Figure 1: DEX synergistically enhances the NTHi-induced IRAK-M expression in vitro and in vivo.

(a) IRAK-M mRNA expression in human bronchial epithelial BEAS-2B cells treated with DEX (0.01, 0.1, 1, 10, 100 or 1,000 nM) for 1 h, followed by the stimulation of NTHi for 5 h. (b) IRAK-M mRNA expression in human lung epithelial A549 cells stimulated with DEX (100 nM) for 1 h, followed by NTHi for indicated time. (c) A549 cells were treated with DEX (100 nM) for 1 h, followed by NTHi for 12 h. IRAK-M protein was detected by anti-IRAK-M antibody. (d) IRAK-M protein expression was quantified from three independent experiments. (e) IRAK-M mRNA expression in primary NHBE cells treated with DEX (10, 100 or 1,000 nM) for 1 h and subjected to NTHi stimulation for 5 h. (f) Human peripheral blood CD14⁺ monocytes were differentiated to macrophages by culturing them with granulocyte–macrophage colony-stimulating factor for 7 days. IRAK-M mRNA was determined after the macrophages were treated with DEX (100 nM) for 1 h, followed by stimulation of NTHi for 5 h. (g) Mice were stimulated with DEX (1 mg kg⁻¹, intraperitoneally) for 2 h and intratracheally inoculated with NTHi (1 × 10⁷ c.f.u.) for 24 h. The mRNA expression in alveolar macrophages was assessed by qPCR. (h) The IRAK-M mRNA expression in lung was assessed by qPCR. (i) Immunostaining of mouse lung with control IgG or anti-IRAK-M antibody by LSAB (Labelled Streptavidin Biotin) staining system (Scale bar, 50 μm; magnification, × 400). (j) Treatment-blind observers scored the IRAK-M expression from the histology results. (k) The lung sections were stained by using indicated antibodies. The arrows and arrowheads indicate the epithelial cells and macrophages merged with IRAK-M expression, respectively. (Scale bar, 50 μm; magnification, × 400. Data in a,b,d-h, n=3; j, n=6) are mean±s.d. *P<0.05; t-test.