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A Transcriptome-Based Approach to Identify Functional Modules Within and Across Primary Human Immune Cells

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A Transcriptome-Based Approach to Identify Functional Modules Within and Across Primary Human Immune Cells PLOS ONE RESEARCH ARTICLE A transcriptome-based approach to identify functional modules within and across primary human immune cells Saraï Mola1, Sylvain Foisy1, Gabrielle Boucher1, FrancËois Major2,3,4, Claudine Beauchamp1, Mohamad Karaky1, Philippe Goyette1, Sylvie Lesage5,6, John 1,4 D. RiouxID * 1 Centre de recherche, Institut de cardiologie de MontreÂal, MontreÂal, QueÂbec, Canada, 2 Unite de recherche en ingeÂnierie des ARN, Institut de recherche en immunologie et en canceÂrologie, MontreÂal, QueÂbec, Canada, a1111111111 3 DeÂpartement d'informatique et de recherche opeÂrationnelle, Universite de MontreÂal, MontreÂal, QueÂbec, a1111111111 Canada, 4 DeÂpartement de biochimie et meÂdecine moleÂculaire, Universite de MontreÂal, MontreÂal, QueÂbec, a1111111111 Canada, 5 Centre de recherche, HoÃpital Maisonneuve-Rosemont, MontreÂal, QueÂbec, Canada, 6 DeÂpartement de microbiologie, infectiologie et immunologie, Universite de MontreÂal, MontreÂal, QueÂbec, a1111111111 Canada a1111111111 * [email protected] Abstract OPEN ACCESS Citation: Mola S, Foisy S, Boucher G, Major F, Genome-wide transcriptomic analyses have provided valuable insight into fundamental biol- Beauchamp C, Karaky M, et al. (2020) A ogy and disease pathophysiology. Many studies have taken advantage of the correlation in transcriptome-based approach to identify the expression patterns of the transcriptome to infer a potential biologic function of uncharac- functional modules within and across primary human immune cells. PLoS ONE 15(5): e0233543. terized genes, and multiple groups have examined the relationship between co-expression, https://doi.org/10.1371/journal.pone.0233543 co-regulation, and gene function on a broader scale. Given the unique characteristics of Editor: Gualtiero I. Colombo, Centro Cardiologico immune cells circulating in the blood, we were interested in determining whether it was possi- Monzino, ITALY ble to identify functional co-expression modules in human immune cells. Specifically, we Received: October 23, 2019 sequenced the transcriptome of nine immune cell types from peripheral blood cells of healthy donors and, using a combination of global and targeted analyses of genes within co-expres- Accepted: May 7, 2020 sion modules, we were able to determine functions for these modules that were cell lineage- Published: May 29, 2020 specific or shared among multiple cell lineages. In addition, our analyses identified transcrip- Copyright: © 2020 Mola et al. This is an open tion factors likely important for immune cell lineage commitment and/or maintenance. access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Introduction Data Availability Statement: All of the results per The human immune system protects us from microbes (bacteria, viruses, fungi, and parasites) cell type can be shared without restrictions and are included as a Supplementary file. A table containing that penetrate the physical and chemical barriers of the body. It fulfills surveillance functions in the aggregated data is included in the paper. I can order to detect and eliminate aberrant cells that result from infection, cancer, and senescence. confirm that the CERDNT (MHI Ethics Committee) In contrast, the immune system is also at the heart of pathogenic syndromes and chronic dis- will not allow depositing individual-level data. eases that involve virtually all organ systems (e.g. cardiovascular, gastrointestinal, respiratory, Researchers can request access to the individual- musculoskeletal), and are associated with significant morbidity and mortality [1±5]. level data from the current study by contacting the Montreal Heart Institute ethics committee at the Given the central importance of the immune system in human health and disease, extensive following institutional email address: <cer. work by numerous research groups over the past 100+ years has provided a detailed functional [email protected]> understanding of the cells and organs within the immune system. This includes how the PLOS ONE | https://doi.org/10.1371/journal.pone.0233543 May 29, 2020 1 / 30 PLOS ONE Using transcriptomics to identify key functions in human immune cells Funding: This work was supported by grants from multiple different cell populations, which make up the complex immune system, differentiate the Canadian Institutes of Health Research: (1) The from a common pluripotent progenitor cell, how different cells communicate through an exten- ``CIHR Emerging Team in Integrative Biology of sive system of cytokines, chemokines, and associated receptors, and how signaling pathways Inflammatory Diseases'' [CIHR #102170] with co- funding from Crohn's and Colitis Canada made can regulate cell response to various stimuli [6±8]. This functional understanding has been possible via a generous donation from the Ross made possible by a combination of human and animal model systems, sensitive and specific McMaster Memorial Fund; (2) the IMAGINE-SPOR reagents, such as antibodies directed against cell surface and intracellular proteins, and the Chronic Disease Network [CIHR #SCA-145105]. development of technological platforms to isolate and study-specific cell populations based on Salary support to Saraï Mola was provided by the multi-parameter flow cytometry, fluorescence-activated cell sorting, and mass cytometry [5, 9]. Canada Research Chair in Genetics and Genomic Genome-wide transcriptomic-based approaches have been instrumental in the identification Medicine awarded to John David Rioux [CRC#230625]. Sylvie Lesage holds a Research of molecular classifiers of leukemias, interferon and granulopoiesis signatures in the blood of Scholar Emeritus award from the Fonds de autoimmune patients, cytokine response pathways of T cells in psoriasis and the activation of recherche du QueÂbec ± Sante (FRQ-S). This work macrophages, to name a few [10±12]. Transcriptomic analyses have also been employed to per- was supported in part by the infrastructure at form surveys of the different cell populations within the myeloid and lymphoid lineages of the Compute Quebec/Canada. mouse immune system in different contexts (e.g. age, differentiation and activation states, tissue Competing interests: The authors have declared location) [13, 14]. More recently, the use of single-cell RNA sequencing to evaluate the tran- that no competing interests exist. scriptome has enabled the identification of previously unrecognized rare cell subsets of the Abbreviations: TF, transcription factor; WGCNA, human immune system [15±18]. Together, these studies suggest that genome-wide transcrip- weighted gene co-expression network analysis; tomics is an important tool for studying how the human immune system responds to different TEG, top expressing gene; MRG, module conditions (e.g. stimuli, genotypes, health status, response to therapy). representative gene. The identification of gene sets that robustly represent different conditions has the potential to become powerful biomarkers, even without fully understanding the impact of the observed transcriptomic changes. The functional interpretation of changes in transcriptome between different conditions, however, remains an important challenge. A common approach to addressing this problem is to first perform gene co-expression analyses, for identifying gene sets that potentially are involved in common biological functions, followed by annotation- based analyses (e.g. enrichment of annotation terms, guilt-by-association) for attributing spe- cific functions to the observed gene sets [19±21]. The success of this approach depends on numerous factors such as the complexity of the samples being tested, the true extent of the dif- ferences in the transcriptomes between the different conditions, the degree of experimental variability in the collection, processing, and sequencing of the samples [19±21]. In the current study, we were interested in testing this approach in the context of circulating human immune cells because of the well-characterized and distinctive nature of the different immune cells in terms of cell-specific markers (and thus genes expressed) and functions, as well as the relative ease by which highly purified immune cell populations can be isolated from peripheral blood. Specifically, we analyzed the transcriptomes of nine distinct immune cell populations from the peripheral blood of twelve healthy volunteers using a combination of global and targeted approaches to functionally annotate modules of co-expressed genes. We were able to attribute functions to most of the modules that were either cell population-specific or shared across more than one cell population. In addition, we identified TFs associated with lymphoid vs. myeloid lineages, T and B cell fates, many of which were previously known to be involved in lineage differentiation, as well as others that we propose as new candidates for hav- ing a role in lineage commitment and/or lineage maintenance. Materials and methods Subject selection criteria, ethics committee approval, and blood sample procedures The project was submitted to and approved by the MHI Institutional Ethics Committee «Comite d'eÂthique de la recherche et du deÂveloppement des nouvelles technologies ». PLOS ONE | https://doi.org/10.1371/journal.pone.0233543 May 29, 2020 2 / 30 PLOS ONE Using transcriptomics to identify key functions in human immune cells Volunteers were then solicited
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