Alveolar type II (ATII) cells are essential for the maintenance of the alveolar homeostasis. However, knowledge of the expression of the miRNAs and miRNA-regulated networks which control homeostasis and coordinate diverse functions of murine ATII cells is limited. Therefore, we asked how miRNAs expressed in ATII cells might contribute to the regulation of signaling pathways. We purified "untouched by antibodies" ATII cells using a flow cytometric sorting method with a highly autofluorescent population of lung cells. TaqMan® miRNA low-density arrays were performed on sorted cells and intersected with miRNA profiles of ATII cells isolated according to a previously published protocol. Of 293 miRNAs expressed in both ATII preparations, 111 showed equal abundances. The target mRNAs of bona fide ATII miRNAs were used for pathway enrichment analysis. This analysis identified nine signaling pathways with known functions in fibrosis and/or epithelial-to-mesenchymal transition (EMT). In particular, a subset of 19 miRNAs was found to target 21 components of the TGF-β signaling pathway. Three of these miRNAs (miR-16-5p, -17-5p and -30c-5p) were down-modulated by TGF-β1 stimulation in human A549 cells, and concomitant up-regulation of associated mRNA targets (BMPR2, JUN, RUNX2) was observed. These results suggest an important role for miRNAs in maintaining the homeostasis of the TGF-β signaling pathway in ATII cells under physiological conditions.

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.

Mechanisms of injury and repair in alveolar epithelial cells (AECs) are critically involved in the progression of various lung diseases including idiopathic pulmonary fibrosis (IPF). Homeobox only protein x (HOPX) contributes to the formation of distal lung during development. In adult lung, alveolar epithelial type (AT) I cells express HOPX and lineage-labeled Hopx+ cells give rise to both ATI and ATII cells after pneumonectomy. However, the cell function of HOPX-expressing cells in adult fibrotic lung diseases has not been investigated. In this study, we have established a flow cytometry-based method to evaluate HOPX-expressing cells in the lung. HOPX expression in cultured ATII cells increased over culture time, which was accompanied by a decrease of proSP-C, an ATII marker. Moreover, HOPX expression was increased in AECs from bleomycin-instilled mouse lungs in vivo. Small interfering RNA-based knockdown of Hopx resulted in suppressing ATII-ATI trans-differentiation and activating cellular proliferation in vitro. In IPF lungs, HOPX expression was decreased in whole lungs and significantly correlated to a decline in lung function and progression of IPF. In conclusion, HOPX is upregulated during early alveolar injury and repair process in the lung. Decreased HOPX expression might contribute to failed regenerative processes in end-stage IPF lungs.

The alveolar epithelium represents a major site of tissue destruction during lung injury. It consists of alveolar epithelial type I (ATI) and type II (ATII) cells. ATII cells are capable of self-renewal and exert progenitor function for ATI cells upon alveolar epithelial injury. Cell differentiation pathways enabling this plasticity and allowing for proper repair, however, are poorly understood. Here, we applied proteomics, expression analysis and functional studies in primary murine ATII cells to identify proteins and molecular mechanisms involved in alveolar epithelial plasticity. Mass spectrometry of cultured ATII cells revealed a reduction of carbonyl reductase 2 (CBR2) and an increase in enolase 1 (ENO1) and protein disulfide-isomerase associated 3 (PDIA3) protein expression during ATII-to-ATI cell trans-differentiation. This was accompanied by increased Wnt/β-catenin signaling, as analyzed by qRT-PCR and immunoblotting. Notably, ENO1 and PDIA3, along with T1α (podoplanin; an ATI cell marker), exhibited decreased protein expression upon pharmacological and molecular Wnt/β-catenin inhibition in cultured ATII cells, whereas CBR2 levels were stabilized. Moreover, we analyzed primary ATII cells from mice with bleomycin-induced lung injury, a model exhibiting activated Wnt/β-catenin signaling in vivo. We observed reduced CBR2 significantly correlating with surfactant protein C (SFTPC), whereas ENO1 and PDIA3 along with T1α were increased in injured ATII cells. Finally, siRNA-mediated knockdown of ENO1, as well as PDIA3, in primary ATII cells led to reduced T1α expression, indicating diminished cell trans-differentiation. Our data thus identified proteins involved in ATII-to-ATI cell trans-differentiation and suggest a Wnt/β-catenin-driven functional role of ENO1 and PDIA3 in alveolar epithelial cell plasticity in lung injury and repair.

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease with poor prognosis and limited therapeutic options. The incidence of IPF increases with age, and ageing-related mechanisms such as cellular senescence have been proposed as pathogenic drivers. The lung alveolar epithelium represents a major site of tissue injury in IPF and senescence of this cell population is probably detrimental to lung repair. However, the potential pathomechanisms of alveolar epithelial cell senescence and the impact of senolytic drugs on senescent lung cells and fibrosis remain unknown. Here we demonstrate that lung epithelial cells exhibit increased P16 and P21 expression as well as senescence-associated β-galactosidase activity in experimental and human lung fibrosis tissue and primary cells.Primary fibrotic mouse alveolar epithelial type (AT)II cells secreted increased amounts of senescence-associated secretory phenotype (SASP) factors in vitro, as analysed using quantitative PCR, mass spectrometry and ELISA. Importantly, pharmacological clearance of senescent cells by induction of apoptosis in fibrotic ATII cells or ex vivo three-dimensional lung tissue cultures reduced SASP factors and extracellular matrix markers, while increasing alveolar epithelial markers.These data indicate that alveolar epithelial cell senescence contributes to lung fibrosis development and that senolytic drugs may be a viable therapeutic option for IPF.

The rapid expansion of the elderly population has led to the recent epidemic of age-related diseases, including increased incidence and mortality of chronic lung diseases, such as Idiopathic Pulmonary Fibrosis (IPF). Cellular senescence is a major hallmark of aging and has a higher occurrence in IPF. The lung epithelium represents a major site of tissue injury, cellular senescence and aberrant activity of developmental pathways such as the WNT/β-catenin pathway in IPF. The potential impact of WNT/β-catenin signaling on alveolar epithelial senescence in general as well as in IPF, however, remains elusive. Here, we characterized alveolar epithelial cells of aged mice and assessed the contribution of chronic WNT/β-catenin signaling on alveolar epithelial type (AT) II cell senescence. Whole lungs from old (16-24 months) versus young (3 months) mice had relatively less epithelial (EpCAM+) but more inflammatory (CD45+) cells, as assessed by flow cytometry. Compared to young ATII cells, old ATII cells showed decreased expression of the ATII cell marker Surfactant Protein C along with increased expression of the ATI cell marker Hopx, accompanied by increased WNT/β-catenin activity. Notably, when placed in an organoid assay, old ATII cells exhibited decreased progenitor cell potential. Chronic canonical WNT/β-catenin activation for up to 7 days in primary ATII cells as well as alveolar epithelial cell lines induced a robust cellular senescence, whereas the non-canonical ligand WNT5A was not able to induce cellular senescence. Moreover, chronic WNT3A treatment of precision-cut lung slices (PCLS) further confirmed ATII cell senescence. Simultaneously, chronic but not acute WNT/β-catenin activation induced a profibrotic state with increased expression of the impaired ATII cell marker Keratin 8. These results suggest that chronic WNT/β-catenin activity in the IPF lung contributes to increased ATII cell senescence and reprogramming. In the fibrotic environment, WNT/β-catenin signaling thus might lead to further progenitor cell dysfunction and impaired lung repair.

The cell type specific sequences of transcriptional programs during lung regeneration have remained elusive. Using time-series single cell RNA-seq of the bleomycin lung injury model, we resolved transcriptional dynamics for 28 cell types. Trajectory modeling together with lineage tracing revealed that airway and alveolar stem cells converge on a unique Krt8 + transitional stem cell state during alveolar regeneration. These cells have squamous morphology, feature p53 and NFkB activation and display transcriptional features of cellular senescence. The Krt8+ state appears in several independent models of lung injury and persists in human lung fibrosis, creating a distinct cell-cell communication network with mesenchyme and macrophages during repair. We generated a model of gene regulatory programs leading to Krt8+ transitional cells and their terminal differentiation to alveolar type-1 cells. We propose that in lung fibrosis, perturbed molecular checkpoints on the way to terminal differentiation can cause aberrant persistence of regenerative intermediate stem cell states.

Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial lung disease, characterized by damage of lung epithelial cells, excessive deposition of extracellular matrix in the lung interstitium, and enhanced activation and proliferation of fibroblasts. S100a4, also termed FSP-1 (fibroblast-specific protein-1), was previously considered as a marker of fibroblasts but recent findings in renal and liver fibrosis indicated that M2 macrophages are an important cellular source of S100a4. Thus, we hypothesized that also in pulmonary fibrosis, M2 macrophages produce and secrete S100a4, and that secreted S100a4 induces the proliferation and activation of fibroblasts. To prove this hypothesis, we comprehensively characterized two established mouse models of lung fibrosis: infection of IFN-γR-/- mice with MHV-68 and intratracheal application of bleomycin to C57BL/6 mice. We further provide in vitro data using primary macrophages and fibroblasts to investigate the mechanism by which S100A4 exerts its effects. Finally, we inhibit S100a4 in vivo in the bleomycin-induced lung fibrosis model by treatment with niclosamide. Our data suggest that S100a4 is produced and secreted by M2 polarized alveolar macrophages and enhances the proliferation and activation of lung fibroblasts. Inhibition of S100a4 might represent a potential therapeutic strategy for pulmonary fibrosis.

Proteasome inhibition has been shown to prevent development of fibrosis in several organs including the lung. However, effects of proteasome inhibitors on lung fibrosis are controversial and cytotoxic side effects of the overall inhibition of proteasomal protein degradation cannot be excluded. Therefore, we hypothesized that local lung-specific application of a novel, selective proteasome inhibitor, oprozomib (OZ), provides antifibrotic effects without systemic toxicity in a mouse model of lung fibrosis. Oprozomib was first tested on the human alveolar epithelial cancer cell line A549 and in primary mouse alveolar epithelial type II cells regarding its cytotoxic effects on alveolar epithelial cells and compared to the FDA approved proteasome inhibitor bortezomib (BZ). OZ was less toxic than BZ and provided high selectivity for the chymotrypsin-like active site of the proteasome. In primary mouse lung fibroblasts, OZ showed significant anti-fibrotic effects, i.e. reduction of collagen I and α smooth muscle actin expression, in the absence of cytotoxicity. When applied locally into the lungs of healthy mice via instillation, OZ was well tolerated and effectively reduced proteasome activity in the lungs. In bleomycin challenged mice, however, locally applied OZ resulted in accelerated weight loss and increased mortality of treated mice. Further, OZ failed to reduce fibrosis in these mice. While upon systemic application OZ was well tolerated in healthy mice, it rather augmented instead of attenuated fibrotic remodelling of the lung in bleomycin challenged mice. To conclude, low toxicity and antifibrotic effects of OZ in pulmonary fibroblasts could not be confirmed for pulmonary fibrosis of bleomycin-treated mice. In light of these data, the use of proteasome inhibitors as therapeutic agents for the treatment of fibrotic lung diseases should thus be considered with caution.

Currently, there is no pharmacological treatment targeting defective tissue repair in chronic disease. Here, we used a transcriptomics-guided drug target discovery strategy using gene signatures of smoking-associated chronic obstructive pulmonary disease (COPD) and from mice chronically exposed to cigarette smoke, identifying druggable targets expressed in alveolar epithelial progenitors, of which we screened the function in lung organoids. We found several drug targets with regenerative potential, of which EP and IP prostanoid receptor ligands had the most profound therapeutic potential in restoring cigarette smoke-induced defects in alveolar epithelial progenitors in vitro and in vivo. Mechanistically, we found, using single-cell RNA sequencing analysis, that circadian clock and cell cycle/apoptosis signaling pathways were differentially expressed in alveolar epithelial progenitor cells in patients with COPD and in a relevant model of COPD, which was prevented by prostaglandin E2 or prostacyclin mimetics. We conclude that specific targeting of EP and IP receptors offers therapeutic potential for injury to repair in COPD.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease, characterized by distorted lung architecture and loss of respiratory function. Alveolar epithelial cell injury and hyperplasia, enhanced extracellular matrix deposition, and (myo)fibroblast activation are features of IPF. Wnt/beta-catenin signaling has been shown to determine epithelial cell fate during development. As aberrant reactivation of developmental signaling pathways has been suggested to contribute to IPF pathogenesis, we hypothesized that Wnt/beta-catenin signaling is activated in epithelial cells in IPF. Thus, we quantified and localized the expression and activity of the Wnt/beta-catenin pathway in IPF.

Lymphotoxin β-receptor (LTβR) 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 LTβR signalling drives chronic tissue damage particularly in the lung, the mechanism(s) that regulate this process, and whether LTβR blockade might be of therapeutic value have remained unclear. Here we demonstrate increased expression of LTβR ligands in adaptive and innate immune cells, enhanced non-canonical NF-κB signalling, and enriched LTβR 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 LTβR 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 LTβR signalling dampened epithelial non-canonical activation of NF-κB, reduced TGFβ signalling in airways, and induced regeneration by preventing epithelial cell death and activating WNT/β-catenin signalling in alveolar epithelial progenitor cells. These findings suggest that inhibition of LTβR signalling represents a viable therapeutic option that combines prevention of tertiary lymphoid structures1 and inhibition of apoptosis with tissue-regenerative strategies.

Lung diseases impose a huge economic and health burden worldwide. A key aspect of several adult lung diseases, such as idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD), including emphysema, is aberrant tissue repair, which leads to an accumulation of damage and impaired respiratory function. Currently, there are few effective treatments available for these diseases and their incidence is rising. The planar cell polarity (PCP) pathway is critical for the embryonic development of many organs, including kidney and lung. We have previously shown that perturbation of the PCP pathway impairs tissue morphogenesis, which disrupts the number and shape of epithelial tubes formed within these organs during embryogenesis. However, very little is known about the role of the PCP pathway beyond birth, partly because of the perinatal lethality of many PCP mouse mutant lines. Here, we investigate heterozygous Looptail (Lp) mice, in which a single copy of the core PCP gene, Vangl2, is disrupted. We show that these mice are viable but display severe airspace enlargement and impaired adult lung function. Underlying these defects, we find that Vangl2Lp/+ lungs exhibit altered distribution of actin microfilaments and abnormal regulation of the actin-modifying protein cofilin. In addition, we show that Vangl2Lp/+ lungs exhibit many of the hallmarks of tissue damage, including an altered macrophage population, abnormal elastin deposition and elevated levels of the elastin-modifying enzyme, Mmp12, all of which are observed in emphysema. In vitro, disruption of VANGL2 impairs directed cell migration and reduces the rate of repair following scratch wounding of human alveolar epithelial cells. Moreover, using population data from a birth cohort of young adults, all aged 31, we found evidence of an interactive effect between VANGL2 and smoking on lung function. Finally, we show that PCP genes VANGL2 and SCRIB are significantly downregulated in lung tissue from patients with emphysema. Our data reveal an important novel role for the PCP pathway in adult lung homeostasis and repair and shed new light on the genetic factors which may modify destructive lung diseases such as emphysema.