Arrestin beta 1 Regulates Alveolar Progenitor Renewal and Lung Fibrosis.

The molecular mechanisms that regulate progressive pulmonary fibrosis remain poorly understood. Type 2 alveolar epithelial cells (AEC2s) function as adult stem cells in the lung. We previously showed that there is a loss of AEC2s and a failure of AEC2 renewal in the lungs of idiopathic pulmonary fibrosis (IPF) patients. We also reported that beta-arrestins are the key regulators of fibroblast invasion, and beta-arrestin 1 and 2 deficient mice exhibit decreased mortality, decreased matrix deposition, and increased lung function in bleomycin-induced lung fibrosis. However, the role of beta-arrestins in AEC2 regeneration is unclear. In this study, we investigated the role and mechanism of Arrestin beta 1 (ARRB1) in AEC2 renewal and in lung fibrosis. We used conventional deletion as well as cell type-specific deletion of ARRB1 in mice and found that Arrb1 deficiency in fibroblasts protects mice from lung fibrosis, and the knockout mice exhibit enhanced AEC2 regeneration in vivo, suggesting a role of fibroblast-derived ARRB1 in AEC2 renewal. We further found that Arrb1-deficient fibroblasts promotes AEC2 renewal in 3D organoid assays. Mechanistically, we found that CCL7 is among the top downregulated cytokines in Arrb1 deficient fibroblasts and CCL7 inhibits AEC2 regeneration in 3D organoid experiments. Therefore, fibroblast ARRB1 mediates AEC2 renewal, possibly by releasing chemokine CCL7, leading to fibrosis in the lung.

Lipid Deficiency Contributes to Impaired Alveolar Progenitor Cell Function in Aging and Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is an aging-associated interstitial lung disease resulting from repeated epithelial injury and inadequate epithelial repair. Alveolar type II cells (AEC2) are progenitor cells that maintain epithelial homeostasis and repair the lung after injury. In the current study, we assessed lipid metabolism in AEC2s from human lungs of IPF patients and healthy donors, as well as AEC2s from bleomycin-injured young and old mice. Through single cell RNA sequencing (scRNA-seq), we observed that lipid metabolism-related genes were downregulated in IPF AEC2s and bleomycin-injured mouse AEC2s. Aging aggravated this decrease and hindered recovery of lipid metabolism gene expression in AEC2s after bleomycin injury. Pathway analyses revealed down-regulation of genes related to lipid biosynthesis and fatty acid -oxidation in AEC2s from IPF lungs and bleomycin-injured, aged mouse lungs compared to the respective controls. We confirmed decreased cellular lipid content in AEC2s from IPF lungs and bleomycin-injured, aged mouse lungs using immunofluorescence staining and flow cytometry. We further show that lipid metabolism was associated with AEC2 progenitor function. Lipid supplementation and peroxisome proliferator activated receptor gamma (PPARγ) activation promoted progenitor renewal capacity of both human and mouse AEC2s in 3D organoid cultures. Lipid supplementation also increased AEC2 proliferation and expression of SFTPC in AEC2s. In summary, we identified a lipid metabolism deficiency in AEC2s from lungs of patients with IPF and bleomycin-injured aged mice. Restoration of lipid metabolism homeostasis in AEC2s might promote AEC2 progenitor function and offer new opportunities for therapeutic approaches to IPF. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

HER2 drives lung fibrosis by activating a metastatic cancer signature in invasive lung fibroblasts.

Progressive tissue fibrosis, including idiopathic pulmonary fibrosis (IPF), is characterized by excessive recruitment of fibroblasts to sites of tissue injury and unremitting extracellular matrix deposition associated with severe morbidity and mortality. However, the molecular mechanisms that control progressive IPF have yet to be fully determined. Previous studies suggested that invasive fibroblasts drive disease progression in IPF. Here, we report profiling of invasive and noninvasive fibroblasts from IPF patients and healthy donors. Pathway analysis revealed that the activated signatures of the invasive fibroblasts, the top of which was ERBB2 (HER2), showed great similarities to those of metastatic lung adenocarcinoma cancer cells. Activation of HER2 in normal lung fibroblasts led to a more invasive genetic program and worsened fibroblast invasion and lung fibrosis, while antagonizing HER2 signaling blunted fibroblast invasion and ameliorated lung fibrosis. These findings suggest that HER2 signaling may be a key driver of fibroblast invasion and serve as an attractive target for therapeutic intervention in IPF.

The ZIP8/SIRT1 axis regulates alveolar progenitor cell renewal in aging and idiopathic pulmonary fibrosis.

Type 2 alveolar epithelial cells (AEC2s) function as progenitor cells in the lung. We have shown previously that failure of AEC2 regeneration results in progressive lung fibrosis in mice and is a cardinal feature of idiopathic pulmonary fibrosis (IPF). In this study, we identified deficiency of a specific zinc transporter, SLC39A8 (ZIP8), in AEC2s from both IPF lungs and lungs of old mice. Loss of ZIP8 expression was associated with impaired renewal capacity of AEC2s and enhanced lung fibrosis. ZIP8 regulation of AEC2 progenitor function was dependent on SIRT1. Replenishment with exogenous zinc and SIRT1 activation promoted self-renewal and differentiation of AEC2s from lung tissues of IPF patients and old mice. Deletion of Zip8 in AEC2s in mice resulted in impaired AEC2 renewal, increased susceptibility to bleomycin injury, and development of spontaneous lung fibrosis. Therapeutic strategies to restore zinc metabolism and appropriate SIRT1 signaling could improve AEC2 progenitor function and mitigate ongoing fibrogenesis.

Mesenchymal growth hormone receptor deficiency leads to failure of alveolar progenitor cell function and severe pulmonary fibrosis.

Recent studies have identified impaired type 2 alveolar epithelial cell (ATII) renewal in idiopathic pulmonary fibrosis (IPF) human organoids and severe fibrosis when ATII is defective in mice. ATIIs function as progenitor cells and require supportive signals from the surrounding mesenchymal cells. The mechanisms by which mesenchymal cells promote ATII progenitor functions in lung fibrosis are incompletely understood. We identified growth hormone receptor (GHR) is mainly expressed in mesenchymal cells, and its expression is substantially decreased in IPF lungs. Higher levels of GHR expression correlated with better lung function in patients with IPF. Profibrotic mesenchymal cells retarded ATII growth and were associated with suppressed vesicular GHR expression. Vesicles enriched with Ghr promote ATII proliferation and diminished pulmonary fibrosis in mesenchymal Ghr-deficient mice. Our findings demonstrate a previously unidentified mesenchymal paracrine signaling coordinated by GHR that is capable of supporting ATII progenitor cell renewal and limiting the severity of lung fibrosis.

PD-L1 on invasive fibroblasts drives fibrosis in a humanized model of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with unremitting extracellular matrix deposition, leading to a distortion of pulmonary architecture and impaired gas exchange. Fibroblasts from IPF patients acquire an invasive phenotype that is essential for progressive fibrosis. Here, we performed RNA sequencing analysis on invasive and noninvasive fibroblasts and found that the immune checkpoint ligand CD274 (also known as PD-L1) was upregulated on invasive lung fibroblasts and was required for the invasive phenotype of lung fibroblasts, is regulated by p53 and FAK, and drives lung fibrosis in a humanized IPF model in mice. Activating CD274 in IPF fibroblasts promoted invasion in vitro and pulmonary fibrosis in vivo. CD274 knockout in IPF fibroblasts and targeting CD274 by FAK inhibition or CD274-neutralizing antibodies blunted invasion and attenuated fibrosis, suggesting that CD274 may be a novel therapeutic target in IPF.

Innate Recognition of the Microbiota by TLR1 Promotes Epithelial Homeostasis and Prevents Chronic Inflammation.

There is cross-talk between the intestinal epithelium and the microbiota that functions to maintain a tightly regulated microenvironment and prevent chronic inflammation. This communication is partly mediated through the recognition of bacterial proteins by host-encoded innate receptors, such as TLRs. However, studies examining the role of TLR signaling on colonic homeostasis have given variable and conflicting results. Despite its critical role in mediating immunity during enteric infection of the small intestine, TLR1-mediated recognition of microbiota-derived ligands and their influence on colonic homeostasis has not been well studied. In this study, we demonstrate that defective TLR1 recognition of the microbiome by epithelial cells results in disruption of crypt homeostasis specifically within the secretory cell compartment, including a defect in the mucus layer, ectopic Paneth cells in the colon, and an increase in the number of rapidly dividing cells at the base of the crypt. As a consequence of the perturbed epithelial barrier, we found an increase in mucosal-associated and translocated commensal bacteria and chronic low-grade inflammation characterized by an increase in lineage-negative Sca1+Thy1hi innate lymphoid-like cells that exacerbate inflammation and worsen outcomes in a model of colonic injury and repair. Our findings demonstrate that sensing of the microbiota by TLR1 may provide key signals that regulate the colonic epithelium, thereby limiting inflammation through the prevention of bacterial attachment to the mucosa and exposure to the underlying immune system.

Single-Cell Deconvolution of Fibroblast Heterogeneity in Mouse Pulmonary Fibrosis.

Fibroblast heterogeneity has long been recognized in mouse and human lungs, homeostasis, and disease states. However, there is no common consensus on fibroblast subtypes, lineages, biological properties, signaling, and plasticity, which severely hampers our understanding of the mechanisms of fibrosis. To comprehensively classify fibroblast populations in the lung using an unbiased approach, single-cell RNA sequencing was performed with mesenchymal preparations from either uninjured or bleomycin-treated mouse lungs. Single-cell transcriptome analyses classified and defined six mesenchymal cell types in normal lung and seven in fibrotic lung. Furthermore, delineation of their differentiation trajectory was achieved by a machine learning method. This collection of single-cell transcriptomes and the distinct classification of fibroblast subsets provide a new resource for understanding the fibroblast landscape and the roles of fibroblasts in fibrotic diseases.

MicroRNA-29c Prevents Pulmonary Fibrosis by Regulating Epithelial Cell Renewal and Apoptosis.

Successful repair and renewal of alveolar epithelial cells (AECs) are critical in prohibiting the accumulation of myofibroblasts in pulmonary fibrogenesis. MicroRNAs (miRNAs) are multifocal regulators involved in lung injury and repair. However, the contribution of miRNAs to AEC2 renewal and apoptosis is incompletely understood. We report that miRNA-29c (miR-29c) expression is lower in AEC2s of individuals with idiopathic pulmonary fibrosis than in healthy lungs. Epithelial cells overexpressing miR-29c show higher proliferative rates and viability. miR-29c protects epithelial cells from apoptosis by targeting forkhead box O3a (Foxo3a). Both overexpression of miR-29c conventionally and AEC2s specifically lead to less fibrosis and better recovery in vivo. Furthermore, deficiency of miR-29c in AEC2s results in higher apoptosis and reduced epithelial renewal. Interestingly, a gene network including a subset of apoptotic genes was coregulated by both Toll-like receptor 4 and miR-29c. Taken together, miR-29c maintains epithelial integrity and promotes recovery from lung injury, thereby attenuating lung fibrosis in mice.