Fig 2: DEX suppresses the NTHi-induced proinflammatory mediator expression in Irak-m+/+ but not in Irak-m-/- mice.WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg-1, intraperitoneally) for 2 h, followed by intratracheal inoculation with NTHi (1 × 107 c.f.u. per lung) for 24 h. (a) The mRNA expression in the mouse lung tissue was analysed by qPCR. (b) The protein level of proinflammatory mediators in BAL fluid was determined by enzyme-linked immunosorbent assay. (c) The mRNA expression in alveolar macrophages was analysed by qPCR. Data in a,c, n=3; b, n=9 are mean±s.d. *P<0.05, NS, non-significant; t-test.

Fig 3: A schematic representation of suppression of inflammation by GCs via the upregulation of IRAK-M expression.

Fig 4: IRAK-M suppresses the NTHi-induced proinflammatory mediator expression.(a) The proteins from BEAS-2B cells stably overexpressing IRAK-M and its control pcDNA3.1 (Mock) cells were detected by immunoblot with indicated antibodies. (b) The mRNA expression was detected by qPCR after the stable cells were treated with DEX (100 nM) and NTHi. (c) BEAS-2B cells were transfected with siRNA control or IRAK-M for 72 h. IRAK-M protein expression was detected by immunoblot with anti-IRAK-M antibodies (SC, Santa Cruz Biotechnology sc-100389; PS, ProSci #2355). (d) siRNA-transfected BEAS-2B cells were infected with NTHi followed by qPCR. (e) The mRNA level was calculated by following: (NTHi-induced mRNA at each concentration of DEX)/(NTHi-induced mRNA without DEX). Data in b,d,e, n=3) are mean±s.d. *P<0.05; t-test.

Fig 5: 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.