PGAP3 regulates human bronchial epithelial cell mRNAs present in asthma and respiratory virus reference data sets.

PGAP3 is a glycosylphosphatidylinositol (GPI) phospholipase gene localized within chromosome 17q12-21, a region highly linked to asthma. Although much is known about the function of other chromosome 17q12-21 genes expressed at increased levels in bronchial epithelium such as ORMDL3 and GSDMB, little is known about the function of increased PGAP3 expression in bronchial epithelium in the context of asthma. The aim of this study was therefore to determine whether increased PGAP3 expression in human bronchial epithelial cells regulated expression of mRNA pathways important to the pathogenesis of asthma by utilizing RNA-sequencing and bioinformatic analysis. We performed RNA-sequencing on normal human bronchial epithelial cells transfected with PGAP3 for 24 and 48 hours. PGAP3 regulated genes were compared to asthma and respiratory virus (influenza A, rhinovirus, respiratory syncytial virus) reference data sets to identify PGAP3 target genes and pathways. Approximately 9% of the upregulated PGAP3-induced genes were found in an asthma reference data set, 41% in a rhinovirus reference data set, 33% in an influenza A reference data set, and 3% in a respiratory syncytial virus reference data set. PGAP3 significantly upregulated the expression of several genes associated with the innate immune response and viral signatures of respiratory viruses associated with asthma exacerbations. Two of the highest expressed genes induced by PGAP3 are RSAD2, OASL, and IFN-λ, which are anti-viral genes associated with asthma. PGAP3 also upregulated the antiviral gene BST2, which like PGAP3 is a GPI-anchored protein. We conclude that PGAP3 expression in human bronchial epithelial cells regulates expression of genes known to be linked to asthma, and also regulates the bronchial epithelial expression of genes pertinent to the pathogenesis of respiratory viral triggered asthma exacerbations.

Lymphotoxin beta receptor signaling directly controls airway smooth muscle deregulation and asthmatic lung dysfunction.

BACKGROUND: Dysregulation of airway smooth muscle cells (ASM) is central to the severity of asthma. Which molecules dominantly control ASM in asthma is unclear. High levels of the cytokine LIGHT (aka TNFSF14) have been linked to asthma severity and lower baseline predicted FEV1 percentage, implying that signals through its receptors might directly control ASM dysfunction. OBJECTIVE: Our study sought to determine whether signaling via lymphotoxin beta receptor (LTßR) or herpesvirus entry mediator from LIGHT dominantly drives ASM hyperreactivity induced by allergen. METHODS: Conditional knockout mice deficient for LTßR or herpesvirus entry mediator in smooth muscle cells were used to determine their role in ASM deregulation and airway hyperresponsiveness (AHR) in vivo. Human ASM were used to study signals induced by LTßR. RESULTS: LTßR was strongly expressed in ASM from normal and asthmatic subjects compared to several other receptors implicated in smooth muscle deregulation. Correspondingly, conditional deletion of LTßR only in smooth muscle cells in smMHCCreLTßRfl/fl mice minimized changes in their numbers and mass as well as AHR induced by house dust mite allergen in a model of severe asthma. Intratracheal LIGHT administration independently induced ASM hypertrophy and AHR in vivo dependent on direct LTßR signals to ASM. LIGHT promoted contractility, hypertrophy, and hyperplasia of human ASM in vitro. Distinguishing LTßR from the receptors for IL-13, TNF, and IL-17, which have also been implicated in smooth muscle dysregulation, LIGHT promoted NF-κB-inducing kinase-dependent noncanonical nuclear factor kappa-light-chain enhancer of activated B cells in ASM in vitro, leading to sustained accumulation of F-actin, phosphorylation of myosin light chain kinase, and contractile activity. CONCLUSIONS: LTßR signals directly and dominantly drive airway smooth muscle hyperresponsiveness relevant for pathogenesis of airway remodeling in severe asthma.

Single-base editing of rs12603332 on chromosome 17q21 with a cytosine base editor regulates ORMDL3 and ATF6α expression.

BACKGROUND: Genetic association studies have demonstrated that the SNP rs12603332 located on chromosome 17q21 is highly associated with the risk of the development of asthma. METHODS: To determine whether SNP rs1260332 is functional in regulating levels of ORMDL3 expression, we used a Cytosine Base Editor (CBE) plasmid DNA or a CBE mRNA to edit the rs12603332 C risk allele to the T non-risk allele in a human lymphocyte cell line (i.e., Jurkat cells) and in primary human CD4 T cells that carry the C risk alleles. RESULTS: Jurkat cells with the rs12603332 C risk allele expressed significantly higher levels of ORMDL3 mRNA, as well as the ORMDL3 regulated gene ATF6α as assessed by qPCR compared to Jurkat clones with the T non-risk allele. In primary human CD4 T cells, we edited 90 ± 3% of the rs12603332-C risk allele to the T non-risk allele and observed a reduction in ORMDL3 and ATF6α expression. Bioinformatic analysis predicted that the non-risk allele rs12603332-T could be the central element of the E-box binding motif (CANNTG) recognized by the E47 transcription factor. An EMSA assay confirmed the bioinformatics prediction demonstrating that a rs12603332-T containing probe bound to the transcription factor E47 in vitro. CONCLUSIONS: SNP rs12603332 is functional in regulating the expression of ORMDL3 as well as ORMDL3 regulated gene ATF6α expression. In addition, we demonstrate the use of CBE technology in functionally interrogating asthma-associated SNPs using studies of primary human CD4 cells.

ORMDL3 expression in ASM regulates hypertrophy, hyperplasia via TPM1 and TPM4, and contractility.

ORM1-like 3 (ORMDL3) has strong genetic linkage to childhood onset asthma. To determine whether ORMDL3 selective expression in airway smooth muscle (ASM) influences ASM function, we used Cre-loxP techniques to generate transgenic mice (hORMDL3Myh11eGFP-cre), which express human ORMDL3 selectively in smooth muscle cells. In vitro studies of ASM cells isolated from the bronchi of hORMDL3Myh11eGFP-cre mice demonstrated that they developed hypertrophy (quantitated by FACS and image analysis), developed hyperplasia (assessed by BrdU incorporation), and expressed increased levels of tropomysin proteins TPM1 and TPM4. siRNA knockdown of TPM1 or TPM4 demonstrated their importance to ORMDL3-mediated ASM proliferation but not hypertrophy. In addition, ASM derived from hORMDL3Myh11eGFP-cre mice had increased contractility to histamine in vitro, which was associated with increased levels of intracellular Ca2+; increased cell surface membrane Orai1 Ca2+ channels, which mediate influx of Ca2+ into the cytoplasm; and increased expression of ASM contractile genes sarco/endoplasmic reticulum Ca2+ ATPase 2b and smooth muscle 22. In vivo studies of hORMDL3Myh11eGFP-cre mice demonstrated that they had a spontaneous increase in ASM and airway hyperreactivity (AHR). ORMDL3 expression in ASM thus induces changes in ASM (hypertrophy, hyperplasia, increased contractility), which may explain the contribution of ORMDL3 to the development of AHR in childhood onset asthma, which is highly linked to ORMDL3 on chromosome 17q12-21.

TL1A Promotes Lung Tissue Fibrosis and Airway Remodeling.

Lung fibrosis and tissue remodeling are features of chronic diseases such as severe asthma, idiopathic pulmonary fibrosis, and systemic sclerosis. However, fibrosis-targeted therapies are currently limited. We demonstrate in mouse models of allergen- and bleomycin-driven airway inflammation that neutralization of the TNF family cytokine TL1A through Ab blocking or genetic deletion of its receptor DR3 restricted increases in peribronchial smooth muscle mass and accumulation of lung collagen, primary features of remodeling. TL1A was found as a soluble molecule in the airways and expressed on the surface of alveolar macrophages, dendritic cells, innate lymphoid type 2 cells, and subpopulations of lung structural cells. DR3 was found on CD4 T cells, innate lymphoid type 2 cells, macrophages, fibroblasts, and some epithelial cells. Suggesting in part a direct activity on lung structural cells, administration of recombinant TL1A into the naive mouse airways drove remodeling in the absence of other inflammatory stimuli, innate lymphoid cells, and adaptive immunity. Correspondingly, human lung fibroblasts and bronchial epithelial cells were found to express DR3 and responded to TL1A by proliferating and/or producing fibrotic molecules such as collagen and periostin. Reagents that disrupt the interaction of TL1A with DR3 then have the potential to prevent deregulated tissue cell activity in lung diseases that involve fibrosis and remodeling.

Integration of Clinical and Molecular Features into Prediction Models for Outcomes in Endometrial Cancer.

Endometrial cancer recurrence carries a poor prognosis. The rising incidence of endometrial cancer calls for improvements in treatment of advanced and recurrent diseases. Efforts have been made to molecularly characterize endometrial cancer with the goal of improving therapies. The study presented here describes the utilization of molecular features of endometrial cancer tumors that are likely to recur, along with clinical characteristics utilized together to predict recurrence. This work further studies recurrent endometrial cancers to group them into "clusters" based on the tumor's molecular makeups with the ultimate aim to focus therapy on the molecular pathways potentially leading to recurrence.

An integrated prediction model of recurrence in endometrial endometrioid cancers.

Objectives: Endometrial cancer incidence and mortality are rising in the US. Disease recurrence has been shown to have a significant impact on mortality. However, to date, there are no accurate and validated prediction models that would discriminate which individual patients are likely to recur. Reliably predicting recurrence would be of benefit for treatment decisions following surgery. We present an integrated model constructed with comprehensive clinical, pathological and molecular features designed to discriminate risk of recurrence for patients with endometrioid endometrial adenocarcinoma. Subjects and methods: A cohort of endometrioid endometrial cancer patients treated at our institution was assembled. Clinical characteristics were extracted from patient charts. Primary tumors from these patients were obtained and total tissue RNA extracted for RNA sequencing. A prediction model was designed containing both clinical characteristics and molecular profiling of the tumors. The same analysis was carried out with data derived from The Cancer Genome Atlas for replication and external validation. Results: Prediction models derived from our institutional data predicted recurrence with high accuracy as evidenced by areas under the curve approaching 1. Similar trends were observed in the analysis of TCGA data. Further, a scoring system for risk of recurrence was devised that showed specificities as high as 81% and negative predictive value as high as 90%. Lastly, we identify specific molecular characteristics of patient tumors that may contribute to the process of disease recurrence. Conclusion: By constructing a comprehensive model, we are able to reliably predict recurrence in endometrioid endometrial cancer. We devised a clinically useful scoring system and thresholds to discriminate risk of recurrence. Finally, the data presented here open a window to understanding the mechanisms of recurrence in endometrial cancer.

Population Substructure Has Implications in Validating Next-Generation Cancer Genomics Studies with TCGA.

In the era of large genetic and genomic datasets, it has become crucially important to validate results of individual studies using data from publicly available sources, such as The Cancer Genome Atlas (TCGA). However, how generalizable are results from either an independent or a large public dataset to the remainder of the population? The study presented here aims to answer that question. Utilizing next generation sequencing data from endometrial and ovarian cancer patients from both the University of Iowa and TCGA, genomic admixture of each population was analyzed using STRUCTURE and ADMIXTURE software. In our independent data set, one subpopulation was identified, whereas in TCGA 4⁻6 subpopulations were identified. Data presented here demonstrate how different the genetic substructures of the TCGA and University of Iowa populations are. Validation of genomic studies between two different population samples must be aware of, account for and be corrected for background genetic substructure.

A Prediction Model for Preoperative Risk Assessment in Endometrial Cancer Utilizing Clinical and Molecular Variables.

The utility of comprehensive surgical staging in patients with low risk disease has been questioned. Thus, a reliable means of determining risk would be quite useful. The aim of our study was to create the best performing prediction model to classify endometrioid endometrial cancer (EEC) patients into low or high risk using a combination of molecular and clinical-pathological variables. We then validated these models with publicly available datasets. Analyses between low and high risk EEC were performed using clinical and pathological data, gene and miRNA expression data, gene copy number variation and somatic mutation data. Variables were selected to be included in the prediction model of risk using cross-validation analysis; prediction models were then constructed using these variables. Model performance was assessed by area under the curve (AUC). Prediction models were validated using appropriate datasets in The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. A prediction model with only clinical variables performed at 88%. Integrating clinical and molecular data improved prediction performance up to 97%. The best prediction models included clinical, miRNA expression and/or somatic mutation data, and stratified pre-operative risk in EEC patients. Integrating molecular and clinical data improved the performance of prediction models to over 95%, resulting in potentially useful clinical tests.

The miR-503 cluster is coordinately under-expressed in endometrial endometrioid adenocarcinoma and targets many oncogenes, cell cycle genes, DNA repair genes and chemotherapy response genes.

BACKGROUND: The miR-503 miRNA cluster, located at Xq23.1, is composed of six miRNAs; miR-424, miR-503, miR-542, miR-450a-1, miR-450a-2 and miR-450b. Numerous studies have focused on the relationship of one or two members of the cluster and various human cancers. Here, we suggest that the entire cluster as a single coordinately expressed polycistron transcribed from a single promoter in endometrial endometrioid adenocarcinoma (EEA). SUBJECTS AND METHODS: A tissue panel composed of twenty histologically confirmed endometrial endometrioid adenocarcinomas (EEA) and four benign endometrium was assembled under informed consent. Expression of each member of the miR-503 cluster was determined by quantitative PCR and differences in expression between EEA and benign tissues were assessed via the standard ΔΔCt method. In addition, the role of promoter methylation status in miRNA expression was examined in Ishikawa H cells following exposure to the cytidine analog Decitabine. RESULTS: Expression of each member of the miR-503 cluster is significantly downregulated in EEA in our tumor sample. Both in our tumor sample and in The Cancer Genome Atlas (TCGA) there is evidence of highly correlated expression further supporting the idea that the miR-503 cluster is a polycistron. Looking at each member of the miR-503 cluster we were able to identify 55 unique experimentally validated target genes which include a substantial number of genes involved in carcinogenesis, DNA damage response, cell cycle regulation and chemotherapeutic response. We also found preliminary evidence that regulation of the miR-503 cluster is governed by methylation of the promoter in EEA. CONCLUSION: The totality of the data presented here strongly suggest that the miR-503 cluster as a whole merits further investigation as an important potential therapeutic target in EEA.

ß2 integrins rather than ß1 integrins mediate Alternaria-induced group 2 innate lymphoid cell trafficking to the lung.

BACKGROUND: Group 2 innate lymphoid cells (ILC2s) expand in the lungs of mice during type 2 inflammation induced by the fungal allergen Alternaria alternata. The increase in ILC2 numbers in the lung has been largely attributed to local proliferation and whether ILC2s migrate from the circulation to the lung after Alternaria exposure is unknown. OBJECTIVE: We examined whether human (lung, lymph node, and blood) and mouse lung ILC2s express ß1 and ß2 integrin adhesion molecules and whether these integrins are required for trafficking of ILC2s into the lungs of mice. METHODS: Human and mouse ILC2s were assessed for surface expression of ß1 and ß2 integrin adhesion molecules by using flow cytometry. The role of ß1 and ß2 integrins in ILC2 trafficking to the lungs was assessed by in vivo blocking of these integrins before airway exposure to Alternaria in mice. RESULTS: Both human and mouse lung ILC2s express high levels of ß1 and ß2 integrin adhesion receptors. Intranasal administration of Alternaria challenge reduced ILC2 numbers in the bone marrow and concurrently increased blood and lung ILC2 numbers. In vivo blocking of ß2 integrins (CD18) significantly reduced ILC2 numbers in the lungs but did not alter ILC2 proliferation, apoptosis, and function. In contrast, in vivo blocking of ß1 integrins or α4 integrins did not affect lung ILC2 numbers. CONCLUSION: ILC2 numbers increase in the mouse lung not only through local proliferation but also through trafficking from the circulation into the lung using ß2 rather than ß1 or α4 integrins.

Orosomucoid-like 3 (ORMDL3) upregulates airway smooth muscle proliferation, contraction, and Ca2+ oscillations in asthma.

BACKGROUND: Airway hyperresponsiveness is a major feature of asthma attributed predominantly to an extrinsic immune/inflammatory response increasing airway smooth muscle (ASM) contractility. OBJECTIVE: We investigated whether increased ASM expression of orosomucoid-like 3 (ORMDL3), a gene on chromosome 17q21 highly linked to asthma, induced increased ASM proliferation and contractility in vitro and influenced airway contractility and calcium flux in ASM in precision-cut lung slices (PCLSs) from wild-type and hORMDL3Zp3-Cre mice (which express increased levels of human ORMDL3 [hORMDL3]). METHODS: Levels of ASM proliferation and contraction were assessed in ASM cells transfected with ORMDL3 in vitro. In addition, airway contractility and calcium oscillations were quantitated in ASM cells in PCLSs derived from naive wild-type and naive hORMDL3Zp3-Cre mice, which do not have a blood supply. RESULTS: Increased ASM expression of ORMDL3 in vitro resulted in increased ASM proliferation and contractility. PCLSs derived from naive hORMDL3Zp3-Cre mice, which do not have airway inflammation, exhibit increased airway contractility with increased calcium oscillations in ASM cells. Increased ASM ORMDL3 expression increases levels of ASM sarcoplasmic reticulum Ca2+ ATPase 2b (SERCA2b), which increases ASM proliferation and contractility. CONCLUSION: Overall, these studies provide evidence that an intrinsic increase in ORMDL3 expression in ASM can induce increased ASM proliferation and contractility, which might contribute to increased airway hyperresponsiveness in the absence of airway inflammation in asthmatic patients.

Chromosome 17q21 Genes ORMDL3 and GSDMB in Asthma and Immune Diseases.

Chromosome 17q21 contains a cluster of genes including ORMDL3 and GSDMB, which have been highly linked to asthma in genome-wide association studies. ORMDL3 is localized to the endoplasmic reticulum and regulates downstream pathways including sphingolipids, metalloproteases, remodeling genes, and chemokines. ORMDL3 inhibits serine palmitoyl-CoA transferase, the rate-limiting enzyme for sphingolipid biosynthesis. In addition, ORMDL3 activates the ATF6α branch of the unfolded protein response which regulates SERCA2b and IL-6, pathways of potential importance to asthma. The SNP-linking chromosome 17q21 to asthma is associated with increased ORMDL3 and GSDMB expression. Mice expressing either increased levels of human ORMDL3, or human GSDMB, have an asthma phenotype characterized by increased airway responsiveness and increased airway remodeling (increased smooth muscle and fibrosis) in the absence of airway inflammation. GSDMB regulates expression of 5-LO and TGF-ß1 which are known pathways involved in the pathogenesis of asthma. GSDMB is one of four members of the GSDM family (GSDMA, GSDMB, GSDMC, and GSDMD). GSDMD (located on chromosome 8q24 and not linked to asthma) has emerged as a key mediator of pyroptosis. GSDMD is a key component of the NLPR3 inflammasome and is required for its activation. GSDMD undergoes proteolytic cleavage by caspase-1 to release its N-terminal fragment, which in turn mediates pyroptosis and IL-1ß secretion. Chromosome 17q21 has not only been linked to asthma but also to type 1 diabetes, inflammatory bowel disease, and primary biliary cirrhosis suggesting that future insights into the biology of genes located in this region will increase our understanding of these diseases.