Single-Cell Profiling Reveals Immune Aberrations in Progressive Idiopathic Pulmonary Fibrosis.

RATIONALE: Changes in peripheral blood cell populations have been observed but not detailed at single-cell resolution in idiopathic pulmonary fibrosis (IPF). OBJECTIVES: To provide an atlas of the changes in the peripheral immune system in stable and progressive IPF. METHODS: Peripheral blood mononuclear cells (PBMCs) from IPF patients and controls were profiled using 10x Chromium 5' single-cell RNA sequencing (scRNA-seq). Flow cytometry was used for validation. Protein concentrations of Regulatory T-cells (Tregs) and Monocytes chemoattractants were measured in plasma and lung homogenates from patients and controls. MEASUREMENTS AND MAIN RESULTS: Thirty-eight PBMC samples from 25 patients with IPF and 13 matched controls yielded 149,564 cells that segregated into 23 subpopulations. Classical monocytes were increased in progressive and stable IPF compared to controls (32.1%, 25.2%, 17.9%, respectively, p<0.05). Total lymphocytes were decreased in IPF vs controls, and in progressive vs stable IPF (52.6% vs 62.6%, p=0.035). Tregs were increased in progressive vs stable IPF (1.8% vs 1.1% of all PBMC, p=0.007), although not different than controls, and may be associated with decreased survival (P=0.009 in Kaplan-Meier analysis; P=0.069 after adjusting for age, sex, and baseline FVC). Flow cytometry analysis confirmed this finding in an independent cohort of IPF patients. Fraction of Tregs out of all T cells was also increased in two cohorts of lung scRNA-seq. CCL22 and CCL18, ligands for CCR4 and CCR8 Treg chemotaxis receptors, were increased in IPF. CONCLUSIONS: The single-cell atlas of the peripheral immune system in IPF, reveals an outcome-predictive increase in classical monocytes and Tregs, as well as evidence for a lung-blood immune recruitment axis involving CCL7 (for classical monocytes) and CCL18/CCL22 (for Tregs).

Single Cell Analysis Reveals Novel Immune Perturbations in Fibrotic Hypersensitivity Pneumonitis.

Rationale: Fibrotic hypersensitivity pneumonitis is a debilitating interstitial lung disease driven by incompletely understood immune mechanisms. Objectives: To elucidate immune aberrations in fibrotic hypersensitivity pneumonitis in single-cell resolution. Methods: Single-cell 5' RNA sequencing was conducted on peripheral blood mononuclear cells and bronchoalveolar lavage cells obtained from 45 patients with fibrotic hypersensitivity pneumonitis, 63 idiopathic pulmonary fibrosis, 4 non-fibrotic hypersensitivity pneumonitis, and 36 healthy controls in the United States and Mexico. Analyses included differential gene expression (Seurat), transcription factor activity imputation (DoRothEA-VIPER), and trajectory analyses (Monocle3/Velocyto-scVelo-CellRank). Measurements and Main Results: Overall, 501,534 peripheral blood mononuclear cells from 110 patients and controls and 88,336 bronchoalveolar lavage cells from 19 patients were profiled. Compared to controls, fibrotic hypersensitivity pneumonitis has elevated classical monocytes (adjusted-p=2.5e-3) and are enriched in CCL3hi/CCL4hi and S100Ahi classical monocytes (adjusted-p<2.2e-16). Trajectory analyses demonstrate that S100Ahi classical monocytes differentiate into SPP1hi lung macrophages associated with fibrosis. Compared to both controls and idiopathic pulmonary fibrosis, fibrotic hypersensitivity pneumonitis patient cells are significantly enriched in GZMhi cytotoxic T cells. These cells exhibit transcription factor activities indicative of TGFß and TNFα/NFκB pathways. These results are publicly available at https://ildimmunecellatlas.org. Conclusions: Single-cell transcriptomics of fibrotic hypersensitivity pneumonitis patients uncovered novel immune perturbations, including previously undescribed increases in GZMhi cytotoxic CD4+ and CD8+ T cells - reflecting this disease's unique inflammatory T-cell driven nature - as well as increased S100Ahi and CCL3hi/CCL4hi classical monocytes also observed in idiopathic pulmonary fibrosis. Both cell populations may guide the development of new biomarkers and therapeutic interventions.

Anti-inflammatory roles of type I interferon signaling in the lung.

Excessive or persistent inflammation may have detrimental effects on lung structure and function. Currently, our understanding of conserved host mechanisms that control the inflammatory response remains incompletely understood. In this study, we investigated the role of type I interferon signaling in the inflammatory response against diverse clinically relevant stimuli. Using mice deficient in type I interferon signaling (IFNAR1-/-), we demonstrate that the absence of interferon signaling resulted in a robust and persistent inflammatory response against Pseudomonas aeruginosa, lipopolysaccharide, and chemotherapeutic agent bleomycin. The elevated inflammatory response in IFNAR1-/- mice was manifested as elevated myeloid cells, such as macrophages and neutrophils, in the bronchoalveolar lavage. The inflammatory cell response in the IFNAR1-/- mice persisted to 14 days and there is impaired recovery and fibrotic remodeling of the lung in IFNAR1-/- mice after bleomycin injury. In the Pseudomonas infection model, the elevated inflammatory cell response led to improved bacterial clearance in IFNAR1-/- mice, although there was similar lung injury and survival. We performed RNA sequencing of lung tissue in wild-type and IFNAR1-/- mice after LPS and bleomycin injury. Our unbiased analysis identified differentially expressed genes between IFNAR1-/- and wild-type mice, including previously unknown regulation of nucleotide-binding oligomerization domain (NOD)-like receptor signaling, retinoic acid-inducible gene-I (RIG-I) signaling, and necroptosis pathway by type I interferon signaling in both models. These data provide novel insights into the conserved anti-inflammatory mechanisms of the type I interferon signaling.NEW & NOTEWORTHY Type I interferons are known for their antiviral activities. In this study, we demonstrate a conserved anti-inflammatory role of type I interferon signaling against diverse stimuli in the lung. We show that exacerbated inflammatory response in the absence of type I interferon signaling has both acute and chronic consequences in the lung including structural changes.

Saracatinib, a Selective Src Kinase Inhibitor, Blocks Fibrotic Responses in Preclinical Models of Pulmonary Fibrosis.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and often fatal disorder. Two U.S. Food and Drug Administration-approved antifibrotic drugs, nintedanib and pirfenidone, slow the rate of decline in lung function, but responses are variable and side effects are common. Objectives: Using an in silico data-driven approach, we identified a robust connection between the transcriptomic perturbations in IPF disease and those induced by saracatinib, a selective Src kinase inhibitor originally developed for oncological indications. Based on these observations, we hypothesized that saracatinib would be effective at attenuating pulmonary fibrosis. Methods: We investigated the antifibrotic efficacy of saracatinib relative to nintedanib and pirfenidone in three preclinical models: 1) in vitro in normal human lung fibroblasts; 2) in vivo in bleomycin and recombinant Ad-TGF-ß (adenovirus transforming growth factor-ß) murine models of pulmonary fibrosis; and 3) ex vivo in mice and human precision-cut lung slices from these two murine models as well as patients with IPF and healthy donors. Measurements and Main Results: In each model, the effectiveness of saracatinib in blocking fibrogenic responses was equal or superior to nintedanib and pirfenidone. Transcriptomic analyses of TGF-ß-stimulated normal human lung fibroblasts identified specific gene sets associated with fibrosis, including epithelial-mesenchymal transition, TGF-ß, and WNT signaling that was uniquely altered by saracatinib. Transcriptomic analysis of whole-lung extracts from the two animal models of pulmonary fibrosis revealed that saracatinib reverted many fibrogenic pathways, including epithelial-mesenchymal transition, immune responses, and extracellular matrix organization. Amelioration of fibrosis and inflammatory cascades in human precision-cut lung slices confirmed the potential therapeutic efficacy of saracatinib in human lung fibrosis. Conclusions: These studies identify novel Src-dependent fibrogenic pathways and support the study of the therapeutic effectiveness of saracatinib in IPF treatment.

Integrated Single-Cell Atlas of Endothelial Cells of the Human Lung.

BACKGROUND: Cellular diversity of the lung endothelium has not been systematically characterized in humans. We provide a reference atlas of human lung endothelial cells (ECs) to facilitate a better understanding of the phenotypic diversity and composition of cells comprising the lung endothelium. METHODS: We reprocessed human control single-cell RNA sequencing (scRNAseq) data from 6 datasets. EC populations were characterized through iterative clustering with subsequent differential expression analysis. Marker genes were validated by fluorescent microscopy and in situ hybridization. scRNAseq of primary lung ECs cultured in vitro was performed. The signaling network between different lung cell types was studied. For cross-species analysis or disease relevance, we applied the same methods to scRNAseq data obtained from mouse lungs or from human lungs with pulmonary hypertension. RESULTS: Six lung scRNAseq datasets were reanalyzed and annotated to identify >15 000 vascular EC cells from 73 individuals. Differential expression analysis of EC revealed signatures corresponding to endothelial lineage, including panendothelial, panvascular, and subpopulation-specific marker gene sets. Beyond the broad cellular categories of lymphatic, capillary, arterial, and venous ECs, we found previously indistinguishable subpopulations; among venous EC, we identified 2 previously indistinguishable populations: pulmonary-venous ECs (COL15A1neg) localized to the lung parenchyma and systemic-venous ECs (COL15A1pos) localized to the airways and the visceral pleura; among capillary ECs, we confirmed their subclassification into recently discovered aerocytes characterized by EDNRB, SOSTDC1, and TBX2 and general capillary EC. We confirmed that all 6 endothelial cell types, including the systemic-venous ECs and aerocytes, are present in mice and identified endothelial marker genes conserved in humans and mice. Ligand-receptor connectome analysis revealed important homeostatic crosstalk of EC with other lung resident cell types. scRNAseq of commercially available primary lung ECs demonstrated a loss of their native lung phenotype in culture. scRNAseq revealed that endothelial diversity is maintained in pulmonary hypertension. Our article is accompanied by an online data mining tool (www.LungEndothelialCellAtlas.com). CONCLUSIONS: Our integrated analysis provides a comprehensive and well-crafted reference atlas of ECs in the normal lung and confirms and describes in detail previously unrecognized endothelial populations across a large number of humans and mice.

An endothelial microRNA-1-regulated network controls eosinophil trafficking in asthma and chronic rhinosinusitis.

BACKGROUND: Airway eosinophilia is a prominent feature of asthma and chronic rhinosinusitis (CRS), and the endothelium plays a key role in eosinophil trafficking. To date, microRNA-1 (miR-1) is the only microRNA known to be regulated in the lung endothelium in asthma models. OBJECTIVE: We sought to determine the role of endothelial miR-1 in allergic airway inflammation. METHODS: We measured microRNA and mRNA expression using quantitative RT-PCR. We used ovalbumin and house dust mite models of asthma. Endothelium-specific overexpression of miR-1 was achieved through lentiviral vector delivery or induction of a transgene. Tissue eosinophilia was quantified by using Congo red and anti-eosinophil peroxidase staining. We measured eosinophil binding with a Sykes-Moore adhesion chamber. Target recruitment to RNA-induced silencing complex was assessed by using anti-Argonaute2 RNA immunoprecipitation. Surface P-selectin levels were measured by using flow cytometry. RESULTS: Serum miR-1 levels had inverse correlations with sputum eosinophilia, airway obstruction, and number of hospitalizations in asthmatic patients and sinonasal tissue eosinophilia in patients with CRS. IL-13 stimulation decreased miR-1 levels in human lung endothelium. Endothelium-specific overexpression of miR-1 reduced airway eosinophilia and asthma phenotypes in murine models and inhibited IL-13-induced eosinophil binding to endothelial cells. miR-1 recruited P-selectin, thymic stromal lymphopoietin, eotaxin-3, and thrombopoietin receptor to the RNA-induced silencing complex; downregulated these genes in the lung endothelium; and reduced surface P-selectin levels in IL-13-stimulated endothelial cells. In our asthma and CRS cohorts, miR-1 levels correlated inversely with its target genes. CONCLUSION: Endothelial miR-1 regulates eosinophil trafficking in the setting of allergic airway inflammation. miR-1 has therapeutic potential in asthmatic patients and patients with CRS.

Role of dual-specificity protein phosphatase DUSP10/MKP-5 in pulmonary fibrosis.

Mitogen-activated protein kinase (MAPK) phosphatase 5 (MKP-5) is a member of the dual-specificity family of protein tyrosine phosphatases that negatively regulates p38 MAPK and the JNK. MKP-5-deficient mice exhibit improved muscle repair and reduced fibrosis in an animal model of muscular dystrophy. Here, we asked whether the effects of MKP-5 on muscle fibrosis extend to other tissues. Using a bleomycin-induced model of pulmonary fibrosis, we found that MKP-5-deficient mice were protected from the development of lung fibrosis, expressed reduced levels of hydroxyproline and fibrogenic genes, and displayed marked polarization towards an M1-macrophage phenotype. We showed that the profibrogenic effects of the transforming growth factor-ß1 (TGF-ß1) were inhibited in MKP-5-deficient lung fibroblasts. MKP-5-deficient fibroblasts exhibited enhanced p38 MAPK activity, impaired Smad3 phosphorylation, increased Smad7 levels, and decreased expression of fibrogenic genes. Myofibroblast differentiation was attenuated in MKP-5-deficient fibroblasts. Finally, we found that MKP-5 expression was increased in idiopathic pulmonary fibrosis (IPF)-derived lung fibroblasts but not in whole IPF lungs. These data suggest that MKP-5 plays an essential role in promoting lung fibrosis. Our results couple MKP-5 with the TGF-ß1 signaling machinery and imply that MKP-5 inhibition may serve as a therapeutic target for human lung fibrosis.

Integrating multiomics longitudinal data to reconstruct networks underlying lung development.

A comprehensive understanding of the dynamic regulatory networks that govern postnatal alveolar lung development is still lacking. To construct such a model, we profiled mRNA, microRNA, DNA methylation, and proteomics of developing murine alveoli isolated by laser capture microdissection at 14 predetermined time points. We developed a detailed comprehensive and interactive model that provides information about the major expression trajectories, the regulators of specific key events, and the impact of epigenetic changes. Intersecting the model with single-cell RNA-Seq data led to the identification of active pathways in multiple or individual cell types. We then constructed a similar model for human lung development by profiling time-series human omics data sets. Several key pathways and regulators are shared between the reconstructed models. We experimentally validated the activity of a number of predicted regulators, leading to new insights about the regulation of innate immunity during lung development.

A role for telomere length and chromosomal damage in idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis is a fatal lung disease characterized by a progressive formation of fibroblastic foci in the interstitium. This disease is strongly associated with telomere dysfunction but the extent of telomere shortening and consequent chromosomal damage within IPF lungs and with regional disease severity remains unknown. METHODS: Explanted IPF lungs (n = 10) were collected from transplant surgeries with six samples per lung analysed to capture the regional heterogeneity ranging from mild to severe disease. Non-used donor lungs (n = 6) were collected as "healthy" controls. Structural changes related to disease severity (microCT surface density), relative telomere length (real-time qPCR), and quantitative histology of chromosomal damage (γ-H2A.X) and extracellular matrix (elastin, total collagen, collagen 1, and collagen 3) were measured. A multivariate linear mixed-effects model controlling for subject was used to identify association of disease severity or fibrotic markers with telomere length and chromosomal damage. RESULTS: We observed shorter telomere length (p = 0.001) and increased chromosomal damage (p = 0.018) in IPF lungs compared to controls. In IPF lungs, telomere length was associated with total collagen (p < 0.001) but not with structural changes of disease severity. Chromosomal damage was positively associated with increased elastin (p = 0.006) and negatively with structural disease severity (p = 0.046). Extensive γ-H2A.X staining was also present in airway epithelial cells. CONCLUSIONS: Telomere length and chromosomal damage are involved in IPF with regional variation in telomere length and chromosomal damage associated with pathological changes in tissue structure and the extracellular matrix.

SH2 Domain-Containing Phosphatase-2 Is a Novel Antifibrotic Regulator in Pulmonary Fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a chronic fatal lung disease with dismal prognosis and no cure. The potential role of the ubiquitously expressed SH2 domain-containing tyrosine phosphatase-2 (SHP2) as a therapeutic target has not been studied in IPF. OBJECTIVES: To determine the expression, mechanistic role, and potential therapeutic usefulness of SHP2 in pulmonary fibrosis. METHODS: The effects of SHP2 overexpression and inhibition on fibroblast response to profibrotic stimuli were analyzed in vitro in primary human and mouse lung fibroblasts. In vivo therapeutic effects were assessed in the bleomycin model of lung fibrosis by SHP2-lentiviral administration and transgenic mice carrying a constitutively active SHP2 mutation. MEASUREMENTS AND MAIN RESULTS: SHP2 was down-regulated in lungs and lung fibroblasts obtained from patients with IPF. Immunolocalization studies revealed that SHP2 was absent within fibroblastic foci. Loss of SHP2 expression or activity was sufficient to induce fibroblast-to-myofibroblast differentiation in primary human lung fibroblasts. Overexpression of constitutively active SHP2 reduced the responsiveness of fibroblasts to profibrotic stimuli, including significant reductions in cell survival and myofibroblast differentiation. SHP2 effects were mediated through deactivation of fibrosis-relevant tyrosine kinase and serine/threonine kinase signaling pathways. Mice carrying the Noonan syndrome-associated gain-of-function SHP2 mutation (SHP2D61G/+) were resistant to bleomycin-induced pulmonary fibrosis. Restoration of SHP2 levels in vivo through lentiviral delivery blunted bleomycin-induced pulmonary fibrosis. CONCLUSIONS: Our data suggest that SHP2 is an important regulator of fibroblast differentiation, and its loss as observed in IPF facilitates profibrotic phenotypic changes. Augmentation of SHP2 activity or expression should be investigated as a novel therapeutic strategy for IPF.

Chitinase 3-like-1 regulates both visceral fat accumulation and asthma-like Th2 inflammation.

RATIONALE: Obesity, especially truncal obesity, is a risk factor for asthma incidence, prevalence, and severity. Chitinase 3-like-1 (Chi3l1) is an evolutionarily conserved moiety that plays a critical role in antipathogen and Th2 responses. However, the mechanisms that underlie the association between asthma and obesity and the role(s) of Chi3l1 in fat accumulation have not been defined. OBJECTIVES: To determine whether Chi3l1 is regulated by a high-fat diet (HFD) and simultaneously plays an important role(s) in the pathogenesis of asthma and obesity. METHODS: We evaluated the regulation of Chi3l1 by an HFD and Th2 inflammation. We also used genetically modified mice to define the roles of Chi3l1 in white adipose tissue (WAT) accumulation and Th2 inflammation and blockers of sirtuin 1 (Sirt1) to define its roles in these responses. Finally, the human relevance of these findings was assessed with a case-control study involving obese and lean control subjects and those with asthma. MEASUREMENTS AND MAIN RESULTS: These studies demonstrate that an HFD and aeroallergen challenge augment the expression of WAT and pulmonary Chi3l1. Chi3l1 also played a critical role in WAT accumulation and lung Th2 inflammation. In addition, Chi3l1 inhibited Sirt1 expression, and the deficient visceral fat and Th2 responses in Chi3l1 null mice were reversed by Sirt1 inhibition. Finally, serum and sputum Chi3l1 were positively associated with truncal adiposity, and serum Chi3l1 was associated with persistent asthma and low lung function in obese subjects with asthma. CONCLUSIONS: Chi3l1 is induced by an HFD and Th2 inflammation, and simultaneously contributes to the genesis of obesity and asthma.

Role of chitin and chitinase/chitinase-like proteins in inflammation, tissue remodeling, and injury.

The 18 glycosyl hydrolase family of chitinases is an ancient gene family that is widely expressed from prokaryotes to eukaryotes. In mammals, despite the absence of endogenous chitin, a number of chitinases and chitinase-like proteins (C/CLPs) have been identified. However, their roles have only recently begun to be elucidated. Acidic mammalian chitinase (AMCase) inhibits chitin-induced innate inflammation; augments chitin-free, allergen-induced Th2 inflammation; and mediates effector functions of IL-13. The CLPs BRP-39/YKL-40 (also termed chitinase 3-like 1) inhibit oxidant-induced lung injury, augments adaptive Th2 immunity, regulates apoptosis, stimulates alternative macrophage activation, and contributes to fibrosis and wound healing. In accord with these findings, levels of YKL-40 in the lung and serum are increased in asthma and other inflammatory and remodeling disorders and often correlate with disease severity. Our understanding of the roles of C/CLPs in inflammation, tissue remodeling, and tissue injury in health and disease is reviewed below.

Chitinase 1 is a biomarker for and therapeutic target in scleroderma-associated interstitial lung disease that augments TGF-ß1 signaling.

Interstitial lung disease (ILD) with pulmonary fibrosis is an important manifestation in systemic sclerosis (SSc, scleroderma) where it portends a poor prognosis. However, biomarkers that predict the development and or severity of SSc-ILD have not been validated, and the pathogenetic mechanisms that engender this pulmonary response are poorly understood. In this study, we demonstrate in two different patient cohorts that the levels of chitotriosidase (Chit1) bioactivity and protein are significantly increased in the circulation and lungs of SSc patients compared with demographically matched controls. We also demonstrate that, compared with patients without lung involvement, patients with ILD show high levels of circulating Chit1 activity that correlate with disease severity. Murine modeling shows that in comparison with wild-type mice, bleomycin-induced pulmonary fibrosis was significantly reduced in Chit1⁻/⁻ mice and significantly enhanced in lungs from Chit1 overexpressing transgenic animals. In vitro studies also demonstrated that Chit1 interacts with TGF-ß1 to augment fibroblast TGF-ß receptors 1 and 2 expression and TGF-ß-induced Smad and MAPK/ERK activation. These studies indicate that Chit1 is potential biomarker for ILD in SSc and a therapeutic target in SSc-associated lung fibrosis and demonstrate that Chit1 augments TGF-ß1 effects by increasing receptor expression and canonical and noncanonical TGF-ß1 signaling.

Noninvasive assessment of the lung inflammation-fibrosis axis by targeted imaging of CMKLR1.

Precision management of fibrotic lung diseases is challenging due to their diverse clinical trajectories and lack of reliable biomarkers for risk stratification and therapeutic monitoring. Here, we validated the accuracy of CMKLR1 as an imaging biomarker of the lung inflammation-fibrosis axis. By analyzing single-cell RNA sequencing datasets, we demonstrated CMKLR1 expression as a transient signature of monocyte-derived macrophages (MDMφ) enriched in patients with idiopathic pulmonary fibrosis (IPF). Consistently, we identified MDMφ as the major driver of the uptake of CMKLR1-targeting peptides in a murine model of bleomycin-induced lung fibrosis. Furthermore, CMKLR1-targeted positron emission tomography in the murine model enabled quantification and spatial mapping of inflamed lung regions infiltrated by CMKLR1-expressing macrophages and emerged as a robust predictor of subsequent lung fibrosis. Last, high CMKLR1 expression by bronchoalveolar lavage cells identified an inflammatory endotype of IPF with poor survival. Our investigation supports the potential of CMKLR1 as an imaging biomarker for endotyping and risk stratification of fibrotic lung diseases.

Mice heterozygous for mutation in Atm, the gene involved in ataxia-telangiectasia, have heightened susceptibility to cancer.

Ataxia-telangiectasia is characterized by radiosensitivity, genome instability and predisposition to cancer. Heterozygous carriers of ATM, the gene defective in ataxia-telangiectasia, have a higher than normal risk of developing breast and other cancers. We demonstrate here that Atm 'knock-in' (Atm-Delta SRI) heterozygous mice harboring an in-frame deletion corresponding to the human 7636del9 mutation show an increased susceptibility to developing tumors. In contrast, no tumors are observed in Atm knockout (Atm(+/-)) heterozygous mice. In parallel, we report the appearance of tumors in 6 humans from 12 families who are heterozygous for the 7636del9 mutation. Expression of ATM cDNA containing the 7636del9 mutation had a dominant-negative effect in control cells, inhibiting radiation-induced ATM kinase activity in vivo and in vitro. This reduces the survival of these cells after radiation exposure and enhances the level of radiation-induced chromosomal aberrations. These results show for the first time that mouse carriers of a mutated Atm that are capable of expressing Atm have a higher risk of cancer. This finding provides further support for cancer predisposition in human ataxia-telangiectasia carriers.

SH2 Domain-Containing Phosphatase-SHP2 Attenuates Fibrotic Responses through Negative Regulation of Mitochondrial Metabolism in Lung Fibroblasts.

BACKGROUND: We have previously shown that SHP2 downregulation may predispose fibroblasts to differentiate into myofibroblasts and proposed a role for SHP2 downregulation in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Recent data have shown that SHP2 localizes to the mitochondrial intercristae, and its overexpression enhances mitochondrial metabolism leading to oxidative stress and senescence. OBJECTIVE: To determine the effect of SHP2 on fibrotic responses. METHODS AND RESULTS: Primary mouse lung fibroblasts derived from mice carrying a conditional knock-in mutation (D61G/+), rendering the SHP2 catalytic domain constitutively active, had reduced proliferation (1.6-fold, p < 0.05), migration (2-fold, p < 0.05), as well as reduced responsiveness of TGFB-1 induced fibroblasts-to-myofibroblasts differentiation, compared to wild-type ones. Electron microscope analysis revealed that SHP2 D61G/+ mouse lung fibroblasts were characterized by mitochondrial abnormalities, including swollen mitochondria with disrupted electron-lucent cristae and an increased number of autophagosomes compared to wild-type ones. SHP2 D61G/+ MLFs exhibited increased protein levels of autophagy markers, including LC3B-II and p-62, evidence that was confirmed by immunofluorescence analysis. Mitochondrial function analysis revealed that stable (genotype D61G/+) overexpression of SHP2 led to impaired mitochondrial function, as assessed by decreased mitochondrial membrane potential (1.29-fold, p < 0.05), coupling efficiency (1.82 fold, p < 0.05), oxygen consumption rate (1.9-fold, p < 0.05), and increased reactive oxygen species production both at baseline (1.75-fold, p < 0.05) and following H2O2 stimulation (1.63-fold, p < 0.05) compared to wild-type ones (SHP2+/+). SHP2 D61G/+ mouse lung fibroblasts showed enhanced AMPK activity, as well as decreased activation of the mTORC1 signaling pathway, potentially leading to ineffective mitochondrial metabolism and increased autophagy. CONCLUSIONS: SHP2 attenuates fibrotic responses in fibroblast cell lines through negative regulation of mitochondrial metabolism and induction of autophagy. SHP2 activation may represent a promising therapeutic strategy for patients with fibrotic lung diseases.

Single-cell profiling reveals immune aberrations in progressive idiopathic pulmonary fibrosis.

Rationale: Changes in peripheral blood cell populations have been observed but not detailed at single-cell resolution in idiopathic pulmonary fibrosis (IPF). Objectives: To provide an atlas of the changes in the peripheral immune system in stable and progressive IPF. Methods: Peripheral blood mononuclear cells (PBMCs) from IPF patients and controls were profiled using 10x Chromium 5' single-cell RNA sequencing (scRNA-seq). Flow cytometry was used for validation. Protein concentrations of Regulatory T-cells (Tregs) and Monocytes chemoattractants were measured in plasma and lung homogenates from patients and controls. Measurements and Main Results: Thirty-eight PBMC samples from 25 patients with IPF and 13 matched controls yielded 149,564 cells that segregated into 23 subpopulations, corresponding to all expected peripheral blood cell populations. Classical monocytes were increased in progressive and stable IPF compared to controls (32.1%, 25.2%, 17.9%, respectively, p<0.05). Total lymphocytes were decreased in IPF vs controls, and in progressive vs stable IPF (52.6% vs 62.6%, p=0.035). Tregs were increased in progressive IPF (1.8% vs 1.1%, p=0.007), and were associated with decreased survival (P=0.009 in Kaplan-Meier analysis). Flow cytometry analysis confirmed this finding in an independent cohort of IPF patients. Tregs were also increased in two cohorts of lung scRNA-seq. CCL22 and CCL18, ligands for CCR4 and CCR8 Treg chemotaxis receptors, were increased in IPF. Conclusions: The single-cell atlas of the peripheral immune system in IPF, reveals an outcome-predictive increase in classical monocytes and Tregs, as well as evidence for a lung-blood immune recruitment axis involving CCL7 (for classical monocytes) and CCL18/CCL22 (for Tregs).

Peripheral Blood Single-Cell Sequencing Uncovers Common and Specific Immune Aberrations in Fibrotic Lung Diseases.

Rationale and Objectives: The extent and commonality of peripheral blood immune aberrations in fibrotic interstitial lung diseases are not well characterized. In this study, we aimed to identify common and distinct immune aberrations in patients with idiopathic pulmonary fibrosis (IPF) and fibrotic hypersensitivity pneumonitis (FHP) using cutting-edge single-cell profiling technologies. Methods: Single-cell RNA sequencing was performed on patients and healthy controls' peripheral blood and bronchoalveolar lavage samples using 10X Genomics 5' gene expression and V(D)J profiling. Cell type composition, transcriptional profiles, cellular trajectories and signaling, and T and B cell receptor repertoires were studied. The standard Seurat R pipeline was followed for cell type composition and differential gene expression analyses. Transcription factor activity was imputed using the DoRothEA-VIPER algorithm. Pseudotime analyses were conducted using Monocle3, while RNA velocity analyses were performed with Velocyto, scVelo, and CellRank. Cell-cell connectomics were assessed using the Connectome R package. V(D)J analyses were conducted using CellRanger and Immcantation frameworks. Across all analyses, disease group differences were assessed using the Wilcoxon rank-sum test. Measurements and Main Results: 327,990 cells from 83 samples were profiled. Overall, changes in monocytes were common to IPF and FHP, whereas lymphocytes exhibited disease-specific aberrations. Both diseases displayed enrichment of CCL3 hi /CCL4 hi CD14+ monocytes (p<2.2e-16) and S100A hi CD14+ monocytes (p<2.2e-16) versus controls. Trajectory and RNA velocity analysis suggested that pro-fibrotic macrophages observed in BAL originated from peripheral blood monocytes. Lymphocytes exhibited disease-specific aberrations, with CD8+ GZMK hi T cells and activated B cells primarily enriched in FHP patients. V(D)J analyses revealed unique T and B cell receptor complementarity-determining region 3 (CDR3) amino acid compositions (p<0.05) in FHP and significant IgA enrichment in IPF (p<5.2e-7). Conclusions: We identified common and disease-specific immune mechanisms in IPF and FHP; S100A hi monocytes and SPP1 hi macrophages are common to IPF and FHP, whereas GMZK hi T lymphocytes and T and B cell receptor repertoires were unique in FHP. Our findings open novel strategies for the diagnosis and treatment of IPF and FHP.

Unagi: Deep Generative Model for Deciphering Cellular Dynamics and In-Silico Drug Discovery in Complex Diseases.

Human diseases are characterized by intricate cellular dynamics. Single-cell sequencing provides critical insights, yet a persistent gap remains in computational tools for detailed disease progression analysis and targeted in-silico drug interventions. Here, we introduce UNAGI, a deep generative neural network tailored to analyze time-series single-cell transcriptomic data. This tool captures the complex cellular dynamics underlying disease progression, enhancing drug perturbation modeling and discovery. When applied to a dataset from patients with Idiopathic Pulmonary Fibrosis (IPF), UNAGI learns disease-informed cell embeddings that sharpen our understanding of disease progression, leading to the identification of potential therapeutic drug candidates. Validation via proteomics reveals the accuracy of UNAGI's cellular dynamics analyses, and the use of the Fibrotic Cocktail treated human Precision-cut Lung Slices confirms UNAGI's predictions that Nifedipine, an antihypertensive drug, may have antifibrotic effects on human tissues. UNAGI's versatility extends to other diseases, including a COVID dataset, demonstrating adaptability and confirming its broader applicability in decoding complex cellular dynamics beyond IPF, amplifying its utility in the quest for therapeutic solutions across diverse pathological landscapes.