Apremilast mitigates interleukin (IL)-13-induced inflammatory response and mucin production in human nasal epithelial cells (hNECs)

Abstract

Interleukin (IL)-13-associated inflammatory response is important for the pathogenesis of allergic rhinitis (AR). Apremilast is a phosphodiesterase-4 (PDE4) inhibitor approved for psoriasis treatment. Here, we investigated the potential effects of Apremilast against IL-13-induced injury in human nasal epithelial cells (hNECs). Firstly, Apremilast ameliorated oxidative stress in IL-13-challenged cells by decreasing the levels of reactive oxygen species (ROS) and the production of malondialdehyde (MDA). Secondly, Apremilast inhibited the expressions of IL-6 and IL-8. Moreover, Apremilast inhibited the expressions of the chemokines colony-stimulating factor 2 (CSF2) and chemokine ligand 11 (CCL11). Interestingly, exposure to IL-13 increased the expressions of mucin 4 and mucin 5AC (MUC5AC), which was ameliorated by treatment with Apremilast. Interestingly, we found that Apremilast inhibited the phosphorylation of c-Jun-N-terminal kinase (JNK). Importantly, Apremilast reduced the levels of c-fos and c-Jun, the two AP-1 subfamilies. The luciferase reporter assay demonstrates that Apremilast reduced the transcriptional activity of activator protein 1 (AP-1). Lastly, we found that Apremilast prevented the activation of nuclear factor kappa-B (NF-κB) by decreasing the levels of nuclear NF-κB p65 and the luciferase activity of the NF-κB reporter. In summary, we conclude that Apremilast possesses a protective effect against IL-13-induced inflammatory response and mucin production in hNECs by inhibiting the activity of AP-1 and NF-κB.

Keywords: Apremilast; IL-13; NF-κB; allergic rhinitis; mucin.

Figure 1.

Cytotoxicity of Apremilast in human nasal epithelial cells (hNECs). Cells were stimulated with Apremilast (0, 0.25, 0.5, 2.5, 5, 25, 50, 100 μM) for 24 hours. (a). Molecular structure of Apremilast; (b). Cell viability was measured by the CCK-8 assay (*, **, ***, P < 0.05, 0.01, 0.001 vs. Vehicle group)

Figure 2.

Apremilast attenuated IL-13-induced oxidative stress in hNECs. Cells were stimulated with IL-13 (10 ng/ml) in the presence and absence of Apremilast (2.5, 5 μM) for 24 hours. (a). The levels of ROS; (b). The levels of MDA (***, P < 0.001 vs. vehicle group; #, ##, P < 0.05, 0.01 vs. IL-13 group)

Figure 3.

Apremilast inhibited IL-13-induced expression of pro-inflammatory cytokines in hNECs. Cells were stimulated with IL-13 (10 ng/ml) in the presence and absence of Apremilast (2.5, 5 μM) for 24 hours. (a). mRNA of IL-6 and IL-8; (b). Secretions of IL-6 and IL-8 (***, P < 0.001 vs. vehicle group; #, ##, P < 0.05, 0.01 vs. IL-13 group)

Figure 4.

Apremilast suppressed IL-13-induced expressions of CSF2 and CCL11 in hNECs. Cells were stimulated with IL-13 (10 ng/ml) in the presence and absence of Apremilast (2.5, 5 μM) for 24 hours. (a). mRNA of CSF2 and CCL11; (b). Secretions of CSF2 and CCL11 as measured with ELISA (***, P < 0.001 vs. vehicle group; #, ##, P < 0.05, 0.01 vs. IL-13 group)

Figure 5.

Apremilast inhibited the expression of Mucin 4 and Mucin 5AC (MUC5AC) in hNECs. Cells were stimulated with IL-13 (10 ng/ml) in the presence and absence of Apremilast (2.5, 5 μM) for 24 hours. (a). mRNA of Mucin 4 and MUC5AC; (b). Secretions of Mucin 4 and MUC5AC (***, P < 0.001 vs. vehicle group; #, ##, P < 0.05, 0.01 vs. IL-13 group)

Figure 6.

Apremilast mitigated the activation of JNK in IL-13 challenged hNECs. Cells were stimulated with IL-13 (10 ng/ml) in the presence and absence of Apremilast (2.5, 5 μM) for 24 hours. The levels of p-JNK and total JNK were measured by western blot analysis (***, P < 0.001 vs. vehicle group; #, ##, P < 0.05, 0.01 vs. IL-13 group)

Figure 7.

Apremilast prevented activation of the transcriptional factor AP-1. Cells were stimulated with IL-13 (10 ng/ml) in the presence and absence of Apremilast (2.5, 5 μM) for 24 hours. (a). The expressions of c-fos and c-Jun; (b). Luciferase activity of AP-1 (***, P < 0.001 vs. vehicle group; #, ##, P < 0.05, 0.01 vs. IL-13 group)

Figure 8.

Apremilast ameliorated IL-13- induced activation of the transcriptional factor NF-κB. Cells were stimulated with IL-13 (10 ng/ml) in the presence and absence of Apremilast (2.5, 5 μM) for 24 hours. (a). Protein level of nuclear NF-κB p65;(b)Luciferase activity of NF-κB (***, P < 0.001 vs. vehicle group; #, ##, P < 0.05, 0.01 vs. IL-13 group)