Huang, Steven K.

Abstract Idiopathic pulmonary fibrosis (IPF) is characterized by the sustained activation of interstitial fibroblasts leading to excessive collagen deposition and progressive organ failure. Epigenetic and metabolic abnormalities have been shown to contribute to the persistent activated state of scar-forming fibroblasts. However, how epigenetic changes regulate fibroblast metabolic responses to promote fibroblast activation and progressive fibrosis remains largely unknown. Here, we show that the epigenetic regulator CBX5 (chromobox protein homolog 5) is critical to the transition of quiescent fibroblasts to activated collagen-producing fibroblasts in response to bleomycin-induced lung injury. Loss of mesenchymal CBX5 attenuated fibrosis development, and this effect was accompanied by the downregulation of pathogenic fibroblast genes, including Cthrc1, Col1a1, and Spp1, and by the upregulation of metabolic genes with antifibrotic activity such as Ppara and Pparg. Single-cell RNA sequencing and immunohistochemistry analyses revealed that CBX5 expression was enriched in pathogenic fibroblasts and fibroblastic foci of IPF lungs. Bulk RNA-sequencing analysis combined with metabolic assessments demonstrated that CBX5 silencing in IPF fibroblasts potently inhibited transforming growth factor–stimulated glycolysis while enhancing AMPK signaling and mitochondrial metabolism. Finally, interruption of the CBX5 pathway in IPF fibroblasts in vitro and in IPF lung explants ex vivo synergistically potentiated the activation of metformin-induced AMP-activated protein kinase activation and inhibited collagen secretion. Collectively, our findings identify CBX5 as an epigenetic regulator linking metabolic maladaptation to the persistent activated state of lung fibroblasts during IPF progression.

Idiopathic pulmonary fibrosis (IPF) is an aging-associated disease characterized by myofibroblast accumulation and progressive lung scarring. To identify transcriptional gene programs driving persistent lung fibrosis in aging, we performed RNA-Seq on lung fibroblasts isolated from young and aged mice during the early resolution phase after bleomycin injury. We discovered that, relative to injured young fibroblasts, injured aged fibroblasts exhibited a profibrotic state characterized by elevated expression of genes implicated in inflammation, matrix remodeling, and cell survival. We identified the proviral integration site for Moloney murine leukemia virus 1 (PIM1) and its target nuclear factor of activated T cells-1 (NFATc1) as putative drivers of the sustained profibrotic gene signatures in injured aged fibroblasts. PIM1 and NFATc1 transcripts were enriched in a pathogenic fibroblast population recently discovered in IPF lungs, and their protein expression was abundant in fibroblastic foci. Overexpression of PIM1 in normal human lung fibroblasts potentiated their fibrogenic activation, and this effect was attenuated by NFATc1 inhibition. Pharmacological inhibition of PIM1 attenuated IPF fibroblast activation and sensitized them to apoptotic stimuli. Interruption of PIM1 signaling in IPF lung explants ex vivo inhibited prosurvival gene expression and collagen secretion, suggesting that targeting this pathway may represent a therapeutic strategy to block IPF progression.

Abstract Idiopathic pulmonary fibrosis (IPF) is a progressive disease thought to result from impaired lung repair following injury and is strongly associated with aging. While vascular alterations have been associated with IPF previously, the contribution of lung vasculature during injury resolution and fibrosis is not well understood. To compare the role of endothelial cells (ECs) in resolving and non‐resolving models of lung fibrosis, we applied bleomycin intratracheally to young and aged mice. We found that injury in aged mice elicited capillary rarefaction, while injury in young mice resulted in increased capillary density. ECs from the lungs of injured aged mice relative to young mice demonstrated elevated pro‐fibrotic and reduced vascular homeostasis gene expression. Among the latter, Nos3 (encoding the enzyme endothelial nitric oxide synthase, eNOS) was transiently upregulated in lung ECs from young but not aged mice following injury. Young mice deficient in eNOS recapitulated the non‐resolving lung fibrosis observed in aged animals following injury, suggesting that eNOS directly participates in lung fibrosis resolution. Activation of the NO receptor soluble guanylate cyclase in human lung fibroblasts reduced TGFβ‐induced pro‐fibrotic gene and protein expression. Additionally, loss of eNOS in human lung ECs reduced the suppression of TGFβ‐induced lung fibroblast activation in 2D and 3D co‐cultures. Altogether, our results demonstrate that persistent lung fibrosis in aged mice is accompanied by capillary rarefaction, loss of EC identity, and impaired eNOS expression. Targeting vascular function may thus be critical to promote lung repair and fibrosis resolution in aging and IPF.

Abstract Rationale Prostaglandin (PG) E2, a cyclooxygenase-derived lipid mediator, is a potent down-regulator of fibroblast activation in normal lung fibroblasts. Although fibroblasts from patients with idiopathic pulmonary fibrosis are known to exhibit a defect in PGE2 synthesis, there is little information about their responsiveness to this lipid mediator. Objectives To compare responses to PGE2 in normal, usual interstitial pneumonia (UIP), and other diffuse parenchymal lung disease (DPLD) fibroblasts. Methods Fibroblasts were grown in vitro from well characterized control (n = 7), UIP (n = 17), or other DPLD (n = 13) lung tissue. The effects of PGE2 on fibroblast proliferation and collagen expression were determined. Measurements and Main Results Only 3 of 12 UIP fibroblast lines exhibited PGE2-mediated inhibition of both collagen synthesis and cell proliferation, as opposed to 6 of 6 nonfibrotic control cell lines. The degree of PGE2 resistance in DPLD fibroblasts was quite variable, with UIP cells exhibiting the greatest degree of resistance to PGE2, whereas other DPLD fibroblasts manifested a degree of resistance intermediate to control and UIP. The resistance to suppression of collagen expression correlated with worse lung function. Molecular mechanisms for resistance included altered E prostanoid receptor profiles and diminished expression of the downstream kinase, protein kinase A. Conclusions The recognition that UIP fibroblasts manifest variable refractoriness to PGE2 suppression sheds new light on the activation phenotype of these cells and on the pathogenesis of fibrotic lung disease.