B77-27 Mitoception: A Novel Strategy to Alleviate Pulmonary Fibrosis

Background

Dysregulated epithelial-mesenchymal crosstalk mediates the progression in pulmonary fibrosis. We hypothesize that mitochondrial dysfunction is a critical driver in this process. In this study, we use a mitoception-based approach to determine whether restoring mitochondrial function can attenuate profibrotic signaling and shift fibroblasts toward a less fibrogenic phenotype.

Methods

To study mitochondrial dysfunction, we compared explant idiopathic pulmonary fibrosis (IPF) to healthy donor lung tissues using immunohistochemistry and gene expression assays (qRT-PCR). To assess the impact of mitochondrial transfer on fibrosis, patient-derived normal and IPF fibroblasts were treated with mitochondria (5 or 15 µg) isolated from non-fibrotic, Alveolar Type II-like epithelial cells (A549) at different time-points (3, 6, and 24 hours). After treatment, cells were collected for qRT-PCR and Western blot analysis of mitochondrial and fibrotic markers. Mitochondrial membrane potential (Δ𝜓;m) was assessed using Mitotracker CMX Red dye (200 nM).

Results

TGF-β expression was significantly elevated in fibrotic lung tissues, consistent with heightened inflammatory and profibrotic activity (Figure 1). However, transcription factors for mitochondrial biogenesis (TFAM and NRF2), and upstream regulator, PGC-1α, were significantly reduced relative to normal lung tissue. Concomitantly, complex I (NDUFB6) and IV (COX5B) subunits were also reduced in fibrotic tissue. A time and dose-dependent changes in the fibrotic and mitochondrial markers were observed with transfer of mitochondrial from non-fibrotic epithelial cells to IPF and normal fibroblast. In the IPF-derived fibroblasts, mitochondrial transfer led to reduction in fibrotic markers (FN1, COL1A1, and COL3A1, α-SMA) at 6 hours with both 5- and 15-µg concentration. Decreased TGF-β expression was also observed following mitochondrial treatment with most pronounced effect at 24h treatment. The mRNA expression of mitochondrial biogenesis markers (TFAM and NRF2) and mitochondrial complexes (COX5B, ATP5A1) were significantly upregulated after 6 and 24h of treatment at 15µg concentration in both normal and IPF fibroblasts. These molecular changes correlated with 24h Δ𝜓;m measurements, which increased in a dose-dependent manner in both normal and IPF fibroblasts, indicating sustained improvement in mitochondrial functional capacity.

Conclusion

These data suggest that mitochondrial transfer from non-fibrotic Alveolar Type-II like epithelial cells to patient-derived primary fibroblasts promotes sustained improvement in mitochondrial function and suppresses profibrotic signaling, providing a strong rationale for future mechanistic studies targeting mitochondrial-fibrotic signaling pathways in pulmonary fibrosis.

This abstract is funded by: University of Kentucky