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.
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
and
(FABP4
), and one highly expressing
and
(SPP1
). SPP1
macrophages in fibrotic lower lobes showed highly upregulated
and
expression. Low-level local proliferation of SPP1
macrophages in normal lungs was strikingly increased in IPF lungs.Co-localisation and causal modelling supported the role for these highly proliferative SPP1
macrophages in activation of IPF myofibroblasts in lung fibrosis. These data suggest that SPP1
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.
Idiopathic pulmonary fibrosis (IPF) and systemic sclerosis-associated interstitial lung disease (SSc-ILD) differ in the predominant demographics and identified genetic risk alleles of effected ...patients, however both diseases frequently progress to respiratory failure and death. Contrasting advanced SSc-ILD to IPF provides insight to the role dysregulated immunity may play in pulmonary fibrosis. To analyze cell-type specific transcriptome commonalities and differences between IPF and SSc-ILD, we compared single-cell RNA-sequencing (scRNA-seq) of 21 explanted lung tissue specimens from patients with advanced IPF, SSc-ILD, and organ donor controls. Comparison of IPF and SSc-ILD tissue identified divergent patterns of interferon signaling, with interferon-gamma signaling upregulated in the
and
macrophages, cytotoxic T cells, and natural kill cells of IPF, while type I interferon signaling and production was upregulated in the corresponding SSc-ILD populations. Plasmacytoid dendritic cells were found in diseased lungs only, and exhibited upregulated cellular stress pathways in SSc-ILD compared to IPF. Alveolar type I cells were dramatically decreased in both IPF and SSc-ILD, with a distinct transcriptome signature separating these cells by disease.
aberrant basaloid cells exhibiting markers of cellular senescence and epithelial-mesenchymal transition were identified in SSc-ILD for the first time. In summary, our study utilizes the enriched capabilities of scRNA-seq to identify key divergent cell types and pathways between IPF and SSc-ILD, providing new insights into the shared and distinct mechanisms between idiopathic and autoimmune interstitial lung diseases.
Despite recent improvements in management of idiopathic pulmonary arterial hypertension, mortality remains high. Understanding the alterations in the transcriptome–phenotype of the key lung cells ...involved could provide insight into the drivers of pathogenesis. In this study, we examined differential gene expression of cell types implicated in idiopathic pulmonary arterial hypertension from lung explants of patients with idiopathic pulmonary arterial hypertension compared to control lungs. After tissue digestion, we analyzed all cells from three idiopathic pulmonary arterial hypertension and six control lungs using droplet-based single cell RNA-sequencing. After dimensional reduction by t-stochastic neighbor embedding, we compared the transcriptomes of endothelial cells, pericyte/smooth muscle cells, fibroblasts, and macrophage clusters, examining differential gene expression and pathways implicated by analysis of Gene Ontology Enrichment. We found that endothelial cells and pericyte/smooth muscle cells had the most differentially expressed gene profile compared to other cell types. Top differentially upregulated genes in endothelial cells included novel genes: ROBO4, APCDD1, NDST1, MMRN2, NOTCH4, and DOCK6, as well as previously reported genes: ENG, ORAI2, TFDP1, KDR, AMOTL2, PDGFB, FGFR1, EDN1, and NOTCH1. Several transcription factors were also found to be upregulated in idiopathic pulmonary arterial hypertension endothelial cells including SOX18, STRA13, LYL1, and ELK, which have known roles in regulating endothelial cell phenotype. In particular, SOX18 was implicated through bioinformatics analyses in regulating the idiopathic pulmonary arterial hypertension endothelial cell transcriptome. Furthermore, idiopathic pulmonary arterial hypertension endothelial cells upregulated expression of FAM60A and HDAC7, potentially affecting epigenetic changes in idiopathic pulmonary arterial hypertension endothelial cells. Pericyte/smooth muscle cells expressed genes implicated in regulation of cellular apoptosis and extracellular matrix organization, and several ligands for genes showing increased expression in endothelial cells. In conclusion, our study represents the first detailed look at the transcriptomic landscape across idiopathic pulmonary arterial hypertension lung cells and provides robust insight into alterations that occur in vivo in idiopathic pulmonary arterial hypertension lungs.
Studies identifying nonspecific interstitial pneumonia (NSIP) as the predominant histopathology in systemic sclerosis-associated interstitial lung disease (SSc-ILD) have primarily utilised surgical ...lung biopsies in early disease. These case series may only reflect the histopathology of early disease and differ from the histopathology of advanced disease in those with respiratory failure.
Patients receiving a lung transplant for a diagnosis of SSc at a single centre from 2000-2021 were included for retrospective analysis. All explanted lungs underwent histopathology review as part of routine care.
127 patients with SSc received a native lung transplant during the study period. Usual interstitial pneumonia (UIP) was identified in 111 explants (87.4%), NSIP in 45 (35.4%) explants, organising pneumonia in 11 explants (8.7%), and lymphocytic bronchitis in 2 explants (1.6%). Areas of both UIP and NSIP were identified in 37 explants (29.1%), with only 9 explants (7.1%) showing neither UIP nor NSIP. Aspiration was identified on histology in 49 (38.6%) explants. Pathology results were available from a prior surgical lung biopsy for 19 patients, with 11 patients maintaining the same primary pathology on biopsy and explant (2 NSIP, 9 UIP) and 8 patients showing different pathology at the timepoints, all of whom had UIP on explant. Most patients (101, 79.5%) had evidence of pulmonary hypertension and vasculopathy on explant.
UIP is the predominant histopathology in patients with SSc receiving a lung transplant, with many patients concurrently having both NSIP and UIP or showing progression from NSIP to UIP over time before transplant.
Objective
Systemic sclerosis–associated interstitial lung disease (SSc‐ILD) is the leading cause of death in patients with SSc with unclear pathogenesis and limited treatment options. Evidence ...strongly supports an important role for profibrotic secreted phosphoprotein 1 (SPP1)–expressing macrophages in SSc‐ILD. This study was undertaken to define the transcriptome and chromatin structural changes of SPP1 SSc‐ILD macrophages in order to better understand their role in promoting fibrosis and to identify transcription factors associated with open chromatin driving their altered phenotype.
Methods
We performed single‐cell RNA sequencing (scRNA‐Seq) on 11 explanted SSc‐ILD and healthy control lung samples, as well as single‐cell assay for transposase‐accessible chromatin sequencing on 5 lung samples to define altered chromatin accessibility of SPP1 macrophages. We predicted transcription factors regulating SPP1 macrophages using single‐cell regulatory network inference and clustering (SCENIC) and determined transcription factor binding sites associated with global alterations in SPP1 chromatin accessibility using Signac/Seurat.
Results
We identified distinct macrophage subpopulations using scRNA‐Seq analysis in healthy and SSc‐ILD lungs and assessed gene expression changes during the change of healthy control macrophages into SPP1 macrophages. Analysis of open chromatin validated SCENIC predictions, indicating that microphthalmia‐associated transcription factor, transcription factor EB, activating transcription factor 6, sterol regulatory element binding transcription factor 1, basic helix‐loop‐helix family member E40, Kruppel‐like factor 6, ETS variant transcription factor 5, and/or members of the activator protein 1 family of transcription factors regulate SPP1 macrophage differentiation.
Conclusion
Our findings shed light on the underlying changes in chromatin structure and transcription factor regulation of profibrotic SPP1 macrophages in SSc‐ILD. Similar alterations in SPP1 macrophages may underpin fibrosis in other organs involved in SSc and point to novel targets for the treatment of SSc‐ILD, specifically targeting profibrotic macrophages.
Multiple observations indicate a role for lymphocytes in driving autoimmunity in SSc. While T and NK cells have been studied in SSc whole blood and bronchoalveolar lavage fluid, their role remains ...unclear, partly because no studies have analysed these cell types in SSc-interstitial lung disease (ILD) lung tissue. This research aimed to identify and analyse the lymphoid subpopulations in SSc-ILD lung explants.
Lymphoid populations from 13 SSc-ILD and 6 healthy control (HC) lung explants were analysed using Seurat following single-cell RNA sequencing. Lymphoid clusters were identified by their differential gene expression. Absolute cell numbers and cell proportions in each cluster were compared between cohorts. Additional analyses were performed using pathway analysis, pseudotime and cell ligand-receptor interactions.
Activated CD16+ NK cells, CD8+ tissue resident memory T cells and Treg cells were proportionately higher in SSc-ILD compared with HC lungs. Activated CD16+ NK cells in SSc-ILD showed upregulated granzyme B, IFN-γ and CD226. Amphiregulin, highly upregulated by NK cells, was predicted to interact with epidermal growth factor receptor on several bronchial epithelial cell populations. Shifts in CD8+ T cell populations indicated a transition from resting to effector to tissue resident phenotypes in SSc-ILD.
SSc-ILD lungs show activated lymphoid populations. Activated cytotoxic NK cells suggest they may kill alveolar epithelial cells, while their expression of amphiregulin suggests they may also induce bronchial epithelial cell hyperplasia. CD8+ T cells in SSc-ILD appear to transition from resting to the tissue resident memory phenotype.
BACKGROUND: Pulmonary hypertension (PH) is a common complication of systemic sclerosis (SSc) and a leading cause of mortality among patients with this disease. PH can also occur as an idiopathic ...condition (idiopathic pulmonary arterial hypertension). Investigation of transcriptomic alterations in vascular populations is critical to elucidating cellular mechanisms underlying pathobiology of SSc-associated and idiopathic PH. METHODS: We analyzed single-cell RNA sequencing profiles of endothelial and perivascular mesenchymal populations from explanted lung tissue of patients with SSc-associated PH (n=16), idiopathic pulmonary arterial hypertension (n=3), and healthy controls (n=15). Findings were validated by immunofluorescence staining of explanted human lung tissue. RESULTS: Three disease-associated endothelial populations emerged. Two angiogenic endothelial cell (EC) subtypes markedly expanded in SSc-associated PH lungs: tip ECs expressing canonical tip markers PGF and APLN and phalanx ECs expressing genes associated with vascular development, endothelial barrier integrity, and Notch signaling. Gene regulatory network analysis suggested enrichment of Smad1 and PPAR-γ (peroxisome proliferator-activated receptor-γ) regulon activities in these 2 populations, respectively. Mapping of potential ligand-receptor interactions highlighted Notch, apelin-APJ, and angiopoietin-Tie signaling pathways between angiogenic ECs and perivascular cells. Transitional cells, expressing both endothelial and pericyte/smooth muscle cell markers, provided evidence for the presence of endothelial-to-mesenchymal transition. Transcriptional programs associated with arterial endothelial dysfunction implicated VEGF-A (vascular endothelial growth factor-A), TGF-β1, angiotensin, and TNFSF12/TWEAK in the injury/remodeling phenotype of PH arterial ECs. CONCLUSIONS: These data provide high-resolution insights into the complexity and plasticity of the pulmonary endothelium in SSc-associated PH and idiopathic pulmonary arterial hypertension and provide direct molecular insights into soluble mediators and transcription factors driving PH vasculopathy.
Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease of unknown etiology. The genes TOLLIP and MUC5B play important roles in lung host defense, which is an immune process influenced by ...oxidative signaling. Whether polymorphisms in TOLLIP and MUC5B modify the effect of immunosuppressive and antioxidant therapy in individuals with IPF is unknown.
To determine whether single-nucleotide polymorphisms (SNPs) within TOLLIP and MUC5B modify the effect of interventions in subjects participating in the Evaluating the Effectiveness of Prednisone, Azathioprine, and N-Acetylcysteine in Patients with Idiopathic Pulmonary Fibrosis (PANTHER-IPF) clinical trial.
SNPs within TOLLIP (rs5743890/rs5743894/rs5743854/rs3750920) and MUC5B (rs35705950) were genotyped. Interaction modeling was conducted with multivariable Cox regression followed by genotype-stratified survival analysis using a composite endpoint of death, transplantation, hospitalization, or a decline of ≥ 10% in FVC.
Significant interaction was observed between N-acetylcysteine (NAC) therapy and rs3750920 within TOLLIP (P interaction = 0.001). After stratifying by rs3750920 genotype, NAC therapy was associated with a significant reduction in composite endpoint risk (hazard ratio, 0.14; 95% confidence interval, 0.02-0.83; P = 0.03) in those with a TT genotype, but a nonsignificant increase in composite endpoint risk (hazard ratio, 3.23; 95% confidence interval, 0.79-13.16; P = 0.10) was seen in those with a CC genotype. These findings were then replicated in an independent IPF cohort.
NAC may be an efficacious therapy for individuals with IPF with an rs3750920 (TOLLIP) TT genotype, but it was associated with a trend toward harm in those with a CC genotype. A genotype-stratified prospective clinical trial should be conducted before any recommendation regarding the use of off-label NAC to treat IPF.
Patients with interstitial lung disease (ILD) may have features of connective tissue disease (CTD), but lack findings diagnostic of a specific CTD. A recent European Respiratory Society/American ...Thoracic Society research statement proposed criteria for patients with interstitial pneumonia with autoimmune features (IPAF).We applied IPAF criteria to patients with idiopathic interstitial pneumonia and undifferentiated CTD-ILD (UCTD). We then characterised the clinical, serological and morphological features of the IPAF cohort, compared outcomes to other ILD cohorts and validated individual IPAF domains using survival as an endpoint.Of 422 patients, 144 met IPAF criteria. Mean age was 63.2 years with a slight female predominance. IPAF cohort survival was marginally better than patients with idiopathic pulmonary fibrosis, but worse than CTD-ILD. A non-usual interstitial pneumonia pattern was associated with improved survival, as was presence of the clinical domain. A modified IPAF cohort of those meeting the clinical domain and a radiographic or histological feature within the morphological domain displayed survival similar to those with CTD-ILD.IPAF is common among patients with idiopathic interstitial pneumonia and UCTD. Specific IPAF features can identify subgroups with differential survival. Further research is needed to replicate these findings and determine whether patients meeting IPAF criteria benefit from immunosuppressive therapy.
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