Shilo, K. | Search bioRxiv

bioRxiv2026

Transcriptional and spatial profiling of fibroblasts from human lungs highlights CTHRC1+ cells as fibrogenic signaling hubs in fibrosis

Lung fibroblasts are key regulators of tissue homeostasis and extracellular matrix (ECM) remodeling, and their aberrant activation drives the progressive parenchymal scarring characteristic of idiopathic pulmonary fibrosis (IPF), a fatal disease with limited therapeutic options. Despite their central pathogenic role, lung fibroblasts are difficult to isolate due to their embedded position within the ECM, and standard in vitro culture conditions may lead to the loss of their native functional and transcriptional characteristics, hampering the study of fibroblast behavior in disease. The transcriptional heterogeneity of lung fibroblast subtypes and the extent to which culture-induced alterations diverge from native tissue signatures remain poorly understood. Here, we integrated single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics of lung tissue from IPF patients and age-matched healthy donors with transcriptomic profiling of cultured fibroblasts collected at passages 1 and 6 after isolation using three optimized protocols: whole lung cell suspension (WLCS), negative fraction enrichment, and outgrowth. Tissue-based analysis identified six transcriptionally distinct mesenchymal subtypes: alveolar, adventitial, inflammatory, peribronchial, CTHRC1+ and smooth muscle cell (SMC). The fibroblast subtype CTHRC1+ represented the most transcriptionally activated pro-fibrotic subtype, showing the greatest upregulation of ECM biosynthesis genes, a prominent role in intercellular communication, and preferential enrichment within fibroblastic foci in IPF lung tissue. Pseudotime trajectory analysis supported a directional transcriptional continuum from alveolar and inflammatory fibroblasts toward the CTHRC1+ state, driven by coordinated activation of pro-fibrotic transcription factors, including RUNX2, CREB3L1, and SCX. In vitro culture progressively reshaped fibroblast transcriptional identity relative to native tissue, with increased collagen and matrix metalloproteinase (MMP) expression during passaging, loss of distinct CTHRC1+ fibroblasts, and gain of alveolar fibroblasts displaying pro-fibrotic activation across all isolation protocols. These findings provide a high-resolution transcriptional map of lung fibroblast heterogeneity in IPF and highlight critical limitations of standard in vitro culture systems for recapitulating native fibroblast diversity, with important implications for the development and evaluation of fibroblast-targeted therapeutic strategies in IPF.

molecular biology↗

bioRxiv2018

Is IκBζ constitutively expressed in mammalian airway epithelium?

I{kappa}B{zeta} is a transcriptional factor induced primarily in immune cells upon Toll-like receptor (TLR) activation that drives important cytokine responses. Recent studies have demonstrated constitutive I{kappa}B{zeta} expression in the epithelial cells of mouse skin and eyes, possibly reflecting the activation of TLRs by pathogen-associated molecular patterns (PAMPs). In this context, another mucosal surface, the lung epithelium, may not be as actively exposed to the external environment as the skin and the conjunctiva, especially since the lower lung airways are typically conceived to be sterile. Whether I{kappa}B{zeta} expression in the lungs is constitutive or induced remains largely unexplored. This is especially important since I{kappa}B{zeta} has been shown to promote the expression of protective cytokine and antimicrobial peptide responses, supporting a role for I{kappa}B{zeta} in lung host defense. We hence evaluated I{kappa}B{zeta} expression in airway epithelia of both humans and mice using immunostaining with antiserum raised against recombinant I{kappa}B{zeta} in our laboratory. We observed positive signal in the nuclei of ciliated epithelial cells lining the central airways. Airway cells of gnotobiotic mice also stained positive, suggesting that I{kappa}B{zeta} expression does not require induction by bacterial PAMPs. Unexpectedly, we also observed staining in the lung epithelia of I{kappa}B{zeta} knockout mice, indicating possible false positive signals from our immunohistochemistry experiments. In this context, 2D gel analysis followed by mass spectrometry revealed that our I{kappa}B{zeta} antiserum also detected a nuclear protein lamin B1. Nevertheless, immunoblotting tissue homogenates from gnotobiotic mouse lungs and primary human airway epithelial cells showed the appropriate 86 kDa band for I{kappa}B{zeta}. Together, these results demonstrate constitutive I{kappa}B{zeta} expression in airway epithelium, suggesting that lung epithelial cells may depend upon I{kappa}B{zeta} expression for airway protection.

immunology↗