Abstract Rationale Alveolar epithelial cell loss is a hallmark of idiopathic pulmonary fibrosis (IPF). Type I alveolar epithelial cells (AT1) line the lung alveoli to facilitate gas exchange. In IPF, repeat injury leads to AT1 cell loss, releasing pro-inflammatory factors. In preclinical models, including bleomycin, AT1 cells are lost through apoptosis, an effect that is diminished in LPA1-/- mice. Here, we wanted to determine whether, 1) LPA directly mediated human AT1 apoptosis through LPA1 and 2) conditioned media from human IPF-derived precision cut lung slice (PCLS) cultures could induce apoptosis in an LPA1-sensitive manner. Methods Human primary epithelial cells (HPAEpiCs, ScienCell) were plated at 15k/96-well and adhered overnight. In culture, HPAEpiCs terminally differentiate into AT1 cells. Cells were pretreated with vehicle or 300 nM PIPE-791 for 8h. LPA was added to the cells, incubated for 24h, then immunostained for podoplanin (Pdpn), cleaved caspase 3 (Asp175; Cc3), phalloidin, and Hoechst. Apoptosis was measured as a %Cc3+/Pdpn+/Hoechst+ of total Pdpn+/Hoechst+ counts. For conditioned media studies, PCLS were cultured in vehicle or 300 nM PIPE-791. Conditioned media was collected at study end and frozen (-80oC). Separately, AT1 cells were preincubated with vehicle or PIPE-791 for 8h, followed by 50% exchange of culture media for conditioned media. If needed, PIPE-791 was added to reach a 300 nM final concentration. Results Direct addition of LPA significantly increased the %Cc3+/Pdpn+ cells, indicating apoptosis. This increase was inhibited by PIPE-791. AT1 cells exposed to normal-PCLS conditioned media did not show a significant difference in the %Cc3+/Pdpn+ cells compared to complete growth media. Exposure of AT1 cells to IPF-PCLS conditioned media, however, significantly increased Cc3+/Pdpn+ cells and was inhibited by PIPE-791. Interestingly, conditioned media from PIPE-791 treated IPF-PCLS cultures did not increase the %Cc3+/Pdpn+. AT1 differentiation was not affected by any of the aforementioned treatments. Conclusions Alveolar epithelial cell loss is an established hallmark of IPF and may occur by way of apoptosis. Using cultured human AT1 cells, we determined that cleaved caspase 3 is induced by direct addition of either LPA or IPF-PCLS conditioned media, and prevented by the LPA1-selective antagonist, PIPE-791. Of note, conditioned media from IPF-PCLS cultures treated with PIPE-791 did not increase the %Cc3+/Pdpn+ cells, suggesting LPA1 is involved in yet undefined pro-apoptotic factors released by the IPF-PCLS. Thus, while LPA1’s role in fibroblast activation is well-established, we postulate that antagonizing LPA1 prevents additional mechanisms involved in IPF initiation and progression. This abstract is funded by: Contineum Therapeutics
Poon et al. (Fri,) studied this question.