Figure 3. Bacterial invasion and NF-κB activation in airways of pIgR−/− mice.
(a) Immunofluorescent detection of bacteria (fluorescent in situ hybridization (FISH) probe for 16S rRNA, red, top panels) or NF-κB (phospho-p65 (Ser276, red, bottom panels), IgA (green, top panels) and DAPI (blue) from 12-month-old WT and pIgR−/− mice (original magnification, × 1,000). In WT mice, bacteria with bound SIgA were identified within the airway lumen (yellow on merged image, identified by arrow), whereas bacteria intercalated within the epithelium were identified in pIgR−/− mice (red, identified by arrow). Bottom panels show phopsho-p65 localized to nuclei (arrows). Scale bar, 50 μm. (b) Box and whisker plot (showing median, 25th–75th percentile and range) for intraepithelial (intercalated) and luminal bacteria in airways of 12-month-old WT and pIgR−/− mice as identified by FISH for bacterial DNA. Seven mice per group; *P<0.001. (c) Western blotting and densitometry for p65 component of NF-κB (normalized to p84) in nuclear protein extracts from lungs of 12-month-old WT and pIgR−/− mice. Six mice per group; *P<0.05 (Student's t-test). (d) KC protein levels in BAL fluid. Six mice per group; *P<0.05 compared with 2-month-old pIgR−/− mice and age-matched WT controls; **P<0.05 compared with all other groups (two-way analysis of variance (ANOVA)). (e) Total bacterial DNA was quantified from lung tissue in WT and pIgR−/− mice using quantitative PCR (qPCR) and primers specific for V1 region of prokaryotic 16S rRNA. Ten mice per group. (f) Distribution of bacterial phyla and classes in lung tissue as determined by 16S sequencing from lungs of 12-month-old WT and pIgR−/− mice (three mice studied per group but only two WT mice had sufficient bacterial DNA amplification for detailed analysis).