Oncostatin M promotes mucosal epithelial barrier dysfunction, and its expression is increased in patients with eosinophilic mucosal disease

Abstract

Background: Epithelial barrier dysfunction is thought to play a role in many mucosal diseases, including asthma, chronic rhinosinusitis (CRS), and eosinophilic esophagitis.

Objective: The objective of this study was to investigate the role of oncostatin M (OSM) in epithelial barrier dysfunction in human mucosal disease.

Methods: OSM expression was measured in tissue extracts, nasal secretions, and bronchoalveolar lavage fluid. The effects of OSM stimulation on barrier function of normal human bronchial epithelial cells and nasal epithelial cells cultured at the air-liquid interface were assessed by using transepithelial electrical resistance and fluorescein isothiocyanate-dextran flux. Dual-color immunofluorescence was used to evaluate the integrity of tight junction structures in cultured epithelial cells.

Results: Analysis of samples from patients with CRS showed that OSM mRNA and protein levels were highly increased in nasal polyps compared with those seen in control uncinate tissue (P < .05). OSM levels were also increased in bronchoalveolar lavage fluid of allergic asthmatic patients after segmental allergen challenge and in esophageal biopsy specimens from patients with eosinophilic esophagitis. OSM stimulation of air-liquid interface cultures resulted in reduced barrier function, as measured by decreased transepithelial electrical resistance and increased fluorescein isothiocyanate-dextran flux (P < .05). Alterations in barrier function by OSM were reversible, and the viability of epithelial cells was unaffected. OSM levels in lysates of nasal polyps and uncinate tissue positively correlated with levels of α2-macroglobulin, a marker of epithelial leak, in localized nasal secretions (r = 0.4855, P < .05).

Conclusions: These results suggest that OSM might play a role in epithelial barrier dysfunction in patients with CRS and other mucosal diseases.

Keywords: Oncostatin M; atopic asthma; chronic rhinosinusitis; eosinophilic esophagitis; epithelial barrier; tight junctions; transepithelial electrical resistance.

Figure 1. OSM was elevated in CRs

(A) OSM mRNA was increased in nasal polyps compared to UT from control, CRSsNP and CRSwNP patients, (1.76 ± .85 vs. .06 ± .013 respectively, p< .01, n=11-13, Kruskal-Wallis test). (B) OSM was increased in nasal polyp tissue compared to control UT as measured by Luminex, (3.38 pg/mg ± 1.95 vs. 47.49 pg/mg ± 17.98 respectively, p<.01, n=11-19, Kruskal-Wallis test). (C) OSM protein was increased in nasal lavage of CRSwNP patients compared to CRSsNP, (78.50pg ± 44.16 vs. 327.1pg ± 78.83 respectively, n=10-40, Kruskal-Wallis test). (D) OSM protein was increased in culture supernatants of nasal polyp tissue compared to UT from CRSsNP and CRSwNP, (19.1ng ± 2.6ng vs. 4.7ng ± 1.1 and 4.9ng ± 0.89 respectively, n=4-23, Kruskal-Wallis test).

Figure 2. OSM decreased barrier function in airway epithelium

ALI cultures of NHBE cells were grown until fully differentiated at day 21 of culture, and then the cells were left unstimulated, or stimulated with OSM at the specified concentration. (A) OSM stimulation resulted in a concentration dependent decrease of TEER, data is presented as the change in resistance over the 48 hour stimulation normalized to resistance at 0h to show the percentage of resistance lost during stimulation (((0h TEER- 48h TEER)/ 0h TEER)*100) (p< .0001, n=7-11, Kruskal-Wallis test). (B) Dextran flux across the cell layer was increased with OSM stimulation, suggesting decreased barrier function following OSM stimulation (p< .001, n=5, Kruskal-Wallis test). Data represent mean ± SEM.

Figure 3. OSM disrupted organization of tight junctions

NHBE were left unstimulated, or stimulated with indicated concentrations of OSM for 48hrs. NEC were either left unstimulated, or stimulated with 100ng/mL OSM. The tight junction protein occludin is stained in green, cell nuclei are in blue. Representative of NHBE, n=3, and NEC, n=3.

Figure 4. OSM expression correlated with a marker of epithelial leak in vivo

(A) ALI cultures of NEC from IT from control, CRSsNP, and CRSwNP patients show no difference in TEER (n=7-11). Data represent mean ± SEM. (B) OSM levels in nasal polyps and UT from CRSsNP patients correlated with epithelial leak as determined by assessment of α2-macroglobulin levels in adjacent nasal secretions (Pearson r=.4855, n=11-13, p<.05). Grey dots signify nasal polyp tissue and black dots signify UT of CRSsNP patients.

Figure 5. OSM levels were increased in EoE and upon allergen challenge in allergic asthmatics

(A) OSM mRNA was increased in esophageal biopsies of EoE patients compared to control, (6.4e-005 ± 1.8e-005 vs. .00024 ± 4.9e-005 respectively, p<.01, n=8-13, Mann-Whitney U test). (B) OSM protein was increased in BAL of allergic asthmatics following segmental allergen challenge compared to BAL challenged with saline, (.56 ± .56 vs. 22.8 ± 6.3 respectively, p<.0001, n=16, Mann-Whitney U test. (C) OSM levels in BAL following allergen challenge in allergic asthmatics correlate with levels of HSA, (Pearson r=.8062, p<.001).

Figure 6. Proposed model for the role of OSM in epithelial barrier dysfunction in CRS

Airway epithelium develops barrier function through a complex network of intercellular junctions that adhere the cells to each other and the cytoskeleton, illustrated on the left side of the figure. Barrier dysfunction, as illustrated on the right side of the figure, may occur following exposure to OSM and lead to a chronic inflammatory state due to the presence of environmental factors that would not otherwise have access to the tissue.