IL-1ß Induces a Proinflammatory Fibroblast Microenvironment that Impairs Lung Progenitors' Function.

Chronic obstructive pulmonary disease (COPD) is characterized by a persistent inflammatory state in the lungs and defective tissue repair. Although the inflammatory response in patients with COPD is well characterized and known to be exaggerated during exacerbations, its contribution to lung injury and abnormal repair is still unclear. In this study, we aimed to investigate how the inflammatory microenvironment affects the epithelial progenitors and their supporting mesenchymal niche cells involved in tissue repair of the distal lung. We focused on IL-1ß, a key inflammatory mediator that is increased during exacerbations of COPD, and used an organoid model of lung epithelial cells and fibroblasts to assess the effect of IL-1ß treatment on these cells' transcriptome and secreted factors. Whereas direct treatment of the lung organoids with IL-1ß promoted organoid growth, this switched toward inhibition when it was added as fibroblast pretreatment followed by organoid treatment. We then investigated the IL-1ß-driven mechanisms in the fibroblasts and found an inflammatory response related to (C-X-C motif) ligand (CXCL) chemokines; we confirmed that these chemokines were responsible for the impaired organoid growth and found that targeting their C-X-C chemokine receptors 1/2 (CXCR1/2) receptors or the IL-1ß intracellular signaling reduced the proinflammatory response and restored organoid growth. These data demonstrate that IL-1ß alters the fibroblasts' state by promoting a distinct inflammatory response, switching their supportive function on epithelial progenitors toward an inhibitory one in an organoid assay. These results imply that chronic inflammation functions as a shift toward inhibition of repair, thereby contributing to chronic inflammatory diseases like COPD.

Diesel exhaust particles distort lung epithelial progenitors and their fibroblast niche.

Chronic obstructive pulmonary disease (COPD) is a progressive lung disease characterized by inflammation and impaired tissue regeneration, and is reported as the fourth leading cause of death worldwide by the Centers for Disease Control and Prevention (CDC). Environmental pollution and specifically motor vehicle emissions are known to play a role in the pathogenesis of COPD, but little is still known about the molecular mechanisms that are altered following diesel exhaust particles (DEP) exposure. Here we used lung organoids derived from co-culture of alveolar epithelial progenitors and fibroblasts to investigate the effect of DEP on the epithelial-mesenchymal signaling niche in the distal lung, which is essential for tissue repair. We found that DEP treatment impaired the number as well as the average diameter of both airway and alveolar type of lung organoids. Bulk RNA-sequencing of re-sorted epithelial cells and fibroblasts following organoid co-culture shows that the Nrf2 pathway, which regulates antioxidants' activity, was upregulated in both cell populations in response to DEP; and WNT/ß-catenin signaling, which is essential to promote epithelial repair, was downregulated in DEP-exposed epithelial cells. We show that pharmacological treatment with anti-oxidant agents such as N-acetyl cysteine (NAC) or Mitoquinone mesylate (MitoQ) reversed the effect of DEP on organoids growth. Additionally, a WNT/ß-catenin activator (CHIR99021) successfully restored WNT signaling and promoted organoid growth upon DEP exposure. We propose that targeting oxidative stress and specific signaling pathways affected by DEP in the distal lung may represent a strategy to restore tissue repair in COPD.

WNT Signalling in Lung Physiology and Pathology.

The main physiological function of the lung is gas exchange, mediated at the interface between the alveoli and the pulmonary microcapillary network and facilitated by conducting airway structures that regulate the transport of these gases from and to the alveoli. Exposure to microbial and environmental factors such as allergens, viruses, air pollution, and smoke contributes to the development of chronic lung diseases such as asthma, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), and lung cancer. Respiratory diseases as a cluster are the commonest cause of chronic disease and of hospitalization in children and are among the three most common causes of morbidity and mortality in the adult population worldwide. Many of these chronic respiratory diseases are associated with inflammation and structural remodelling of the airways and/or alveolar tissues. They can often only be treated symptomatically with no disease-modifying therapies that normalize the pathological tissue destruction driven by inflammation and remodelling. In search for novel therapeutic strategies for these diseases, several lines of evidence revealed the WNT pathway as an emerging target for regenerative strategies in the lung. WNT proteins, their receptors, and signalling effectors have central regulatory roles under (patho)physiological conditions underpinning lung function and (chronic) lung diseases and we summarize these roles and discuss how pharmacological targeting of the WNT pathway may be utilized for the treatment of chronic lung diseases.

Inhibition of LTßR signalling activates WNT-induced regeneration in lung.

Lymphotoxin ß-receptor (LTßR) signalling promotes lymphoid neogenesis and the development of tertiary lymphoid structures1,2, which are associated with severe chronic inflammatory diseases that span several organ systems3-6. How LTßR signalling drives chronic tissue damage particularly in the lung, the mechanism(s) that regulate this process, and whether LTßR blockade might be of therapeutic value have remained unclear. Here we demonstrate increased expression of LTßR ligands in adaptive and innate immune cells, enhanced non-canonical NF-κB signalling, and enriched LTßR target gene expression in lung epithelial cells from patients with smoking-associated chronic obstructive pulmonary disease (COPD) and from mice chronically exposed to cigarette smoke. Therapeutic inhibition of LTßR signalling in young and aged mice disrupted smoking-related inducible bronchus-associated lymphoid tissue, induced regeneration of lung tissue, and reverted airway fibrosis and systemic muscle wasting. Mechanistically, blockade of LTßR signalling dampened epithelial non-canonical activation of NF-κB, reduced TGFß signalling in airways, and induced regeneration by preventing epithelial cell death and activating WNT/ß-catenin signalling in alveolar epithelial progenitor cells. These findings suggest that inhibition of LTßR signalling represents a viable therapeutic option that combines prevention of tertiary lymphoid structures1 and inhibition of apoptosis with tissue-regenerative strategies.

Wnt/ß-catenin signaling is critical for regenerative potential of distal lung epithelial progenitor cells in homeostasis and emphysema.

Wnt/ß-catenin signaling regulates progenitor cell fate decisions during lung development and in various adult tissues. Ectopic activation of Wnt/ß-catenin signaling promotes tissue repair in emphysema, a devastating lung disease with progressive loss of parenchymal lung tissue. The identity of Wnt/ß-catenin responsive progenitor cells and the potential impact of Wnt/ß-catenin signaling on adult distal lung epithelial progenitor cell function in emphysema are poorly understood. Here, we used TCF/Lef:H2B/GFP reporter mice to investigate the role of Wnt/ß-catenin signaling in lung organoid formation. We identified an organoid-forming adult distal lung epithelial progenitor cell population characterized by a low Wnt/ß-catenin activity, which was enriched in club and alveolar epithelial type (AT)II cells. Endogenous Wnt/ß-catenin activity was required for the initiation of multiple subtypes of distal lung organoids derived from the Wntlow epithelial progenitors. Further ectopic Wnt/ß-catenin activation specifically led to an increase in alveolar organoid number; however, the subsequent proliferation of alveolar epithelial cells in the organoids did not require constitutive Wnt/ß-catenin signaling. Distal lung epithelial progenitor cells derived from the mouse model of elastase-induced emphysema exhibited reduced organoid forming capacity. This was rescued by Wnt/ß-catenin signal activation, which largely increased the number of alveolar organoids. Together, our study reveals a novel mechanism of lung epithelial progenitor cell activation in homeostasis and emphysema.

Mesenchymal WNT-5A/5B Signaling Represses Lung Alveolar Epithelial Progenitors.

Chronic obstructive pulmonary disease (COPD) represents a worldwide concern with high morbidity and mortality, and is believed to be associated with accelerated ageing of the lung. Alveolar abnormalities leading to emphysema are a key characteristic of COPD. Pulmonary alveolar epithelial type 2 cells (AT2) produce surfactant and function as progenitors for type 1 cells. Increasing evidence shows elevated WNT-5A/B expression in ageing and in COPD that may contribute to the disease process. However, supportive roles for WNT-5A/B in lung regeneration were also reported in different studies. Thus, we explored the role of WNT-5A/B on alveolar epithelial progenitors (AEPs) in more detail. We established a Precision-Cut-Lung Slices (PCLS) model and a lung organoid model by co-culturing epithelial cells (EpCAM+/CD45-/CD31-) with fibroblasts in matrigel in vitro to study the impact of WNT-5A and WNT-5B. Our results show that WNT-5A and WNT-5B repress the growth of epithelial progenitors with WNT-5B preferentially restraining the growth and differentiation of alveolar epithelial progenitors. We provide evidence that both WNT-5A and WNT-5B negatively regulate the canonical WNT signaling pathway in alveolar epithelium. Taken together, these findings reveal the functional impact of WNT-5A/5B signaling on alveolar epithelial progenitors in the lung, which may contribute to defective alveolar repair in COPD.