Research Area: This Application Addresses the Broad Challenge Area (15) Translational Science

Research Area: This Application Addresses the Broad Challenge Area (15) Translational Science

5. Research Design and Methods, 5.1 Research Area: This application addresses the broad challenge area “(15) Translational Science” and specific challenge topic, “15-HL-102: Develop new therapeutic strategies for heart, lung, and blood diseases based on microRNA technology”. The project title is “microRNA Regulation of Human Airway Epithelial Cell Phenotype”.

5.2 Challenge and Potential Impact: It is estimated that >22 million people in the USA currently have asthma and that >15 million people have chronic obstructive pulmonary disease (COPD), which is the fourth leading cause of death (data from Centers for Disease Control and Prevention). As well, there are other less prevalent, but individually devastating, obstructive lung diseases such as cystic fibrosis (CF), non-CF bronchiectasis and primary ciliary dyskinesia (PCD). These conditions place massive burdens on health care resources and immeasurably degrade human spirit and potential. All of them are focused on the lung airways, which become characteristically inflamed and remodeled, causing functional impairment. The airway epithelium is strategically located at the lung-environment interface. In addition to its well-known barrier and protective functions, it is now widely appreciated that the epithelium initiates, integrates and orchestrates host immune responses and airway remodeling via secretion of regulatory factors within complex feedback loops {}. Phenotypic changes in the airway such as enhanced epithelial shedding and turnover, deficient repair, mucous secretory (goblet) cell hyperplasia and squamous metaplasia are variably present and are integral to the progression of inflammatory airway diseases. Despite recent advances, many basic mechanisms regulating altered structure and function of hBE cells remain poorly understood, and there are no specific therapies directed at preventing or reversing disease-related phenotypic changes in the airway epithelium.

In the last decade, “omics” have revolutionized our understanding of biology and disease, specifically including numerous microarray studies of airway mRNA in asthma and COPD {14248} {14244}. We now appreciate that non-coding, small RNA’s including microRNAs (miRNAs) are critical regulators of genomes and genes. Major effects of miRNA’s are to destabilize mRNA and to inhibit protein translation, but there are also examples of miRNA positive regulation of transcription. In any case, a regulatory network between miRNAs and gene expression is clearly involved in almost every aspect of biology from stem cell maintenance and embryonic development / cell differentiation to cancer (reviewed in {14264}). Recent, but limited, studies provide insight that miRNAs are important in lung development {14253} {14293}, lung inflammation {14238}, the biological response to tobacco smoking {14287}, and lung cancer {14250}. However, we do not know the full miRNA repertoire of the airway epithelium, nor do we have a comprehensive understanding the miRNA-mRNA regulatory networks controlling hBE cell phenotype in health and disease. A unique combination of resources and talents at the University of North Carolina is poised to fill this major lung biology knowledge gap. The Randell laboratory is a leading provider of primary human bronchial epithelial (hBE) cells in models recapitulating their normal structure and function and has extensive knowledge of airway epithelial cell biology {}. The Hammond and Hayes laboratories provide cutting edge expertise in miRNA biology and bioinformatics, respectively {14223} {14253} {14252}. Supported by provocative preliminary data that miRNA expression changes dramatically as a function of hBE cell differentiation, these groups have combined forces with a plan to rapidly create new data that will significantly advance our understanding of miRNA regulation of airway epithelial phenotype during normal growth and differentiation and under a series of highly relevant injury/repair conditions.

Figure 1. Time course of hBE cell differentiation in a replicate of ALI cultures used for preliminary miRNA arrays. Formalin paraffin sections stained with H&E, or AB-PAS for glucoconjugates (blue), illustrate the progression from poorly- to well-differentiated.

5.3 Approach, Strategy: Our basic hypothesis is that miRNAs are critical regulators of hBE cell differentiation and the response to injury, that miRNAs become altered in pathologic states, and that modulating miRNAs can change hBE cell phenotype for therapeutic benefit. This hypothesis is based on initial studies showing dramatic regulation of miRNAs as a function of hBE cell differentiation in vitro (Figures 1-3). In this experiment, primary hBE cells were harvested from explanted lungs of six individual tissue donors. The initial primary cell culture stage was on plastic, where the cells predictably de-differentiated and proliferated but were unable to polarize and re-differentiate. The cells were then passaged from plastic to a porous support and were grown at an air-liquid interface (ALI) where they again proliferated but were now able to fully differentiate, recapitulating the normal pseudostratified airway epithelial morphology (Figure 1). Total RNA was harvested on days 5, 14 and 35 after seeding under ALI conditions, representing distinct differentiation stages. The 3’ hydroxyl group of miRNA was fluorescently labeled with P-cytidyl-uridyl-Cy3 using T4-RNA ligase and RNA was hybridized to home-spotted microarray slides, consisting of 527 unique oligonucleotides complementary to putative human miRNAs in the Sanger miRBase {14252}. The slides were scanned using the 532-nM channel on a Genepix scanner and raw expression data was extracted and analyzed using GeneCluster, TreeView and Significance Analysis of Microarrays (SAM). Median centered and normalized miRNA expression data of hBE cell cultures from 6 individuals at day 5, 14 and 35, subjected to unsupervised hierarchical clustering (Figure 2), revealed complete separation of the day 5 group from the later time points. The day 14 and 35 groups also tended to cluster but there was some admixture. Significance Analysis of Microarrays (SAM) software called 49 genes as significantly different at a 0% false discovery rate (Figure 3). The predominant expression pattern was a steady increase in expression of several miRNAs from day 5 to day 35, but there were examples of equivalent increases at day 14 and 35. Three miRNAs definitively decreased from day 5 to day 35. Manual observation of the data revealed additional borderline and/or inconsistent changes in several miRNAs.

Figure 2. Unsupervised hierarchical clustering of hBE cell miRNA expression data. Cultures from 6 individuals (1-6) at day (D) 5, 14 and 35 were studied.

Furthermore, many miRNAs not reliably detected by array technology likely changed but were not visible to us. Manual searching of the literature for functions of highly regulated miRNAs indicated several associated with cellular differentiation in other systems, tumor suppressors, and those with variable expression in different cancers. However, other strongly regulated miRNAs with unknown function were present. These results strongly indicate a key role for miRNAs in regulation of hBE cell proliferation and differentiation and illustrate significant gaps in our knowledge.

Figure 3. Expression map of miRNAs called as significant by SAM. Cultures from 6 individuals (1-6) at day (D) 5, 14 and 35 were studied. Genes (rows) are median centered and clustered (tree not shown). Arrays (columns) are median centered but not clustered.

The above results underlie the strategy for this proposal. Namely, that: 1) miRNAs are important regulators of the phenotype of the airway epithelium; 2) technology exists enabling comprehensive miRNA analysis through both unbiased discovery using deep sequencing, and via state-of-the-art microarray and RT-PCR technology for known miRNAs, and 3) in vitro studies are not plagued by the uncertainty of variable non-epithelial cell contributions to the expression pattern, and are amenable to genetic manipulation for functional testing. Our team has the optimal combination of resources and experience. The hBE cells themselves, which are prohibitively expensive when purchased commercially, will be provided to this project from our existing collection at no expense, which enables more ambitious experimentation. It is important to note, based on our prior array experience that there can be significant variability in miRNA and mRNA expression in hBE cells from human to human, which can be compounded by experimental variability. Our ability to simultaneously study cells from a representative sample of 6 people minimizes one source of experimental variability. Furthermore, we will use triplicate cultures from each donor within each group and will pool RNA to minimize contributions from one untoward, outlier culture well. The cells will be employed in state-of-of-the-art models of key disease processes that are either already well established in our labs or easily achievable. Our team includes world-class, expert co-investigators in both miRNA molecular biology and bioinformatics, which are key to achieving the experimental goals.

We note our strategy to perform mRNA expression analysis in the identical set of cultures as for the miRNA analyses, which entails considerable extra effort. Theoretically, we could have explored existing databases for the mRNA data. However, such data does not exist for all the experimental conditions, and it is likely that specific technical differences between different laboratories would inhibit direct comparisons. Thus, we think that using the identical samples is the optimal strategy to determine the miRNA regulatory network governing phenotypic changes in hBE cells in health and disease. Finally, we already have RNA samples for 2 of the seven sets of experiments in hand, which is compatible with the goal of the ARRA to quickly increase the use of Core facilities, create new hires and generate novel data that accelerates the pace of scientific discovery.

Specific Aim 1) Develop a comprehensive portrait of miRNA expression in hBE cells during normal differentiation and in a spectrum of relevant injury/repair conditions.

Figure 4. Wound repair model. A) Complementary pair of probes to wound ALI cultures. B) Probes placed in 12 mm diameter Millicell inserts. C) hBE cells 24 hours after wounding, repairing cells (left of dash) showing nuclear localization of EGR-1 transcription factor.

Rationale: miRNAs play a vital regulatory role in many cell processes and their expression is highly cell type- and differentiation stage-specific. Human bronchial epithelial cells are central participants in the pathogenesis of several extremely important lung diseases and, despite provocative early evidence in the literature and preliminary data in this proposal, there is minimal miRNA data in this key cell type. In vitro model systems recapitulating in vivo structure and function are critical milestone tests for therapeutic development and translation. We are uniquely poised to provide a comprehensive picture of the miRNA repertoire in hBE cells and their expression patter and cell localization during normal differentiation and in a series of highly relevant disease models. We will also perform mRNA arrays from the identical cell cultures in order to elucidate miRNA:gene expression networks.

Aim 1A. Identify novel hBE cell miRNAs by creating libraries and performing deep sequencing from: 1) the ALI hBE cell differentiation time course; 2) the response of well differentiated cells to wounding; 3) the response to acute and chronic exposure to bacterial products; 4) exposure to the Th2-type cytokine IL-13, which induces mucous secretory cell hyperplasia; 5) exposure to cigarette smoke gas phase; 6) simulated ambient ozone exposures and 7) squamous metaplasia induced by retinoic acid deficiency.

Methods: We begin this section with a brief general discussion of the cell culture model and overall experimental design and then a short piece on each of the 7 experimental conditions. Since the downstream analytical methods are common to each of the groups, we follow with a single section with methods for unbiased miRNA discovery by deep sequencing.

The ALI hBE cell culture methods have been described in detail previously {}. For all experiments, cells from 6 different tissue donors will be employed. Within each donor, each experimental group (time point, exposure etc.) will consist of 3 replicate wells for RNA harvest, which results in excess RNA, but prevents one potentially bad well from ruining a data point and allows for replicates in downstream quantitative analyses (qRT-PCR). Each well is inspected at each media change, culling any that are unusual. In experiments involving potentially toxic exposures, we assess lactate dehydrogenase release into the apical media and basolateral inflammatory cytokine (IL-8, Gro) production. Replicate wells are also prepared for histology (H&E, other stians and in situ hybridization) using fixation with 4% formaldehyde followed by paraffin embedding and blocks for frozen sections. RNA is extracted using a guanidinum:chloroform/phenol protocol optimized for hBE cells to prevent glycoconjugate (mucous) contamination of RNA. An aliquot is assessed by spectrophotometry (Nano Drop) and using LabChips on an Agilent 2100 Bioanalyzer.

The ALI hBE cell differentiation time course. As illustrated above in the Strategy section (Figure 1), the time course of hBE cell growth at an ALI recapitulates a process similar to wound repair in vivo in which poorly-differentiated cells undergo a phase of rapid proliferation, followed by polarization, and ultimately differentiation into the characteristic airway epithelial cell types, namely basal, secretory and ciliated cells. As noted above, we have chosen 3 time points, days 5, 14 and 35 that represent distinct stages in the process. Cells at day 5 are almost uniformly squamoid and highly proliferative with little expression of differentiation markers, whereas by Day 14 apical membrane polarization and mucous secretory cell development are evident. By Day 35 large numbers of ciliated cells are present as well as fully mature basal and secretory cells. There is a single study of mRNA expression during the ALI hBE cell time course in the literature {} but no miRNA data. Cell culture, RNA harvest and preparation of histology sections of triplicate specimens from an n = 6 different tissue donors is already complete and ready for uniform downstream processing as described below.

The response of well differentiated cells to wounding. The normal airway epithelium in vivo is mitogenically quiescent but responds dramatically to wounding via cell de-differentiation, migration, proliferation, and re-differentiation to ultimately restore an intact epithelium, which is critical to prevent airway obliteration. We have studied hBE cell wound repair {} and have recently developed a unique device for wounding ALI cultures that enables subsequent harvest of just the non-wounded and wound-repairing cell compartments (Figure 4). To our knowledge there are no miRNA or mRNA gene array studies of hBE cell wound repair. To fill this knowledge gap we will collect RNA for analysis of miRNA and mRNA expression at time 0, and in both cell compartments at 8, 24 and 48 hours after wounding, and subject them to uniform analysis as below.

Figure 5. hBE cell mRNA expression after P. aeruginosa. A) The acute response of naive cells- 372 probesets changed > 1.5 fold with p<0.01. B) The response of adapted cells- only 72 probesets met these criteria.
Figure 6. Cigarette smoke model. ALI hBE cells exposed to air or smoke for 3 consecutive days modestly released IL-8 and LDH, indicating appropriate dosing. N=4 wells/group, mean + SD, *p< 0.05.

The response to acute and chronic exposure to bacterial products. Repeated and/or chronic infection is characteristic of COPD, CF, non-CF bronchiectasis and PCD. The airway epithelium occupies the interface between the body and luminal infectious agents and is strategically positioned to detect and respond to danger and to orchestrate the host response. There are several studies of the hBE cell transcriptional response to pathogens but many are in cell lines and/or in poorly differentiated cells on plastic. Furthermore, no studies have assessed adaptation to chronic infection, although this is undoubtedly an important feature of the host response. We performed mRNA arrays following acute and chronic exposure of ALI hBE cells to P. aeruginosa bacteria and detected potent induction of inflammatory cytokines and other genes but, interestingly, the cells exhibited partial tolerance (decreased responsiveness) after repeat challenges (Figure 5). We will now perform unbiased detection using deep sequencing and arrays to test the hypothesis that miRNAs are involved in the ALI hBE cell response to acute and chronic bacterial product exposure. We will study cells 4 hours after an initial challenge (acute response) and 24 hours after three challenges (the new baseline at 72 hours, representing adaptation to chronic exposure) and 4 hours after a fourth challenge (76 hours, representing modified responses in adapted cells). As with the other experimental protocols, the cells will be subjected to uniform downstream analysis as below.

Exposure to the Th2-type cytokine IL-13, which induces mucous secretory cell hyperplasia. Increased mucus production is a feature of multiple lung diseases and is thought to contribute to airway obstruction, especially in acute asthma exacerbations and fatal asthma. Inefficient mucus clearance due to imbalanced, excessive mucous glycoprotein production and deficient ion and water transport creates mucus stasis and infection characteristic of CF and COPD. However, there are no specific therapies directed at preventing or reversing excess mucus production. To understand the role of miRNAs in mucus hyper-production, we will expose ALI hBE cells to 10 ng/ml 1L-13, a Th2-type cytokine known to induce mucous secretory cell hyperplasia. IL-13 treatment will take place every 48 hours beginning at day 25, for up to 10 days, which is a standard protocol in this field, and RNA and histology samples will be harvested at 8, 24, 48 and 240 hours.