Background: Idiopathic pulmonary fibrosis (IPF) is the most common type of interstitial lung disease and is also a fatal condition, characterized by the dysfunction of both alveolar type I cells (AT1) and alveolar type II cells (AT2). As AT2 cells are deemed the adult stem cell (ASC), the potential way to stimulate their proliferation and differentiation is one therapeutic strategy for IPF.

Methods: Utilizing scRNA sequencing of the lung tissue of IPF patients and healthy donors, we plotted the AT2 cells and found the genes specifically expressed in AT2 cells. Then, the bioinformatic prediction was further validated by the Human Protein Atlas website. Subsequently, we isolated the AT2 cells from newborn mice by MACS and validated whether Bex2 is expressed in AT2 cells. By differentiation of AT1 cells, the expression of Bex2 is also assessed by immunoblotting. Followed by establishing the IPF mouse model via Bleomycin, we assessed the expression levels of Bex2, and then tried to elevate the cellular level of Bex2 by the trachea titration of AAV-Bex2. Tissue staining by HE, Masson, and Sirius Red was performed to validate whether the elevated Bex2 could alleviate IPF. Lastly, we established Bex2 conditional knockout mice with the deficiency of the Bex2 gene from the AT2 cells, and found whether there is spontaneous IPF, and whether AAV-Bex2 delivery from the trachea could alleviate the disease progression by increasing AT2 cells.

Results: scRNA sequencing revealed that Bex2 is only expressed in AT2 cells, which is further validated by the Human Protein Atlas. By isolating AT2 cells and inhibiting their differentiation, we observed increased transcription and translation of Bex2 in AT2 cells, however, loss of Bex2 will lead to the differentiation of AT2 cells to AT1 cells. In IPF mouse models, we found that Bex2 expression levels are decreased, accompanied by exacerbated lung tissue damage, which was validated by HE, Masson, and Sirius Red staining. With AAV-Bex2 delivery, the AT2 cells could remain intact even in the presence of bleomycin, accompanied by intact lung tissue and AT2 cells. Lastly, mice with Bex2 conditional knockout from the AT2 cells showed spontaneous IPF even at a very young age (1 month) with damaged tissue and loss of AT2 cells.

Conclusion: Increased expression of Bex2 in AT2 cells possesses the potential for IPF treatment, and Bex2 may be the potential regulator gene for the maintenance of AT2 cells to counteract IPF progression.