Activation of NF-𝜅;B Promotes Distal Lung Organoid Formation

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Abstract Rational: Alveolar regeneration is a process that involves the mobilization of different facultative progenitor stem cells, such as alveolar type 2 (AT2) cells and distal lung epithelial cells. Multiple studies have linked proinflammatory cytokines such as IL-1β to alveolar regeneration, but the mechanisms regulating this process are incompletely understood. We hypothesized that NF-κB activation mediates alveolar regeneration by promoting proliferation of distal lung progenitor cells. Materials/Methods: Single cell suspensions were generated from the lungs of C57BL6, SHHcre/IKKβflox/flox(constitutive epithelial IKKβ deletion in the lungs), or SPCcreERT2/IKKβflox/flox(inducible IKKβ deletion in AT2 cells). CD326+ epithelial cells were sorted using magnetic beads (Miltenyi), and 5,000 cells were seeded in 50% Matrigel as 50 ul droplets. Serum-free, feeder-free media was used, and organoids were cultured with and without IL-1β (for the first 3 days of culture), a small-molecule NF-κB inhibitor (BMS-345541), or vehicle throughout the duration of experiments. At day 14, organoids were fixed, then imaged and quantified using ImageJ. Organoids were stained for AT2 and AT1 cells using proSPC and Hopx antibodies, respectively. Results: The presence of IL-1β in serum-free feeder-free media (SFM) resulted in increased organoid formation. To test whether NF-κB was involved, we used a chemical inhibitor at 5 and 25 μM) and found that there were almost no organoids formation with 25 μM and organoid formation was reduced by 71.6% with 5 μM compared to vehicle control. In addition, we used a global epithelial knockout of IKKβ and identified a substantial reduction in organoids formation compared to littermate controls mice. Further, AT2-specific NF-κB inhibition reduced organoid formation by 37.3% compared to controls. In addition, there was no difference in the size and appearance of organoids cultured without IL-1β, nor in those derived from IKKβ knockout mice when compared to the wild-type control. Lastly, staining with markers for AT1 and AT2 showed no differences in the composition of AT2 or AT1 cells across all IKKβ knockout models compared to the wild-type controls. Conclusion: In conclusion, inhibiting NF-κB in distal lung epithelial progenitor cells led to decreased organoid formation. These findings suggest that NF-κB activation is important at initiating alveolar regeneration by promoting epithelial cell proliferation. Future studies will investigate the role of NF-κB in alveolar regeneration in vivo.

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