Jose D. Herazo-Maya

Peripheral Blood Mononuclear Cell Gene Expression Profiles Predict Poor Outcome in Idiopathic Pulmonary Fibrosis

Genome-scale transcriptomic profiling of peripheral blood mononuclear cells from patients with idiopathic pulmonary fibrosis reveals that decreased expression of CD28, ICOS, LCK, and ITK predicts mortality. Gene Signature Predicts Mortality Idiopathic pulmonary fibrosis (IPF) is a fatal disease that progresses at different rates. Although no therapies exist, giving patients a more accurate prognosis is highly desirable. To this end, Herazo-Maya and colleagues searched the genomes of cells circulating in the blood of IPF patients and found that four genes may be indicators of poor outcome. Patients were recruited into discovery or replication cohorts from two different medical centers in the United States and followed until death or completion of the study. In both groups, genetic material was isolated from the patients’ peripheral blood mononuclear cells (PBMCs) and analyzed for increased or decreased expression. These gene expression profiles were then correlated with transplant-free survival (TFS). In the discovery cohort, Herazo-Maya et al. found that underexpression of the genes CD28, ICOS, LCK, and ITK was associated with decreased TFS. These findings were confirmed in the replication cohort. This “genomic model” incorporating the four genes was combined with the clinical outputs age, gender, and forced vital capacity to create an even stronger predictor of poor outcome. The authors suggest that the decreased expression of these genes might be linked to lower percentages of CD4+CD28+ T cells in the PBMC population, which could contribute to a mechanistic understanding of why some IPF patients progress differently than others. The findings of this study have the potential to affect the care of patients with IPF as well as the understanding of disease mechanism. However, the combined genomic and clinical predictor will need to be validated in additional independent cohorts before translation. 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.

Wnt Coreceptor Lrp5 Is a Driver of Idiopathic Pulmonary Fibrosis

RATIONALE Wnt/β-catenin signaling has been implicated in lung fibrosis, but how this occurs and whether expression changes in Wnt pathway components predict disease progression is unknown. OBJECTIVES To determine whether the Wnt coreceptor Lrp5 drives pulmonary fibrosis in mice and is predictive of disease severity in humans. METHODS We examined mice with impaired Wnt signaling caused by loss of the Wnt coreceptor Lrp5 in models of lung fibrosis induced by bleomycin or an adenovirus encoding an active form of transforming growth factor (TGF)-β. We also analyzed gene expression in peripheral blood mononuclear cells (PBMC) from patients with idiopathic pulmonary fibrosis (IPF). MEASUREMENTS AND MAIN RESULTS In patients with IPF, analysis of peripheral blood mononuclear cells revealed that elevation of positive regulators, Lrp5 and 6, was independently associated with disease progression. LRP5 was also associated with disease severity at presentation in an additional cohort of patients with IPF. Lrp5 null mice were protected against bleomycin-induced pulmonary fibrosis, an effect that was phenocopied by direct inhibition of β-catenin signaling by the small molecular inhibitor of β-catenin responsive transcription. Transplantation of Lrp5 null bone marrow cells into wild-type mice did not limit fibrosis. Instead, Lrp5 loss was associated with reduced TGF-β production by alveolar type 2 cells and leukocytes. Consistent with a role of Lrp5 in the activation of TGF-β, Lrp5 null mice were not protected against lung fibrosis induced by TGF-β. CONCLUSIONS We show that the Wnt coreceptor, Lrp5, is a genetic driver of lung fibrosis in mice and a marker of disease progression and severity in humans with IPF. Evidence that TGF-β signaling can override a loss in Lrp5 has implications for patient selection and timing of Wnt pathway inhibitors in lung fibrosis.

Blockade of the programmed death-1 pathway restores sarcoidosis CD4(+) T-cell proliferative capacity.

RATIONALE Effective therapeutic interventions for chronic, idiopathic lung diseases remain elusive. Normalized T-cell function is an important contributor to spontaneous resolution of pulmonary sarcoidosis. Up-regulation of inhibitor receptors, such as programmed death-1 (PD-1) and its ligand, PD-L1, are important inhibitors of T-cell function. OBJECTIVES To determine the effects of PD-1 pathway blockade on sarcoidosis CD4(+) T-cell proliferative capacity. METHODS Gene expression profiles of sarcoidosis and healthy control peripheral blood mononuclear cells were analyzed at baseline and follow-up. Flow cytometry was used to measure ex vivo expression of PD-1 and PD-L1 on systemic and bronchoalveolar lavage-derived cells of subjects with sarcoidosis and control subjects, as well as the effects of PD-1 pathway blockade on cellular proliferation after T-cell receptor stimulation. Immunohistochemistry analysis for PD-1/PD-L1 expression was conducted on sarcoidosis, malignant, and healthy control lung specimens. MEASUREMENTS AND MAIN RESULTS Microarray analysis demonstrates longitudinal increase in PDCD1 gene expression in sarcoidosis peripheral blood mononuclear cells. Immunohistochemistry analysis revealed increased PD-L1 expression within sarcoidosis granulomas and lung malignancy, but this was absent in healthy lungs. Increased numbers of sarcoidosis PD-1(+) CD4(+) T cells are present systemically, compared with healthy control subjects (P < 0.0001). Lymphocytes with reduced proliferative capacity exhibited increased proliferation with PD-1 pathway blockade. Longitudinal analysis of subjects with sarcoidosis revealed reduced PD-1(+) CD4(+) T cells with spontaneous clinical resolution but not with disease progression. CONCLUSIONS Analogous to the effects in other chronic lung diseases, these findings demonstrate that the PD-1 pathway is an important contributor to sarcoidosis CD4(+) T-cell proliferative capacity and clinical outcome. Blockade of the PD-1 pathway may be a viable therapeutic target to optimize clinical outcomes.

Thyroid hormone inhibits lung fibrosis in mice by improving epithelial mitochondrial function

Thyroid hormone (TH) is critical for the maintenance of cellular homeostasis during stress responses, but its role in lung fibrosis is unknown. Here we found that the activity and expression of iodothyronine deiodinase 2 (DIO2), an enzyme that activates TH, were higher in lungs from patients with idiopathic pulmonary fibrosis than in control individuals and were correlated with disease severity. We also found that Dio2-knockout mice exhibited enhanced bleomycin-induced lung fibrosis. Aerosolized TH delivery increased survival and resolved fibrosis in two models of pulmonary fibrosis in mice (intratracheal bleomycin and inducible TGF-β1). Sobetirome, a TH mimetic, also blunted bleomycin-induced lung fibrosis. After bleomycin-induced injury, TH promoted mitochondrial biogenesis, improved mitochondrial bioenergetics and attenuated mitochondria-regulated apoptosis in alveolar epithelial cells both in vivo and in vitro. TH did not blunt fibrosis in Ppargc1a- or Pink1-knockout mice, suggesting dependence on these pathways. We conclude that the antifibrotic properties of TH are associated with protection of alveolar epithelial cells and restoration of mitochondrial function and that TH may thus represent a potential therapy for pulmonary fibrosis.

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

BackgroundThe 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.ResultsIn 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.ConclusionsIdentification 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.

Validation of the prognostic value of MMP‐7 in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with poor prognosis and variable clinical course. Although matrix metalloproteinase‐7 (MMP‐7) is emerging as an important IPF biomarker, reproducibility across studies is unclear. We aimed to determine whether a previously reported prognostic threshold for MMP‐7 was predictive of mortality in an independent cohort of IPF patients.

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 All-comers with Idiopathic Pulmonary Fibrosis diagnosis were enrolled in a six-cohort study. Peripheral blood mononuclear cells or whole blood was collected at baseline from 425 participants and during follow up from 98 patients. The 52-gene signature was measured by the nCounter® analysis system in four cohorts and extracted from microarray data in two others. The Scoring Algorithm for Molecular Subphenotypes (SAMS) was used to classify patients into low or high risk groups based on a 52-gene signature. Mortality and transplant-free survival were studied using Competing risk and Cox proportional-hazard models, respectively. Time course data and response to anti-fibrotic drugs were analyzed using linear mixed-effect models. Findings The application of SAMS to the 52-gene signature identified two groups of IPF patients (low and high risk) with significant differences in mortality or transplant-free survival in each of the six cohorts (HR 2·03–4·37). Pooled data revealed similar results for mortality (HR:2·18, 95%CI:1·53–3·09, P<0·0001) or transplant-free survival (HR:2·04, 95%CI: 1·52–2·74, P<0·0001). Adding 52-gene risk profiles to the Gender, Age and Physiology (GAP) 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 transplant-free survival (HR:3·18· 95%CI 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 anti-fibrotic drugs was associated with a significant (P=0·005) 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.

Extracellular Mitochondrial DNA Is Generated by Fibroblasts and Predicts Death in Idiopathic Pulmonary Fibrosis

Rationale: Idiopathic pulmonary fibrosis (IPF) involves the accumulation of &agr;‐smooth muscle actin‐expressing myofibroblasts arising from interactions with soluble mediators such as transforming growth factor‐&bgr;1 (TGF‐&bgr;1) and mechanical influences such as local tissue stiffness. Whereas IPF fibroblasts are enriched for aerobic glycolysis and innate immune receptor activation, innate immune ligands related to mitochondrial injury, such as extracellular mitochondrial DNA (mtDNA), have not been identified in IPF. Objectives: We aimed to define an association between mtDNA and fibroblast responses in IPF. Methods: We evaluated the response of normal human lung fibroblasts (NHLFs) to stimulation with mtDNA and determined whether the glycolytic reprogramming that occurs in response to TGF‐&bgr;1 stimulation and direct contact with stiff substrates, and spontaneously in IPF fibroblasts, is associated with excessive levels of mtDNA. We measured mtDNA concentrations in bronchoalveolar lavage (BAL) from subjects with and without IPF, as well as in plasma samples from two longitudinal IPF cohorts and demographically matched control subjects. Measurements and Main Results: Exposure to mtDNA augments &agr;‐smooth muscle actin expression in NHLFs. The metabolic changes in NHLFs that are induced by interactions with TGF‐&bgr;1 or stiff hydrogels are accompanied by the accumulation of extracellular mtDNA. These findings replicate the spontaneous phenotype of IPF fibroblasts. mtDNA concentrations are increased in IPF BAL and plasma, and in the latter compartment, they display robust associations with disease progression and reduced event‐free survival. Conclusions: These findings demonstrate a previously unrecognized and highly novel connection between metabolic reprogramming, mtDNA, fibroblast activation, and clinical outcomes that provides new insight into IPF.

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.

Plexin C1 deficiency permits synaptotagmin 7-mediated macrophage migration and enhances mammalian lung fibrosis.

Pulmonary fibrosis is a progressive and often fatal condition that is believed to be partially orchestrated by macrophages. Mechanisms that control migration of these cells into and within the lung remain undefined. We evaluated thecontributions of the semaphorinreceptor, plexinC1 (PLXNC1), andtheexocytic calcium sensor, synaptotagmin 7 (Syt7), in these processes. Weevaluated the role of PLXNC1 in macrophagemigration by using Boyden chambers and scratch tests, characterized its contribution to experimentally induced lung fibrosis inmice, and defined themechanism for our observations. Our findings reveal that relative to controlparticipants, patients with idiopathic pulmonary fibrosis demonstrate excessive monocyte migration and underexpression of PLXNC1 in the lungs and circulation, a finding that is recapitulated in the setting of scleroderma‐related interstitial lung disease. Relative to wild type, PLXNC1‐/‐ mouse macrophages are excessively migratory, and PLXNC1‐/‐ mice show exacerbated collagen accumulation in response to either inhaled bleomycin or inducible lung targeted TGF‐β1 overexpression. These findings are ameliorated by replacement of PLXNC1 on bone marrow–derived cells or by genetic deletion of Syt7. These data demonstrate the previously unrecognized observation that PLXNC1 deficiency permits Syt7‐mediated macrophage migration and enhances mammalian lung fibrosis.—Peng, X., Moore, M., Mathur, A., Zhou, Y., Sun, H., Gan, Y., Herazo‐Maya, J. D., Kaminski, N., Hu, X., Pan, H., Ryu, C., Osafo‐Addo, A., Homer, R. J., Feghali‐Bostwick, C., Fares, W.H., Gulati, M.,Hu, B., Lee, C.‐G., Elias, J. A.,Herzog, E. L. Plexin C1 deficiency permits synaptotagmin 7–mediated macrophage migration and enhances mammalian lung fibrosis. FASEB J. 30, 4056–4070 (2016). www.fasebj.org