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Up to 20% of idiopathic interstitial lung disease is familial, referred to as Familial Pulmonary Fibrosis (FPF). We performed an integrated analysis of FPF genetic risk by comprehensively evaluating for genetic rare variants (RVs) in a large cohort of FPF kindreds.We performed whole-exome sequencing and/or candidate gene sequencing from affected individuals in 569 FPF kindreds, followed by co-segregation analysis in large kindreds, gene burden analysis, gene-based risk scoring, cell type enrichment analysis, and co-expression network construction.We found that 14.9% - 23.4% of genetic risk in kindreds could be explained by RVs in genes previously linked to FPF, predominantly telomere related genes. We identified new candidate genes in a small number of families, including SYDE1, SERPINB8, GPR87, and NETO1, and developed tools for evaluation and prioritization of RV-containing genes across kindreds. Several pathways were enriched for RV-containing genes in FPF, including focal adhesion and mitochondrial complex I assembly. By combining single cell transcriptomics with prioritized candidate genes, we discovered that expression of RV-containing genes was enriched in smooth muscle cells, type II alveolar epithelial cells, and endothelial cells.In the most comprehensive FPF genetic study to date, we defined the prevalence of RVs in known FPF-related genes and identified new candidate genes and pathways relevant to FPF. However, we did not identify new RV-containing genes shared across multiple kindreds, thereby suggesting that heterogeneous genetic variants involving a variety of genes and pathways mediate genetic risk in most FPF kindreds.

Relatives of patients with familial interstitial pneumonia are at increased risk for pulmonary fibrosis, and develop preclinical pulmonary fibrosis (PrePF).We defined the incidence and progression of new onset PrePF; its relationship to survival among first degree relatives of families with familial interstitial pneumonia.This is a cohort study of family members with familial interstitial pneumonia who were initially screened with a questionnaire and chest HRCT scan, and approximately 4 years later, the evaluation was repeated. 493 asymptomatic first-degree relatives of patients with familial interstitial pneumonia were evaluated at baseline, and 296 (60%) of the original subjects participated in subsequent evaluation.The median interval between HRCTs was 3.9 years (IQ range 3.5-4.4). 252 subjects who agreed to repeat evaluation were originally determined not to have PrePF at baseline; 16 developed PrePF. A conservative estimate of the annual incidence of PrePF is 1,023 per 100, 000 person years (95% CI 511-1831). Of 44 subjects with PrePF at baseline, 38.4% subjects had worsening dyspnea compared to 15.4% of those without PrePF (P=0.002). Usual interstitial pneumonia (UIP) by HRCT (P0.0002) and baseline quantitative fibrosis score (P0.001) are also associated with worsening dyspnea. PrePF at the initial screen is associated with decreased survival (P0.001).The incidence of PrePF in this at-risk population is at least 100-fold higher than that reported for sporadic idiopathic pulmonary fibrosis (IPF). While PrePF and IPF represent distinct entities, our study demonstrates that PrePF, like IPF, is progressive and associated with decreased survival.

Loss of secretory IgA (SIgA) is common in chronic obstructive pulmonary disease (COPD) small airways and likely contributes to disease progression. We hypothesized that loss of SIgA results from reduced expression of pIgR (polymeric immunoglobulin receptor), a chaperone protein needed for SIgA transcytosis, in the COPD small airway epithelium. pIgR-expressing cells were defined and quantified at single-cell resolution in human airways using RNA

Immune cells have been implicated in idiopathic pulmonary fibrosis (IPF), but the phenotypes and effector mechanisms of these cells remain incompletely characterized. We performed mass cytometry to quantify immune cell subsets in lungs of 12 patients with IPF and 15 organ donors without chronic lung disease and used existing single-cell RNA-sequencing data to investigate transcriptional profiles of immune cells overrepresented in IPF. Among myeloid cells, we found increased numbers of alveolar macrophages (AMØs) and dendritic cells (DCs) in IPF, as well as a subset of monocyte-derived DCs. In contrast, monocyte-like cells and interstitial macrophages were reduced in IPF. Transcriptomic profiling identified an enrichment for IFN-γ response pathways in AMØs and DCs from IPF, as well as antigen processing in DCs and phagocytosis in AMØs. Among T cells, we identified three subsets of memory T cells that were increased in IPF, including CD4(+) and CD8(+) resident memory T cells (T(RM)) and CD8(+) effector memory cells. The response to the IFN-γ pathway was enriched in CD4 T(RM) and CD8 T(RM) cells in IPF, together with T cell activation and immune response–regulating signaling pathways. Increased AMØs, DCs, and memory T cells were present in IPF lungs compared with control subjects. In IPF, these cells possess an activation profile indicating increased IFN-γ signaling and upregulation of adaptive immunity in the lungs. Together, these studies highlight critical features of the immunopathogenesis of IPF.

BackgroundPulmonary hypertension (PH) in heart failure with preserved ejection fraction (HFpEF) is a common and highly morbid syndrome, but mechanisms driving PH-HFpEF are not well understood. We sought to determine whether a well-accepted murine model of HFpEF also displays features of PH in HFpEF, and we sought to identify pathways that might drive early remodeling of the pulmonary vasculature in HFpEF.MethodsEight week old male and female C57/BL6J mice were given either L-NAME and high fat diet (HFD) or control water/diet for 2,5, and 12 weeks. Bulk RNA sequencing and single cell RNA sequencing was performed to identify early and cell-specific pathways that might regulate pulmonary vascular remodeling in PH-HFpEF. Finally, clodronate liposome and IL1β antibody treatments were utilized to deplete macrophages or IL1β, respectively, to assess their impact on pulmonary vascular remodeling in HFpEF.ResultsMice given L-NAME/HFD developed PH, small vessel muscularization, and right heart dysfunction after 2 weeks of treatment. Inflammation-related gene ontologies were over-represented in bulk RNA sequencing analysis of whole lungs, with an increase in CD68+ cells in both murine and human PH-HFpEF lungs. Cytokine profiling of mouse lung and plasma showed an increase in IL1β, which was confirmed in plasma from patients with HFpEF. Single cell sequencing of mouse lungs also showed an increase in M1-like, pro-inflammatory populations of Ccr2+ monocytes and macrophages, and transcript expression of IL1β was primarily restricted to myeloid-type cells. Finally, clodronate liposome treatment prevented the development of PH in L-NAME/HFD treated mice, and IL1β antibody treatment also attenuated PH in L-NAME/HFD treated mice.ConclusionsOur study demonstrated that a well-accepted model of HFpEF recapitulates features of pulmonary vascular remodeling commonly seen in patients with HFpEF, and we identified myeloid cell derived IL1β as an important contributor to PH in HFpEF.

Common genetic variants confer substantial risk for chronic lung diseases, including pulmonary fibrosis (PF). Defining the genetic control of gene expression in a cell-type-specific and context-dependent manner is critical for understanding the mechanisms through which genetic variation influences complex traits and disease pathobiology. To this end, we performed single-cell RNA-sequencing of lung tissue from 67 PF and 49 unaffected donors. Employing a pseudo-bulk approach, we mapped expression quantitative trait loci (eQTL) across 38 cell types, observing both shared and cell type-specific regulatory effects. Further, we identified disease-interaction eQTL and demonstrated that this class of associations is more likely to be cell-type specific and linked to cellular dysregulation in PF. Finally, we connected PF risk variants to their regulatory targets in disease-relevant cell types. These results indicate that cellular context determines the impact of genetic variation on gene expression, and implicates context-specific eQTL as key regulators of lung homeostasis and disease.

Background Interstitial lung abnormalities (ILA) may represent undiagnosed early-stage or subclinical interstitial lung disease (ILD). ILAs are often observed incidentally in patients who subsequently develop clinically overt ILD. There is limited information on consensus definitions for, and the appropriate evaluation of, ILA. Early recognition of patients with ILD remains challenging, yet critically important. Expert consensus could inform early recognition and referral. Research Question Can consensus-based expert recommendations be identified to guide clinicians in the recognition, referral, and follow-up of patients with or at risk of developing early ILDs? Study Design and Methods Pulmonologists and radiologists with expertise in ILD participated in two iterative rounds of surveys. The surveys aimed to establish consensus regarding ILA reporting, identification of patients with ILA, and identification of populations that might benefit from screening for ILD. Recommended referral criteria and follow-up processes were also addressed. Threshold for consensus was defined a priori as ≥ 75% agreement or disagreement. Results Fifty-five experts were invited and 44 participated; consensus was reached on 39 of 85 questions. The following clinically important statements achieved consensus: honeycombing and traction bronchiectasis or bronchiolectasis indicate potentially progressive ILD; honeycombing detected during lung cancer screening should be reported as potentially significant (eg, with the Lung CT Screening Reporting and Data System “S-modifier” [which indicates clinically significant or potentially significant noncancer findings]), recommending referral to a pulmonologist in the radiology report; high-resolution CT imaging and full pulmonary function tests should be ordered if nondependent subpleural reticulation, traction bronchiectasis, honeycombing, centrilobular ground-glass nodules, or patchy ground-glass opacity are observed on CT imaging; patients with honeycombing or traction bronchiectasis should be referred to a pulmonologist irrespective of FVC and diffusion capacity values; and patients with systemic sclerosis should be screened with pulmonary function tests for early-stage ILD. Interpretation Guidance for identifying clinically relevant ILA, with subsequent referral and follow-up, was established. These results lay the foundation for developing practical guidance on managing patients with ILA.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by limited treatment options and high mortality. A better understanding of the molecular drivers of IPF progression is needed. OBJECTIVE: To identify and validate molecular determinants of IPF survival. METHODS: A staged genome-wide association study (GWAS) was performed using paired genomic and survival data. Stage I cases were drawn from centers across the US and Europe and stage II cases from Vanderbilt University. Cox proportional hazards regression was used to identify gene variants associated with differential transplant-free survival (TFS). Stage I variants with nominal significance (p

Rationale: Idiopathic pulmonary fibrosis is a rare, irreversible, and progressive disease of the lungs. Common genetic variants, in addition to non-genetic factors, have been consistently associated with IPF. Rare variants identified by candidate gene, family-based, and exome studies have also been reported to associate with IPF. However, the extent to which rare variants genome-wide may contribute to the risk of IPF remains unknown. Objectives: We used whole-genome sequencing to investigate the role of rare variants, genome-wide, on IPF risk. Methods: As part of the Trans-Omics for Precision Medicine Program, we sequenced 2,180 cases of IPF. Association testing focused on the aggregated effect of rare variants (minor allele frequency ≤0.01) within genes or regions. We also identified individual variants that are influential within genes and estimated the heritability of IPF based on rare and common variants. Measurements and Main Results: Rare variants in both TERT and RTEL1 were significantly associated with IPF. A single rare variant in each of the TERT and RTEL1 genes was found to consistently influence the aggregated test statistics. There was no significant evidence of association with other previously reported rare variants. The SNP-heritability of IPF was estimated to be 32% (s.e. 3%). Conclusions: Rare variants within the TERT and RTEL1 genes and well-established common variants have the largest contribution to IPF risk overall. Efforts in risk profiling or development of therapies for IPF that focus on TERT, RTEL1, common variants, and environmental risk factors are likely to have the largest impact on this complex disease.

OBJECTIVE: A higher proportion of circulating memory CD4(+) T cells is associated with prevalent diabetes mellitus in persons with HIV (PWH) and HIV-negative persons. We assessed whether circulating T cell subsets could also identify individuals who will subsequently develop diabetes. DESIGN: This is a longitudinal follow-up study of PWH and similar HIV-negative individuals from the Veterans Aging Cohort Study who provided peripheral mononuclear blood cells between 2005 and 2007. METHODS: We quantified T cell subsets using flow cytometry and functional assays to identify CD4(+) and CD8(+) naïve, activated, senescent, memory (central, effector, and effector RA(+)), and T(H)1, T(H)2, and T(H)17-phenotype cells. The occurrence of an incident diabetes diagnosis (i.e., after baseline blood draw) was adjudicated by a two-physician chart review. Cox proportional hazards models adjusted for traditional risk factors, cytomegalovirus serostatus, and plasma inflammatory biomarkers assessed the relationship between T cell subsets and incident diabetes. RESULTS: 1837 participants (1259 PWH) without diabetes at baseline were included; 69% were black, 95% were male, and median follow-up was 8.6 years. Higher baseline frequencies of CD4(+) T effector memory RA(+) (T(EMRA)) cells defined as CD45RA(+) CD27(−) (p=0.04) and senescent T cells defined as CD4(+) CD28(−) (p=0.04) were associated with incident diabetes in PWH only. CONCLUSIONS: Higher frequencies of CD4(+) T(EMRA) and CD4(+) CD28(−) T cells were associated with incident diabetes in PWH only after adjustment for other factors. Additional studies are necessary to assess whether these cells act in blood via inflammatory mediators or reflect T cell populations in metabolically active tissues.

Determining how alveoli are formed and maintained is critical to understanding lung organogenesis and regeneration after injury. While technological barriers have heretofore limited real-time observation of alveologenesis, we have now used scanned oblique plane illumination microscopy of living lung slices to observe specific cellular behaviors at high resolution over several days. Contrary to the prevailing paradigm that alveoli form by airspace subdivision via ingrowing septa, we find that alveoli form by ballooning epithelial outgrowth supported by stable mesenchymal ring structures. Our systematic analysis allowed creation of a computational model of finely-timed cellular structural changes that drive alveologenesis under normal conditions or with perturbed intercellular Wnt signaling. This new paradigm and platform can be leveraged for mechanistic studies and screening for therapies to promote lung regeneration.One-Sentence SummaryLong-term live analysis of neonatal lungs supports a dynamic epithelial outgrowth model for alveologenesis.

Rationale: Human herpesviruses Epstein-Barr virus and cytomegalovirus are frequently detectable in the lungs of patients with idiopathic pulmonary fibrosis (IPF) and could contribute to disease pat...

Supplemental Digital Content is available in the text., 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.

During alveolar repair, alveolar type 2 (AT2) epithelial cell progenitors rapidly proliferate and differentiate into flat type 1 alveolar epithelial cells. Failure of normal alveolar repair mechanisms can lead to loss of alveolar structure (emphysema) or development of fibrosis, depending on the type and severity of injury. To test if β1-containing integrins are required during repair following acute injury, we administeredE. colilipopolysaccharide (LPS) by intratracheal injection to mice with a post-developmental deletion of β1 integrin in AT2 cells. While control mice recovered from LPS injury without structural abnormalities, β1-deficient mice had more severe inflammation and developed emphysema. In addition, recovering alveoli were repopulated with an abundance of rounded epithelial cells co-expressing type 2, type 1, and mixed intermediate cell state markers, with few mature type 1 cells. β1-deficient AT2 cells showed persistently increased proliferation after injury, which was blocked by inhibiting NF-κB activation in these cells. Lineage tracing experiments revealed that β1-deficient AT2 cells failed to differentiate into mature type 1 alveolar epithelial cells. Together, these findings demonstrate that functional alveolar repair after injury with terminal alveolar epithelial differentiation requires β1-containing integrins.

RationaleIdiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by limited treatment options and high mortality. Novel therapies and prognostic biomarkers are needed.ObjectiveTo identify and validate molecular determinants of IPF survival.MethodsA staged genome-wide association study (GWAS) was performed using paired genomic and survival data. Stage I cases were drawn from centers across the US and Europe and stage II cases from Vanderbilt University. Cox proportional hazards regression was used to identify gene variants associated with differential transplant-free survival (TFS). Stage I variants with nominal significance (p−5) were advanced for stage II testing and meta-analyzed to identify those reaching genome-wide significance (p−8). Downstream analyses were performed for genes and proteins associated with variants reaching genome-wide significance.Main ResultsAfter quality controls, 1481 stage I cases and 397 stage II cases were included in the analysis. After filtering, 9,075,629 variants were tested in stage I, with 158 meeting advancement criteria. Four variants associated with TFS with consistent effect direction were identified in stage II, including one in an intron of proprotein convertase subtilisin/kexin type 6 (PCSK6) reaching genome-wide significance (HR 4.11; 95%CI 2.54-6.67; p=9.45×10−9). PCSK6 protein was highly expressed in IPF lung parenchyma and negatively correlated with survival. Peripheral blood PCSK6 gene expression and plasma concentration were associated with reduced transplant-free survival.ConclusionsWe identified four novel variants associated with IPF survival, including one in PCSK6 that reached genome-wide significance. Downstream analyses suggested that PCSK6 protein may serve as prognostic biomarker in IPF and potential therapeutic target.

Subcutaneous adipose tissue (SAT) is a critical regulator of systemic metabolic homeostasis. Persons with HIV (PWH) have an increased risk of metabolic diseases and significant alterations in the SAT immune environment compared with the general population. We generated a comprehensive SAT atlas to characterize cellular compositional and transcriptional changes in 59 PWH with a spectrum of metabolic health. Glucose intolerance was associated with increased lipid-associated macrophages and CD4+and CD8+T effector memory cells, and decreased perivascular macrophages. We observed a coordinated intercellular regulatory program which enriched for genes related to inflammation and lipid-processing across multiple cell types as glucose intolerance increased. Increased CD4+effector memory tissue resident cells most strongly associated with altered expression of adipocyte genes critical for lipid metabolism and cellular regulation. Many of these findings were present in a separate group of 32 diabetic HIV-negative persons, suggesting these changes are not specific to HIV.

In the human lung, terminal bronchioles (TBs), the most distal conducting airways, open to respiratory bronchioles (RBs) that lead to the alveolar region where gas exchange takes place. This transition occurs in pulmonary lobules, lung tissue units supplied by pre-TBs, which give rise to TBs. Accumulating evidence suggests that remodeling and loss of pre-TBs and TBs underlies progressive airflow limitation in chronic obstructive pulmonary disease (COPD), the third leading cause of death worldwide. Understanding the nature of these changes at the single-cell level has so far been limited by poor accessibility of pre-TBs and TBs. Here, we introduce a novel method of region-precise airway dissection, which enables capture of the entire anatomical continuum of peripheral airways, from pre-TBs to RBs, and the associated alveolar region within the lobule. This approach allowed us to identify terminal airway-enriched secretory cells (TASCs), a unique epithelial cell population of distal airways expressing secretoglobin 3A2 (SCGB3A2) and/or surfactant protein B (SFTPB). TASCs were enriched in TBs, particularly, in areas of TB-RB transition and exhibited an intermediate, broncho-alveolar molecular pattern. TASC frequency was markedly decreased in pre-TBs and TBs of COPD patients compared to those in non-diseased lungs, accompanied by changes in cellular composition of vascular and immune microenvironments. In vitro regeneration assays identified basal cells (BCs) of pre-TBs and TBs as a cellular origin of TASCs in the human lung. Generation of TASCs by these region-specific progenitors was suppressed by IFN-γ signaling that was augmented in distal airways of COPD patients. Thus, altered maintenance of region-specific cellular organization of pre-TBs and TBs represents a key component of distal airway pathology in COPD.

Stem cells undergo dynamic changes in response to injury to regenerate lost cells. However, the identity of transitional states and the mechanisms that drive their trajectories remain understudied. Using lung organoids, multiple in vivo repair models, single-cell transcriptomics and lineage tracing, we find that alveolar type-2 epithelial cells undergoing differentiation into type-1 cells acquire pre-alveolar type-1 transitional cell state (PATS) en route to terminal maturation. Transitional cells undergo extensive stretching during differentiation, making them vulnerable to DNA damage. Cells in the PATS show an enrichment of TP53, TGFβ, DNA-damage-response signalling and cellular senescence. Gain and loss of function as well as genomic binding assays revealed a direct transcriptional control of PATS by TP53 signalling. Notably, accumulation of PATS-like cells in human fibrotic lungs was observed, suggesting persistence of the transitional state in fibrosis. Our study thus implicates a transient state associated with senescence in normal epithelial tissue repair and its abnormal persistence in disease conditions.

Lung organogenesis requires precise timing and coordination to effect spatial organization and function of the parenchymal cells. To provide a systematic broad-based view of the mechanisms governing the dynamic alterations in parenchymal cells over crucial periods of development, we performed a single-cell RNA-sequencing time-series yielding 102,571 epithelial, endothelial and mesenchymal cells across nine time points from embryonic day 12 to postnatal day 14 in mice. Combining computational fate-likelihood prediction with RNA in situ hybridization and immunofluorescence, we explore lineage relationships during the saccular to alveolar stage transition. The utility of this publicly searchable atlas resource (www.sucrelab.org/lungcells) is exemplified by discoveries of the complexity of type 1 pneumocyte function and characterization of mesenchymal Wnt expression patterns during the saccular and alveolar stages – wherein major expansion of the gas-exchange surface occurs. We provide an integrated view of cellular dynamics in epithelial, endothelial and mesenchymal cell populations during lung organogenesis.

BackgroundLoss of secretory immunoglobulin A (SIgA) is common in COPD small airways and likely contributes to disease progression. We hypothesized loss of SIgA results from reduced expression of pIgR, a chaperone protein needed for SIgA transcytosis, in the COPD small airway epithelium.MethodspIgR-expressing cells were defined and quantified at single-cell resolution in human airways using RNA in-situ hybridization, immunostaining, and single-cell RNA sequencing. Complementary studies in mice utilized immunostaining, primary murine tracheal epithelial cell (MTEC) culture, and transgenic mice with secretory or ciliated cell-specific knockout of pIgR. SIgA degradation by human neutrophil elastase or secreted bacterial proteases from non-typeable Haemophilus influenzae (NTHi) was evaluated in vitro.ResultsWe found that secretory cells are the predominant cell type responsible for pIgR expression in human and murine airways. Loss of SIgA in small airways was not associated with a reduction in secretory cells but rather a reduction in pIgR protein expression despite intact PIGR mRNA expression. Neutrophil elastase and NTHi-secreted proteases are both capable of degrading SIgA in vitro and may also contribute to a deficient SIgA immunobarrier in COPD.InterpretationLoss of the SIgA immunobarrier in small airways of patients severe COPD is complex and likely results from both pIgR-dependent defects in IgA transcytosis and SIgA degradation.Key MessagesWhat is the key question? Localized SIgA deficiency in small airways is an established driver of COPD pathogenesis, but the mechanism of loss remains unclear. We hypothesized loss of SIgA is due to reduced numbers of pIgR-expressing cells in SIgA-deficient small airways.What is the bottom line? pIgR is primarily expressed by secretory cells in human and murine airways. Although numbers of secretory cells are similar between SIgA-deficient and SIgA-replete airways in COPD, there is reduced expression of pIgR protein, but not mRNA, in SIgA-deficient airways. Additionally, host and bacterial proteases degrade SIgA in vitro, suggesting loss of SIgA may relate to both impaired transcytosis and increased degradation.Why read on? This study highlights the complexity of SIgA immunobarrier maintenance and suggests that strategies aimed at restoring the SIgA immunobarrier will need to account for both impaired transcytosis and degradation by host and/or bacterial proteases.

Pulmonary fibrosis is characterized by loss of normal alveoli, accumulation of pathologic activated fibroblasts, and exuberant extracellular matrix deposition that over time can lead to progressive loss of respiratory function and death. This loss of respiratory function is associated with the loss of alveolar type 1 cells (AT1) that play a crucial role in gas exchange and the depletion of the alveolar type 2 cells (AT2) that act as progenitor cells to regenerate the AT1 and AT2 cell populations during repair. Understanding the mechanisms that regulate normal alveolar repair and those associated with pathologic repair is essential to identify potential therapeutic targets to treat or delay progression of fibrotic diseases. The Hippo/YAP developmental signaling pathway has been implicated as a regulator of normal alveolar development and repair. In idiopathic pulmonary fibrosis, aberrant activation of YAP/TAZ has been demonstrated in both the alveolar epithelium and activated fibroblasts associated with increased fibrotic remodeling, and there is emerging interest in this pathway as a target for antifibrotic therapies. In this review, we summarize current evidence as to the role of the Hippo-YAP/TAZ pathway in alveolar development, homeostasis, and repair, and highlight key questions that must be resolved to determine effective strategies to modulate YAP/TAZ signaling to prevent progressive pulmonary fibrosis and enhance adaptive alveolar repair.

Precision-cut lung slices (PCLS) are increasingly utilized for ex vivo disease modeling, but a high-resolution characterization of cellular phenotype stability in PCLS has not been reported. Comparing the single-cell transcriptomic profile of human PCLS after five days of culture to freshly isolated human lung tissue, we found striking changes in endothelial cell and alveolar epithelial cell programs, reflecting both injury and pathways activated in static culture, while immune cell frequencies and programs remained largely intact and similar to the native lung. These cellular dynamics should be considered when utilizing PCLS as a model of the human lung.

Integrins - the principal extracellular matrix (ECM) receptors of the cell - promote cell adhesion, migration, and proliferation, which are key events for cancer growth and metastasis. To date, most integrin-targeted cancer therapeutics have disrupted integrin-ECM interactions, which are viewed as critical for integrin functions. However, such agents have failed to improve cancer patient outcomes. We show that the highly expressed integrin β1 subunit is required for lung adenocarcinoma development in a carcinogen-induced mouse model. Likewise, human lung adenocarcinoma cell lines with integrin β1 deletion failed to form colonies in soft agar and tumors in mice. Mechanistically, we demonstrate that these effects do not require integrin β1-mediated adhesion to ECM but are dependent on integrin β1 cytoplasmic tail-mediated activation of focal adhesion kinase (FAK). These studies support a critical role for integrin β1 in lung tumorigenesis that is mediated through constitutive, ECM binding-independent signaling involving the cytoplasmic tail.

OBJECTIVE: Fat redistribution from subcutaneous adipose tissue (SAT) to the abdominal viscera, pericardium, liver and skeletal muscle contributes to the rising burden of cardiometabolic disease among persons with HIV (PWH). Prior studies found SAT inflammation in PWH impairs lipid storage and persists despite plasma viral suppression on antiretroviral therapy (ART). In this study, we identify SAT immune-related genes associated with ectopic fat deposition in PWH on long-term ART. DESIGN AND METHODS: A total of 92 PWH with well-controlled viremia underwent computed tomography (CT) imaging and abdominal SAT biopsy for gene expression analysis. SAT gene expression was measured using a NanoString panel of 255 immune-related genes. Associations between gene expression and CT measurements of the volume and attenuation (radiodensity) of metabolically relevant ectopic fat depots were assessed using multivariable linear regression and network analysis. RESULTS: Greater SAT volume was associated with higher visceral and pericardial adipose tissue volume, but lower skeletal muscle attenuation. Lower SAT attenuation, a measure of lipid content, was associated with lower VAT attenuation. Hierarchical clustering identified a subset of macrophage-related genes in SAT, including CCL2, CCL22, CCL13, CCR1, CD86, CD163, IL-6, IL-10, MRC1, and TREM2, that were associated with increased lipid deposition in multiple ectopic depots. CONCLUSION: Altered expression of macrophage-related genes in SAT is associated with differences in ectopic fat depot morphometrics among PWH on long-term ART, including in the pericardial and visceral compartments. These findings provide basis for future studies to assess host, virus, and treatment factors shaping the SAT immune environment and its effects on morphometric changes and metabolic comorbidities in PWH.

Patients with chronic lung disease (CLD) have an increased risk for severe coronavirus disease-19 (COVID-19) and poor outcomes. Here, we analyze the transcriptomes of 611,398 single cells isolated from healthy and CLD lungs to identify molecular characteristics of lung cells that may account for worse COVID-19 outcomes in patients with chronic lung diseases. We observe a similar cellular distribution and relative expression of SARS-CoV-2 entry factors in control and CLD lungs. CLD AT2 cells express higher levels of genes linked directly to the efficiency of viral replication and the innate immune response. Additionally, we identify basal differences in inflammatory gene expression programs that highlight how CLD alters the inflammatory microenvironment encountered upon viral exposure to the peripheral lung. Our study indicates that CLD is accompanied by changes in cell-type-specific gene expression programs that prime the lung epithelium for and influence the innate and adaptive immune responses to SARS-CoV-2 infection., Patients with chronic lung disease (CLD) have an increased risk for severe coronavirus disease-19 and poor outcomes. Here the authors compare the transcriptomes of single cells isolated from healthy and CLD lungs to identify molecular characteristics of lung cells that may account for worse COVID-19 outcomes in these patients.

Rationale: Alveolar epithelial cell (AEC) injury and dysregulated repair are implicated in the pathogenesis of pulmonary fibrosis. Endoplasmic reticulum (ER) stress in AEC has been observed in idiopathic pulmonary fibrosis (IPF), a disease of aging. Objectives: To investigate a causal role for ER stress in the pathogenesis of pulmonary fibrosis (PF) and therapeutic potential of ER stress inhibition in PF. Methods: The role of ER stress in AEC dysfunction and fibrosis was studied in mice with tamoxifen (Tmx)-inducible deletion of ER chaperone Grp78, a key regulator of ER homeostasis, in alveolar type II (AT2) cells, progenitors of distal lung epithelium, and in IPF lung slice cultures. Measurements and Main Results: Grp78 deletion caused weight loss, mortality, lung inflammation, and spatially heterogeneous fibrosis characterized by fibroblastic foci, hyperplastic AT2 cells, and increased susceptibility of old and male mice, all features of IPF. Fibrosis was more persistent in more severely injured Grp78 knockout (KO) mice. Grp78 KO AT2 cells showed evidence of ER stress, apoptosis, senescence, impaired progenitor capacity, and activation of TGF-β (transforming growth factor-β)/SMAD signaling. Glucose-regulated protein 78 is reduced in AT2 cells from old mice and patients with IPF, and ER stress inhibitor tauroursodeoxycholic acid ameliorates ER stress and fibrosis in Grp78 KO mouse and IPF lung slice cultures. Conclusions: These results support a causal role for ER stress and resulting epithelial dysfunction in PF and suggest ER stress as a potential mechanism linking aging to IPF. Modulation of ER stress and chaperone function may offer a promising therapeutic approach for pulmonary fibrosis.

Summary Background There is an urgent need for biomarkers to better stratify patients with idiopathic pulmonary fibrosis by risk for lung transplantation allocation who have the same clinical presentation. We aimed to investigate whether a specific immune cell type from patients with idiopathic pulmonary fibrosis could identify those at higher risk of poor outcomes. We then sought to validate our findings using cytometry and electronic health records. Methods We first did a discovery analysis with transcriptome data from the Gene Expression Omnibus at the National Center for Biotechnology Information for 120 peripheral blood mononuclear cell (PBMC) samples of patients with idiopathic pulmonary fibrosis. We estimated percentages of 13 immune cell types using statistical deconvolution, and investigated the association of these cell types with transplant-free survival. We validated these results using PBMC samples from patients with idiopathic pulmonary fibrosis in two independent cohorts (COMET and Yale). COMET profiled monocyte counts in 45 patients with idiopathic pulmonary fibrosis from March 12, 2010, to March 10, 2011, using flow cytometry; we tested if increased monocyte count was associated with the primary outcome of disease progression. In the Yale cohort, 15 patients with idiopathic pulmonary fibrosis (with five healthy controls) were classed as high risk or low risk from April 28, 2014, to Aug 20, 2015, using a 52-gene signature, and we assessed whether monocyte percentage (measured by cytometry by time of flight) was higher in high-risk patients. We then examined complete blood count values in the electronic health records (EHR) of 45 068 patients with idiopathic pulmonary fibrosis, systemic sclerosis, hypertrophic cardiomyopathy, or myelofibrosis from Stanford (Jan 01, 2008, to Dec 31, 2015), Northwestern (Feb 15, 2001 to July 31, 2017), Vanderbilt (Jan 01, 2008, to Dec 31, 2016), and Optum Clinformatics DataMart (Jan 01, 2004, to Dec 31, 2016) cohorts, and examined whether absolute monocyte counts of 0·95 K/μL or greater were associated with all-cause mortality in these patients. Findings In the discovery analysis, estimated CD14+ classical monocyte percentages above the mean were associated with shorter transplant-free survival times (hazard ratio [HR] 1·82, 95% CI 1·05–3·14), whereas higher percentages of T cells and B cells were not (0·97, 0·59–1·66; and 0·78, 0·45–1·34 respectively). In two validation cohorts (COMET trial and the Yale cohort), patients with higher monocyte counts were at higher risk for poor outcomes (COMET Wilcoxon p=0·025; Yale Wilcoxon p=0·049). Monocyte counts of 0·95 K/μL or greater were associated with mortality after adjusting for forced vital capacity (HR 2·47, 95% CI 1·48–4·15; p=0·0063), and the gender, age, and physiology index (HR 2·06, 95% CI 1·22–3·47; p=0·0068) across the COMET, Stanford, and Northwestern datasets). Analysis of medical records of 7459 patients with idiopathic pulmonary fibrosis showed that patients with monocyte counts of 0·95 K/μL or greater were at increased risk of mortality with lung transplantation as a censoring event, after adjusting for age at diagnosis and sex (Stanford HR=2·30, 95% CI 0·94–5·63; Vanderbilt 1·52, 1·21–1·89; Optum 1·74, 1·33–2·27). Likewise, higher absolute monocyte count was associated with shortened survival in patients with hypertrophic cardiomyopathy across all three cohorts, and in patients with systemic sclerosis or myelofibrosis in two of the three cohorts. Interpretation Monocyte count could be incorporated into the clinical assessment of patients with idiopathic pulmonary fibrosis and other fibrotic disorders. Further investigation into the mechanistic role of monocytes in fibrosis might lead to insights that assist the development of new therapies. Funding Bill & Melinda Gates Foundation, US National Institute of Allergy and Infectious Diseases, and US National Library of Medicine.

Idiopathic pulmonary fibrosis (IPF) is a disease of older adults leading to progressive dyspnea and reduced exercise capacity, typically resulting in death within 3–5 years of diagnosis. Underlying genetic susceptibility combined with environmental insults is proposed to trigger a chronic wound repair response, leading to activation of the fibrotic cascade. Perturbations in several molecular pathways mediate vulnerability of the alveolar epithelium to injurious agents, including the unfolded protein response, autophagy, mitophagy, and cellular senescence. These cellular responses are intricately intertwined and link genetic susceptibility to the progressive fibrotic phenotype. Ongoing studies investigating these pathways in type II alveolar epithelial cells show promise for identifying new targeted interventions that could prevent or halt the progression of IPF.

Background: Thousands of biomarker tests are either available or under development for lung diseases. In many cases, adoption of these tests into clinical practice is outpacing the generation and evaluation of sufficient data to determine clinical utility and ability to improve health outcomes. There is a need for a systematically organized report that provides guidance on how to understand and evaluate use of biomarker tests for lung diseases. Methods: We assembled a diverse group of clinicians and researchers from the American Thoracic Society and leaders from the National Heart, Lung, and Blood Institute with expertise in various aspects of precision medicine to review the current status of biomarker tests in lung diseases. Experts summarized existing biomarker tests that are available for lung cancer, pulmonary arterial hypertension, idiopathic pulmonary fibrosis, asthma, chronic obstructive pulmonary disease, sepsis, acute respiratory distress syndrome, cystic fibrosis, and other rare lung diseases. The group identified knowledge gaps that future research studies can address to efficiently translate biomarker tests into clinical practice, assess their cost-effectiveness, and ensure they apply to diverse, real-life populations. Results: We found that the status of biomarker tests in lung diseases is highly variable depending on the disease. Nevertheless, biomarker tests in lung diseases show great promise in improving clinical care. To efficiently translate biomarkers into tests used widely in clinical practice, researchers need to address specific clinical unmet needs, secure support for biomarker discovery efforts, conduct analytical and clinical validation studies, ensure tests have clinical utility, and facilitate appropriate adoption into routine clinical practice. Conclusions: Although progress has been made toward implementation of precision medicine for lung diseases in clinical practice in certain settings, additional studies focused on addressing specific unmet clinical needs are required to evaluate the clinical utility of biomarkers; ensure their generalizability to diverse, real-life populations; and determine their cost-effectiveness.

SummaryLung organogenesis requires precisely timed shifts in the spatial organization and function of parenchymal cells, especially during the later stages of lung development. To investigate the mechanisms governing lung parenchymal dynamics during development, we performed a single cell RNA sequencing (scRNA-seq) time-series yielding 92,238 epithelial, endothelial, and mesenchymal cells across 8 time points from embryonic day 12 (E12) to postnatal day 14 (P14) in mice. We combined new computational analyses with RNAin situhybridization to explore transcriptional velocity, fate likelihood prediction, and spatiotemporal localization of cell populations during the transition between the saccular and alveolar stages. We interrogated this atlas to illustrate the complexity of type 1 pneumocyte function during the saccular and alveolar stages, and we demonstrate an integrated view of the cellular dynamics during lung development.

While transient fibroblast activation is a normal and adaptive aspect of injury-repair in many contexts, persistent fibroblast activation is a hallmark of idiopathic pulmonary fibrosis (IPF) and other chronic fibrotic lung diseases. The mechanisms regulating persistent fibroblast activation in IPF have not been fully elucidated. In the lungs of IPF patients and in mice with experimental lung fibrosis, we observed that expression of the thromboxane-prostanoid receptor (TBXA2R) was increased in fibroblasts. Genetic deletion of TBXA2R, but not inhibition of thromboxane synthase, protected mice from bleomycin-induced lung fibrosis, suggesting an alternative ligand activates profibrotic TBXA2R signaling. We found that F2-isoprostanes (F2-IsoPs), nonezymatic products of arachidonic acid metabolism generated in the setting of reactive oxygen species (ROS), are persistently elevated in experimental lung fibrosis and act as an alternative TBXA2R ligand. Further studies demonstrated that F2-isoprostanes signal through TBXA2R to activate Smad signaling, revealing TBXA2R as a previously unrecognized regulator of the transforming growth factor beta (TGF-β) pathway. Further, treatment with the small-molecule TBXA2R antagonist ifetroban protected mice from lung fibrosis in three pre-clinical models: bleomycin treatment, Hermansky-Pudlak Syndrome, and radiation-induced fibrosis. Importantly, treatment with ifetroban during the fibrotic phase of bleomycin injury markedly enhanced fibrotic resolution. Together, these studies implicate TBXA2R signaling as a crucial mediator linking oxidative stress to fibroblast activation and indicate that TBXA2R antagonists could be efficacious for treatment of IPF and other chronic fibrotic disorders.