Identification of Evolutionarily Conserved Md1 Splice Variants That Regulate Innate Immunity through Differential Induction of NF-?B This information is current as of September 26, 2021. Sergio Candel, Sylwia D. Tyrkalska, Diana García-Moreno, José Meseguer and Victoriano Mulero J Immunol 2016; 197:1379-1388; Prepublished online 11 July 2016; doi: 10.4049/jimmunol.1502052 Downloaded from http://www.jimmunol.org/content/197/4/1379 Supplementary http://www.jimmunol.org/content/suppl/2016/07/11/jimmunol.150205 http://www.jimmunol.org/ Material 2.DCSupplemental References This article cites 67 articles, 24 of which you can access for free at: http://www.jimmunol.org/content/197/4/1379.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 26, 2021 • No Triage! Every submission reviewed by practicing scientists • 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. The Journal of Immunology Identification of Evolutionarily Conserved Md1 Splice Variants That Regulate Innate Immunity through Differential Induction of NF-кB Sergio Candel,1 Sylwia D. Tyrkalska,1 Diana Garcı´a-Moreno, Jose´ Meseguer, and Victoriano Mulero Although in mammals the TLR4/myeloid differentiation factor (MD)2/CD14 complex is responsible for the recognition of bacterial LPS, and it is known that the RP105/MD1 complex negatively regulates TLR4 signaling, the evolutionary history of LPS recognition remains enigmatic. Thus, zebrafish has orthologs of mammalian TLR4 (Tlr4a and Tlr4b), RP105, and MD1, but MD2 and CD14 seem to be absent from all fish genomes available to date. In addition, and to make the story more intriguing, zebrafish Tlr4a and Tlr4b do not recognize LPS, whereas the zebrafish Rp105/Md1 complex unexpectedly participates in the regulation of innate im- Downloaded from munity and viral resistance. In this work, we report the identification of two novel splice variants of Md1, which are expressed at similar levels as full-length Md1 in the main immune-related organs of zebrafish and are highly induced upon viral infection. One of these splice variants, which is also expressed by mouse macrophages, lacks three conserved cysteine residues that have been shown to form disulfide bonds that are crucial for the three-dimensional structure of the MD-2–related lipid recognition domain of Md1. Functional studies in zebrafish demonstrate that this evolutionarily conserved splice variant shows higher antiviral activity than full-length Md1, but reduced proinflammatory activity, due to an impaired ability to activate the master regulator of inflamma- http://www.jimmunol.org/ tion, NF-kB. These results uncover a previously unappreciated evolutionarily conserved Md1 splice variant with important functions in the regulation of innate immunity and the antiviral response in zebrafish, and point to the need for additional functional studies in mammals on this little explored molecule. The Journal of Immunology, 2016, 197: 1379–1388. he recognition of conserved molecular structures of All TLRs share the same structure, defined by the presence of an pathogenic microorganisms, called pathogen-associated extracellular domain containing leucine-rich repeats, a conserved T molecular patterns (PAMPs), by pattern recognition re- pattern of juxtamembrane cysteine residues, and an intracellular ceptors (PRRs) is crucial for mounting an efficient and rapid de- Toll/IL-1R domain in the cytosolic part of the protein, the fensive response in both invertebrates and vertebrate organisms C-terminal, that initiates signal transduction (1, 5). by guest on September 26, 2021 (1, 2). TLRs, a multigene family of type I transmembrane pro- TLR4 was the first TLR described and characterized in mammals teins, are the best characterized group of PRRs, and are highly (6, 7), where it was shown to be the PRR responsible for signaling conserved in both the invertebrate and vertebrate lineages (3, 4). in response to LPS, the complex glycolipid that is the major component of the Gram-negative bacteria outer membrane (8, 9). Some accessory molecules have been identified and shown to be Departamento de Biologı´a Celular e Histologı´a, Facultad de Biologı´a, Universidad de Murcia, Instituto Murciano de Investigacio´n Biosanitaria-Arrixaca, 30100 Murcia, essential for the regulation of the TLR4 function and, therefore, Spain for the maintenance of the fine balance necessary to respond 1S.C. and S.D.T. contributed equally to this work. against pathogens, but avoiding an exacerbated response. Thus, ORCIDs: 0000-0001-7919-6584 (S.C.); 0000-0002-1521-2327 (D.G.-M.); 0000- myeloid differentiation factor (MD)2 (also named LY96), LPS- 0001-9527-0211 (V.M.). binding protein, CD14, and CD36 are accessory molecules re- Received for publication September 16, 2015. Accepted for publication June 12, quired for the TLR4 function by mediating ligand delivery and/or 2016. recognition (10–14), whereas MD1 and RP105 negatively regulate This work was supported by the Spanish Ministry of Economy and Competitiveness TLR4 signaling (15–17). MD1, also named lymphocyte Ag 86, (Grants BIO2011-23400 and BIO2014-52655-R [to V.M.] and a Ph.D. fellowship [to S.C.], all cofunded with Fondos Europeos de Desarrollo Regional/European Re- directly interacts with LPS (18) and with RP105 (19), which gional Development Funds) and by European 7th Framework Initial Training Net- presents the same structure as TLR4, but lacks the intracellular work FishForPharma Ph.D. Fellowship PITG-GA-2011-289209 (to S.D.T.). Toll/IL-1R domain signaling domain (20). Thus, the MD1-RP105 The sequences presented in this article have been submitted to the European complex acts as physiologically negative regulator of TLR4 sig- Nucleotide Archive (http://www.ebi.ac.uk/ena/) under accession numbers LN875559 (zebrafish md1_tv1), LN875560 (zebrafish md1_tv2), and LN875561 naling through its direct interaction with the MD2–TLR4 complex (mouse Md1_tv2). and the subsequent inhibition of LPS binding (15–17). Address correspondence and reprint requests to Prof. Victoriano Mulero, Departa- In zebrafish, two orthologs of mammalian TLR4, Tlr4a and mento de Biologı´a Celular e Histologı´a, Facultad de Biologı´a, Universidad de Tlr4b (also known as Tlr4ba and Tlr4bb), have been cloned and Murcia, 30100 Murcia, Spain. E-mail address: [email protected] characterized (21, 22), as well as homologs of MD1 and RP105 The online version of this article contains supplemental material. (23). However, MD2 and CD14 seem to be absent in the zebrafish Abbreviations used in this article: MD, myeloid differentiation factor; ML, MD2- related lipid recognition; MO, morpholino; ORF, open reading frame; PAMP, genome (24), as well as in other fish genomes available. Even pathogen-associated molecular pattern; PRR, pattern recognition receptor; RT-qPCR, more intriguing is the fact that zebrafish Tlr4a and Tlr4b do not 5 quantitative RT-PCR; SVCV, spring viremia of carp virus; TCID50,10 tissue culture recognize LPS (25, 26). Furthermore, it has recently been de- infectious dose; VaDNA, Vibrio anguillarum genomic DNA. scribed that Md1 physically interacts with Tlr4a, Tlr4b, and Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 Rp105 in zebrafish, and that it participates in the regulation of www.jimmunol.org/cgi/doi/10.4049/jimmunol.1502052 1380 IDENTIFICATION OF NOVEL Md1 SPLICE VARIANTS innate immunity and viral resistance (23). However, many pieces sequences were determined using the Simple Modular Architecture Re- are still necessary to solve this puzzle and to elucidate the role search Tool, from the European Molecular Biology Laboratory Web site played by Tlr4 and its accessory molecules from fish to mammals. (http://smart.embl-heidelberg.de/) (46, 47). Finally, three-dimensional structure predictions were performed using the PSIPRED Protein Se- New gene functions are normally achieved through redundancy, quence Analysis Workbench (48, 49) and the Protein Homology/AnalogY which can occur through two different mechanisms: gene duplication Recognition Engine (Phyre 2) (50, 51) and visualized with the open- (the emergence of additional genomic copies) (27–29) and alternative source Java viewer Jmol 14.0.13 (http://jmol.sourceforge.net/). Confi- splicing (an increase in transcript diversity) (30). Gene duplication dence values for the three-dimensional structural predictions were also provided by Phyre 2. Moreover, The ModFOLD Quality Assessment and alternative splicing are inversely correlated evolutionary mech- Server (version 4.0) has been used to check the accuracy of the models anisms because an inverse correlation between the size of a gene’s (52). family and its use of alternatively spliced isoforms has been found (31). Alternative splicing is a cellular mechanism in eukaryotes Morpholinos and RNA injection that produces multiple mature mRNA isoforms from a pre-mRNA