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.

CXCR3-Independent Role of CXCL10 in Alveolar Epithelial Repair.

The alveolar Type II epithelial (AEC2) cells act as stem cells in the lung for alveolar epithelial maintenance and repair. Chemokine CXCL10 is expressed in injured tissues, modulating multiple cellular functions. AEC2s, previously reported to release chemokines to recruit leukocytes, were found in our study to secrete CXCL10 after bleomycin injury. We found that Sftpc-Cxcl10 transgenic mice were protected from bleomycin injury. The transgenic mice showed an increase in the AEC2 population in the lung by flow cytometry analysis. Both endogenous and exogenous CXCL10 promoted the colony formation efficiency of AEC2s in a 3D organoid growth assay. We identified that the regenerative effect of CXCL10 was CXCR3 independent using Cxcr3-deficient mice, but it was related to the TrkA pathway. Binding experiments showed that CXCL10 interacted with TrkA directly and reversibly. This study demonstrates a previously unidentified AEC2 autocrine signaling of CXCL10 to promote their regeneration and proliferation, probably involving a CXCR3-independent TrkA pathway.

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/).

Reciprocal interactions between alveolar progenitor dysfunction and aging promote lung fibrosis.

Aging is a critical risk factor in idiopathic pulmonary fibrosis (IPF). Dysfunction and loss of type 2 alveolar epithelial cells (AEC2s) with failed regeneration is a seminal causal event in the pathogenesis of IPF, although the precise mechanisms for their regenerative failure and demise remain unclear. To systematically examine the genomic program changes of AEC2s in aging and after lung injury, we performed unbiased single-cell RNA-seq analyses of lung epithelial cells from uninjured or bleomycin-injured young and old mice, as well as from lungs of IPF patients and healthy donors. We identified three AEC2 subsets based on their gene signatures. Subset AEC2-1 mainly exist in uninjured lungs, while subsets AEC2-2 and AEC2-3 emerged in injured lungs and increased with aging. Functionally, AEC2 subsets are correlated with progenitor cell renewal. Aging enhanced the expression of the genes related to inflammation, stress responses, senescence, and apoptosis. Interestingly, lung injury increased aging-related gene expression in AEC2s even in young mice. The synergistic effects of aging and injury contributed to impaired AEC2 recovery in aged mouse lungs after injury. In addition, we also identified three subsets of AEC2s from human lungs that formed three similar subsets to mouse AEC2s. IPF AEC2s showed a similar genomic signature to AEC2 subsets from bleomycin-injured old mouse lungs. Taken together, we identified synergistic effects of aging and AEC2 injury in transcriptomic and functional analyses that promoted fibrosis. This study provides new insights into the interactions between aging and lung injury with interesting overlap with diseased IPF AEC2 cells.

Basal Cell-derived WNT7A Promotes Fibrogenesis at the Fibrotic Niche in Idiopathic Pulmonary Fibrosis.

Loss of epithelial integrity, bronchiolarization, and fibroblast activation are key characteristics of idiopathic pulmonary fibrosis (IPF). Prolonged accumulation of basal-like cells in IPF may impact the fibrotic niche to promote fibrogenesis. To investigate their role in IPF, basal cells were isolated from IPF explant and healthy donor lung tissues. Single-cell RNA sequencing was used to assess differentially expressed genes in basal cells. Basal cell and niche interaction was demonstrated with the sLP-mCherry niche labeling system. Luminex assays were used to assess cytokines secreted by basal cells. The role of basal cells in fibroblast activation was studied. Three-dimensional organoid culture assays were used to interrogate basal cell effects on AEC2 (type 2 alveolar epithelial cell) renewal capacity. Perturbation was used to investigate WNT7A function in vitro and in a repetitive bleomycin model in vivo. We found that WNT7A is highly and specifically expressed in basal-like cells. Proteins secreted by basal cells can be captured by neighboring fibroblasts and AEC2s. Basal cells or basal cell-conditioned media activate fibroblasts through WNT7A. Basal cell-derived WNT7A inhibits AEC2 progenitor cell renewal in three-dimensional organoid cultures. Neutralizing antibodies against WNT7A or a small molecule inhibitor of Frizzled signaling abolished basal cell-induced fibroblast activation and attenuated lung fibrosis in mice. In summary, basal cells and basal cell-derived WNT7A are key components of the fibrotic niche, providing a unique non-stem cell function of basal cells in IPF progression and a novel targeting strategy for IPF.

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.

Mitogen-activated Protein Kinase-activated Protein Kinase 2 Inhibition Attenuates Fibroblast Invasion and Severe Lung Fibrosis.

Severe pulmonary fibrosis such as idiopathic pulmonary fibrosis (IPF) is characterized by the accumulation of extracellular matrix and fibroblast activation. Targeting fibroblast activation has contributed to the development of antifibrotic therapeutics for patients with IPF. Mitogen-activated protein kinase-activated protein kinase 2 (MK2), downstream in the transforming growth factor-ß/p38 mitogen-activated protein kinase pathway, has been implicated in inflammatory and fibrosing diseases. Increased concentrations of activated MK2 were expressed in IPF lung and in the mouse bleomycin model of lung fibrosis. The aim of the present study was to determine the role and the mechanisms of MK2 in fibroblast invasion and lung fibrosis. Our results showed that an MK2 inhibitor (MMI-0100) was able to inhibit the invasive capacity of lung fibroblasts isolated from patients with IPF, as well as fibroblasts isolated from both wild-type mice and mice with overexpressing hyaluronan synthase 2 (HAS2) in the myofibroblast compartment. We previously showed that hyaluronan and HAS2 regulate fibroblast invasion and lung fibrosis in vivo. The results of the present study showed that MMI-0100 reduced transforming growth factor-ß-induced hyaluronan production in human and mouse fibroblasts in vitro and that HAS2 mediated MK2 activation, suggesting a feed-forward loop in fibroblast activation. More importantly, MK2 inhibition attenuated hyaluronan accumulation and reduced collagen content in bleomycin-injured mouse lungs in vivo. Conditional deletion of MK2 in fibroblasts attenuated bleomycin-induced lung fibrosis. These data provide evidence that MK2 has a role in fibroblast invasion and fibrosis and may be a novel therapeutic target in pulmonary fibrosis.

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.

Hyaluronan and TLR4 promote surfactant-protein-C-positive alveolar progenitor cell renewal and prevent severe pulmonary fibrosis in mice.

Successful recovery from lung injury requires the repair and regeneration of alveolar epithelial cells to restore the integrity of gas-exchanging regions within the lung and preserve organ function. Improper regeneration of the alveolar epithelium is often associated with severe pulmonary fibrosis, the latter of which involves the recruitment and activation of fibroblasts, as well as matrix accumulation. Type 2 alveolar epithelial cells (AEC2s) are stem cells in the adult lung that contribute to the lung repair process. The mechanisms that regulate AEC2 renewal are incompletely understood. We provide evidence that expression of the innate immune receptor Toll-like receptor 4 (TLR4) and the extracellular matrix glycosaminoglycan hyaluronan (HA) on AEC2s are important for AEC2 renewal, repair of lung injury and limiting the extent of fibrosis. Either deletion of TLR4 or HA synthase 2 in surfactant-protein-C-positive AEC2s leads to impaired renewal capacity, severe fibrosis and mortality. Furthermore, AEC2s from patients with severe pulmonary fibrosis have reduced cell surface HA and impaired renewal capacity, suggesting that HA and TLR4 are key contributors to lung stem cell renewal and that severe pulmonary fibrosis is the result of distal epithelial stem cell failure.

Transcription factor TBX4 regulates myofibroblast accumulation and lung fibrosis.

Progressive tissue fibrosis is a major cause of the morbidity and mortality associated with repeated epithelial injuries and accumulation of myofibroblasts. Successful treatment options are limited by an incomplete understanding of the molecular mechanisms that regulate myofibroblast accumulation. Here, we employed in vivo lineage tracing and real-time gene expression transgenic reporting methods to analyze the early embryonic transcription factor T-box gene 4 (TBX4), and determined that TBX4-lineage mesenchymal progenitors are the predominant source of myofibroblasts in injured adult lung. In a murine model, ablation of TBX4-expressing cells or disruption of TBX4 signaling attenuated lung fibrosis after bleomycin-induced injury. Furthermore, TBX4 regulated hyaluronan synthase 2 production to enable fibroblast invasion of matrix both in murine models and in fibroblasts from patients with severe pulmonary fibrosis. These data identify TBX4 as a mesenchymal transcription factor that drives accumulation of myofibroblasts and the development of lung fibrosis. Targeting TBX4 and downstream factors that regulate fibroblast invasiveness could lead to therapeutic approaches in lung fibrosis.

Hyaluronan synthase 2 regulates fibroblast senescence in pulmonary fibrosis.

Dysregulated repair of lung injury often results in lung fibrosis characterized by unremitting deposition of matrix components including glycosaminoglycan hyaluronan (HA). HA is mainly produced by hyaluronan synthases (HAS) in mesenchymal cells. We previously demonstrated that over-expression of HAS2 in mesenchymal cells in mice regulates the invasiveness of fibroblasts and promotes severe lung fibrosis. The mechanisms that control the resolution of lung fibrosis are unknown. We propose that a critical step in resolving fibrosis is the induction of senescence in fibrotic fibroblasts and hyaluronan synthase 2 may regulate this process. We found that fibrotic fibroblasts developed the characteristics of replicative senescence in culture and that HAS2 expression was dramatically down-regulated. Furthermore, down-regulation of HAS2 initiated and regulated fibroblast senescence through a p27-CDK2-SKP2 pathway. Deletion of HAS2 in mouse mesenchymal cells increased the cellular senescence of fibroblasts in bleomycin-induced mouse lung fibrosis in vivo. These data suggest that HAS2 may be a critical regulator of the fate of pulmonary fibrosis and we propose a model where over-expression of HAS2 promotes an invasive phenotype resulting in severe fibrosis and down-regulation of HAS2 promotes resolution. Targeting HAS2 to induce fibroblast senescence could be an attractive approach to resolve tissue fibrosis.

Methylation-mediated BMPER expression in fibroblast activation in vitro and lung fibrosis in mice in vivo.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease. Although the pathogenesis is poorly understood, evidence suggests that genetic and epigenetic alterations, such as DNA methylation, may play a key role. Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-ß (TGF-ß) superfamily and are important regulators in IPF. Here we identified BMP endothelial cell precursor-derived regulator (BMPER) as a key regulator of fibroblast activation. BMPER is a secreted glycoprotein that binds directly to BMPs and may regulate TGF-ß/BMP signaling, but its role in lung fibrosis is not clear. BMPER is highly expressed in human IPF lung fibroblasts compared to normal lung fibroblasts. Demethylation agent 5'-azacytidine decreased BMPER expression in fibroblasts, and attenuated the invasion and migration of IPF lung fibroblasts. Furthermore, siRNA-mediated reduction of BMPER in the human lung fibroblasts impaired cell migration and invasion. 5'-azacytidine treatment additionally regulated BMPER expression and reduced lung fibrosis in mice in vivo. These findings demonstrate that methylation of specific genes in fibroblasts may offer a new therapeutic strategy for IPF by modulating fibroblast activation.

A macrophage subpopulation recruited by CC chemokine ligand-2 clears apoptotic cells in noninfectious lung injury.

CC chemokine ligand-2 (CCL2)/monocyte chemoattractant protein (MCP)-1 expression is upregulated during pulmonary inflammation, and the CCL2-CCR2 axis plays a critical role in leukocyte recruitment and promotion of host defense against infection. The role of CCL2 in mediating macrophage subpopulations in the pathobiology of noninfectious lung injury is unknown. The goal of this study was to examine the role of CCL2 in noninfectious acute lung injury. Our results show that lung-specific overexpression of CCL2 protected mice from bleomycin-induced lung injury, characterized by significantly reduced mortality, reduced neutrophil accumulation, and decreased accumulation of the inflammatory mediators IL-6, CXCL2 (macrophage inflammatory protein-2), and CXCL1 (keratinocyte-derived chemokine). There were dramatic increases in the recruitment of myosin heavy chain (MHC) II IA/IE(int)CD11c(int) cells, exudative macrophages, and dendritic cells in Ccl2 transgenic mouse lungs both at baseline and after bleomycin treatment compared with levels in wild-type mice. We further demonstrated that MHCII IA/IE(int)CD11c(int) cells engulfed apoptotic cells during acute lung injury. Our data suggested a previously undiscovered role for MHCII IA/IE(int)CD11c(int) cells in apoptotic cell clearance and inflammation resolution.

MicroRNA-127 inhibits lung inflammation by targeting IgG Fcγ receptor I.

The molecular mechanisms of acute lung injury are incompletely understood. MicroRNAs (miRNAs) are crucial biological regulators that act by suppressing their target genes and are involved in a variety of pathophysiologic processes. miR-127 appears to be downregulated during lung injury. We set out to investigate the role of miR-127 in lung injury and inflammation. Expression of miR-127 significantly reduced cytokine release by macrophages. Looking into the mechanisms of regulation of inflammation by miR-127, we found that IgG FcγRI (CD64) was a target of miR-127, as evidenced by reduced CD64 protein expression in macrophages overexpressing miR-127. Furthermore, miR-127 significantly reduced the luciferase activity with a reporter construct containing the native 3' untranslated region of CD64. Importantly, we demonstrated that miR-127 attenuated lung inflammation in an IgG immune complex model in vivo. Collectively, these data show that miR-127 targets macrophage CD64 expression and promotes the reduction of lung inflammation. Understanding how miRNAs regulate lung inflammation may represent an attractive way to control inflammation induced by infectious or noninfectious lung injury.