Thymic Mesenchymal Cells Have a Distinct Transcriptomic Profile Julien Patenaude and Claude Perreault This information is current as J Immunol 2016; 196:4760-4770; Prepublished online 29 of October 1, 2021. April 2016; doi: 10.4049/jimmunol.1502499 http://www.jimmunol.org/content/196/11/4760 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2016/04/29/jimmunol.150249 Material 9.DCSupplemental References This article cites 65 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/196/11/4760.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on October 1, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Thymic Mesenchymal Cells Have a Distinct Transcriptomic Profile Julien Patenaude and Claude Perreault In order to understand the role of mesenchymal cells (MCs) in the adult thymus, we performed whole transcriptome analyses of primary thymic, bone, and skin MCs. These three MC populations shared expression of 2850 core MC genes involved in generic processes including interactions with tissue-resident macrophages. Moreover, we discovered that 2036 genes were differentially expressed, by at least 5-fold, in the three MC populations. Genes preferentially expressed in thymic MCs are instrumental in clearance of apoptotic thymocytes by macrophages, maintenance of a noninflammatory milieu, and attraction-expansion of thymocyte progen- itors. Thymic and bone MCs share other sets of differentially expressed genes implicated in resolution of inflammation and expansion of hematolymphoid progenitors. Consistent with the fact that thymic and skin MCs have to support epithelial cells, they express at higher levels genes mediating epithelial cell adhesion to basement membrane and mesenchymal–epithelial cross-talk. Differentially expressed genes preferentially expressed by bone MCs are connected to formation and remodeling of bone, whereas those prefer- Downloaded from entially expressed in skin MCs are involved in skin and hair follicle homeostasis. We conclude that MCs from different organs display substantial heterogeneity and that the transcriptome of thymic MCs is exquisitely suited for interactions with epithelial and hematolymphoid cells in an environment with a high apoptosis rate. The Journal of Immunology, 2016, 196: 4760–4770. esenchymal cells (MCs) derive mainly from the me- MCs. During development, thymic MCs regulate the proliferation soderm and, to a lesser extent, from the neural crest of thymic epithelial cells through production of fibroblast growth http://www.jimmunol.org/ M (ectoderm). In the adult organism, they are found in all factor (FGF)-7 and -10, insulin-like growth factor (IGF)-1 and -2, tissues and organs, where they play mechanical and metabolic and retinoic acid (7–11). However, little is known of the role of roles. MCs are the major producers of extracellular matrix, which thymic MCs during postnatal life, aside from the fact that CD248+ provides a structural framework, a scaffold for cell migration, and MCs play a role in revascularizing thymuses during infection- serves as a reservoir for cytokines and growth factors used by dependent regeneration and that FSP1+ MCs are essential for epithelial and hematopoietic parenchymal cells (1). Bone marrow the maintenance of the medullary thymic epithelium (12, 13). MCs have attracted extraordinary attention because they are key In order to gain insights into the function of thymic MCs, we components of the hematopoietic stem cell niche, and they contain elected to compare their transcriptome to that of bone and skin mesenchymal stem and progenitor cells that display an amazing MCs. Two reasons led us to select bone and skin MCs for this study. by guest on October 1, 2021 ability to regulate immune responses and coordinate tissue re- First, we included bone MCs as a reference because they have been generation (2–6). However, because extramedullary MCs are seen studied most extensively and represent by far the best characterized as supporting players, their role in normal adult physiology has MC population. Second, we reasoned that the function of MCs received much less attention than that of parenchymal (e.g., epi- might be influenced by the nature of their neighboring parenchymal thelial or hematopoietic) cells. This is notably the case for thymic cells: thymic and skin MCs have to support epithelial cells, whereas thymic and bone MCs have to support hematolymphoid cells. The Institute for Research in Immunology and Cancer, University of Montreal, Montreal, transcriptome is a critical component of systems-level under- Quebec H3C 3J7, Canada; and Department of Medicine, University of Montreal, standing of cell biology, and it can be reliably tackled in its entirety Montreal, Quebec H3C 3J7, Canada using relatively modest cell numbers (14). In the last decade, ORCID: 0000-0001-9453-7383 (C.P.). studies by the Immunological Genome Project (http://www.immgen. Received for publication November 30, 2015. Accepted for publication March 28, org/) have illustrated the relevance of transcriptome analyses by 2016. unraveling large gene networks in primary myeloid, lymphoid, and This work was supported by Grant MOP 42384 from the Canadian Institute of Health Research. The Institute for Research in Immunology and Cancer is supported in part stromal cells from various hematolymphoid organs (15). Never- by the Canada Foundation for Innovation and the Fonds de la Recherche en Sante´ du theless, several pioneer studies addressing the question of MCs’ Que´bec. transcriptomic heterogeneity are fraught with limitations because The RNA-sequencing data presented in this article have been submitted to the Gene they were based on microarray analyses performed on in vitro– Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession numbers GSE73175 and GSE75598. cultured MCs (16, 17). This approach is indeed problematic be- Address correspondence and reprint requests to Prof. Claude Perreault, Institute for cause plastic adherence and in vitro culture induce major changes Research in Immunology and Cancer, P.O. Box 6128, Station Centre-Ville, Montreal, in the phenotype and biology of MCs (6). Furthermore, relative to QC H3C 3J7, Canada. E-mail address: [email protected] RNA-sequencing (RNA-seq), microarrays display lower sensitiv- The online version of this article contains supplemental material. ity and dynamic range coupled to higher technical variations (18, Abbreviations used in this article: 7-AAD, 7-aminoactinomycin D; DEG, differen- 19). We therefore elected to explore the question of MC hetero- tially expressed gene; ETP, early thymocyte progenitor; FC, fold change; FGF, fibro- blast growth factor; GEO, Gene Expression Omnibus; HSC, hematopoietic stem cell; geneity by studying the transcriptome of primary freshly harvested IGF, insulin-like growth factor; IPA, Ingenuity Pathway Analysis; Lin, lineage; MC, MCs using RNA-seq. mesenchymal cell; RNA-seq, RNA-sequencing; RPKM, reads per kilobase of exon We identified three main gene sets in MCs: 6270 housekeeping per million mapped reads; TEC, thymic epithelial cell. genes, 2850 core MC genes, and 2036 differentially expressed Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 genes (DEGs) that were preferentially expressed in one or two MC www.jimmunol.org/cgi/doi/10.4049/jimmunol.1502499 The Journal of Immunology 4761 populations. Core MC genes are involved in generic MC functions, Sca-1+ cells. For intracellular staining, cell viability was assessed using the whereas DEGs are connected to organ-specific features such as Live/Dead fixable blue dead cell stain kit (Invitrogen). Cells were fixed/ clearance of apoptotic cells (thymic MCs), osteoclastogenesis permeabilized with the Cytofix/Cytoperm Plus kit with GolgiPlug (BD Biosciences) and stained with PE anti-CXCL12 Ab (R&D Systems) or PE (bone MCs), and hair follicle homeostasis (skin MCs). Our work anti-Nestin Ab (R&D Systems). MCs were sorted on a three-laser FACSAria provides a systems-level representation of MC heterogeneity and a (BD Biosciences) or analyzed on a three-laser LSR II (BD Biosciences) global framework to explain how MCs may adapt to organ-specific using FACSDiva software (BD Biosciences), as described (24). functions. RNA extraction and high-throughput RNA-seq RNA extraction and RNA-seq were performed as described (25, 26). Output Materials and Methods data were mapped to the Mus muculus (mm10) reference genome using Mice ELANDv2 alignment tool from the CASAVA 1.8.2 software, and transcript levels were expressed as reads per kilobase of exon per million mapped C57BL/6 mice purchased from The Jackson Laboratory (Bar Harbor, ME) reads (RPKM) (27).