Figure 5.

Epithelial inflammation in inhibitory κB kinase β transactivated (IKTA) lungs results in NF-κB activation and decreased fibulin-5 expression by interstitial fibroblasts. A: Representative images of control (CTRL) and IKTA lung at postnatal day (PN) 2 immunostained for phosphoserine 276 in the transcription factor p65 (RelA) component of NF-κB as a marker of NF-κB activation. Arrow denotes cells in the interstitium with phospho-p65 staining. B and C: Lung fibroblasts were isolated from PN2 NF-κB–GFP–luciferase (NGL) mice and cultured in the presence of bronchoalveolar lavage (BAL) fluid from adult control or IKTA mice that were treated with doxycycline in drinking water (1 g/L) for 7 days (50 μL of BAL per 500 μL of culture medium). B: Gene expression of elastin assembly components in cells treated with control or IKTA BAL. C: Representative immunostaining for green fluorescent protein as an indicator of NF-κB activation in NGL fibroblasts treated with control or IKTA BAL. D and E: PN2 NGL fibroblasts were treated with IKTA BAL or lipopolysaccharide (LPS) (800 ng/mL). Luciferase expression (D) was quantified as a measure of NF-κB activation and gene expression of select elastin assembly components (E) was assessed using quantitative RT-PCR (RT-qPCR). F: Adenoviral vectors expressing a dominant negative IκB-α (DN IκB Adv) or control (Cre recombinase; CTRL Adv) (1500 multiplicity of infection) were added to PN2 NGL fibroblasts treated with 800 ng/mL of Escherichia coli LPS for 24 hours, and Fbln5 expression was then quantified by RT-qPCR. Data are expressed as means ± SEM. n = 4 per group. ^∗P < 0.05 versus control BAL-treated cells (B), versus untreated control cells (E and F). Original magnification, ×200 (C); ×400 (A). TO-PRO-3, Life Science Technologies (Grand Island, NY).