Unveiling the Role of NF-kB in Distal Lung Epithelial Regeneration

Introduction: During alveolar injury, various processes are involved in facilitating repair and regeneration. Alveolar type 2 (AT2) cells can self-renew, proliferate, and differentiate into alveolar type 1 (AT1) cells. Prior studies have demonstrated that proinflammatory cytokines can promote epithelial cell proliferation, but the exact mechanism has not been fully elucidated. Our hypothesis is that activation of NF-kB initiates the proliferation of alveolar epithelial cells, which mediates the process of injury repair.

Materials and

Methods: Single cell suspensions were generated from the lungs of C57B6 or Sftpc-rtTA/tet-o-IKK2DN (dominant negative IKK2). Cd326+ epithelial cells were sorted using magnetic beads (Miltenyi), and 5,000 cells seeded in 50% Matrigel as 50 ul droplets. Serum-free, feeder-free media was used to enrich for alveolar type 2 cells. Organoids were cultured with doxycycline, a small-molecule NF-kB inhibitor (BMS-345541), or respective vehicles throughout the duration of experiments. Organoids were then imaged and quantified using ImageJ.

Results, Conclusions, and Discussions:

Results: The inclusion of IL-1b in the media for the first 3 days of culture increased the number of organoids from wild-type mice compared to IL-1b deficient media. Treatment of organoids with BMS-345541 (to inhibit Nfkb) starting at day 0 dramatically reduced the number of organoids compared to vehicle-treated wells. Using a genetic model of a dominant-negative IKK2 (to conditionally inhibit Nfkb), we observed a dose-dependent diminution of organoid size and number.

Conclusion and discussion: IL-1b signaling mediated through the NF-kB pathway mediates AT2 organoid formation and promotes AT2 proliferation.

Acknowledgements (Optional): NIH-NHLBI and T32: 5T32HL094296-15