Figure 2: Inhibition of PDE4 leads to suppression of NTHi-induced inflammation in vitro and in vivo.

(a) A549 cells were pretreated with Rolipram (10 μM) for 1 h, followed by NTHi stimulation, and NF-κB-promoter activity was determined. (b) mRNA expression of pro-inflammatory cytokines was measured in A549 cells pretreated with Rolipram for 1 h and stimulated with NTHi. mRNA expression of pro-inflammatory cytokines was measured in (c) lung and (d) middle ear tissues from C57BL/6J mice preinoculated with Rolipram (10 mg/kg) for 2 h and inoculated with NTHi for 9 h. Haematoxylin and eosin staining of (e) lung and (f) middle ear tissues from C57BL/6J mice preinoculated with Rolipram for 2 h and inoculated with NTHi for 9 h (magnification × 100 in e; × 400 in f, respectively). Scale bars, 100 μm in e; 20 μm in f. (g) Thickness of middle ear mucosa in C57BL/6J mice preinoculated with Rolipram and inoculated with NTHi was measured from 15 middle ear tissue sections per experimental group. (h) Mice were transtympanically inoculated with NTHi with or without Rolipram pretreatment, and tympanic cavity was observed and recorded under the otoscope. (i) IL-1β mRNA expression in HMEEC (left panel) or mouse middle ear tissues (right panel) was measured after NTHi treatment pretreated with or without Roflumilast (1 μM for in vitro, 5 mg kg⁻¹ for in vivo). Data in a–d, g and i are mean±s.d. (n=3 in a–d, i and 15 in g). *P<0.05. Statistical analysis was performed using Student’s t-test. CON, control. Data are representative of three or more independent experiments.