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Epithelial Endoplasmic Reticulum Stress Enhances the Risk of Muc5b-associated Lung Fibrosis

Dobrinskikh, Evgenia; Hennessy, Corinne E; Kurche, Jonathan S; Kim, Eunjoo; Estrella, Alani M; Cardwell, Jonathan; Yang, Ivana V; Schwartz, David A
The gain-of-function minor allele of the MUC5B (mucin 5B, oligomeric mucus/gel-forming) promoter (rs35705950) is the strongest risk factor for idiopathic pulmonary fibrosis (IPF), a devastating fibrotic lung disease that leads to progressive respiratory failure in adults. We have previously demonstrated that Muc5b overexpression in mice worsens lung fibrosis after bleomycin exposure and have hypothesized that excess Muc5b promotes endoplasmic reticulum (ER) stress and apoptosis, stimulating fibrotic lung injury. Here, we report that ER stress pathway members ATF4 (activating transcription factor 4) and ATF6 coexpress with MUC5B in epithelia of the distal IPF airway and honeycomb cyst and that this is more pronounced in carriers of the gain-of-function MUC5B promoter variant. Similarly, in mice exposed to bleomycin, Muc5b expression is temporally associated with markers of ER stress. Using bulk and single-cell RNA sequencing in bleomycin-exposed mice, we found that pathologic ER stress-associated transcripts Atf4 and Ddit3 (DNA damage inducible transcript 3) were elevated in alveolar epithelia of SFTPC-Muc5b transgenic (SFTPC-Muc5bTg) mice relative to wild-type (WT) mice. Activation of the ER stress response inhibits protein translation for most genes by phosphorylation of Eif2α (eukaryotic translation initiation factor 2 alpha), which prevents guanine exchange by Eif2B and facilitates translation of Atf4. The integrated stress response inhibitor (ISRIB) facilitates interaction of phosphorylated Eif2α with Eif2B, overcoming translation inhibition associated with ER stress and reducing Atf4. We found that a single dose of ISRIB diminished Atf4 translation in SFTPC-Muc5bTg mice after bleomycin injury. Moreover, ISRIB resolved the exaggerated fibrotic response of SFTPC-Muc5bTg mice to bleomycin. In summary, we demonstrate that MUC5B and Muc5b expression is associated with pathologic ER stress and that restoration of normal translation with a single dose of ISRIB promotes lung repair in bleomycin-injured Muc5b-overexpressing mice.
PMID: 36108173
ISSN: 1535-4989
CID: 5399602

Molecular Signatures of Idiopathic Pulmonary Fibrosis

Konigsberg, Iain R; Borie, Raphael; Walts, Avram D; Cardwell, Jonathan; Rojas, Mauricio; Metzger, Fabian; Hauck, Stefanie M; Fingerlin, Tasha E; Yang, Ivana V; Schwartz, David A
Molecular patterns and pathways in idiopathic pulmonary fibrosis (IPF) have been extensively investigated but few studies have assimilated multi-omic platforms to provide an integrative understanding of molecular patterns that are relevant in IPF. Herein, we combine coding and non-coding transcriptome, DNA methylome, and proteome from IPF and healthy lung tissue to identify molecules and pathways associated with this disease. RNA sequencing, Illumina MethylationEPIC array, and liquid chromatography-mass spectrometry (LC-MS) proteomic data were collected on lung tissue from 24 IPF cases and 14 control subjects. Significant differential features were identified using linear models adjusting for age and sex, inflation and bias where appropriate. Data Integration Analysis for Biomarker discovery using a Latent component method for Omics studies (DIABLO) was used for integrative multi-omic analysis. We identified 4,643 differentially expressed transcripts aligning to 3,439 genes, 998 differentially abundant proteins, 2,500 differentially methylated regions (DMRs), and 1,269 differentially expressed lncRNAs that were significant after correcting for multiple tests (false discovery rate [FDR]<0.05). Unsupervised hierarchical clustering using 20 coding mRNA, protein, methylation, and lncRNA features with highest loadings on the top latent variable from the four datasets demonstrates perfect separation of IPF and control lungs. Our analysis confirmed previously validated molecules and pathways known to be dysregulated in disease, and implicated novel molecular features as potential drivers and modifiers of disease. For example, four proteins, 18 DMRs, and 10 lncRNAs were found to have strong correlations (|r|>0.8) with MMP7. Therefore, using a systems biology approach, we have identified novel molecular relationships in IPF.
PMID: 34038697
ISSN: 1535-4989
CID: 4887962

The MUC5B-associated variant rs35705950 resides within an enhancer subject to lineage- and disease-dependent epigenetic remodeling

Gally, Fabienne; Sasse, Sarah K; Kurche, Jonathan S; Gruca, Margaret A; Cardwell, Jonathan H; Okamoto, Tsukasa; Chu, Hong W; Hou, Xiaomeng; Poirion, Olivier B; Buchanan, Justin; Preissl, Sebastian; Ren, Bing; Colgan, Sean P; Dowell, Robin D; Yang, Ivana V; Schwartz, David A; Gerber, Anthony N
The G/T transversion rs35705950, located approximately 3 kb upstream of the MUC5B start site, is the cardinal risk factor for idiopathic pulmonary fibrosis (IPF). Here, we investigate the function and chromatin structure of this -3 kb region and provide evidence that it functions as a classically defined enhancer subject to epigenetic programming. We use nascent transcript analysis to show that RNA polymerase II loads within 10 bp of the G/T transversion site, definitively establishing enhancer function for the region. By integrating Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) analysis of fresh and cultured human airway epithelial cells with nuclease sensitivity data, we demonstrate that this region is in accessible chromatin that affects the expression of MUC5B. Through applying paired single-nucleus RNA- and ATAC-seq to frozen tissue from IPF lungs, we extend these findings directly to disease, with results indicating that epigenetic programming of the -3 kb enhancer in IPF occurs in both MUC5B-expressing and nonexpressing lineages. In aggregate, our results indicate that the MUC5B-associated variant rs35705950 resides within an enhancer that is subject to epigenetic remodeling and contributes to pathologic misexpression in IPF.
PMID: 33320836
ISSN: 2379-3708
CID: 4838032

Preclinical Pulmonary Fibrosis (PrePF) Circulating Protein Biomarkers

Mathai, Susan K; Cardwell, Jonathan; Metzger, Fabian; Powers, Julia; Walts, Avram D; Kropski, Jonathan A; Eickelberg, Oliver; Hauck, Stefanie M; Yang, Ivana V; Schwartz, David A
PMID: 32755483
ISSN: 1535-4970
CID: 4555002

Chronic Hypersensitivity Pneumonitis, an Interstitial Lung Disease with Distinct Molecular Signatures

Furusawa, Haruhiko; Cardwell, Jonathan H; Okamoto, Tsukasa; Walts, Avram D; Konigsberg, Iain R; Kurche, Jonathan S; Bang, Tami J; Schwarz, Marvin I; Brown, Kevin K; Kropski, Jonathan A; Rojas, Mauricio; Cool, Carlyne D; Lee, Joyce S; Wolters, Paul J; Yang, Ivana V; Schwartz, David A
Rationale: Chronic hypersensitivity pneumonitis (CHP) is caused by an immune response to antigen inhalation and is characterized by variable histopathological and clinical features. A subset of subjects with CHP have usual interstitial pneumonia and appear to be clinically similar to subjects with idiopathic pulmonary fibrosis (IPF).Objectives: To determine the common and unique molecular features of CHP and IPF.Methods: Transcriptome analysis of lung samples from CHP (n = 82), IPF (n = 103), and unaffected controls (n = 103) was conducted. Differential gene expression was determined adjusting for sex, race, age, and smoking history and using false discovery rate to control for multiple comparisons.Measurements and Main Results: When compared with controls, we identified 413 upregulated and 317 downregulated genes in CHP and 861 upregulated and 322 downregulated genes in IPF. Concordantly upregulated or downregulated genes in CHP and IPF were related to collagen catabolic processes and epithelial development, whereas genes specific to CHP (differentially expressed in CHP when compared with control and not differentially expressed in IPF) were related to chemokine-mediated signaling and immune responsiveness. Using weighted gene coexpression network analysis, we found that among subjects with CHP, genes involved in adaptive immunity or epithelial cell development were associated with improved or reduced lung function, respectively, and that MUC5B expression was associated with epithelial cell development. MUC5B expression was also associated with lung fibrosis and honeycombing.Conclusions: Gene expression analysis of CHP and IPF identified signatures common to CHP and IPF, as well as genes uniquely expressed in CHP. Select modules of gene expression are characterized by distinct clinical and pathological features of CHP.
PMID: 32602730
ISSN: 1535-4970
CID: 4837962

Epigenome-Wide Association Study of DNA Methylation and Adult Asthma in the Agricultural Lung Health Study

Hoang, Thanh T; Sikdar, Sinjini; Xu, Cheng-Jian; Lee, Mi Kyeong; Cardwell, Jonathan; Forno, Erick; Imboden, Medea; Jeong, Ayoung; Madore, Anne-Marie; Qi, Cancan; Wang, Tianyuan; Bennett, Brian D; Ward, James M; Parks, Christine G; Beane-Freeman, Laura E; King, Debra; Motsinger-Reif, Alison; Umbach, David M; Wyss, Annah B; Schwartz, David A; Celedón, Juan C; Laprise, Catherine; Ober, Carole; Probst-Hensch, Nicole; Yang, Ivana V; Koppelman, Gerard H; London, Stephanie J
Epigenome-wide studies of methylation in children support a role for epigenetic mechanisms in asthma. Studies in adults are rare, and few have examined non-atopic asthma. We conducted the largest epigenome-wide association study of blood DNA methylation in adults in relation to non-atopic and atopic asthma.We measured DNA methylation in blood using the Illumina MethylationEPIC array among 2286 participants in a case-control study of current adult asthma nested within a U.S. agricultural cohort. Atopy was defined by serum specific immunoglobulin E. Participants were categorised as atopy without asthma (n=185), non-atopic asthma (n=673), atopic asthma (n=271), or a reference group of neither atopy nor asthma (n=1157). Analyses were conducted using logistic regression.No associations were observed with atopy without asthma. Numerous CpGs were differentially methylated in non-atopic asthma (8 at family-wise error rate [FWER] p<9×10-8; 524 at False Discovery Rate [FDR]<0.05) and implicated 382 novel genes. More CpGs were identified in atopic asthma (181 at FWER; 1086 at FDR) and implicated 569 novel genes. 104 FDR CpGs overlapped. 35% of CpGs in non-atopic asthma and 91% in atopic asthma replicated in studies of whole blood, eosinophils, airway epithelium, or nasal epithelium. Implicated genes were enriched in pathways related to the nervous system or inflammation.We identified numerous, distinct differentially methylated CpGs in non-atopic and atopic asthma. Many CpGs from blood replicated in asthma-relevant tissues. These circulating biomarkers reflect risk and sequelae of disease and implicate novel genes associated with non-atopic and atopic asthma.
PMID: 32381493
ISSN: 1399-3003
CID: 4554982

Nasal DNA methylation profiling of asthma and rhinitis

Qi, Cancan; Jiang, Yale; Yang, Ivana V; Forno, Erick; Wang, Ting; Vonk, Judith M; Gehring, Ulrike; Smit, Henriëtte A; Milanzi, Edith B; Carpaij, Orestes A; Berg, Marijn; Hesse, Laura; Brouwer, Sharon; Cardwell, Jonathan; Vermeulen, Cornelis J; Acosta-Pérez, Edna; Canino, Glorisa; Boutaoui, Nadia; van den Berge, Maarten; Teichmann, Sarah A; Nawijn, Martijn C; Chen, Wei; Celedón, Juan C; Xu, Cheng-Jian; Koppelman, Gerard H
BACKGROUND:Epigenetic signatures in the nasal epithelium, which is a primary interface with the environment and an accessible proxy for the bronchial epithelium, might provide insights into mechanisms of allergic disease. OBJECTIVE:We aimed to identify and interpret methylation signatures in nasal epithelial brushes associated with rhinitis and asthma. METHODS:Nasal epithelial brushes were obtained from 455 children at the 16-year follow-up of the Dutch Prevention and Incidence of Asthma and Mite Allergy birth cohort study. Epigenome-wide association studies were performed on children with asthma, rhinitis, and asthma and/or rhinitis (AsRh) by using logistic regression, and the top results were replicated in 2 independent cohorts of African American and Puerto Rican children. Significant CpG sites were related to environmental exposures (pets, active and passive smoking, and molds) during secondary school and were correlated with gene expression by RNA-sequencing (n = 244). RESULTS:The epigenome-wide association studies identified CpG sites significantly associated with rhinitis (n = 81) and AsRh (n = 75), but not with asthma. We significantly replicated 62 of 81 CpG sites with rhinitis and 60 of 75 with AsRh, as well as 1 CpG site with asthma. Methylation of cg03565274 was negatively associated with AsRh and positively associated with exposure to pets during secondary school. DNA methylation signals associated with AsRh were mainly driven by specific IgE-positive subjects. DNA methylation related to gene transcripts that were enriched for immune pathways and expressed in immune and epithelial cells. Nasal CpG sites performed well in predicting AsRh. CONCLUSIONS:We identified replicable DNA methylation profiles of asthma and rhinitis in nasal brushes. Exposure to pets may affect nasal epithelial methylation in relation to asthma and rhinitis.
PMID: 31953105
ISSN: 1097-6825
CID: 4554972

A2ML1 and otitis media: novel variants, differential expression, and relevant pathways

Larson, Eric D; Magno, Jose Pedrito M; Steritz, Matthew J; Llanes, Erasmo Gonzalo D V; Cardwell, Jonathan; Pedro, Melquiadesa; Roberts, Tori Bootpetch; Einarsdottir, Elisabet; Rosanes, Rose Anne Q; Greenlee, Christopher; Santos, Rachel Ann P; Yousaf, Ayesha; Streubel, Sven-Olrik; Santos, Aileen Trinidad R; Ruiz, Amanda G; Lagrana-Villagracia, Sheryl Mae; Ray, Dylan; Yarza, Talitha Karisse L; Scholes, Melissa A; Anderson, Catherine B; Acharya, Anushree; Gubbels, Samuel P; Bamshad, Michael J; Cass, Stephen P; Lee, Nanette R; Shaikh, Rehan S; Nickerson, Deborah A; Mohlke, Karen L; Prager, Jeremy D; Cruz, Teresa Luisa G; Yoon, Patricia J; Abes, Generoso T; Schwartz, David A; Chan, Abner L; Wine, Todd M; Cutiongco-de la Paz, Eva Maria; Friedman, Norman; Kechris, Katerina; Kere, Juha; Leal, Suzanne M; Yang, Ivana V; Patel, Janak A; Tantoco, Ma Leah C; Riazuddin, Saima; Chan, Kenny H; Mattila, Petri S; Reyes-Quintos, Maria Rina T; Ahmed, Zubair M; Jenkins, Herman A; Chonmaitree, Tasnee; Hafrén, Lena; Chiong, Charlotte M; Santos-Cortez, Regie Lyn P
A genetic basis for otitis media is established, however, the role of rare variants in disease etiology is largely unknown. Previously a duplication variant within A2ML1 was identified as a significant risk factor for otitis media in an indigenous Filipino population and in US children. In this report exome and Sanger sequencing was performed using DNA samples from the indigenous Filipino population, Filipino cochlear implantees, US probands, Finnish, and Pakistani families with otitis media. Sixteen novel, damaging A2ML1 variants identified in otitis media patients were rare or low-frequency in population-matched controls. In the indigenous population, both gingivitis and A2ML1 variants including the known duplication variant and the novel splice variant c.4061 + 1 G>C were independently associated with otitis media. Sequencing of salivary RNA samples from indigenous Filipinos demonstrated lower A2ML1 expression according to the carriage of A2ML1 variants. Sequencing of additional salivary RNA samples from US patients with otitis media revealed differentially expressed genes that are highly correlated with A2ML1 expression levels. In particular, RND3 is upregulated in both A2ML1 variant carriers and high-A2ML1 expressors. These findings support a role for A2ML1 in keratinocyte differentiation within the middle ear as part of otitis media pathology and the potential application of ROCK inhibition in otitis media.
PMID: 31009165
ISSN: 1098-1004
CID: 4554952

Resequencing Study Confirms That Host Defense and Cell Senescence Gene Variants Contribute to the Risk of Idiopathic Pulmonary Fibrosis

Moore, Camille; Blumhagen, Rachel Z; Yang, Ivana V; Walts, Avram; Powers, Julie; Walker, Tarik; Bishop, Makenna; Russell, Pamela; Vestal, Brian; Cardwell, Jonathan; Markin, Cheryl R; Mathai, Susan K; Schwarz, Marvin I; Steele, Mark P; Lee, Joyce; Brown, Kevin K; Loyd, James E; Crapo, James D; Silverman, Edwin K; Cho, Michael H; James, Judith A; Guthridge, Joel M; Cogan, Joy D; Kropski, Jonathan A; Swigris, Jeffrey J; Bair, Carol; Kim, Dong Soon; Ji, Wonjun; Kim, Hocheol; Song, Jin Woo; Maier, Lisa A; Pacheco, Karin A; Hirani, Nikhil; Poon, Azin S; Li, Feng; Jenkins, R Gisli; Braybrooke, Rebecca; Saini, Gauri; Maher, Toby M; Molyneaux, Philip L; Saunders, Peter; Zhang, Yingze; Gibson, Kevin F; Kass, Daniel J; Rojas, Mauricio; Sembrat, John; Wolters, Paul J; Collard, Harold R; Sundy, John S; O'Riordan, Thomas; Strek, Mary E; Noth, Imre; Ma, Shwu-Fan; Porteous, Mary K; Kreider, Maryl E; Patel, Namrata B; Inoue, Yoshikazu; Hirose, Masaki; Arai, Toru; Akagawa, Shinobu; Eickelberg, Oliver; Fernandez, Isis Enlil; Behr, Jürgen; Mogulkoc, Nesrin; Corte, Tamera J; Glaspole, Ian; Tomassetti, Sara; Ravaglia, Claudia; Poletti, Venerino; Crestani, Bruno; Borie, Raphael; Kannengiesser, Caroline; Parfrey, Helen; Fiddler, Christine; Rassl, Doris; Molina-Molina, Maria; Machahua, Carlos; Worboys, Ana Montes; Gudmundsson, Gunnar; Isaksson, Helgi J; Lederer, David J; Podolanczuk, Anna J; Montesi, Sydney B; Bendstrup, Elisabeth; Danchel, Vivi; Selman, Moises; Pardo, Annie; Henry, Michael T; Keane, Michael P; Doran, Peter; Vašáková, Martina; Sterclova, Martina; Ryerson, Christopher J; Wilcox, Pearce G; Okamoto, Tsukasa; Furusawa, Haruhiko; Miyazaki, Yasunari; Laurent, Geoffrey; Baltic, Svetlana; Prele, Cecilia; Moodley, Yuben; Shea, Barry S; Ohta, Ken; Suzukawa, Maho; Narumoto, Osamu; Nathan, Steven D; Venuto, Drew C; Woldehanna, Merte L; Kokturk, Nurdan; de Andrade, Joao A; Luckhardt, Tracy; Kulkarni, Tejaswini; Bonella, Francesco; Donnelly, Seamus C; McElroy, Aoife; Armstong, Michelle E; Aranda, Alvaro; Carbone, Roberto G; Puppo, Francesco; Beckman, Kenneth B; Nickerson, Deborah A; Fingerlin, Tasha E; Schwartz, David A
Rationale: Several common and rare genetic variants have been associated with idiopathic pulmonary fibrosis, a progressive fibrotic condition that is localized to the lung. Objectives: To develop an integrated understanding of the rare and common variants located in multiple loci that have been reported to contribute to the risk of disease. Methods: We performed deep targeted resequencing (3.69 Mb of DNA) in cases (n = 3,624) and control subjects (n = 4,442) across genes and regions previously associated with disease. We tested for associations between disease and 1) individual common variants via logistic regression and 2) groups of rare variants via sequence kernel association tests. Measurements and Main Results: Statistically significant common variant association signals occurred in all 10 of the regions chosen based on genome-wide association studies. The strongest risk variant is the MUC5B promoter variant rs35705950, with an odds ratio of 5.45 (95% confidence interval, 4.91-6.06) for one copy of the risk allele and 18.68 (95% confidence interval, 13.34-26.17) for two copies of the risk allele (P = 9.60 × 10-295). In addition to identifying for the first time that rare variation in FAM13A is associated with disease, we confirmed the role of rare variation in the TERT and RTEL1 gene regions in the risk of IPF, and found that the FAM13A and TERT regions have independent common and rare variant signals. Conclusions: A limited number of common and rare variants contribute to the risk of idiopathic pulmonary fibrosis in each of the resequencing regions, and these genetic variants focus on biological mechanisms of host defense and cell senescence.
PMID: 31034279
ISSN: 1535-4970
CID: 4554962

FUT2 Variants Confer Susceptibility to Familial Otitis Media

Santos-Cortez, Regie Lyn P; Chiong, Charlotte M; Frank, Daniel N; Ryan, Allen F; Giese, Arnaud P J; Bootpetch Roberts, Tori; Daly, Kathleen A; Steritz, Matthew J; Szeremeta, Wasyl; Pedro, Melquiadesa; Pine, Harold; Yarza, Talitha Karisse L; Scholes, Melissa A; Llanes, Erasmo Gonzalo D V; Yousaf, Saira; Friedman, Norman; Tantoco, Ma Leah C; Wine, Todd M; Labra, Patrick John; Benoit, Jeanne; Ruiz, Amanda G; de la Cruz, Rhodieleen Anne R; Greenlee, Christopher; Yousaf, Ayesha; Cardwell, Jonathan; Nonato, Rachelle Marie A; Ray, Dylan; Ong, Kimberly Mae C; So, Edward; Robertson, Charles E; Dinwiddie, Jordyn; Lagrana-Villagracia, Sheryl Mae; Gubbels, Samuel P; Shaikh, Rehan S; Cass, Stephen P; Einarsdottir, Elisabet; Lee, Nanette R; Schwartz, David A; Gloria-Cruz, Teresa Luisa I; Bamshad, Michael J; Yang, Ivana V; Kere, Juha; Abes, Generoso T; Prager, Jeremy D; Riazuddin, Saima; Chan, Abner L; Yoon, Patricia J; Nickerson, Deborah A; Cutiongco-de la Paz, Eva Maria; Streubel, Sven-Olrik; Reyes-Quintos, Maria Rina T; Jenkins, Herman A; Mattila, Petri; Chan, Kenny H; Mohlke, Karen L; Leal, Suzanne M; Hafrén, Lena; Chonmaitree, Tasnee; Sale, Michele M; Ahmed, Zubair M
Non-secretor status due to homozygosity for the common FUT2 variant c.461G>A (p.Trp154∗) is associated with either risk for autoimmune diseases or protection against viral diarrhea and HIV. We determined the role of FUT2 in otitis media susceptibility by obtaining DNA samples from 609 multi-ethnic families and simplex case subjects with otitis media. Exome and Sanger sequencing, linkage analysis, and Fisher exact and transmission disequilibrium tests (TDT) were performed. The common FUT2 c.604C>T (p.Arg202∗) variant co-segregates with otitis media in a Filipino pedigree (LOD = 4.0). Additionally, a rare variant, c.412C>T (p.Arg138Cys), is associated with recurrent/chronic otitis media in European-American children (p = 1.2 × 10-5) and US trios (TDT p = 0.01). The c.461G>A (p.Trp154∗) variant was also over-transmitted in US trios (TDT p = 0.01) and was associated with shifts in middle ear microbiota composition (PERMANOVA p < 10-7) and increased biodiversity. When all missense and nonsense variants identified in multi-ethnic US trios with CADD > 20 were combined, FUT2 variants were over-transmitted in trios (TDT p = 0.001). Fut2 is transiently upregulated in mouse middle ear after inoculation with non-typeable Haemophilus influenzae. Four FUT2 variants-namely p.Ala104Val, p.Arg138Cys, p.Trp154∗, and p.Arg202∗-reduced A antigen in mutant-transfected COS-7 cells, while the nonsense variants also reduced FUT2 protein levels. Common and rare FUT2 variants confer susceptibility to otitis media, likely by modifying the middle ear microbiome through regulation of A antigen levels in epithelial cells. Our families demonstrate marked intra-familial genetic heterogeneity, suggesting that multiple combinations of common and rare variants plus environmental factors influence the individual otitis media phenotype as a complex trait.
PMID: 30401457
ISSN: 1537-6605
CID: 4554942