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The Activation of Human Dermal Microvascular Cells by Poly(I:C)

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The Activation of Human Dermal Microvascular Cells by Poly(I:C) The Activation of Human Dermal Microvascular Cells by Poly(I:C), Lipopolysaccharide, Imiquimod, and ODN2395 Is Mediated by the Fli1/FOXO3A This information is current as Pathway of September 24, 2021. Lukasz Stawski, Grace Marden and Maria Trojanowska J Immunol published online 15 November 2017 http://www.jimmunol.org/content/early/2017/11/14/jimmun ol.1601968 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2017/11/14/jimmunol.160196 Material 8.DCSupplemental 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 by guest on September 24, 2021 • Fast Publication! 4 weeks from acceptance to publication *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 © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published November 15, 2017, doi:10.4049/jimmunol.1601968 The Journal of Immunology The Activation of Human Dermal Microvascular Cells by Poly(I:C), Lipopolysaccharide, Imiquimod, and ODN2395 Is Mediated by the Fli1/FOXO3A Pathway Lukasz Stawski, Grace Marden, and Maria Trojanowska Endothelial cell (EC) dysfunction has been associated with inflammatory and autoimmune diseases; however, the factors contributing to this dysfunction have not been fully explored. Because activation of TLRs has been implicated in autoimmune diseases, the goal of this study was to determine the effects of TLR ligands on EC function. Human dermal microvascular ECs (HDMECs) treated with TLR3 [Poly(I:C)], TLR4 (LPS), and TLR7 (imiquimod) agonists showed decreased proliferation and a reduced total number of branching tubules in three-dimensional human dermal organoid ex vivo culture. In contrast, the TLR9 ligand class C, ODN2395, increased angiogenesis. The antiproliferative effects of TLR3, TLR4, and TLR7 ligands correlated with significant downregulation of Downloaded from a key regulator of vascular homeostasis, Fli1, whereas TLR9 increased Fli1 levels. Furthermore, Poly(I:C) and LPS induced endothelial to mesenchymal transition that was reversed by the pretreatment with TGF-b neutralizing Ab or re-expression of Fli1. We showed that Fli1 was required for the HDMEC proliferation by transcriptionally repressing FOXO3A. In contrast to TLR9, which suppressed activation of the FOXO3A pathway, TLR3, TLR4, and TLR7 ligands activated FOXO3A as indicated by decreased phosphorylation and increased nuclear accumulation. The inverse correlation between Fli1 and FOXO3A was also observed in the vasculature of scleroderma patients. This work revealed opposing effects of TLR9 and TLR3, TLR4, and TLR7 http://www.jimmunol.org/ on the key angiogenic pathways, Fli1 and FOXO3A. Our results provide a mechanistic insight into the regulation of angiogenesis by TLRs and confirm a central role of Fli1 in regulating vascular homeostasis. The Journal of Immunology, 2018, 200: 000–000. ndothelial cells (ECs) control important vascular func- gands released by injured or stressed cells. TLRs are expressed tions, including blood pressure, permeability, coagulation, ubiquitously in immune cells and are less widespread in nonimmune E and angiogenesis (1). Various pathological conditions cells such as fibroblasts, epithelial cells, or ECs. The presence of including autoimmune diseases, such as systemic lupus eryth- TLRs on ECs depends on EC origin or location and may reflect ematosus and systemic sclerosis (SSc), are associated with dys- specialized functions of different ECs (5). In response to their li- functional endothelium (2, 3). In addition to their important role in gands, TLRs can activate the NF-kB signaling pathway and trigger by guest on September 24, 2021 regulating vascular homeostasis, ECs participate in inflammatory secretion of the proinflammatory cytokines, including IL-6, or the response by increasing production of proinflammatory factors production of type 1 IFNs (4). Recent studies demonstrated the including MCP-1, IL-6, TNF-a, and IFNs. Whereas inappropriate importance of TLR signaling in the development of several diseases activation of ECs could contribute to tissue damage and defective characterized by endothelial dysfunction, including atherosclerosis, angiogenesis (2), the mechanism underlying the interactions of hypertension, and autoimmune diseases. In particular, endosomal ECs with the immune system, especially the role of TLRs in this TLR3, TLR7, TLR9, which recognize dsRNA, ssRNA, and unmethy- process, is still poorly understood. lated CpG oligonucleotide sequences, as well as plasma membrane Innate immunity constitutes the first line of defense against in- TLR4, essential in recognition of bacterial pathogens, were suggested to vading pathogens and endogenous danger signals. TLRs belong to a be important in the initiation and progression of vascular dys- family of pattern recognition receptors that play a critical role in the function (6–8). Importantly, although TLR receptors share similar sig- activation of the innate immune system (4). TLRs can be recognized naling pathways, they may have divergent effects on cellular by a wide variety of exogenous ligands, including microbial cell wall function. For example, TLR7 and TLR9 displayed opposing ef- components, proteins, and nucleic acids, as well as endogenous li- fects on the development of systemic lupus erythematosus, through different effects on the autoantibody production (9, 10). Section of Rheumatology, School of Medicine, Boston University, Boston, MA Despite significant progress in delineating the role of selected 02118 TLR ligands in EC activation and injury, the signaling pathways ORCIDs: 0000-0001-7782-5420 (G.M.); 0000-0001-9550-7178 (M.T.). involved in these processes are still poorly understood. Received for publication November 21, 2016. Accepted for publication October 17, Relevant to these studies, we have recently observed that ex- 2017. pression levels of Fli1, a key regulator of vascular homeostasis, This work was supported by National Institutes of Health (National Institute of were significantly reduced by treatments with IFN-a in human Arthritis and Musculoskeletal and Skin Diseases) Grant R01 AR42334 to M.T. dermal microvascular ECs (HDMECs) (11). Fli1 is a member of Address correspondence and reprint requests to Prof. Maria Trojanowska, Boston University School of Medicine, 72 East Concord Street, E-5, Boston, MA 02118. the Ets family of transcription factors and is characterized by the E-mail address: [email protected] presence of the evolutionary conserved DNA-binding (ETS) do- The online version of this article contains supplemental material. main. Fli1 has been shown to play a major role in hematopoiesis, Abbreviations used in this article: ChiP, chromatin immunoprecipitation; EC, endo- embryonic development, and vasculogenesis. Additionally, in SSc, thelial cell; HDMEC, human dermal microvascular EC; IHC, immunohistochemical; reduced levels of Fli1 in ECs and fibroblasts (12) were correlated SCR, scramble; siRNA, small interfering RNA; SSc, systemic sclerosis. with vascular dysfunction, inflammation, and upregulated matrix Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 production (13–16). Mice with a conditional knockout of Fli1 in www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601968 2 EFFECT OF TLR LIGANDS ON ENDOTHELIAL CELL ACTIVATION ECs showed abnormal skin vasculature, with greatly compromised Quantitative RT-PCR analysis vessel integrity, and markedly increased vessel permeability (17). Total RNAwas isolated using TRIzol reagent (MRC, Cincinnati, OH). Real- Notably, vascular changes resulting from Fli1 deficiency over- time PCR assays were performed using the StepOnePlus Real-Time PCR lapped to a great extent with the abnormalities observed in the system (Applied Biosystems, Foster City, CA). Briefly, 1 mg of total RNA vasculature of SSc patients, suggesting that persistently reduced was reverse transcribed with random hexamers using the Transcriptor First levels of Fli1 in ECs could be important in disease pathogenesis. Strand cDNA Synthesis kit (Roche Applied Science) according to the manufacturer’s protocol. The amplification mixture (10 ml) contained 1 ml Whereas activation of the immune system in SSc is likely to of cDNA, 0.5 mM of each primer, and 5 ml of SYBR Green PCR Master contribute to vascular pathology, the role of immune mediators in Mix. The primers are listed in Supplemental Table I. Relative changes in 2 the regulation of Fli1 remains largely unexplored. Given the im- the levels of genes of interest were determined by the 2 DDcycle threshold portant role of TLRs in the pathogenesis of autoimmune diseases, method. including SSc, the goal of this study was to determine the role of Immunofluorescence staining on adherent cell cultures selected TLR ligands in modulating EC function focusing on a cross-talk between the TLR and the Fli1 signaling pathways. For immunofluorescence, cultured HDMECs grown on collagen-coated cover slips were treated with Fli1siRNA
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