No abstract

A comprehensive understanding of the changes in gene expression in cell types involved in idiopathic pulmonary fibrosis (IPF) will shed light on the mechanisms underlying the loss of alveolar epithelial cells and development of honeycomb cysts and fibroblastic foci. We sought to understand changes in IPF lung cell transcriptomes and gain insight into innate immune aspects of pathogenesis.We investigated IPF pathogenesis using single-cell RNA-sequencing of fresh lung explants, comparing human IPF fibrotic lower lobes reflecting late disease, upper lobes reflecting early disease and normal lungs.IPF lower lobes showed increased fibroblasts, and basal, ciliated, goblet and club cells, but decreased alveolar epithelial cells, and marked alterations in inflammatory cells. We found three discrete macrophage subpopulations in normal and fibrotic lungs, one expressing monocyte markers, one highly expressing FABP4 and INHBA (FABP4hi), and one highly expressing SPP1 and MERTK (SPP1hi). SPP1hi macrophages in fibrotic lower lobes showed highly upregulated SPP1 and MERTK expression. Low-level local proliferation of SPP1hi macrophages in normal lungs was strikingly increased in IPF lungs.Co-localisation and causal modelling supported the role for these highly proliferative SPP1hi macrophages in activation of IPF myofibroblasts in lung fibrosis. These data suggest that SPP1hi macrophages contribute importantly to lung fibrosis in IPF, and that therapeutic strategies targeting MERTK and macrophage proliferation may show promise for treatment of this disease.

The mechanisms of aging that are involved in the development of idiopathic pulmonary fibrosis (IPF) are still unclear. Although it has been hypothesized that the proliferation and activation of human lung fibroblasts (hLFs) are essential in IPF, no studies have assessed how this process works in an aging lung. Our goal was to elucidate if there were age-related changes on primary hLFs isolated from IPF lungs compared with age-matched controls. We investigated several hallmarks of aging in hLFs from IPF patients and age-matched controls. IPF hLFs have increased cellular senescence with higher expression of β-galactosidase, p21, p16, p53, and cytokines related to the senescence-associated secretory phenotype (SASP) as well as decreased proliferation/apoptosis compared with age-matched controls. Additionally, we observed shorter telomeres, mitochondrial dysfunction, and upon transforming growth factor-β stimulation, increased markers of endoplasmic reticulum stress. Our data suggest that IPF hLFs develop senescence resulting in a decreased apoptosis and that the development of SASP may be an important contributor to the fibrotic process observed in IPF. These results might change the existing paradigm, which describes fibroblasts as aberrantly activated cells, to a cell with a senescence phenotype.

ObjectivesMyofibroblasts are key effector cells in the extracellular matrix remodelling of systemic sclerosis-associated interstitial lung disease (SSc-ILD); however, the diversity of fibroblast populations present in the healthy and SSc-ILD lung is unknown and has prevented the specific study of the myofibroblast transcriptome. We sought to identify and define the transcriptomes of myofibroblasts and other mesenchymal cell populations in human healthy and SSc-ILD lungs to understand how alterations in fibroblast phenotypes lead to SSc-ILD fibrosis.MethodsWe performed droplet-based, single-cell RNA-sequencing with integrated canonical correlation analysis of 13 explanted lung tissue specimens (56 196 cells) from four healthy control and four patients with SSc-ILD, with findings confirmed by cellular indexing of transcriptomes and epitopes by sequencing in additional samples.ResultsExamination of gene expression in mesenchymal cells identified two major, SPINT2hi and MFAP5hi, and one minor, WIF1hi, fibroblast populations in the healthy control lung. Combined analysis of control and SSc-ILD mesenchymal cells identified SPINT2hi, MFAP5hi, few WIF1hi fibroblasts and a new large myofibroblast population with evidence of actively proliferating myofibroblasts. We compared differential gene expression between all SSc-ILD and control mesenchymal cell populations, as well as among the fibroblast subpopulations, showing that myofibroblasts undergo the greatest phenotypic changes in SSc-ILD and strongly upregulate expression of collagens and other profibrotic genes.ConclusionsOur results demonstrate previously unrecognised fibroblast heterogeneity in SSc-ILD and healthy lungs, and define multimodal transcriptome-phenotypes associated with these populations. Our data indicate that myofibroblast differentiation and proliferation are key pathological mechanisms driving fibrosis in SSc-ILD.

SummaryIdiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and usually lethal disease associated with aging. However, the molecular mechanisms of the aging process that contribute to the pathogenesis of IPF have not been elucidated. IPF is characterized by abundant foci of highly active fibroblasts and myofibroblasts resistant to apoptosis. Remarkably, the role of aging in the autophagy activity of lung fibroblasts and its relationship with apoptosis, as adaptive responses, has not been evaluated previously in this disease. In the present study, we analyzed the dynamics of autophagy in primary lung fibroblasts from IPF compared to young and age-matched normal lung fibroblasts. Our results showed that aging contributes for a lower induction of autophagy on basal conditions and under starvation which is mediated by mTOR pathway activation. Treatment with rapamycin and PP242, that target the PI3K/AKT/mTOR signaling pathway, modified starvation-induced autophagy and apoptosis in IPF fibroblasts. Interestingly, we found a persistent activation of this pathway under starvation that contributes to the apoptosis resistance in IPF fibroblasts. These findings indicate that aging affects adaptive responses to stress decreasing autophagy through activation of mTORC1 in lung fibroblasts. The activation of this pathway also contributes to the resistance to cell death in IPF lung fibroblasts.

Pulmonary inflammation, which is characterized by the presence of perivascular macrophages, has been proposed as a key pathogenic driver of pulmonary hypertension (PH), a vascular disease with increasing global significance. However, the mechanisms of expansion of lung macrophages and the role of blood-borne monocytes in PH are poorly understood. Using multicolor flow cytometric analysis of blood in mouse and rat models of PH and patients with PH, an increase in blood monocytes was observed. In parallel, lung tissue displayed increased chemokine transcript expression, including those responsible for monocyte recruitment, such as and, accompanied by an expansion of interstitial lung macrophages. These data indicate that blood monocytes are recruited to lung perivascular spaces and differentiate into inflammatory macrophages. Correspondingly, parabiosis between congenically different hypoxic mice demonstrated that most interstitial macrophages originated from blood monocytes. To define the actions of these cells in PH in vivo, we reduced blood monocyte numbers via genetic deficiency of or in chronically hypoxic male mice and by pharmacologic inhibition of Cxcl1 in monocrotaline-exposed rats. Both models exhibited decreased inflammatory blood monocytes, as well as interstitial macrophages, leading to a substantial decrease in arteriolar remodeling but with a less robust hemodynamic effect. This study defines a direct mechanism by which interstitial macrophages expand in PH. It also demonstrates a pathway for pulmonary vascular remodeling in PH that depends upon interstitial macrophage-dependent inflammation yet is dissociated, at least in part, from hemodynamic consequences, thus offering guidance on future anti-inflammatory therapeutic strategies in this disease.

No abstract

In pre-clinical models of PH CD47 targets cMyc to increase ET-1 signaling. In clinical PH TSP1-CD47 is upregulated, and in both, contributes to pulmonary arterial vasculopathy and dysfunction.