Abstract Rationale Studies have shown increased activation of the NF-κB pathway in the lungs of patients with Idiopathic pulmonary fibrosis (IPF), including in alveolar type 2 (AT2) cells; however, it is unclear how NF-κB pathway signaling contributes to disease pathology. We hypothesized that, in addition to promoting inflammation, NF-κB signaling supports epithelial cell proliferation/and or survival during the injury-repair process. Methods Mice with tamoxifen-inducible IKKβ deletion in AT2 cells (SPCcreERT2/IKKβflox/flox) or mice with constitutive, pan-epithelial deletion of IKKβ (SHHcre/IKKβflox/flox) were used to inhibit canonical NF-κB signaling in vitro and in vivo. For organoid studies, Cd326 epithelial cells were sorted using magnetic beads and cultured in serum-free, feeder-free media to enrich for AT2 cells. In the in vivo fibrosis studies, adult mice were challenged with bleomycin 0.08 IU (or saline controls) delivered by intratracheal instillation, then euthanized at 7, 21, and 42 days post-bleomycin. Lung inflammation was assessed by bronchoalveolar lavage (BAL) cell count and differential. Fibrosis was quantified using the Sircol total collagen assay and the modified Ashcroft score. Apoptosis was measured by TUNEL staining, and proliferation was assessed via Ki67 immunostaining. Results AT2-specific IKKβ deletion reduced organoid colony-forming efficiency (CFE) by 41.4% (from a median CFE of 0.41% to 0.24%, P 0.0108) compared to wildtype controls without effects on organoid size. For comparison, NF-kB inhibition using the dominant-negative IKKβ genetic model reduced CFE by 43.5% and a small-molecule NF-kB inhibitor (BMS-345541) reduced CFE by 71.6% compared to controls. Despite this robust impact on organoid formation, lung fibrosis scores and collagen content did not differ between controls and mice with AT2-specific or pan-epithelial IKKβ deletion at day 21 or 42 post-bleomycin. To assess whether mice with epithelial NF-kB inhibition had early protection against injury and or apoptosis following bleomycin, mice were sacrificed after 7 days. Similar numbers of TUNEL+ epithelial cells and BAL cell counts and differentials were observed across all bleomycin-treated groups. Conclusion Activation of the NF-kB pathway promotes organoid formation; however, this does not translate to differences in lung fibrosis after bleomycin treatment. In addition to suggesting that epithelial NF-kB is not required for epithelial repair in vivo, our findings raise questions about the importance of organoid models for predicting in vivo phenotypes. This abstract is funded by: NIH/NHLBI P01HL092870, R01HL153246 and 5T32HL094296-15
Gninzeko et al. (Fri,) studied this question.