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ACVRL1 Signaling Augments Cd8α+ Dendritic Cell Development

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Cutting Edge: ACVRL1 Signaling Augments CD8 α+ Dendritic Cell Development Rohit Verma, Hemant Jaiswal, Kuldeep Singh Chauhan, Monika Kaushik and Prafullakumar Tailor This information is current as of September 29, 2021. J Immunol 2016; 197:1029-1034; Prepublished online 15 July 2016; doi: 10.4049/jimmunol.1501849 http://www.jimmunol.org/content/197/4/1029 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2016/07/14/jimmunol.150184 Material 9.DCSupplemental References This article cites 31 articles, 12 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/197/4/1029.full#ref-list-1 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 29, 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 © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Th eJournal of Cutting Edge Immunology Cutting Edge: ACVRL1 Signaling Augments CD8a+ Dendritic Cell Development Rohit Verma, Hemant Jaiswal, Kuldeep Singh Chauhan, Monika Kaushik, and Prafullakumar Tailor Dendritic cells (DCs) are a collection of different sub- netic proteins [BMPs], growth-differentiation factor, NODAL, types, each of which is characterized by specific surface activins/inhibins, and anti-Mullerian€ hormone) involves com- markers, gene-expression patterns, and distinct func- binationsoftwotypesofreceptors:typeI(alsoknownas tions. Members of the IFN regulatory factor family play activin receptor–like kinases [ALKs]1–7]) and type II (TGF- critical roles in DC development and functions. Re- bRII, activin receptor II, activin receptor IIB, bone morpho- cently, Irf8 was shown to activate TGF-b signaling, genetic receptor II, and anti-Mullerian€ hormone receptor) which led to exacerbated neuroinflammation in the (7, 8). Activated receptor complex leads to phosphorylation of Downloaded from experimental autoimmune encephalomyelitis mouse receptor-regulated Smad proteins (R-SMADs). Specificity in model. We analyzed the effect of Irf8 on TGF-b/bone the activation of R-SMADs is defined by binding of ligand to morphogenetic protein pathway–specific genes in DCs specific type I receptor during signaling (e.g., SMAD1, and identified Acvrl1, a type I TGF-b superfamily re- SMAD5, and SMAD8 are activated by BMPs through ALK1–3 and ALK6, and TGF-b activates SMAD2–3 through ceptor, as a gene strongly induced by Irf8 expression. http://www.jimmunol.org/ ALK4–5 signaling) (8). Phosphorylated R-SMADs associate Among various DC subtypes, Acvrl1 is differentially with the partner SMAD4 and move to the nucleus where this expressed in CD8a+ DCs. ACVRL1 signaling aug- a+ complex (R-SMAD and SMAD4) can interact with other mented Irf8-directed classical CD8 DC develop- partners to regulate gene transcription. Further, TGF-b sig- ment. Irf8 expression is essential for plasmacytoid naling is also shown to cross-talk with other signaling pathways a+ DC and CD8 DC development, and this study dem- (7). Thus, through multilayered signal-transduction events and onstrates that ACVRL1 signaling plays a pivotal role interactions with other signaling pathways, the TGF-b super- whereby it suppresses plasmacytoid DC development family controls many cellular events, immune functions, and a+ b while enhancing that of CD8 DCs, thus contribut- tumor metastasis (7). TGF- promotes the development of by guest on September 29, 2021 ing to DC diversity development. The Journal of DCs, and TGF-b–null mice have a defect in the development Immunology, 2016, 197: 1029–1034. of epidermal Langerhans cells (LCs) (9, 10). A recent study (11) identified another TGF-b superfamily ligand, BMP7, as endritic cells (DCs) are represented by various sub- an instructive factor for human epidermal LCs. TGF-b1was classes, each of which has differential patterns of reported to upregulate Irf8 during DC development (12). Irf8 D gene expression, anatomical distribution, and ability was shown to enhance transcription of Itgb8 gene that converts for specific cytokine production and Ag presentation (1–3). latent TGF-b to the active form at the interface of APCs and 2 2 CD4+ DC, CD8a+ DC, CD4 CD8a double-negative DC, naive T cells (13). We analyzed the role of Irf8 in the regulation and plasmacytoid DC (pDC) are major DC subtypes in spleen; of TGF-b/BMPsignalinginDCsusingacommercially equivalent populations of these subtypes can be found in dif- available PCR-based array specific to mouse TGFb/BMP sig- ferent body sites, along with tissue-specific DCs (1–4). All DC naling pathway genes. We report that Irf8 expression induces subtypes developed from common DC progenitors in bone Acvrl1 (activin receptor–like kinase 1; also known as ALK1) + marrow (5, 6). Members of the IFN regulatory factor (IRF), gene, which is expressed specifically in the CD8a DC subtype. HLH, BATF, ETS, and STAT families of transcription factors, Further, we demonstrate that ACVRL1 signaling enhances de- + along with other transcription factors, play a critical role in velopment of the CD8a DC subtype. specific DC subtype development (3). Recent studies high- lighted the significance of Notch, Wnt, and TGF-b signaling Materials and Methods pathways in the generation of DC diversity (3). Signaling by All animal work conformed to the guidelines of the institute animal ethics ligands of the TGF-b superfamily (TGF-b,bonemorphoge- committee at the National Institute of Immunology. BXH-2 mice were Laboratory of Innate Immunity, National Institute of Immunology, New Delhi, Delhi The online version of this article contains supplemental material. 110067, India Abbreviations used in this article: ALK, activin receptor–like kinase; BMP, bone mor- Received for publication August 19, 2015. Accepted for publication June 16, 2016. phogenetic protein; cat. no., catalog number; ChIP, chromatin immunoprecipitation; DC, dendritic cell; FL-BMDC, FLT3 ligand–derived bone marrow DC; IRF, IFN- This work was supported by the National Institute of Immunology Core Fund and a regulatory factor; LC, Langerhans cell; pDC, plasmacytoid DC; Q-PCR, quantitative Ramalingaswami Fellowship award (to P.T.). PCR; R-SMAD, receptor-regulated Smad protein; TSS, transcription start site. Address correspondence and reprint requests to Dr. Prafullakumar Tailor, Laboratory of Innate Immunity, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 Delhi 110067, India. E-mail address: [email protected] www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501849 1030 CUTTING EDGE: ACVRL1 SIGNALING ENHANCES CD8a+ DC DEVELOPMENT procured from The Jackson Laboratory and crossed with C57BL/6 female mice and splenic DC populations were analyzed by flow cytometry, as described for three generations to eliminate vertical transmission of ecotropic murine earlier (14–16). Lineage-negative cells were purified from mouse bone mar- leukemia virus. Progeny were crossed to generate mice homozygous for row using a Lineage Cell Depletion Kit (Miltenyi Biotec). CD4+ DCs, 2 2 mutation (IRF8R294C) and littermate controls (IRF8WT). Mice were used after CD8a+ DCs, CD4 CD8a DCs, and B220+ pDCs, as defined earlier (15), genotyping by PCR sequencing, and experiments were performed at 8–12 wk were purified from splenic low-density cells by FACSAria III sorter using anti- of age. FLT3 ligand–derived bone marrow DCs (FL-BMDCs), DC9 cells, CD4–allophycocyanin-Cy7 and anti-B220–allophycocyanin (BD Biosciences), Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021 FIGURE 1. Acvrl1 gene is differentially expressed by CD8a+ DCs. (A) TGF-b/BMP pathway–specific gene-expression profiling was performed on control and 2 2 Irf8-expressing DC9 (Irf8 / background) cells. Acvrl1 gene was highly induced in the Irf8-expressing population. An increase in the Acvrl1 transcript level was validated by Q-PCR analysis of Irf8-expressing DC9 cells. Data are an average of six experiments. (B) Analysis of subtype-specific gene expression from sorted mouse splenic DC subtype populations suggested that Acvrl1 is specifically expressed in CD8a+ DCs. Data are representative of two independent experiments. (C) TGF-b/BMP pathway–specific gene-expression profiling was also performed on BMDCs from BXH-2 (Irf8R294C DC) and littermate control (Irf8WT DC) mice. Acvrl1 and Id2 genes common to analysis in (A) and (C) are shown in bold text. Results were also validated by Q-PCR analysis on BMDCs from BXH-2 (Irf8R294C) and littermate control (Irf8WT) mice. Data are an average of four individual mice. (D) DC9 cells were transduced with retroviruses expressing transcription factors (as shown). Populations were selected for 48 h with puromycin antibiotic, and Acvrl1 transcript levels were measured by Q-PCR. Data are representative of three independent experiments. (E) Schematic diagram shows the organization of human and mouse exons in the Acvrl1 gene (shown up to ∼10 kb). Blue arrows show known TSSs and red arrow shows proposed TSS in mouse. Translation start sites are shown as red bars. Exon (Ex) and Intron-Exon boundary (In-Ex)-specific Q-PCR analysis of Acvrl1 gene transcription from Irf8R294C DC and control Irf8WT DC mice (F) and from control and Irf8-expressing DC9 cells (G) also correlates with the proposed TSS. Results are representative of two independent experiments. Ex12 and Ex13 were analyzed by Q-PCR as control only to check the continuity of transcript. (H)59 RACE analysis demonstrated the expected amplicon from Irf8WT DCs, whereas Irf8R294C DCs show a very faint band correlating with very low transcription.
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  • A Highly Selective Chemical Probe for Activin Receptor-Like Kinases ALK4 and ALK5

    A Highly Selective Chemical Probe for Activin Receptor-Like Kinases ALK4 and ALK5

    bioRxiv preprint doi: https://doi.org/10.1101/2020.01.23.916502; this version posted January 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. A Highly Selective Chemical Probe for Activin Receptor-like Kinases ALK4 and ALK5 Thomas Hanke1, Jong Fu Wong2, Benedict-Tilmann Berger1, Ismahan Abdi1, Lena Marie Berger1, Roberta Tesch1, Claudia Tredup1, Alex N. Bullock2, Susanne Müller1, Stefan Knapp1 1Institute for Pharmaceutical Chemistry and Buchmann Institute for Molecular Life Sciences, Johann Wolfgang Goethe-University, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany. 2Structural Genomics Consortium, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford, OX3 7DQ, UK. *Correspondence: [email protected] Abstract: The transforming growth factor beta-receptor I/activin receptor-like kinase 5 (TGFBR1/ALK5) and its close homologue ALK4 are receptor protein kinases associated with the development of diverse diseases, including cancer, fibrosis, heart diseases and dysfunctional immune response. Therefore, ALK4/5 are among the most studied kinases and several inhibitors have been developed. However, current commercially available inhibitors either lack selectivity or have not been comprehensively characterized, limiting their value for studying ALK4/5 function in cellular systems. To this end, we report the characterization of the 2-oxo-imidazopyridine, TP-008, a potent chemical probe with dual activity for ALK4 and ALK5 as well as the development of a matching negative control compound. TP-008 has excellent cellular potency and strongly abrogates phosphorylation of the substrate SMAD2 (mothers against decapentaplegic homolog 2).
  • Cardiovascular Diseases Genetic Testing Program Information

    Cardiovascular Diseases Genetic Testing Program Information

    Cardiovascular Diseases Genetic Testing Program Description: Congenital Heart Disease Panels We offer comprehensive gene panels designed to • Congenital Heart Disease Panel (187 genes) diagnose the most common genetic causes of hereditary • Heterotaxy Panel (114 genes) cardiovascular diseases. Testing is available for congenital • RASopathy/Noonan Spectrum Disorders Panel heart malformation, cardiomyopathy, arrythmia, thoracic (31 genes) aortic aneurysm, pulmonary arterial hypertension, Marfan Other Panels syndrome, and RASopathy/Noonan spectrum disorders. • Pulmonary Arterial Hypertension (PAH) Panel Hereditary cardiovascular disease is caused by variants in (20 genes) many different genes, and may be inherited in an autosomal dominant, autosomal recessive, or X-linked manner. Other Indications: than condition-specific panels, we also offer single gene Panels: sequencing for any gene on the panels, targeted variant • Confirmation of genetic diagnosis in a patient with analysis, and targeted deletion/duplication analysis. a clinical diagnosis of cardiovascular disease Tests Offered: • Carrier or pre-symptomatic diagnosis identification Arrythmia Panels in individuals with a family history of cardiovascular • Comprehensive Arrhythmia Panel (81 genes) disease of unknown genetic basis • Atrial Fibrillation (A Fib) Panel (28 genes) Gene Specific Sequencing: • Atrioventricular Block (AV Block) Panel (7 genes) • Confirmation of genetic diagnosis in a patient with • Brugada Syndrome Panel (21 genes) cardiovascular disease and in whom a specific