To establish and maintain organ structure and function, tissues need to balance stem cell proliferation and differentiation rates and coordinate cell fate with position. By quantifying and modelling tissue stress and deformation in the mammalian epidermis, we find that this balance is coordinated through local mechanical forces generated by cell division and delamination. Proliferation within the basal stem/progenitor layer, which displays features of a jammed, solid-like state, leads to crowding, thereby locally distorting cell shape and stress distribution. The resulting decrease in cortical tension and increased cell-cell adhesion trigger differentiation and subsequent delamination, reinstating basal cell layer density. After delamination, cells establish a high-tension state as they increase myosin II activity and convert to E-cadherin-dominated adhesion, thereby reinforcing the boundary between basal and suprabasal layers. Our results uncover how biomechanical signalling integrates single-cell behaviours to couple proliferation, cell fate and positioning to generate a multilayered tissue.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease of unknown cause that is characterized by progressive fibrotic lung remodeling. An abnormal emergence of airway epithelial‐like cells within the alveolar compartments of the lung, herein termed bronchiolization, is often observed in IPF. However, the origin of this dysfunctional distal lung epithelium remains unknown due to a lack of suitable human model systems. In this study, we established a human induced pluripotent stem cell (iPSC)‐derived air‐liquid interface (ALI) model of alveolar epithelial type II (ATII)‐like cell differentiation that allows us to investigate alveolar epithelial progenitor cell differentiation in vitro. We treated this system with an IPF‐relevant cocktail (IPF‐RC) to mimic the pro‐fibrotic cytokine milieu present in IPF lungs. Stimulation with IPF‐RC during differentiation increases secretion of IPF biomarkers and RNA sequencing (RNA‐seq) of these cultures reveals significant overlap with human IPF patient data. IPF‐RC treatment further impairs ATII differentiation by driving a shift toward an airway epithelial‐like expression signature, providing evidence that a pro‐fibrotic cytokine environment can influence the proximo‐distal differentiation pattern of human lung epithelial cells. In conclusion, we show for the first time, the establishment of a human model system that recapitulates aspects of IPF‐associated bronchiolization of the lung epithelium in vitro.

Unveiling the molecular mechanisms of tissue remodelling following injury is imperative to elucidate its regenerative capacity and aberrant repair in disease. Using different omics approaches, we identified enhancer of zester homolog 2 (EZH2) as a key regulator of fibrosis in injured lung epithelium. Epithelial injury drives an enrichment of nuclear transforming growth factor‐β‐activated kinase 1 (TAK1) that mediates EZH2 phosphorylation to facilitate its liberation from polycomb repressive complex 2 (PRC2). This process results in the establishment of a transcriptional complex of EZH2, RNA‐polymerase II (POL2) and nuclear actin, which orchestrates aberrant epithelial repair programmes. The liberation of EZH2 from PRC2 is accompanied by an EZH2‐EZH1 switch to preserve H3K27me3 deposition at non‐target genes. Loss of epithelial TAK1, EZH2 or blocking nuclear actin influx attenuates the fibrotic cascade and restores respiratory homeostasis. Accordingly, EZH2 inhibition significantly improves outcomes in a pulmonary fibrosis mouse model. Our results reveal an important non‐canonical function of EZH2, paving the way for new therapeutic interventions in fibrotic lung diseases.

Elevated counts of alveolar macrophages and attenuated phagocytic capacity are associated with COPD. Factors governing macrophage phagocytosis are poorly understood. In this study we aimed to compare the influence of airway epithelial cell secretions from COPD and nonCOPD subjects on macrophage phagocytic activity, and the role of antimicrobial peptides (AMPs).Supernatants from nonCOPD and COPD small airway epithelial cell (SAEC) cultures exposed to Haemophilus influenzae (NTHi), were applied to human monocyte-derived macrophages (MDM) for assessment of their influence on phagocytosis. SAECs were analysed for changes in AMP expression by qRT-PCR and the influence of select AMPs on macrophage phenotype and function was assessed by flow cytometry and metabolic activity assay.Secretions from the apical and basolateral surface of NTHi-exposed SAECs from nonCOPD donors elicited superior phagocytic capacity of MDMs. Moreover, NTHi exposure led to a rapid increase in expression of a range of AMPs by nonCOPD SAECs, but this response was delayed in COPD SAECs. We demonstrate that treatment with AMPs β-defensin 2 and S100A8/A9 improved the phagocytic capacity of MDMs. In depth analysis of S100A8/A9 influence of MDMs revealed a role of this AMP in macrophage phenotype and function. Furthermore, we show that the expression of S100A8 and S100A9 is directly regulated by WNT/β-catenin signalling activity, a known deregulated pathway in COPD.In conclusion, for the first time, we demonstrate that airway epithelium from COPD subjects has a reduced capacity to support the phagocytic function of macrophages in response to acute NTHi exposure, and we identify the WNT/β-catenin signalling-modulated and epithelium-derived S100A8/A9 as a potent regulator of macrophage phenotype and function.

An abnormal emergence of airway epithelial-like cells within the alveolar compartments of the lung, herein termed bronchiolization, is a process often observed in patients suffering from idiopathic pulmonary fibrosis (IPF), a fatal disease characterized by progressive fibrotic lung remodeling. However, the origin of this dysfunctional epithelium remains unknown.In this study, we aimed to investigate the effects of a pro-fibrotic milieu, similar to that found in an IPF lung, on human alveolar epithelial progenitor cell differentiation. We developed an induced pluripotent stem cell (iPSC)-derived air-liquid interface (ALI) model of alveolar type II (ATII)-like cell differentiation and stimulated it with an IPF-relevant cocktail (IPF-RC), composed of cytokines previously reported to be elevated in IPF lungs. iPSC-derived cultures express ATII markers and contain lamellar body-like structures. Stimulation with IPF-RC during the last two weeks of differentiation increases secretion of IPF biomarkers. Transcriptome analysis of IPF-RC treated cultures reveals significant overlap with human IPF data and enrichment of transcripts associated with extracellular matrix organization. IPF-RC stimulation further impairs ATII differentiation by driving a shift towards an airway epithelial-like expression signature.In conclusion, we show for the first time, the establishment of a human model system that recapitulates aspects of IPF-associated bronchiolization in vitro. Our findings reveal how aberrant alveolar epithelial progenitor cell differentiation in a pro-fibrotic environment could contribute to alveolar bronchiolization in the distal IPF lung.SOURCE OF SUPPORTThe research was funded by Boehringer Ingelheim Pharma GmbH & Co. KG.