Digital quantification of p16-positive foci in fibrotic interstitial lung disease is associated with a phenotype of idiopathic pulmonary fibrosis with reduced survival

Abstract

Background Idiopathic pulmonary fibrosis (IPF) is associated with increased expression of cyclin-dependent kinase inhibitors such as p16 and p21, and subsequent induction of cell cycle arrest, cellular senescence, and pro-fibrotic gene expression. We sought to link p16-expression with a diagnosis of IPF or other fibrotic interstitial lung diseases (ILDs), radiographic pattern, senescent foci-specific gene expression, antifibrotic therapy response, and lung transplant (LTx)-free survival. Methods Eighty-six cases of fibrosing ILD were identified with surgical lung biopsy. Immunohistochemistry for p16 was performed on sections with the most active fibrosis. p16-positive foci (loose collection of p16-positive fibroblasts with overlying p16-positive epithelium) were identified on digital slides and quantified. Cases were scored as p16-low (≤ 2.1 foci per 100 mm2) or p16-high (> 2.1 foci per 100 mm2). Twenty-four areas including senescent foci, fibrotic and normal areas were characterized using in situ RNA expression analysis with digital spatial profiling (DSP) in selected cases. Results The presence of p16-positive foci was specific for the diagnosis of IPF, where 50% of cases expressed any level of p16 and 26% were p16-high. There was no relationship between radiographic pattern and p16 expression. However, there was increased expression of cyclin-dependent kinase inhibitors, collagens and matrix remodeling genes within p16-positive foci, and cases with high p16 expression had shorter LTx-free survival. On the other hand, antifibrotic therapy was significantly protective. DSP demonstrated that fibroblastic foci exhibit transcriptional features clearly distinct from that of normal-looking and even fibrotic areas. Conclusions We demonstrated the potential clinical applicability of a standardized quantification of p16-positive fibroblastic foci. This method identifies an IPF phenotype associated with foci-specific upregulation of senescence-associated and matrix remodeling gene expression. While these patients have reduced LTx-free survival, good response to antifibrotic therapies was observed in those who were treated.

An Integrative Multiomics Framework for Identification of Therapeutic Targets in Pulmonary Fibrosis

“…Importantly, deletion of CB 1 R attenuated fibrosis induced expression of 32 genes within the T‐1 and T‐2 tracks (Figure 8H ), which are the activated genes in the lungs of IPF patients from two independent cohorts (Figures 7D and 8H ). Indeed, among these genes such as cell migration induced hyaluronidase 1 ( CEMIP ), [ 31 ] osteopontin ( SPP1 ), [ 32 ] prolyl 4‐hydroxylase subunit alpha 3 (P4HA3 ), [ 33 ] tenascin C ( TNC ), [ 34 ] transmembrane glycoprotein NMB (GPNMB) , [ 35 ] insulin‐like growth factor 1 (IGF1) , [ 36 ] cartilage oligomeric matrix protein ( COMP ), [ 37 ] ADAM metallopeptidase domain 12 ( ADAM12 ), [ 38 ] collagen triple helix repeat containing 1 ( CTHRC1) , [ 39 ] claudin 2 ( CLDN2 ), [ 40 ] latent transforming growth factor beta binding protein 2 ( LTBP2 ) [ 41 ] were proven to involve disease pathology and correlated with disease severity in IPF patients. Accordingly, this further reinstate potential pathologic role of CB 1 R in IPF.…”

Modulating in vitro lung fibroblast activation via senolysis of senescent human alveolar epithelial cells

“…We also confirm and extend the findings of senescent alveolar epithelial cell and fibroblast interaction by characterizing the aberrant and SADD phenotype of bleomycin-treated primary AECs ( Figure 3E ) and by demonstrating a shift toward ECM catabolism and inflammation that is consistent with fibroblast senescence and IPF phenotypes, despite not observing increases in Cdkn1a or Cdkn2a in NHLFs themselves following senescent AEC conditioned medium transfer. Fibroblast senescence in fibroblastic foci is correlated to poor outcomes in IPF [ 50 ], but the variable expression or lack of expression of p16 ink4a in the fibroblastic foci of all IPF patients may suggest that senescence of AECs precedes fibroblast senescence, as AEC senescence has consistently been observed in IPF cells and tissues by our group and others ( Figure 1 ) [ 14 – 16 , 28 ]. Overall, this indicates that AECs may be a primary senescent cell type in IPF, and that fibroblast activation is a secondary consequence of AEC growth arrest after damage or injury.…”

Spatial transcriptomics: recent developments and insights in respiratory research

“…The pathogenesis of IPF is associated with epithelial dysfunction. Another study used the GeoMx DSP platform to explore the transcriptional differences between fibroblastic foci and fibrous and normal areas in IPF cases and identified new fibrogenic biomarkers expressed in fibroblastic foci [ 109 ]. The spatiotemporal analysis brings hope for the development of pulmonary fibrosis.…”