–dominant inflammation.

Figure 1.

The expression of OSM, OSMRβ and IL-4R in tissue. The mRNA expression of OSM in tissue (A); the mRNA expression of OSMRβ in tissue (B); the mRNA expression of IL-4Rα, IL-13Rα1, IL-2Rγ in tissue (C). *P < .05, **P < .01.

Figure 2.

The correlation between IL-4R and OSM, and OSMRβ in tissue. The correlation between IL-4Rα and OSM in tissue (A); the correlation between IL-4Rα, IL-13Rα1 and OSMRβ in tissue (B).

Figure 3.

The distribution of OSMRβ, IL-4Rα in nasal polyp, and the mRNA expression of OSMRβ and IL-4Rα, and the correlation between them in scraping NEC. The distribution of OSMRβ and gp130 in nasal polyp, the two yellow arrows represent the co-stains of OSMRβ and gp130 (A); the distribution of IL-4Rα in nasal polyp (B); the mRNA expression of OSMRβ, IL-4Rα in scraping NEC (C), the correlation between OSMRβ and IL-4Rα in scraping NEC from patients with CRS and control UT. NEC, nasal epithelial cells (D). *P < .05, ***P < .001, ****P < .0001.

Figure 4.

The expression of OSMR, IL-4R in ALI culture NEC from control UT stimulated by OSM or other cytokines. The mRNA expression of OSMRβ, gp130, LIFR in NEC stimulated by 100 ng/ml OSM for 12h, n = 5 (A); the mRNA expression of IL-4Rα, IL-13Rα1 in NEC stimulated by OSM or EREG at a concentration of 100 ng/ml, or by IFN-γ, IL-4, IL-13, IL-17, or IL-6 respectively at a concentration of 10 ng/ml for 12h, n = 5 (B); the protein expression of IL-4Rα detected by flow cytometry in NEC stimulated by OSM (100 ng/ml) for 24h, n = 4 (C); NEC, nasal epithelial cells; *P < .05, **P < .01, ***P < .001.

Figure 5.

The correlation between TSLP and OSM, OSMRβ, and IL-4Rα in tissue, and the expression of TSLP in ALI culture NEC from control UT stimulated by OSM or other cytokines. The mRNA expression of TSLP in tissue (A); the correlation between TSLP and OSM, OSMRβ, and IL-4Rα in tissue (B); the mRNA expression of TSLP in NEC stimulated by OSM or EREG at a concentration of 100 ng/ml, or by IFN-γ, IL-4, IL-13, IL-17, or IL-6 respectively at a concentration of 10 ng/ml, or IL-4 (10 ng/ml) plus OSM (100 ng/ml), or IL-13 (10 ng/ml) plus OSM (100 ng/ml) for 12h, n = 5 (C); NEC, nasal epithelial cells; *P < .05.

Figure 6.

The expression of truncated TSLP in ALI cultured NEC from control UT stimulated by OSM, IL-4, IL-13, IL-4 plus OSM, or IL-13 plus OSM. The release of TSLP in supernatant of NEC stimulated by IL-4 (10 ng/ml), IL-13 (10 ng/ml), OSM (100 ng/ml), IL-4 (10 ng/ml) plus OSM (100 ng/ml), or IL-13 (10 ng/ml) plus OSM (100 ng/ml) in time-dependent manner for 0, 6, 12, 24h, was analyzed by ELISA, n = 4 (A); the expression of native TSLP and TSLP after incubation with PCSK3/Furin for 6h or 24h. The expression of truncated TSLP in lysate of NEC stimulated by IL-4 (10 ng/ml), IL-13(10 ng/ml), OSM (100 ng/ml), IL-4 (10 ng/ml) plus OSM (100 ng/ml), or IL-13 (10 ng/ml) plus OSM (100 ng/ml) for 24h, n =4 (B); the mRNA expression of PCSK3 in the NEC stimulated by IL-4 (10 ng/ml), IL-13 (10 ng/ml), OSM (100 ng/ml), IL-4 (10 ng/ml) plus OSM (100 ng/ml), or IL-13 (10 ng/ml) plus OSM (100 ng/ml) for 12h, n =5 (C); and the mRNA expression of PCSK3 in control UT and NP tissue (D); NEC, nasal epithelial cells; UT; N.D, no detection; *P < .05, **P < .01.

Figure 7.

The release of TSLP in supernatant of ALI cultured NHBE cells stimulated by OSM, IL-4, IL-13, IL-4 plus OSM, or IL-13 plus OSM, and the synergistic effect of IL-4 plus OSM on TSLP was suppressed in NEC from control UT and NHBE cells by an inhibitor of STAT6. The release of TSLP in supernatant of NHBE cells stimulated by IL-4 (10 ng/ml), IL-13(10 ng/ml), OSM (100 ng/ml), IL-4 (10 ng/ml) plus OSM (100 ng/ml), or IL-13 (10 ng/ml) plus OSM (100 ng/ml) for 24h, n = 4 (A). NEC or NHBE cells stimulated by IL-4 (10 ng/ml) plus OSM (100 ng/ml) for 24h, or pretreated with DMSO or STAT6 inhibitor (AS1517499, 50 nM) for 2 hours, then IL-4 (10 ng/ml) plus OSM (100 ng/ml) for further 24h. TSLP release in culture supernatant was analyzed by ELISA, n =4(B). N.D, no detection. *P < .05.

Figure 8.

The expression of IL-4 in tissue, and the correlation between IL-4 and TSLP in tissue. The mRNA expression of IL-4 in tissue (A); the correlation between IL-4 and TSLP in tissue (B).