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).

Mast-cell expressed membrane protein-1 is expressed in classical monocytes and alveolar macrophages in idiopathic pulmonary fibrosis and regulates cell chemotaxis, adhesion, and migration in a TGFß-dependent manner.

Mast-cell expressed membrane protein-1 (MCEMP1) is higher in patients with idiopathic pulmonary fibrosis (IPF) with an increased risk of death. Here we aimed to establish the mechanistic role of MCEMP1 in pulmonary fibrosis. We identified increased MCEMP1 expression in classical monocytes and alveolar macrophages in IPF compared with controls. MCEMP1 is upregulated by transforming growth factor beta (TGFß) at the mRNA and protein levels in monocytic leukemia THP-1 cells. TGFß-mediated MCEMP1 upregulation results from the cooperation of SMAD3 and SP1 via concomitant binding to SMAD3/SP1 cis-regulatory elements within the MCEMP1 promoter. We also found that MCEMP1 regulates TGFß-mediated monocyte chemotaxis, adhesion, and migration. Our results suggest that MCEMP1 may regulate the migration and transition of monocytes to monocyte-derived alveolar macrophages during pulmonary fibrosis development and progression.NEW & NOTEWORTHY MCEMP1 is highly expressed in circulating classical monocytes and alveolar macrophages in IPF, is regulated by TGFß, and participates in the chemotaxis, adhesion, and migration of circulating monocytes by modulating the effect of TGFß in RHO activity.

Risk of 30-Day All-Cause Readmission in Interstitial Lung Disease Patients after COVID-19: National-Level Data.

Rationale: Hospital readmission within 30 days poses challenges for healthcare providers, policymakers, and patients because of its impact on care quality, costs, and outcomes. Patients with interstitial lung disease (ILD) are particularly affected by readmission, which is associated with increased morbidity and mortality and reduced quality of life. Because small sample sizes have hindered previous studies, this study seeks to address this gap in knowledge by examining a large-scale dataset. Objective: To determine the rate and probability of 30-day all-cause readmission and secondary outcomes in patients with coronavirus disease (COVID-19) or ILD admitted to the hospital. Methods: This study is a nested cohort study that used the PearlDiver patient records database. Adult patients (age ⩾18 yr) who were admitted to hospitals in 28 states in the United States with COVID-19 or ILD diagnoses were included. We defined and analyzed two separate cohorts in this study. The first cohort consisted of patients with COVID-19 and was later divided into two groups with or without a history of ILD. The second cohort consisted of patients with ILD and was later divided into groups with COVID-19 or with a non-COVID-19 pneumonia diagnosis at admission. We also studied two other subcohorts of patients with and without idiopathic pulmonary fibrosis within the second cohort. Propensity score matching was employed to match confounders between groups. The Kaplan-Meier log rank test was applied to compare the probabilities of outcomes. Results: We assessed the data of 2,286,775 patients with COVID-19 and 118,892 patients with ILD. We found that patients with COVID-19 with preexisting ILD had an odds ratio of 1.6 for 30-day all-cause readmission. Similarly, an odds ratio of 2.42 in readmission rates was observed among hospitalized individuals with ILD who contracted COVID-19 compared with those who were hospitalized for non-COVID-19 pneumonia. Our study also found a significantly higher probability of intensive care admission among patients in both cohorts. Conclusions: Patients with ILD face heightened rates of hospital readmissions, particularly when ILD is combined with COVID-19, resulting in adverse outcomes such as decreased quality of life and increased healthcare expenses. It is imperative to prioritize preventive measures against COVID-19 and establish effective postdischarge care strategies for patients with ILD.

Expression of PD-1/PD-L1 axis in mediastinal lymph nodes and lung tissue of human and experimental lung fibrosis indicates a potential therapeutic target for idiopathic pulmonary fibrosis.

BACKGROUND: Mediastinal lymph node enlargement is prevalent in patients with idiopathic pulmonary fibrosis (IPF). Studies investigating whether this phenomenon reflects specific immunologic activation are lacking. METHODS: Programmed cell death-1 (PD-1)/ programmed cell death ligand-1 (PD-L1) expression in mediastinal lymph nodes and lung tissues was analyzed. PD-1, PD-L1 mRNA expression was measured in tracheobronchial lymph nodes of mice following bleomycin-induced injury on day 14. Finally, the effect of the PD-1 inhibitor, pembrolizumab, in bleomycin-induced pulmonary fibrosis was investigated. RESULTS: We analyzed mediastinal lymph nodes of thirty-three patients (n = 33, IPF: n = 14, lung cancer: n = 10, concomitant IPF and lung cancer: n = 9) and lung tissues of two hundred nineteen patients (n = 219, IPF: 123, controls: 96). PD-1 expression was increased, while PD-L1 expression was decreased, in mediastinal lymph nodes of patients with IPF compared to lung cancer and in IPF lungs compared to control lungs. Tracheobronchial lymph nodes isolated on day 14 from bleomycin-treated mice exhibited increased size and higher PD-1, PD-L1 mRNA levels compared to saline-treated animals. Pembrolizumab blunted bleomycin-induced lung fibrosis, as indicated by reduction in Ashcroft score and improvement in respiratory mechanics. CONCLUSIONS: Mediastinal lymph nodes of patients with IPF exhibit differential expression profiles than those of patients with lung cancer indicating distinct immune-mediated pathways regulating fibrogenesis and carcinogenesis. PD-1 expression in mediastinal lymph nodes is in line with lung tissue expression. Lower doses of pembrolizumab might exert antifibrotic effects. Clinical trials aiming to endotype patients based on mediastinal lymph node profiling and accordingly implement targeted therapies such as PD-1 inhibitors are greatly anticipated.

Mast-Cell Expressed Membrane Protein-1 (MCEMP1) is expressed in classical monocytes and alveolar macrophages in Idiopathic Pulmonary Fibrosis and regulates cell chemotaxis, adhesion, and migration in a TGFß dependent manner.

Background: Mast-Cell Expressed Membrane Protein-1 (MCEMP1) is higher in Idiopathic Pulmonary Fibrosis (IPF) patients with increased risk of death and poor outcomes. Here we seek to establish the mechanistic role of MCEMP1 in pulmonary fibrosis. Methods: MCEMP1 expression was analyzed by single-cell RNA sequencing, immunofluorescence in Peripheral Blood Mononuclear Cells (PBMC) as well as in lung tissues from IPF patients and controls. Chromatin Immunoprecipitation (ChiP) and Proximity Ligation Assay (PLA) were used to study the transcriptional regulation of MCEMP1 . Transient RNA interference and lentivirus transduction were used to knockdown and knock-in MCEMP1 in THP-1 cells to study chemotaxis, adhesion, and migration. Bulk RNA sequencing was used to identify the mechanisms by which MCEMP1 participates in monocyte function. Active RHO pull-down assay was used to validate bulk RNA sequencing results. Results: We identified increased MCEMP1 expression in classical monocytes and alveolar macrophages in IPF compared to controls. MCEMP1 was upregulated by TGFß at the mRNA and protein levels in THP-1. TGFß-mediated MCEMP1 upregulation results from the cooperation of SMAD3 and SP1 via concomitant binding to SMAD3/SP1 cis -regulatory elements within the MCEMP1 promoter. In terms of its function, we found that MCEMP1 regulates TGFß-mediated monocyte chemotaxis, adhesion, and migration. 400 differentially expressed genes were found to increase after TGFß stimulation of THP-1, further increased in MCEMP1 knock-in cells treated with TGFß and decreased in MCEMP1 knockdown cells treated with TGFß. GO annotation analysis of these genes showed enrichment for positive regulation of RHO GTPase activity and signal transduction. While TGFß enhanced RHO GTPase activity in THP-1 cells, this effect was attenuated following MCEMP1 knockdown. Conclusion: MCEMP1 is highly expressed in circulating classical monocytes and alveolar macrophages in IPF. MCEMP1 is regulated by TGFß and participates in the chemotaxis, adhesion, and migration of circulating monocytes by modulating the effect of TGFß in RHO activity. Our results suggest that MCEMP1 may regulate the migration and transition of monocytes to monocyte-derived alveolar macrophages during pulmonary fibrosis development and progression.

Monocytes and macrophages: emerging mechanisms and novel therapeutic targets in pulmonary fibrosis.

Pulmonary fibrosis results from a plethora of abnormal pathogenetic events. In idiopathic pulmonary fibrosis (IPF), inhalational, environmental, or occupational exposures in genetically and epigenetically predisposed individuals trigger recurrent cycles of alveolar epithelial cell injury, activation of coagulation pathways, chemoattraction, and differentiation of monocytes into monocyte-derived alveolar macrophages (Mo-AMs). When these events happen intermittently and repeatedly throughout the individual's life cycle, the wound repair process becomes aberrant leading to bronchiolization of distal air spaces, fibroblast accumulation, extracellular matrix deposition, and loss of the alveolar-capillary architecture. The role of immune dysregulation in IPF pathogenesis and progression has been underscored in the past mainly after the disappointing results of immunosuppressant use in IPF patients; however, recent reports highlighting the prognostic and mechanistic roles of monocytes and Mo-AMs revived the interest in immune dysregulation in IPF. In this review, we will discuss the role of these cells in the onset and progression of IPF, as well as potential targeted therapies.

Precision medicine advances in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a highly heterogeneous, unpredictable and ultimately lethal chronic lung disease. Over the last decade, two anti-fibrotic agents have been shown to slow disease progression, however, both drugs are administered uniformly with minimal consideration of disease severity and inter-individual molecular, genetic, and genomic differences. Advances in biological understanding of disease endotyping and the emergence of precision medicine have shown that "a one-size-fits-all approach" to the management of chronic lung diseases is no longer appropriate. While precision medicine approaches have revolutionized the management of other diseases such as lung cancer and asthma, the implementation of precision medicine in IPF clinical practice remains an unmet need despite several reports demonstrating a large number of diagnostic, prognostic and theragnostic biomarker candidates in IPF. This review article aims to summarize our current knowledge of precision medicine in IPF and highlight barriers to translate these research findings into clinical practice.

Management of patients with idiopathic pulmonary fibrosis and lung cancer: challenges in clinical practice.

PURPOSE OF REVIEW: Idiopathic pulmonary fibrosis (IPF) is the nonmalignant, chronic lung disease with the worst prognosis. Prevalent comorbidities including lung cancer exert a negative impact on patients' survival. However, there is considerable lack of knowledge on the diagnostic and therapeutic management of patients diagnosed with both clinical entities. This review article presents the main challenges in the management of patients with IPF and lung cancer and highlights future perspectives. RECENT FINDINGS: Recent registries for patients with IPF demonstrated that approximately 10% of patients developed lung cancer. Importantly, incidence of lung cancer was increasing remarkably over time in patients with IPF. Patients with IPF and otherwise technically operable lung cancer who underwent surgical resection had improved survival compared with those who did not undergo surgery. However, specific precautions perioperatively are crucial. Finally, the first randomized-controlled, phase 3 trial (J-SONIC trial) showed no significant difference in exacerbation-free survival for chemotherapy-naive patients with IPF and advanced nonsmall cell lung cancer that were allocated to receive carboplatin and nab-paclitaxel every 3 weeks with or without nintedanib. SUMMARY: Lung cancer is prevalent in IPF. Management of patients with IPF and lung cancer is challenging. A consensus statement aiming to attenuate confusion is greatly anticipated.

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.

Evolution of pulmonary hypertension in interstitial lung disease: a journey through past, present, and future.

Interstitial lung diseases (ILD) are a spectrum of disorders often complicated by pulmonary hypertension (PH) in its course. The pathophysiologic mechanism of WHO group 3 PH is different to other forms of PH. The advent of PH is a harbinger for adverse events like mortality and morbidity, implying that the PH component of disease expedites deteriorated clinical outcomes. In fact, WHO group 3 PH due to ILD has the worse prognosis among all groups of PH. Hence, early detection of PH by a comprehensive screening method is paramount. Given considerable overlap in clinical manifestations between ILD and PH, early detection of PH is often elusive. Despite, the treatment of PH due to ILD has been frustrating until recently. Clinical trials utilizing PAH-specific pulmonary vasodilators have been ongoing for years without desired results. Eventually, the INCREASE study (2018) demonstrated beneficial effect of inhaled Treprostinil to treat PH in ILD. In view of this pioneering development, a paradigm shift in clinical approach to this disease phenotype is happening. There is a renewed vigor to develop a well validated screening tool for early detection and management. Currently inhaled Treprostinil is the only FDA approved therapy to treat this phenotype, but emergence of a therapy has opened a plethora of research toward new drug developments. Regardless of all these recent developments, the overall outlook still remains grim in this condition. This review article dwells on the current state of knowledge of pre-capillary PH due to ILD, especially its diagnosis and management, the recent progresses, and future evolutions in this field.

50-gene risk profiles in peripheral blood predict COVID-19 outcomes: A retrospective, multicenter cohort study.

BACKGROUND: COVID-19 has been associated with Interstitial Lung Disease features. The immune transcriptomic overlap between Idiopathic Pulmonary Fibrosis (IPF) and COVID-19 has not been investigated. METHODS: we analyzed blood transcript levels of 50 genes known to predict IPF mortality in three COVID-19 and two IPF cohorts. The Scoring Algorithm of Molecular Subphenotypes (SAMS) was applied to distinguish high versus low-risk profiles in all cohorts. SAMS cutoffs derived from the COVID-19 Discovery cohort were used to predict intensive care unit (ICU) status, need for mechanical ventilation, and in-hospital mortality in the COVID-19 Validation cohort. A COVID-19 Single-cell RNA-sequencing cohort was used to identify the cellular sources of the 50-gene risk profiles. The same COVID-19 SAMS cutoffs were used to predict mortality in the IPF cohorts. FINDINGS: 50-gene risk profiles discriminated severe from mild COVID-19 in the Discovery cohort (P = 0·015) and predicted ICU admission, need for mechanical ventilation, and in-hospital mortality (AUC: 0·77, 0·75, and 0·74, respectively, P < 0·001) in the COVID-19 Validation cohort. In COVID-19, 50-gene expressing cells with a high-risk profile included monocytes, dendritic cells, and neutrophils, while low-risk profile-expressing cells included CD4+, CD8+ T lymphocytes, IgG producing plasmablasts, B cells, NK, and gamma/delta T cells. Same COVID-19 SAMS cutoffs were also predictive of mortality in the University of Chicago (HR:5·26, 95%CI:1·81-15·27, P = 0·0013) and Imperial College of London (HR:4·31, 95%CI:1·81-10·23, P = 0·0016) IPF cohorts. INTERPRETATION: 50-gene risk profiles in peripheral blood predict COVID-19 and IPF outcomes. The cellular sources of these gene expression changes suggest common innate and adaptive immune responses in both diseases. FUNDING: This work was supported in part by National Institute for Health Research Clinician Scientist Fellowship NIHR: CS-2013-13-017 (TMM); Action for Pulmonary Fibrosis Mike Bray fellowship (PLM); The National Heart, Lung, and Blood Institute (NHLBI) through award K01-HL-130704 (AJ); The University of South Florida (USF) Academic Support Fund and the USF Foundation, Ubben Fibrosis Fund (JHM).

Blood Transcriptomics Predicts Progression of Pulmonary Fibrosis and Associated Natural Killer Cells.

Rationale: Disease activity in idiopathic pulmonary fibrosis (IPF) remains highly variable, poorly understood, and difficult to predict. Objectives: To identify a predictor using short-term longitudinal changes in gene expression that forecasts future FVC decline and to characterize involved pathways and cell types. Methods: Seventy-four patients from COMET (Correlating Outcomes with Biochemical Markers to Estimate Time-Progression in IPF) cohort were dichotomized as progressors (≥10% FVC decline) or stable. Blood gene-expression changes within individuals were calculated between baseline and 4 months and regressed with future FVC status, allowing determination of expression variations, sample size, and statistical power. Pathway analyses were conducted to predict downstream effects and identify new targets. An FVC predictor for progression was constructed in COMET and validated using independent cohorts. Peripheral blood mononuclear single-cell RNA-sequencing data from healthy control subjects were used as references to characterize cell type compositions from bulk peripheral blood mononuclear RNA-sequencing data that were associated with FVC decline. Measurements and Main Results: The longitudinal model reduced gene-expression variations within stable and progressor groups, resulting in increased statistical power when compared with a cross-sectional model. The FVC predictor for progression anticipated patients with future FVC decline with 78% sensitivity and 86% specificity across independent IPF cohorts. Pattern recognition receptor pathways and mTOR pathways were downregulated and upregulated, respectively. Cellular deconvolution using single-cell RNA-sequencing data identified natural killer cells as significantly correlated with progression. Conclusions: Serial transcriptomic change predicts future FVC decline. An analysis of cell types involved in the progressor signature supports the novel involvement of natural killer cells in IPF progression.

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.

Increased monocyte count as a cellular biomarker for poor outcomes in fibrotic diseases: a retrospective, multicentre cohort study.

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.

LCox: a tool for selecting genes related to survival outcomes using longitudinal gene expression data.

Longitudinal genomics data and survival outcome are common in biomedical studies, where the genomics data are often of high dimension. It is of great interest to select informative longitudinal biomarkers (e.g. genes) related to the survival outcome. In this paper, we develop a computationally efficient tool, LCox, for selecting informative biomarkers related to the survival outcome using the longitudinal genomics data. LCox is powerful to detect different forms of dependence between the longitudinal biomarkers and the survival outcome. We show that LCox has improved performance compared to existing methods through extensive simulation studies. In addition, by applying LCox to a dataset of patients with idiopathic pulmonary fibrosis, we are able to identify biologically meaningful genes while all other methods fail to make any discovery. An R package to perform LCox is freely available at https://CRAN.R-project.org/package=LCox.

Joint Models for Time-to-Event Data and Longitudinal Biomarkers of High Dimension.

Joint models for longitudinal biomarkers and time-to-event data are widely used in longitudinal studies. Many joint modeling approaches have been proposed to handle different types of longitudinal biomarkers and survival outcomes. However, most existing joint modeling methods cannot deal with a large number of longitudinal biomarkers simultaneously, such as the longitudinally collected gene expression profiles. In this article, we propose a new joint modeling method under the Bayesian framework, which is able to analyze longitudinal biomarkers of high dimension. Specifically, we assume that only a few unobserved latent variables are related to the survival outcome and the latent variables are inferred using a factor analysis model, which greatly reduces the dimensionality of the biomarkers and also accounts for the high correlations among the biomarkers. Through extensive simulation studies, we show that our proposed method has improved prediction accuracy over other joint modeling methods. We illustrate the usefulness of our method on a dataset of idiopathic pulmonary fibrosis patients in which we are interested in predicting the patients' time-to-death using their gene expression profiles.

BAL Cell Gene Expression Is Indicative of Outcome and Airway Basal Cell Involvement in Idiopathic Pulmonary Fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a fatal disease with a variable and unpredictable course. OBJECTIVES: To determine whether BAL cell gene expression is predictive of survival in IPF. METHODS: This retrospective study analyzed the BAL transcriptome of three independent IPF cohorts: Freiburg (Germany), Siena (Italy), and Leuven (Belgium) including 212 patients. BAL cells from 20 healthy volunteers, 26 patients with sarcoidosis stage III and IV, and 29 patients with chronic obstructive pulmonary disease were used as control subjects. Survival analysis was performed by Cox models and component-wise boosting. Presence of airway basal cells was tested by immunohistochemistry and flow cytometry. MEASUREMENTS AND MAIN RESULTS: A total of 1,582 genes were predictive of mortality in the IPF derivation cohort in univariate analyses adjusted for age and sex at false discovery rate less than 0.05. A nine-gene signature, derived from the discovery cohort (Freiburg), performed well in both replication cohorts, Siena (P < 0.0032) and Leuven (P = 0.0033). nCounter expression analysis confirmed the array results (P < 0.0001). The genes associated with mortality in BAL cells were significantly enriched for genes expressed in airway basal cells. Further analyses by gene expression, flow cytometry, and immunohistochemistry showed an increase in airway basal cells in BAL and tissues of IPF compared with control subjects, but not in chronic obstructive pulmonary disease or sarcoidosis. CONCLUSIONS: Our results identify and validate a BAL signature that predicts mortality in IPF and improves the accuracy of outcome prediction based on clinical parameters. The BAL signature associated with mortality unmasks a potential role for airway basal cells in IPF.

Regularized Latent Class Model for Joint Analysis of High-Dimensional Longitudinal Biomarkers and a Time-to-Event Outcome.

Although many modeling approaches have been developed to jointly analyze longitudinal biomarkers and a time-to-event outcome, most of these methods can only handle one or a few biomarkers. In this article, we propose a novel joint latent class model to deal with high dimensional longitudinal biomarkers. Our model has three components: a class membership model, a survival submodel, and a longitudinal submodel. In our model, we assume that covariates can potentially affect biomarkers and class membership. We adopt a penalized likelihood approach to infer which covariates have random effects and/or fixed effects on biomarkers, and which covariates are informative for the latent classes. Through extensive simulation studies, we show that our proposed method has improved performance in prediction and assigning subjects to the correct classes over other joint modeling methods and that bootstrap can be used to do inference for our model. We then apply our method to a dataset of patients with idiopathic pulmonary fibrosis, for whom gene expression profiles were measured longitudinally. We are able to identify four interesting latent classes with one class being at much higher risk of death compared to the other classes. We also find that each of the latent classes has unique trajectories in some genes, yielding novel biological insights.

BPIFA1 regulates lung neutrophil recruitment and interferon signaling during acute inflammation.

Bpifa1 (BPI fold-containing group A member 1) is an airway host-protective protein with immunomodulatory properties that binds to LPS and is regulated by infectious and inflammatory signals. Differential expression of Bpifa1 has been widely reported in lung disease, yet the biological significance of this observation is unclear. We sought to understand the role of Bpifa1 fluctuations in modulating lung inflammation. We treated wild-type (WT) and Bpifa1-/- mice with intranasal LPS and performed immunological and transcriptomic analyses of lung tissue to determine the immune effects of Bpifa1 deficiency. We show that neutrophil (polymorphonuclear cells, PMNs) lung recruitment and transmigration to the airways in response to LPS is impaired in Bpifa1-/- mice. Transcriptomic analysis revealed a signature of 379 genes that differentiated Bpifa1-/- from WT mice. During acute lung inflammation, the most downregulated genes in Bpifa1-/- mice were Cxcl9 and Cxcl10. Bpifa1-/- mice had lower bronchoalveolar lavage concentrations of C-X-C motif chemokine ligand 10 (Cxcl10) and Cxcl9, interferon-inducible PMN chemokines. This was consistent with lower expression of IFNγ, IFNλ, downstream IFN-stimulated genes, and IFN-regulatory factors, which are important for the innate immune response. Administration of Cxcl10 before LPS treatment restored the inflammatory response in Bpifa1-/- mice. Our results identify a novel role for Bpifa1 in the regulation of Cxcl10-mediated PMN recruitment to the lungs via IFNγ and -λ signaling during acute inflammation.