B cell immune repertoire sequencing in tobacco cigarette smoking, vaping, and chronic obstructive pulmonary disease in the COPDGene cohort.

Rationale: Cigarette smoking (CS) impairs B cell function and antibody production, increasing infection risk. The impact of e-cigarette use ('vaping') and combined CS and vaping ('dual-use') on B cell activity is unclear. Objective: To examine B cell receptor sequencing (BCR-seq) profiles associated with CS, vaping, dual-use, COPD-related outcomes, and demographic factors. Methods: BCR-seq was performed on blood RNA samples from 234 participants in the COPDGene study. We assessed multivariable associations of B cell function measures (immunoglobulin heavy chain (IGH) subclass expression and usage, class-switching, V-segment usage, and clonal expansion) with CS, vaping, dual-use, COPD severity, age, sex, and race. We adjusted for multiple comparisons using the Benjamini-Hochberg method, identifying significant associations at 5% FDR and suggestive associations at 10% FDR. Results: Among 234 non-Hispanic white (NHW) and African American (AA) participants, CS and dual-use were significantly positively associated with increased secretory IgA production, with dual-use showing the strongest associations. Dual-use was positively associated with class switching and B cell clonal expansion, indicating increased B cell activation, with similar trends in those only smoking or only vaping. We observed significant associations between race and IgG antibody usage. AA participants had higher IgG subclass proportions and lower IgM usage compared to NHW participants. Conclusions: CS and vaping additively enhance B cell activation, most notably in dual-users. Self-reported race was strongly associated with IgG isotype usage. These findings highlight associations between B cell activation and antibody transcription, suggesting potential differences in immune and vaccine responses linked to CS, vaping, and race.

Identification of Severe Acute Exacerbations of Chronic Obstructive Pulmonary Disease Subgroups by Machine Learning Implementation in Electronic Health Records.

Rationale: Acute exacerbations of COPD (AECOPD) are heterogeneous. Machine learning (ML) has previously been used to dissect some of the heterogeneity in COPD. The widespread adoption of electronic health records (EHRs) has led to the rapid accumulation of large amounts of patient data as part of routine clinical care. However, it is unclear whether the implementation of ML in EHR-derived data has the potential to identify subgroups of AECOPD. Objectives: Determine whether ML implementation using EHR data from severe AECOPD requiring hospitalization identifies relevant subgroups. Methods: This study used two retrospective cohorts of patients with AECOPD (non-COVID-19 and COVID-19) treated at Yale-New Haven Hospital (YNHHS). K-means clustering was used to identify patient subgroups. Measurements and main results: We identified three subgroups in the non-COVID cohort (n=1,736). Each subgroup had distinct clinical characteristics. The reference subgroup was the largest (n=904), followed by cardio-renal (n = 548) and eosinophilic (n=284). The eosinophilic subgroup had milder severity of AECOPD, including a shorter hospital stay (p<0.01). The cardio-renal subgroup had the highest mortality during (5%) and in the year after hospitalization (30%). Validation of the severe AECOPD classifier in the COVID-19 cohort recapitulated the characteristics seen in the non-COVID cohort. AECOPD subgroups in the COVID-19 cohort had different IL-1 beta, IL-2R, and IL-8 levels (FDR ≤ 0.05. These specific leukocyte and cytokine profiles resulted in inflammatory differences between the AECOPD subgroups based on C-reactive protein levels. Conclusions: Incorporating ML with EHR-data allows the identification of specific clinical and biological subgroups for severe AECOPD.

A Nerve-Fibroblast Axis in Mammalian Lung Fibrosis.

Tissue fibrosis contributes to pathology in vital organs including the lung. Curative therapies are scant. Myofibroblasts, pivotal effector cells in tissue fibrosis, accumulate via incompletely understood interactions with their microenvironment. In an investigative platform grounded in experimental lung biology, we find that sympathetic innervation stimulates fibrotic remodeling via noradrenergic α1-adrenergic receptor engagement in myofibroblasts. We demonstrate the anti-fibrotic potential of targeted sympathetic denervation and pharmacological disruption of noradrenergic neurotransmitter functions mediated by α1-adrenoreceptors (α1-ARs). Using the α1-adrenoreceptor subtype D as a representative α1-AR, we discover direct noradrenergic input from sympathetic nerves to lung myofibroblasts utilizing established mouse models, genetic denervation, pharmacologic interventions, a newly invented transgenic mouse line, advanced tissue mimetics, and samples from patients with diverse forms of pulmonary fibrosis. The discovery of this previously unappreciated nerve-fibroblast axis in the lung demonstrates the crucial contribution of nerves to tissue repair and heralds a novel paradigm in fibrosis research.

Toll-like Receptor 9 Inhibition Mitigates Fibroproliferative Responses in Translational Models of Pulmonary Fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease for which current treatment options only slow clinical progression. Previously, we identified a subset of patients with IPF with an accelerated disease course associated with fibroblast expression of Toll-Like Receptor 9 (TLR9) mediated by interactions with its ligand mitochondrial DNA (mtDNA). OBJECTIVES: We aimed to show that TLR9 activation induces fibroproliferative responses that are abrogated by its antagonism by using two commercially-available indirect inhibitors and a proprietary, selective direct small molecule inhibitor. METHODS: We employed two independent cohorts of patients with IPF, multiple in vitro fibroblast cell culture platforms, an in vivo mouse model, and an ex vivo human precision cut lung slices system to investigate the clinical and biologic significance of TLR9 in this disease. MEASUREMENTS AND MAIN RESULTS: In two independent IPF cohorts, plasma mtDNA activates TLR9 in a manner associated with the expression of MCP-1, IL-6, TNFα, and IP-10 and worsened transplant-free survival. Our cell culture platform showed that TLR9 mediates fibroblast activation via TGFß1 and stiff substrates, and that its antagonism, particularly direct inhibition, ameliorates this process, including production of these TLR9 associated pharmacodynamic endpoints. We further demonstrated that direct TLR9 inhibition mitigates these fibroproliferative responses in our in vivo and ex vivo models of pulmonary fibrosis. CONCLUSIONS: In this novel study, we found that direct TLR9 inhibition mitigates fibroproliferative responses in preclinical models of pulmonary fibrosis. Our work demonstrates the therapeutic potential of direct TLR9 antagonism in IPF and related fibrotic lung diseases.

cGAS Expression is Enhanced in Systemic Sclerosis Associated Interstitial Lung Disease and Stimulates Inflammatory Myofibroblast Activation.

Objective: The lungs of patients with Systemic Sclerosis Associated Interstitial Lung Disease (SSc-ILD) contain inflammatory myofibroblasts arising in association with fibrotic stimuli and perturbed innate immunity. The innate immune DNA binding receptor Cyclic GMP-AMP synthase (cGAS) is implicated in inflammation and fibrosis, but its involvement in SSc-ILD remains unknown. We examined cGAS expression, activity, and therapeutic potential in SSc-ILD using cultured fibroblasts, precision cut lung slices (PCLS), and a well-accepted animal model. Methods: Expression and localization of cGAS, cytokines, and type 1 interferons were evaluated in SSc-ILD lung tissues, bronchoalveolar lavage (BAL), and isolated lung fibroblasts. CGAS activation was assessed in a publicly available SSc-ILD single cell RNA sequencing dataset. Production of cytokines, type 1 interferons, and αSMA elicited by TGFß1 or local substrate stiffness were measured in normal human lung fibroblasts (NHLFs) via qRT-PCR, ELISA, and immunofluorescence. Small molecule cGAS inhibition was tested in cultured fibroblasts, human PCLS, and the bleomycin pulmonary fibrosis model. Results: SSc-ILD lung tissue and BAL are enriched for cGAS, cytokines, and type 1 interferons. The cGAS pathway shows constitutive activation in SSc-ILD fibroblasts and is inducible in NHLFs by TGFß1 or mechanical stimuli. In these settings, and in human PCLS, cGAS expression is paralleled by the production of cytokines, type 1 interferons, and αSMA that are mitigated by a small molecule cGAS inhibitor. These findings are recapitulated in the bleomycin mouse model. Conclusion: cGAS signaling contributes to pathogenic inflammatory myofibroblast phenotypes in SSc-ILD. Inhibiting cGAS or its downstream effectors represents a novel therapeutic approach.

Cell type-specific expression of angiotensin receptors in the human lung with implications for health, aging, and chronic disease.

The renin-angiotensin system is a highly characterized integrative pathway in mammalian homeostasis whose clinical spectrum has been expanded to lung disorders such as chronic obstructive pulmonary disease (COPD)-emphysema, idiopathic pulmonary fibrosis (IPF), and COVID pathogenesis. Despite this widespread interest, specific localization of this receptor family in the mammalian lung is limited, partially due to the imprecision of available antibody reagents. In this study, we establish the expression pattern of the two predominant angiotensin receptors in the human lung, AGTR1 and AGTR2, using complementary and comprehensive bulk and single-cell RNA-sequence datasets that are publicly available. We show these two receptors have distinct localization patterns and developmental trajectories in the human lung, pericytes for AGTR1 and a subtype of alveolar epithelial type 2 cells for AGTR2. In the context of disease, we further pinpoint AGTR2 localization to the COPD-associated subpopulation of alveolar epithelial type 2 (AT2B) and AGTR1 localization to fibroblasts, where their expression is upregulated in individuals with COPD, but not in individuals with IPF. Finally, we examine the genetic variation of the angiotensin receptors, finding AGTR2 associated with lung phenotype (i.e., cystic fibrosis) via rs1403543. Together, our findings provide a critical foundation for delineating this pathway's role in lung homeostasis and constructing rational approaches for targeting specific lung disorders.

Lung Transcriptomics Links Emphysema to Barrier Dysfunction and Macrophage Subpopulations.

RATIONALE: While many studies have examined gene expression in lung tissue, the gene regulatory processes underlying emphysema are still not well understood. Finding efficient non-imaging screening methods and disease-modifying therapies has been challenging, but knowledge of the transcriptomic features of emphysema may help in this effort. OBJECTIVES: Our goals were to identify emphysema-associated biological pathways through transcriptomic analysis of bulk lung tissue, to determine the lung cell types in which these emphysema-associated pathways are altered, and to detect unique and overlapping transcriptomic signatures in blood and lung samples. METHODS: Using RNA-sequencing data from 446 samples in the Lung Tissue Research Consortium (LTRC) and 3,606 blood samples from the COPDGene study, we examined the transcriptomic features of chest computed tomography-quantified emphysema. We also leveraged publicly available lung single-cell RNA-sequencing data to identify cell types showing COPD-associated differential expression of the emphysema pathways found in the bulk analyses. MEASUREMENTS AND MAIN RESULTS: In the bulk lung RNA-seq analysis, 1,087 differentially expressed genes and 34 dysregulated pathways were significantly associated with emphysema. We observed alternative splicing of several genes and increased activity in pluripotency and cell barrier function pathways. Lung tissue and blood samples shared differentially expressed genes and biological pathways. Multiple lung cell types displayed dysregulation of epithelial barrier function pathways, and distinct pathway activities were observed among various macrophage subpopulations. CONCLUSIONS: This study identified emphysema-related changes in gene expression and alternative splicing, cell-type specific dysregulated pathways, and instances of shared pathway dysregulation between blood and lung.

Stem cell migration drives lung repair in living mice.

Tissue repair requires a highly coordinated cellular response to injury. In the lung, alveolar type 2 cells (AT2s) act as stem cells to replenish both themselves and alveolar type 1 cells (AT1s); however, the complex orchestration of stem cell activity after injury is poorly understood. Here, we establish longitudinal imaging of AT2s in murine intact tissues ex vivo and in vivo in order to track their dynamic behavior over time. We discover that a large fraction of AT2s become motile following injury and provide direct evidence for their migration between alveolar units. High-resolution morphokinetic mapping of AT2s further uncovers the emergence of distinct motile phenotypes. Inhibition of AT2 migration via genetic depletion of ArpC3 leads to impaired regeneration of AT2s and AT1s in vivo. Together, our results establish a requirement for stem cell migration between alveolar units and identify properties of stem cell motility at high cellular resolution.

Spatial Transcriptomics Resolve an Emphysema-Specific Lymphoid Follicle B Cell Signature in Chronic Obstructive Pulmonary Disease.

Rationale: Within chronic obstructive pulmonary disease (COPD), emphysema is characterized by a significant yet partially understood B cell immune component. Objectives: To characterize the transcriptomic signatures from lymphoid follicles (LFs) in ever-smokers without COPD and patients with COPD with varying degrees of emphysema. Methods: Lung sections from 40 patients with COPD and ever-smokers were used for LF proteomic and transcriptomic spatial profiling. Formalin- and O.C.T.-fixed lung samples obtained from biopsies or lung explants were assessed for LF presence. Emphysema measurements were obtained from clinical chest computed tomographic scans. High-confidence transcriptional target intersection analyses were conducted to resolve emphysema-induced transcriptional networks. Measurements and Main Results: Overall, 115 LFs from ever-smokers and Global Initiative for Chronic Obstructive Lung Disease (GOLD) 1-2 and GOLD 3-4 patients were analyzed. No LFs were found in never-smokers. Differential gene expression analysis revealed significantly increased expression of LF assembly and B cell marker genes in subjects with severe emphysema. High-confidence transcriptional analysis revealed activation of an abnormal B cell activity signature in LFs (q-value = 2.56E-111). LFs from patients with GOLD 1-2 COPD with emphysema showed significantly increased expression of genes associated with antigen presentation, inflammation, and B cell activation and proliferation. LFs from patients with GOLD 1-2 COPD without emphysema showed an antiinflammatory profile. The extent of centrilobular emphysema was significantly associated with genes involved in B cell maturation and antibody production. Protein-RNA network analysis showed that LFs in emphysema have a unique signature skewed toward chronic B cell activation. Conclusions: An off-targeted B cell activation within LFs is associated with autoimmune-mediated emphysema pathogenesis.

Spatial Transcriptomics Resolve an Emphysema-specific Lymphoid Follicle B Cell Signature in COPD.

RATIONALE: Within chronic obstructive pulmonary disease (COPD), emphysema is characterized by a significant yet partially understood B cell immune component. OBJECTIVE: To characterize the transcriptomic signatures from lymphoid follicles (LFs) in ever-smokers without COPD and COPD patients with varying degrees of emphysema. METHODS: Lung sections from 40 COPD patients and ever-smokers were used for LF proteomic and transcriptomic spatial profiling. Formalin and OCT-fixed lung samples obtained from biopsies or lung explants, were assessed for LF presence. Emphysema measurements were obtained from clinical chest CT scans. High confidence transcriptional (HCT) target intersection analyses were conducted to resolve emphysema-induced transcriptional networks. MEASUREMENTS AND MAIN RESULTS: Overall, 115 LFs from ever-smokers and GOLD 1-2 and GOLD 3-4 patients were analyzed. No LFs were found in never-smokers. Differential gene expression analysis revealed significantly increased expression of LF assembly and B cell markers genes in subjects with severe emphysema. HCT analysis revealed activation of abnormal B cell activity signature in LFs (q-value: 2.56E-111). LFs from GOLD 1-2 COPD patients with emphysema showed significantly increased expression of genes associated with antigen presentation, inflammation, and B cell activation and proliferation. LFs from GOLD 1-2 COPD patients without emphysema showed an anti-inflammatory profile. The extent of centrilobular emphysema was significantly associated with genes involved in B cell maturation and antibody production. Protein-RNA network analysis showed that LFs in emphysema have a unique signature skewed towards chronic B cell activation. CONCLUSIONS: An off-targeted B cell activation within LFs is associated with autoimmune-mediated emphysema pathogenesis.

Alveolar type II epithelial cell FASN maintains lipid homeostasis in experimental COPD.

Alveolar epithelial type II (AEC2) cells strictly regulate lipid metabolism to maintain surfactant synthesis. Loss of AEC2 cell function and surfactant production are implicated in the pathogenesis of the smoking-related lung disease chronic obstructive pulmonary disease (COPD). Whether smoking alters lipid synthesis in AEC2 cells and whether altering lipid metabolism in AEC2 cells contributes to COPD development are unclear. In this study, high-throughput lipidomic analysis revealed increased lipid biosynthesis in AEC2 cells isolated from mice chronically exposed to cigarette smoke (CS). Mice with a targeted deletion of the de novo lipogenesis enzyme, fatty acid synthase (FASN), in AEC2 cells (FasniΔAEC2) exposed to CS exhibited higher bronchoalveolar lavage fluid (BALF) neutrophils, higher BALF protein, and more severe airspace enlargement. FasniΔAEC2 mice exposed to CS had lower levels of key surfactant phospholipids but higher levels of BALF ether phospholipids, sphingomyelins, and polyunsaturated fatty acid-containing phospholipids, as well as increased BALF surface tension. FasniΔAEC2 mice exposed to CS also had higher levels of protective ferroptosis markers in the lung. These data suggest that AEC2 cell FASN modulates the response of the lung to smoke by regulating the composition of the surfactant phospholipidome.

A statistical framework to identify cell types whose genetically regulated proportions are associated with complex diseases.

Finding disease-relevant tissues and cell types can facilitate the identification and investigation of functional genes and variants. In particular, cell type proportions can serve as potential disease predictive biomarkers. In this manuscript, we introduce a novel statistical framework, cell-type Wide Association Study (cWAS), that integrates genetic data with transcriptomics data to identify cell types whose genetically regulated proportions (GRPs) are disease/trait-associated. On simulated and real GWAS data, cWAS showed good statistical power with newly identified significant GRP associations in disease-associated tissues. More specifically, GRPs of endothelial and myofibroblasts in lung tissue were associated with Idiopathic Pulmonary Fibrosis and Chronic Obstructive Pulmonary Disease, respectively. For breast cancer, the GRP of blood CD8+ T cells was negatively associated with breast cancer (BC) risk as well as survival. Overall, cWAS is a powerful tool to reveal cell types associated with complex diseases mediated by GRPs.

In utero delivery of miRNA induces epigenetic alterations and corrects pulmonary pathology in congenital diaphragmatic hernia.

Structural fetal diseases, such as congenital diaphragmatic hernia (CDH) can be diagnosed prenatally. Neonates with CDH are healthy in utero as gas exchange is managed by the placenta, but impaired lung function results in critical illness from the time a baby takes its first breath. MicroRNA (miR) 200b and its downstream targets in the TGF-ß pathway are critically involved in lung branching morphogenesis. Here, we characterize the expression of miR200b and the TGF-ß pathway at different gestational times using a rat model of CDH. Fetal rats with CDH are deficient in miR200b at gestational day 18. We demonstrate that novel polymeric nanoparticles loaded with miR200b, delivered in utero via vitelline vein injection to fetal rats with CDH results in changes in the TGF-ß pathway as measured by qRT-PCR; these epigenetic changes improve lung size and lung morphology, and lead to favorable pulmonary vascular remodeling on histology. This is the first demonstration of in utero epigenetic therapy to improve lung growth and development in a pre-clinical model. With refinement, this technique could be applied to fetal cases of CDH or other forms of impaired lung development in a minimally invasive fashion.

VISTA (PD-1H) Is a Crucial Immune Regulator to Limit Pulmonary Fibrosis.

VISTA (V domain immunoglobulin suppressor of T cell activation, also called PD-1H [programmed death-1 homolog]), a novel immune regulator expressed on myeloid and T lymphocyte lineages, is upregulated in mouse and human idiopathic pulmonary fibrosis (IPF). However, the significance of VISTA and its therapeutic potential in regulating IPF has yet to be defined. To determine the role of VISTA and its therapeutic potential in IPF, the expression profile of VISTA was evaluated from human single-cell RNA sequencing data (IPF Cell Atlas). Inflammatory response and lung fibrosis were assessed in bleomycin-induced experimental pulmonary fibrosis models in VISTA-deficient mice compared with wild-type littermates. In addition, these outcomes were evaluated after VISTA agonistic antibody treatment in the wild-type pulmonary fibrosis mice. VISTA expression was increased in lung tissue-infiltrating monocytes of patients with IPF. VISTA was induced in the myeloid population, mainly circulating monocyte-derived macrophages, during bleomycin-induced pulmonary fibrosis. Genetic ablation of VISTA drastically promoted pulmonary fibrosis, and bleomycin-induced fibroblast activation was dependent on the interaction between VISTA-expressing myeloid cells and fibroblasts. Treatment with VISTA agonistic antibody reduced fibrotic phenotypes accompanied by the suppression of lung innate immune and fibrotic mediators. In conclusion, these results suggest that VISTA upregulation in pulmonary fibrosis may be a compensatory mechanism to limit inflammation and fibrosis, and stimulation of VISTA signaling using VISTA agonists effectively limits the fibrotic innate immune landscape and consequent tissue fibrosis. Further studies are warranted to test VISTA as a novel therapeutic target for the IPF treatment.

Characterization of pulmonary vascular remodeling and MicroRNA-126-targets in COPD-pulmonary hypertension.

BACKGROUND: Despite causing increased morbidity and mortality, pulmonary hypertension (PH) in chronic obstructive pulmonary disease (COPD) patients (COPD-PH) lacks treatment, due to incomplete understanding of its pathogenesis. Hypertrophy of pulmonary arterial walls and pruning of the microvasculature with loss of capillary beds are known features of pulmonary vascular remodeling in COPD. The remodeling features of pulmonary medium- and smaller vessels in COPD-PH lungs are less well described and may be linked to maladaptation of endothelial cells to chronic cigarette smoking (CS). MicroRNA-126 (miR126), a master regulator of endothelial cell fate, has divergent functions that are vessel-size specific, supporting the survival of large vessel endothelial cells and inhibiting the proliferation of microvascular endothelial cells. Since CS decreases miR126 in microvascular lung endothelial cells, we set out to characterize the remodeling by pulmonary vascular size in COPD-PH and its relationship with miR126 in COPD and COPD-PH lungs. METHODS: Deidentified lung tissue was obtained from individuals with COPD with and without PH and from non-diseased non-smokers and smokers. Pulmonary artery remodeling was assessed by ⍺-smooth muscle actin (SMA) abundance via immunohistochemistry and analyzed by pulmonary artery size. miR126 and miR126-target abundance were quantified by qPCR. The expression levels of ceramide, ADAM9, and endothelial cell marker CD31 were assessed by immunofluorescence. RESULTS: Pulmonary arteries from COPD and COPD-PH lungs had significantly increased SMA abundance compared to non-COPD lungs, especially in small pulmonary arteries and the lung microvasculature. This was accompanied by significantly fewer endothelial cell markers and increased pro-apoptotic ceramide abundance. miR126 expression was significantly decreased in lungs of COPD individuals. Of the targets tested (SPRED1, VEGF, LAT1, ADAM9), lung miR126 most significantly inversely correlated with ADAM9 expression. Compared to controls, ADAM9 was significantly increased in COPD and COPD-PH lungs, predominantly in small pulmonary arteries and lung microvasculature. CONCLUSION: Both COPD and COPD-PH lungs exhibited significant remodeling of the pulmonary vascular bed of small and microvascular size, suggesting these changes may occur before or independent of the clinical development of PH. Decreased miR126 expression with reciprocal increase in ADAM9 may regulate endothelial cell survival and vascular remodeling in small pulmonary arteries and lung microvasculature in COPD and COPD-PH.