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.

Integrative genetic and genomic networks identify microRNA associated with COPD and ILD.

Chronic obstructive pulmonary disease (COPD) and interstitial lung disease (ILD) are clinically and molecularly heterogeneous diseases. We utilized clustering and integrative network analyses to elucidate roles for microRNAs (miRNAs) and miRNA isoforms (isomiRs) in COPD and ILD pathogenesis. Short RNA sequencing was performed on 351 lung tissue samples of COPD (n = 145), ILD (n = 144) and controls (n = 64). Five distinct subclusters of samples were identified including 1 COPD-predominant cluster and 2 ILD-predominant clusters which associated with different clinical measurements of disease severity. Utilizing 262 samples with gene expression and SNP microarrays, we built disease-specific genetic and expression networks to predict key miRNA regulators of gene expression. Members of miR-449/34 family, known to promote airway differentiation by repressing the Notch pathway, were among the top connected miRNAs in both COPD and ILD networks. Genes associated with miR-449/34 members in the disease networks were enriched among genes that increase in expression with airway differentiation at an air-liquid interface. A highly expressed isomiR containing a novel seed sequence was identified at the miR-34c-5p locus. 47% of the anticorrelated predicted targets for this isomiR were distinct from the canonical seed sequence for miR-34c-5p. Overexpression of the canonical miR-34c-5p and the miR-34c-5p isomiR with an alternative seed sequence down-regulated NOTCH1 and NOTCH4. However, only overexpression of the isomiR down-regulated genes involved in Ras signaling such as CRKL and GRB2. Overall, these findings elucidate molecular heterogeneity inherent across COPD and ILD patients and further suggest roles for miR-34c in regulating disease-associated gene-expression.

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.

FK506-Binding Protein 11 Is a Novel Plasma Cell-Specific Antibody Folding Catalyst with Increased Expression in Idiopathic Pulmonary Fibrosis.

Antibodies are central effectors of the adaptive immune response, widespread used therapeutics, but also potentially disease-causing biomolecules. Antibody folding catalysts in the plasma cell are incompletely defined. Idiopathic pulmonary fibrosis (IPF) is a fatal chronic lung disease with increasingly recognized autoimmune features. We found elevated expression of FK506-binding protein 11 (FKBP11) in IPF lungs where FKBP11 specifically localized to antibody-producing plasma cells. Suggesting a general role in plasma cells, plasma cell-specific FKBP11 expression was equally observed in lymphatic tissues, and in vitro B cell to plasma cell differentiation was accompanied by induction of FKBP11 expression. Recombinant human FKBP11 was able to refold IgG antibody in vitro and inhibited by FK506, strongly supporting a function as antibody peptidyl-prolyl cis-trans isomerase. Induction of ER stress in cell lines demonstrated induction of FKBP11 in the context of the unfolded protein response in an X-box-binding protein 1 (XBP1)-dependent manner. While deficiency of FKBP11 increased susceptibility to ER stress-mediated cell death in an alveolar epithelial cell line, FKBP11 knockdown in an antibody-producing hybridoma cell line neither induced cell death nor decreased expression or secretion of IgG antibody. Similarly, antibody secretion by the same hybridoma cell line was not affected by knockdown of the established antibody peptidyl-prolyl isomerase cyclophilin B. The results are consistent with FKBP11 as a novel XBP1-regulated antibody peptidyl-prolyl cis-trans isomerase and indicate significant redundancy in the ER-resident folding machinery of antibody-producing hybridoma cells.

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).

Validation of a 52-gene risk profile for outcome prediction in patients with idiopathic pulmonary fibrosis: an international, multicentre, cohort study.

BACKGROUND: The clinical course of idiopathic pulmonary fibrosis (IPF) is unpredictable. Clinical prediction tools are not accurate enough to predict disease outcomes. METHODS: We enrolled patients with IPF diagnosis in a six-cohort study at Yale University (New Haven, CT, USA), Imperial College London (London, UK), University of Chicago (Chicago, IL, USA), University of Pittsburgh (Pittsburgh, PA, USA), University of Freiburg (Freiburg im Breisgau, Germany), and Brigham and Women's Hospital-Harvard Medical School (Boston, MA, USA). Peripheral blood mononuclear cells or whole blood were collected at baseline from 425 participants and from 98 patients (23%) during 4-6 years' follow-up. A 52-gene signature was measured by the nCounter analysis system in four cohorts and extracted from microarray data (GeneChip) in the other two. We used the Scoring Algorithm for Molecular Subphenotypes (SAMS) to classify patients into low-risk or high-risk groups based on the 52-gene signature. We studied mortality with a competing risk model and transplant-free survival with a Cox proportional hazards model. We analysed timecourse data and response to antifibrotic drugs with linear mixed effect models. FINDINGS: The application of SAMS to the 52-gene signature identified two groups of patients with IPF (low-risk and high-risk), with significant differences in mortality or transplant-free survival in each of the six cohorts (hazard ratio [HR] range 2·03-4·37). Pooled data showed similar results for mortality (HR 2·18, 95% CI 1·53-3·09; p<0·0001) or transplant-free survival (2·04, 1·52-2·74; p<0·0001). Adding 52-gene risk profiles to the Gender, Age, and Physiology index significantly improved its mortality predictive accuracy. Temporal changes in SAMS scores were associated with changes in forced vital capacity (FVC) in two cohorts. Untreated patients did not shift their risk profile over time. A simultaneous increase in up score and decrease in down score was predictive of decreased transplant-free survival (3·18, 1·16-8·76; p=0·025) in the Pittsburgh cohort. A simultaneous decrease in up score and increase in down score after initiation of antifibrotic drugs was associated with a significant (p=0·0050) improvement in FVC in the Yale cohort. INTERPRETATION: The peripheral blood 52-gene expression signature is predictive of outcome in patients with IPF. The potential value of the 52-gene signature in predicting response to therapy should be determined in prospective studies. FUNDING: The Pulmonary Fibrosis Foundation, the Harold Amos Medical Faculty Development Program of the Robert Wood Johnson Foundation, and the National Heart, Lung, and Blood Institute of the US National Institutes of Health.

Loss of Twist1 in the Mesenchymal Compartment Promotes Increased Fibrosis in Experimental Lung Injury by Enhanced Expression of CXCL12.

Idiopathic pulmonary fibrosis (IPF) is a disease characterized by the accumulation of apoptosis-resistant fibroblasts in the lung. We have previously shown that high expression of the transcription factor Twist1 may explain this prosurvival phenotype in vitro. However, this observation has never been tested in vivo. We found that loss of Twist1 in COL1A2+ cells led to increased fibrosis characterized by very significant accumulation of T cells and bone marrow-derived matrix-producing cells. We found that Twist1-null cells expressed high levels of the T cell chemoattractant CXCL12. In vitro, we found that the loss of Twist1 in IPF lung fibroblasts increased expression of CXCL12 downstream of increased expression of the noncanonical NF-κB transcription factor RelB. Finally, blockade of CXCL12 with AMD3100 attenuated the exaggerated fibrosis observed in Twist1-null mice. Transcriptomic analysis of 134 IPF patients revealed that low expression of Twist1 was characterized by enrichment of T cell pathways. In conclusion, loss of Twist1 in collagen-producing cells led to increased bleomycin-induced pulmonary fibrosis, which is mediated by increased expression of CXCL12. Twist1 expression is associated with dysregulation of T cells in IPF patients. Twist1 may shape the IPF phenotype and regulate inflammation in fibrotic lung injury.

Expression of RXFP1 Is Decreased in Idiopathic Pulmonary Fibrosis. Implications for Relaxin-based Therapies.

RATIONALE: Relaxin is a hormone that has been considered as a potential therapy for patients with fibrotic diseases. OBJECTIVES: To gauge the potential efficacy of relaxin-based therapies in idiopathic pulmonary fibrosis (IPF), we studied gene expression for relaxin/insulin-like family peptide receptor 1 (RXFP1) in IPF lungs and controls. METHODS: We analyzed gene expression data obtained from the Lung Tissue Research Consortium and correlated RXFP1 gene expression data with cross-sectional clinical and demographic data. We also employed ex vivo donor and IPF lung fibroblasts to test RXFP1 expression in vitro. We tested CGEN25009, a relaxin-like peptide, in lung fibroblasts and in bleomycin injury. MEASUREMENTS AND MAIN RESULTS: We found that RXFP1 is significantly decreased in IPF. In patients with IPF, the magnitude of RXFP1 gene expression correlated directly with diffusing capacity of the lung for carbon monoxide (P < 0.0001). Significantly less RXFP1 was detected in vitro in IPF fibroblasts than in donor controls. Transforming growth factor-ß decreased RXFP1 in both donor and IPF lung fibroblasts. CGEN25009 was effective at decreasing bleomycin-induced, acid-soluble collagen deposition in vivo. The relaxin-like actions of CGEN25009 were abrogated by RXFP1 silencing in vitro, and, in comparison with donor lung fibroblasts, IPF lung fibroblasts exhibited decreased sensitivity to the relaxin-like effects of CGEN25009. CONCLUSIONS: IPF is characterized by the loss of RXFP1 expression. RXFP1 expression is directly associated with pulmonary function in patients with IPF. The relaxin-like effects of CGEN25009 in vitro are dependent on expression of RXFP1. Our data suggest that patients with IPF with the highest RXFP1 expression would be predicted to be most sensitive to relaxin-based therapies.

Integrated Genomics Reveals Convergent Transcriptomic Networks Underlying Chronic Obstructive Pulmonary Disease and Idiopathic Pulmonary Fibrosis.

RATIONALE: Despite shared environmental exposures, idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease are usually studied in isolation, and the presence of shared molecular mechanisms is unknown. OBJECTIVES: We applied an integrative genomic approach to identify convergent transcriptomic pathways in emphysema and IPF. METHODS: We defined the transcriptional repertoire of chronic obstructive pulmonary disease, IPF, or normal histology lungs using RNA-seq (n = 87). MEASUREMENTS AND MAIN RESULTS: Genes increased in both emphysema and IPF relative to control were enriched for the p53/hypoxia pathway, a finding confirmed in an independent cohort using both gene expression arrays and the nCounter Analysis System (n = 193). Immunohistochemistry confirmed overexpression of HIF1A, MDM2, and NFKBIB members of this pathway in tissues from patients with emphysema or IPF. Using reads aligned across splice junctions, we determined that alternative splicing of p53/hypoxia pathway-associated molecules NUMB and PDGFA occurred more frequently in IPF or emphysema compared with control and validated these findings by quantitative polymerase chain reaction and the nCounter Analysis System on an independent sample set (n = 193). Finally, by integrating parallel microRNA and mRNA-Seq data on the same samples, we identified MIR96 as a key novel regulatory hub in the p53/hypoxia gene-expression network and confirmed that modulation of MIR96 in vitro recapitulates the disease-associated gene-expression network. CONCLUSIONS: Our results suggest convergent transcriptional regulatory hubs in diseases as varied phenotypically as chronic obstructive pulmonary disease and IPF and suggest that these hubs may represent shared key responses of the lung to environmental stresses.

Integrative phenotyping framework (iPF): integrative clustering of multiple omics data identifies novel lung disease subphenotypes.

BACKGROUND: The increased multi-omics information on carefully phenotyped patients in studies of complex diseases requires novel methods for data integration. Unlike continuous intensity measurements from most omics data sets, phenome data contain clinical variables that are binary, ordinal and categorical. RESULTS: In this paper we introduce an integrative phenotyping framework (iPF) for disease subtype discovery. A feature topology plot was developed for effective dimension reduction and visualization of multi-omics data. The approach is free of model assumption and robust to data noises or missingness. We developed a workflow to integrate homogeneous patient clustering from different omics data in an agglomerative manner and then visualized heterogeneous clustering of pairwise omics sources. We applied the framework to two batches of lung samples obtained from patients diagnosed with chronic obstructive lung disease (COPD) or interstitial lung disease (ILD) with well-characterized clinical (phenomic) data, mRNA and microRNA expression profiles. Application of iPF to the first training batch identified clusters of patients consisting of homogenous disease phenotypes as well as clusters with intermediate disease characteristics. Analysis of the second batch revealed a similar data structure, confirming the presence of intermediate clusters. Genes in the intermediate clusters were enriched with inflammatory and immune functional annotations, suggesting that they represent mechanistically distinct disease subphenotypes that may response to immunomodulatory therapies. The iPF software package and all source codes are publicly available. CONCLUSIONS: Identification of subclusters with distinct clinical and biomolecular characteristics suggests that integration of phenomic and other omics information could lead to identification of novel mechanism-based disease sub-phenotypes.

FK506-Binding Protein 10, a Potential Novel Drug Target for Idiopathic Pulmonary Fibrosis.

RATIONALE: Increased abundance and stiffness of the extracellular matrix, in particular collagens, is a hallmark of idiopathic pulmonary fibrosis (IPF). FK506-binding protein 10 (FKBP10) is a collagen chaperone, mutations of which have been indicated in the reduction of extracellular matrix stiffness (e.g., in osteogenesis imperfecta). OBJECTIVES: To assess the expression and function of FKBP10 in IPF. METHODS: We assessed FKBP10 expression in bleomycin-induced lung fibrosis (using quantitative reverse transcriptase-polymerase chain reaction, Western blot, and immunofluorescence), analyzed microarray data from 99 patients with IPF and 43 control subjects from a U.S. cohort, and performed Western blot analysis from 6 patients with IPF and 5 control subjects from a German cohort. Subcellular localization of FKBP10 was assessed by immunofluorescent stainings. The expression and function of FKBP10, as well as its regulation by endoplasmic reticulum stress or transforming growth factor-ß1, was analyzed by small interfering RNA-mediated loss-of-function experiments, quantitative reverse transcriptase-polymerase chain reaction, Western blot, and quantification of secreted collagens in the lung and in primary human lung fibroblasts (phLF). Effects on collagen secretion were compared with those of the drugs nintedanib and pirfenidone, recently approved for IPF. MEASUREMENTS AND MAIN RESULTS: FKBP10 expression was up-regulated in bleomycin-induced lung fibrosis and IPF. Immunofluorescent stainings demonstrated localization to interstitial (myo)fibroblasts and CD68(+) macrophages. Transforming growth factor-ß1, but not endoplasmic reticulum stress, induced FKBP10 expression in phLF. The small interfering RNA-mediated knockdown of FKBP10 attenuated expression of profibrotic mediators and effectors, including collagens I and V and α-smooth muscle actin, on the transcript and protein level. Importantly, loss of FKBP10 expression significantly suppressed collagen secretion by phLF. CONCLUSIONS: FKBP10 might be a novel drug target for IPF.

Assessment of microRNA differential expression and detection in multiplexed small RNA sequencing data.

Small RNA sequencing can be used to gain an unprecedented amount of detail into the microRNA transcriptome. The relatively high cost and low throughput of sequencing bases technologies can potentially be offset by the use of multiplexing. However, multiplexing involves a trade-off between increased number of sequenced samples and reduced number of reads per sample (i.e., lower depth of coverage). To assess the effect of different sequencing depths owing to multiplexing on microRNA differential expression and detection, we sequenced the small RNA of lung tissue samples collected in a clinical setting by multiplexing one, three, six, nine, or 12 samples per lane using the Illumina HiSeq 2000. As expected, the numbers of reads obtained per sample decreased as the number of samples in a multiplex increased. Furthermore, after normalization, replicate samples included in distinct multiplexes were highly correlated (R > 0.97). When detecting differential microRNA expression between groups of samples, microRNAs with average expression >1 reads per million (RPM) had reproducible fold change estimates (signal to noise) independent of the degree of multiplexing. The number of microRNAs detected was strongly correlated with the log2 number of reads aligning to microRNA loci (R = 0.96). However, most additional microRNAs detected in samples with greater sequencing depth were in the range of expression which had lower fold change reproducibility. These findings elucidate the trade-off between increasing the number of samples in a multiplex with decreasing sequencing depth and will aid in the design of large-scale clinical studies exploring microRNA expression and its role in disease.

Matrix metalloproteinase-19 promotes metastatic behavior in vitro and is associated with increased mortality in non-small cell lung cancer.

RATIONALE: Lung cancer is the leading cause of cancer death in both men and women in the United States and worldwide. Matrix metalloproteinases (MMPs) have been implicated in the development and progression of lung cancer, but their role in the molecular pathogenesis of lung cancer remains unclear. We have found that MMP19, a relatively novel member of the MMP family, is overexpressed in lung tumors when compared with control subjects. OBJECTIVES: To test the hypothesis that MMP19 plays a significant role in the development and progression of non-small cell lung cancer (NSCLC). METHODS: We have analyzed lung cancer gene expression data, immunostained lung tumors for MMP19, and performed in vitro assays to test the effects of MMP19 in NSCLC cells. MEASUREMENTS AND MAIN RESULTS: We found that MMP19 gene and protein expression is increased in lung cancer tumors compared with adjacent and histologically normal lung tissues. In three independent datasets, increased MMP19 gene expression conferred a poorer prognosis in NSCLC. In vitro, we found that overexpression of MMP19 promotes epithelial-mesenchymal transition, migration, and invasiveness in multiple NSCLC cell lines. Overexpression of MMP19 with a mutation at the catalytic site did not impair epithelial-mesenchymal transition or expression of prometastasis genes. We also found that miR-30 isoforms, a microRNA family predicted to target MMP19, is markedly down-regulated in human lung cancer and regulates MMP19 expression. CONCLUSIONS: Taken together, these findings suggest that MMP19 is associated with the development and progression of NSCLC and may be a potential biomarker of disease severity and outcome.

Peripheral blood mononuclear cell gene expression profiles predict poor outcome in idiopathic pulmonary fibrosis.

We aimed to identify peripheral blood mononuclear cell (PBMC) gene expression profiles predictive of poor outcomes in idiopathic pulmonary fibrosis (IPF) by performing microarray experiments of PBMCs in discovery and replication cohorts of IPF patients. Microarray analyses identified 52 genes associated with transplant-free survival (TFS) in the discovery cohort. Clustering the microarray samples of the replication cohort using the 52-gene outcome-predictive signature distinguished two patient groups with significant differences in TFS. We studied the pathways associated with TFS in each independent microarray cohort and identified decreased expression of "The costimulatory signal during T cell activation" Biocarta pathway and, in particular, the genes CD28, ICOS, LCK, and ITK, results confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). A proportional hazards model, including the qRT-PCR expression of CD28, ICOS, LCK, and ITK along with patient's age, gender, and percent predicted forced vital capacity (FVC%), demonstrated an area under the receiver operating characteristic curve of 78.5% at 2.4 months for death and lung transplant prediction in the replication cohort. To evaluate the potential cellular source of CD28, ICOS, LCK, and ITK expression, we analyzed and found significant correlation of these genes with the PBMC percentage of CD4(+)CD28(+) T cells in the replication cohort. Our results suggest that CD28, ICOS, LCK, and ITK are potential outcome biomarkers in IPF and should be further evaluated for patient prioritization for lung transplantation and stratification in drug studies.

Genetic variants associated with idiopathic pulmonary fibrosis susceptibility and mortality: a genome-wide association study.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a devastating disease that probably involves several genetic loci. Several rare genetic variants and one common single nucleotide polymorphism (SNP) of MUC5B have been associated with the disease. Our aim was to identify additional common variants associated with susceptibility and ultimately mortality in IPF. METHODS: First, we did a three-stage genome-wide association study (GWAS): stage one was a discovery GWAS; and stages two and three were independent case-control studies. DNA samples from European-American patients with IPF meeting standard criteria were obtained from several US centres for each stage. Data for European-American control individuals for stage one were gathered from the database of genotypes and phenotypes; additional control individuals were recruited at the University of Pittsburgh to increase the number. For controls in stages two and three, we gathered data for additional sex-matched European-American control individuals who had been recruited in another study. DNA samples from patients and from control individuals were genotyped to identify SNPs associated with IPF. SNPs identified in stage one were carried forward to stage two, and those that achieved genome-wide significance (p<5 × 10(-8)) in a meta-analysis were carried forward to stage three. Three case series with follow-up data were selected from stages one and two of the GWAS using samples with follow-up data. Mortality analyses were done in these case series to assess the SNPs associated with IPF that had achieved genome-wide significance in the meta-analysis of stages one and two. Finally, we obtained gene-expression profiling data for lungs of patients with IPF from the Lung Genomics Research Consortium and analysed correlation with SNP genotypes. FINDINGS: In stage one of the GWAS (542 patients with IPF, 542 control individuals matched one-by-one to cases by genetic ancestry estimates), we identified 20 loci. Six SNPs reached genome-wide significance in stage two (544 patients, 687 control individuals): three TOLLIP SNPs (rs111521887, rs5743894, rs5743890) and one MUC5B SNP (rs35705950) at 11p15.5; one MDGA2 SNP (rs7144383) at 14q21.3; and one SPPL2C SNP (rs17690703) at 17q21.31. Stage three (324 patients, 702 control individuals) confirmed the associations for all these SNPs, except for rs7144383. Linkage disequilibrium between the MUC5B SNP (rs35705950) and TOLLIP SNPs (rs111521887 [r(2)=0·07], rs5743894 [r(2)=0·16], and rs5743890 [r(2)=0·01]) was low. 683 patients from the GWAS were included in the mortality analysis. Individuals who developed IPF despite having the protective TOLLIP minor allele of rs5743890 carried an increased mortality risk (meta-analysis with fixed-effect model: hazard ratio 1·72 [95% CI 1·24-2·38]; p=0·0012). TOLLIP expression was decreased by 20% in individuals carrying the minor allele of rs5743890 (p=0·097), 40% in those with the minor allele of rs111521887 (p=3·0 × 10(-4)), and 50% in those with the minor allele of rs5743894 (p=2·93 × 10(-5)) compared with homozygous carriers of common alleles for these SNPs. INTERPRETATION: Novel variants in TOLLIP and SPPL2C are associated with IPF susceptibility. One novel variant of TOLLIP, rs5743890, is also associated with mortality. These associations and the reduced expression of TOLLIP in patients with IPF who carry TOLLIP SNPs emphasise the importance of this gene in the disease. FUNDING: National Institutes of Health; National Heart, Lung, and Blood Institute; Pulmonary Fibrosis Foundation; Coalition for Pulmonary Fibrosis; and Instituto de Salud Carlos III.

Cytokine-like factor 1 gene expression is enriched in idiopathic pulmonary fibrosis and drives the accumulation of CD4+ T cells in murine lungs: evidence for an antifibrotic role in bleomycin injury.

Idiopathic pulmonary fibrosis (IPF) is a progressive and typically fatal lung disease. To gain insight into the pathogenesis of IPF, we reanalyzed our previously published gene expression data profiling IPF lungs. Cytokine receptor-like factor 1 (CRLF1) was among the most highly up-regulated genes in IPF lungs, compared with normal controls. The protein product (CLF-1) and its partner, cardiotrophin-like cytokine (CLC), function as members of the interleukin 6 (IL-6) family of cytokines. Because of earlier work implicating IL-6 family members in IPF pathogenesis, we tested whether CLF-1 expression contributes to inflammation in experimental pulmonary fibrosis. In IPF, we detected CLF-1 expression in both type II alveolar epithelial cells and macrophages. We found that the receptor for CLF-1/CLC signaling, ciliary neurotrophic factor receptor (CNTFR), was expressed only in type II alveolar epithelial cells. Administration of CLF-1/CLC to both uninjured and bleomycin-injured mice led to the pulmonary accumulation of CD4(+) T cells. We also found that CLF-1/CLC administration increased inflammation but decreased pulmonary fibrosis. CLF-1/CLC leads to significantly enriched expression of T-cell-derived chemokines and cytokines, including the antifibrotic cytokine interferon-γ. We propose that, in IPF, CLF-1 is a selective stimulus of type II alveolar epithelial cells and may potentially drive an antifibrotic response by augmenting both T-helper-1-driven and T-regulatory-cell-driven inflammatory responses in the lung.