Listen to the WNT; It Talks: WNT7A Drives Epithelial–Mesenchymal Cross-Talk within the Fibrotic Niche in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by aberrant lung epithelial phenotypes, fibroblast activation, distorted immune responses, and increased extracellular matrix deposition leading to a loss of lung function. Honeycomb cysts (HCs) and fibroblast foci represent key histopathological structures of the distal lung in IPF (1). HCs are heterogeneous bronchiolized areas that feature clusters of KRT5⁺ (keratin 5) basal-like cells interspersed with pseudostratified epithelium containing differentiated, hyperplastic epithelial cells, as well as aberrant ciliated cells (2–4). Although HCs are histopathologically well-described and part of the diagnosis of IPF, detailed mechanistic insight about the origin, formation, and functional role of HCs remains elusive. Recent (single-cell) RNA sequencing studies have begun to define cellular subtypes unique to or enriched in IPF, including basaloid KRT5⁻/KRT17⁺ epithelial cells of the distal lung, specifically detected in IPF (5–8). These studies have also identified key genes that contribute to the functional impairment of fibrotic basal cells (9). However, the IPF basal cell still remains poorly characterized, and its contribution to disease pathogenesis remains underinvestigated. In this issue of the Journal, Huang and colleagues (pp. 302–313) describe in detail the RNA repertoire and functional role of basal-like epithelial cells isolated from IPF lungs (10). The authors expand on the paradigm that airway basal cells are key drivers of fibrotic lung disease, analyzing, for the first time, primary basal cells isolated from patients with IPF and controls. Their investigations define specific signaling proteins secreted by IPF basal cells that impair cellular cross-talk in the fibrotic niche, leading to fibroblast activation and alveolar epithelial cell type (AEC) II stem cell failure. The authors ought to be applauded for their comprehensive assessment of primary human basal cells derived from IPF and donor tissue using disease-relevant coculture models. They demonstrate that basal cells induce fibronectin expression in fibroblasts, which was enhanced in IPF-derived cells or when using conditioned media therefrom, thus providing evidence for a functional role of these cells in the fibrotic honeycomb niche. Single-cell analysis of these cells revealed several mRNAs to be enriched in basal cells, including Wnt7A and well-known profibrotic mediators such as Tgf-β1 and Dkk3, another member of the WNT family.

The authors’ rationale for focusing on WNT7A as a part of the WNT signaling pathway is compelling. Unbiased screening approaches and subsequent mechanistic studies have revealed an aberrant WNT signature in IPF and experimental lung fibrosis. Many studies have shown that β-catenin–dependent WNT signaling contributes to tissue fibrosis and can serve as a potential prognostic biomarker, and similar observations have been documented for β-catenin–independent WNT signaling (1, 11–16). Recent single-cell RNA sequencing approaches have provided details of distinct WNT-expressing versus WNT-responsive cell types in the IPF lung (7). These data suggest that cell type–specific responses to a given WNT pathway are controlled by specific spatial niches in the lung, and the current work provides further evidence to this hypothesis.

The study demonstrates that WNT7A is highly expressed in isolated basal cells and that it acts as a main mediator of the functional effects exerted on fibroblasts and AECII cells. Evidence that IPF basal cells exhibit increased WNT7A expression and secretion in situ, however, is limited and based solely on antibody staining, which is notoriously inaccurate for secreted proteins. Perhaps greater sample numbers could be provided in future studies, as well as additional assessments of WNT7A transcript and protein expression in other basal-like cells to corroborate the current findings. Nevertheless, the in vitro data establish a role for WNT7A as a paracrine mediator. Along these lines, extracellular vesicles are well-known mediators of paracrine signaling and cellular cross-talk and have recently been implicated in IPF (17, 18). It is intriguing that WNT proteins, such as WNT5A, have been identified as extracellular vesicle cargo eliciting profibrotic actions (19). Similarly, WNT7A is expressed on extracellular vesicles promoting lung metastasis (20). Transport via extracellular vesicles is of particular interest for WNT proteins, which are modified by lipids and known for their short-ranging effects, as it might significantly expand their range of action in vivo (21).

Following several elegant in vitro experiments establishing a role for WNT7A on fibroblast activation and AECII organoid formation, the authors interrogated whether inhibition of WNT7A can attenuate lung fibrosis in vivo. By using a modified repetitive bleomycin model, the authors use a valuable novel mouse model that is thus far seldom used because of technical challenges (22, 23), in which basal cell numbers increase over time with a concomitant increase in WNT7A protein levels. Neutralizing antibodies against WNT7A, as well as a small-molecule inhibitor of broader WNT/Frizzled signaling, attenuated lung fibrosis in this model. These studies are a first step to deciphering the role of autocrine/paracrine WNT signaling in the fibrotic niche in lung fibrosis. Although these in vivo data are encouraging, it is important to note that these results do not unequivocally show that in vivo treatment exclusively targeted WNT7A secreted by fibrotic basal cells, which, at the time when treatment was initiated, consisted of only 4% of all mouse lung epithelial cells in the experimental model. A deeper characterization of the model, as well as genetic deletion of WNT7A secretion in specific cell types, would be a next logical step to understand the differentiation of KRT5⁺ as well as other basal-like cells, including aberrant basaloid cells, which consistently express high levels of WNT7A across most published data sets. Previous studies have identified transitional Krt8⁺ AECII cells in the bleomycin model, which reportedly are similar to basal-like cells found in IPF and have been shown to exhibit an active β-catenin signature (24). Whether WNT7A exhibits β-catenin–dependent and/or –independent function and whether those are different depending on the receptor expressed on the effector cell need to be clarified in future studies to inform novel therapeutic approaches. Importantly, it remains to be shown whether any of the in vivo therapeutic approaches used in this paper result in a reduction of the potentially disease-driving basal cell population.

The main challenges in the development of therapies targeting WNT signaling include 1) the requirement of this pathway for homeostasis and self-renewal functions and 2) the complexity of the pathways, including more than 19 WNT proteins and at least 10 different (co)receptors. The latter also presents a unique opportunity to develop potential cell- and disease-specific therapies. Further analysis of cell-specific surface receptors responsible for WNT signaling outcome will be crucial to dissect whether WNT signaling is required or sufficient to induce, perpetuate, or attenuate and potentially reverse lung fibrosis. In light of recent technical advancements targeting specific WNT receptors, such as Frizzled 4 antagonists that have made their way into clinical development (25, 26), it seems that we should not only better listen to the WNT but also more deeply explore valuable avenues in modifying the WNT for future therapeutic development serving our patients with IPF.