Identification of Evolutionarily Conserved Md1 Splice Variants

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

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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 Biologıá Celular e Histologıá, Facultad de Biologıá, Universidad de Murcia, Instituto Murciano de Investigacioń 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 Biologıá Celular e Histologıá, Facultad de Biologıá, 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 Standard control (59-CCTCTTACCTCAGTTACAATTTATA-39) and Md1- molecule, resulting in a single gene coding for multiple protein specific (59-TTTCATCACTCTGATCTCACCGCAG-39) morpholinos (MOs) isoforms that differ in their amino acid sequence and, therefore, in (23) were purchased from Gene Tools and resuspended to 1 mM in nuclease-free water. In vitro transcribed RNA was obtained following the their chemical and biological activities (32–34). Different types of manufacturer’s instructions (mMESSAGE mMACHINE Kit; Ambion). alternative splicing have been described in which exons can be MOs (1.7 nmol/egg) and RNA (200 pg/egg) were mixed in microinjection spliced in different arrangements (35, 36), exon skipping being the buffer (0.53 Tango buffer and 0.05% phenol red solution) and micro- most common type of alternative splicing found in teleost fish (37). injected into the yolk sac of one- to eight-cell–stage embryos using a

Several splice variants of TLR4 and its accessory molecules microinjector (Narishige) (0.5–1 nl per embryo). The same amounts of Downloaded from MOs and/or RNA were used in all experimental groups. have already been identified and characterized in mammals, for example, soluble mouse TLR4 (38, 39), MyD88(S) (40), MD2B (41), Infection assays in adult zebrafish and MD2 short (42). In this study, we identify two novel zebrafish For the viral infection assays, the spring viremia of carp virus (SVCV) Md1 splice variants, one of which is conserved in mammals. isolate 56/70 was propagated in Epithelioma Papulosum Cyprini (obtained Using the unique advantages of the zebrafish animal model for from American Type Culture Collection) cells and titrated into 96-well

genetic studies and infection, we show in this work that one splice plates according to Reed and Muench (53). Adult fish were injected i.p. http://www.jimmunol.org/ 5 variant behaves as full-length Md1, whereas the other has stronger with PBS (control) or with 10 tissue culture infectious dose (TCID50) SVCV per fish. Infected fish were dissected, and mRNA levels were antiviral but reduced proinflammatory activities. measured by quantitative RT-PCR (RT-qPCR) in pooled of five heads at 48 h postinfection (54). In parallel, the basal levels of expression of the different md1 variants were also determined in different tissues from Materials and Methods nonchallenged fish. Animals PAMP stimulation and infection assays in embryos/larvae Zebrafish (Danio rerio H.) were obtained from the Zebrafish International Resource Center and mated, staged, raised, and processed, as previously For the stimulation assays, Vibrio anguillarum genomic DNA (VaDNA; 6.5 described (43). The Tg(6xHsa.NFKB:EGFP)sh235 (NF-kB:eGFP for sim-

ng/egg) was mixed in microinjection buffer and microinjected (0.5–1 nl), by guest on September 26, 2021 plicity) line was previously described (44). All adult zebrafish used were as described above. Gene expression was measured by RT-qPCR in from 6 to 18 mo old. Eight-week-old mice (Mus musculus L.) of the dechorionated whole larvae 24 or 48 h postfertilization, as specified in the BALB/c strain were maintained in the Animal Facility of the University of figure legends. For the viral infection assays, groups of 25–30 zebrafish Murcia with a commercial diet and water ad libitum. The experiments larvae were challenged at 3 d postfertilization in 5 ml egg water (60 mg/ml 7 8 performed comply with the Guidelines of the European Union Council sea salts in distilled water) containing 2.5 3 10 –10 TCID50/ml SVCV at (Directive 2010/63/EU) and the Spanish RD 53/2013, and the Bioethical 26˚C. Twenty-four hours later, the virus was diluted by adding 35 ml egg Committee of the University of Murcia (approval number 537/2011). Fish water, and the larvae were monitored every 24 h over a 10-d period for were always anesthetized by immersion in buffered tricaine (200 mg/ml; clinical signs of disease and mortality (55). In addition, 25 larvae were Sigma-Aldrich) before manipulation, whereas mice were killed by cervical collected per experimental group at 48 h postinfection, pooled (20–30), dislocation. and processed for the analysis of gene expression by real-time RT-PCR. Amplification and cloning of the zebrafish and mouse MD1 Analysis of gene expression splice variants Total RNA was extracted from pooled embryos/larvae or from adult tissues For zebrafish, the cDNA sequences of the three Md1 variants were amplified or organs with TRIzol reagent (ThermoFisher Scientific), following the by RT-PCR from samples obtained from adult zebrafish injected i.m. with manufacturer’s instructions, and treated with DNase I, amplification grade 5 mg Escherichia coli 0111-B4 LPS (25) and primers designed from the (1 U/mg RNA; ThermoFisher Scientific). SuperScript III RNase H Reverse previously annotated sequences for zebrafish Md1 (23). For mouse, MD1 Transcriptase (ThermoFisher Scientific) was used to synthesize first-strand variants were also amplified by RT-PCR from cDNA samples obtained cDNA with oligo(dT)18 primer from 1 mg total RNA at 50˚C for 50 min. from bone marrow–derived macrophages obtained using common proce- Real-time quantitative PCR was performed with an ABI PRISM 7500 dures. The different cDNAs were run and extracted from 1.2% agarose gel instrument (Applied Biosystems) using SYBR Green PCR Core Reagents (Sigma-Aldrich), purified using the GenElute Gel Extraction Kit (Sigma- (Applied Biosystems). Reaction mixtures were incubated for 10 min at Aldrich), cloned using the TOPO TA Cloning Kit (Thermofisher Scien- 95˚C, followed by 40 cycles of 15 s at 95˚C, 1 min at 60˚C, and finally 15 s tific), and finally sequenced with an automatic sequencer ABI PRISM 377 at 95˚C, 1 min at 60˚C, and 15 s at 95˚C. For each mRNA, gene expression (Applied Biosystems, Perkin-Elmer). The coding sequences of the three was normalized to the ribosomal protein S11 (rps11) content in each Md1 transcript variants were subcloned into the expression vector pFLAG- sample using the Pfaffl method (56). For analysis of the expression of the CMV-5a (Sigma-Aldrich). All sequences were analyzed using the freeware different splice variants, primers able to hybridize with the specific exon program Chromas Lite 2.01 (Technelysium). boundaries of each of them were designed. All of the primers used in this work are shown in Supplemental Table I. In all cases, each PCR was Sequence analysis of the zebrafish Md1 splice variants performed with triplicate samples and repeated with at least two independent samples. Zebrafish full-length Md1 sequence was compared with other known MD1 sequences, obtained from the Universal Protein Resource database (http:// Western blot assays www.uniprot.org/), and with the newly identified variants by multiple sequence alignment carried out with the ClustalX version 2.1 program (45). The stability of the three Md1 transcript variants was analyzed by means of The m.w. were estimated using the Protein Molecular Weight tool from Western blot. Human embryonic kidney HEK293 cells were purchased from The Sequence Manipulation Suite (http://www.bioinformatics.org/sms/ European Cell Culture Collection and grown at 37˚C in DMEM culture index.html). The domains of the proteins deduced from the nucleotide media (Life Technologies), supplemented with 10% FCS (Life Technologies) The Journal of Immunology 1381 and penicillin/streptomycin (Biochrom). Plasmid DNA was prepared using 3 (Abcam; ab1791) and then developed with ECL reagents (GE Healthcare), the Midi-Prep procedure (Qiagen) and transfected into HEK293 cells with according to the manufacturer’s protocol. LyoVec transfection reagent (Invivogen), according to the manufacturer’s instructions. Briefly, HEK293 cells were plated on 9-cm–diameter Petri Protein determination dishes (2,000,000 cells/dish) and transfected at the same time with 500 ml m The protein concentrations of cell lysates were estimated by the bicin- transfection reagent containing 5 g Md1-FLAG expression constructs. choninic acid protein assay reagent (Pierce) using BSA as a standard. Twenty-four hours after transfection, the cells were incubated with 5 mg/ml actinomycin D (Sigma-Aldrich) and harvested at 0, 3, 6, and 24 h, and In vivo determination of NF-кB activation in zebrafish larvae whole-cell extracts (30 mg protein) were resolved on 12% SDS-PAGE and transferred for 50 min at 200 mA to nitrocellulose membranes (Bio-Rad). At 72 h postfertilization, larvae were anesthetized in tricaine and mounted in Blots were probed with specific Abs to FLAG (Sigma-Aldrich) or to histone 1% (w/v) low-melting–point agarose (Sigma-Aldrich) dissolved in egg Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 1. Identification and molecular characterization of two novel Md1 splice variants in zebrafish. (A) RT-PCR analysis of zebrafish Md1 in samples obtained from the injection site of adult zebrafish injected i.m. with 5 mg ultrapure E. coli 0111-B4 LPS. The picture is representative of three independent experiments. (B) Multiple alignment of Md1, Md1_tv1, and Md1_tv2. The asterisks indicate the amino acids that are common to the three sequences. The predicted signal sequences are boxed, and the ML domain is in bold font. Exons 2 and 3 are highlighted in gray and black, respectively. Red arrowheads indicate the cysteine residues that form disulfide bonds that are crucial for the protein folding. Green arrowheads indicate other evolutionarily conserved amino acid residues. (C) Schematic diagram showing the alternative splicing of zebrafish Md1 pre-mRNA. The exon and intron sizes are indicated. (D) Schematic diagram showing the exon/intron organization of the three Md1 splice variants. Md1_tv1 is generated by skipping the entire exon 2 (a 96-bp deletion), whereas Md1_tv2 is generated by skipping the entire exons 2 and 3 (a 225-bp deletion). The hybridization site for the primers designed to specifically amplify the different variants by RT-qPCR is indicated as well as the amplicons obtained. (E) Three-dimensional structural predictions of zebrafish Md1, Md1_tv1, and Md1_tv2. Note that Md1_tv1 lacks cysteine residue 62, whereas Md1_tv2 lacks cysteine residues 46, 62, 106, and 116. Each color represents an exon of the corresponding mRNA: 1 (blue), 2 (red), 3 (yellow), 4 (green), and 5 (white). (F) Diagrams showing the domain organization of zebrafish Md1, Md1_tv1, and Md1_tv2. The signal peptides are shown as red boxes, the ML domains (SMART accession number SM00737, http://smart. embl.de/) are shown as oval pink boxes, and the ML domains only found by BLAST are shown as gray squares. 1382 IDENTIFICATION OF NOVEL Md1 SPLICE VARIANTS water (57). Images were captured with an epifluorescence Lumar V12 variants lacking the regions encoded by exons 2 or 2 and 3, re- stereomicroscope equipped with a green fluorescent filter, whereas animals spectively. The zebrafish Md1 splice variant lacking the entire were kept in their agar matrixes at 28.5˚C. Stacked images were captured exon 2, subsequently referred to as md1 transcript variant 1 from whole larvae of the NF-кB:eGFP reporter line using 25-mm incre- ments. Stacks were then processed using the free source software ImageJ (md1_tv1) (gene accession number LN875559), had a single open (http://rsbweb.nih.gov/ij) to obtain a maximum intensity projection of the reading frame (ORF) encoding a putative polypeptide of 134 aa, xy axis of the stack. Finally, for the quantification of NF-kB activation, the with a predicted molecular mass of 15.4 kDa. The zebrafish md1 maximum projection was used to measure the GFP fluorescence intensity splice variant lacking both exons 2 and 3, subsequently referred to in the whole larvae using ImageJ. The mean 6 SEM for each treatment is shown in the graphs. as md1 transcript variant 2 (md1_tv2) (gene accession number LN875560), had a single ORF encoding a 91-aa polypeptide, with Statistical analysis a calculated molecular mass of 10.5 kDa (Fig. 1B–D). It has been Data are shown as mean 6 SEM of at least three separate assays for gene previously described that all the proteins with MD2-related lipid expression experiments. Data were analyzed by ANOVA and a Tukey recognition (ML) domains, including MD1, possess three pairs of multiple range test to determine differences between groups, whereas the conserved cysteine residues that have been shown to form disul- t differences between two samples were analyzed by Student test. Log- fide bonds in the ML family members and, therefore, to be crucial rank (Mantel–Cox) test was used for the survival curves. for their protein three-dimensional structure (18, 58) (Supplemental Fig. 1). Those six key cysteine residues were identified in positions Results 34 (exon 1), 46 (exon 1), 62 (exon 2), 106 (exon 3), 116 (exon 3), Identification and characterization of two novel zebrafish Md1 and 158 (exon 5) of the full-length zebrafish Md1, so that, Md1_tv1 splice variants would lack the cysteine residue 62, whereas only residues 34, 46, Downloaded from The analysis of the Md1 expression profile in adult zebrafish by RT- and 158 would be present in Md1_tv2 (Fig. 1B). These data suggest PCR revealed three different amplification products (Fig. 1A), that Md1_tv2, and to some extent Md1-tv1, may present an altered suggesting the existence of alternative splicing in the zebrafish protein-folding pattern, which may affect its function. This idea was md1 gene. The three amplicons were cloned and sequenced, supported by the predicted tridimensional structures for the three finding that whereas the larger cDNA corresponded to the previ- variants, which showed relevant differences between Md1_tv2

ously reported sequence of full-length zebraﬁsh Md1 (23), the and the other two variants, including the loss of the ML domain http://www.jimmunol.org/ other two cDNA fragments corresponded to novel Md1 splice (Fig. 1E, 1F, Supplemental Fig. 2). In addition, comparison of the

FIGURE 2. Identification and molec- by guest on September 26, 2021 ular characterization of a novel MD1 splice variant in mouse. (A) RT-PCR analysis of mouse MD1 using cDNA obtained from bone marrow–derived macrophages (BMM). The picture is representative of three independent experiments. (B) Pairwise alignment of MD1 and MD1_tv2. Asterisks indicate the amino acid residues that are common to both sequences. The predicted signal sequences are boxed, and the ML domain is in bold font. Exons 2 and 3 are highlighted in the full-length MD1 sequence in gray and black, respectively. Black arrowheads indicate the cysteine residues that form disulfide bonds that are crucial for the protein folding, whereas white arrowheads indicate other amino acid residues evolutionarily conserved. (C) Schematic diagram showing the alternative splicing of mouse MD1 pre- mRNA. The exon and intron sizes are indicated. (D) Schematic diagram showing the exon/intron organization of both MD1 splice variants. MD1_tv1 is generated by skipping the entire exons 2 and 3 (a 216-bp deletion). The Journal of Immunology 1383 zebrafish full-length Md1 with other known MD1 sequences from The md1 splice variant lacking exons 2 and 3 is evolutionarily higher vertebrates revealed the presence of 13 conserved aa enco- conserved ded by exons 2 and 3, 7 aa encoded by exon 2, and 6 aa by exon 3 The precedent that several MD2 splice variants have previously (Fig. 1B, Supplemental Fig. 1). Hence, although zebrafish Md1_tv1 been identified in mammals (41, 42) prompted us to investigate lacks only exon 2 and keeps the five key cysteine residues, the whether the new Md1 splice variants identified in zebrafish were absence of the 7 evolutionary conserved aa encoded by exon 2 also evolutionary conserved. To assess this, cDNA from mouse could result in functional differences difficult to predict. bone marrow–derived macrophages was used for the amplification Downloaded from http://www.jimmunol.org/

FIGURE 3. mRNA levels of zebrafish md1, md1_tv1, and md1_tv2 and stability of Md1, Md1_tv1, and Md1_tv2 proteins. (A and B) Zebrafish embryos/larvae of the indicated times postfertilization (A) and adult organs (B) were collected and pooled, and the expression of md1, md1_tv1, and md1_tv2 genes by guest on September 26, 2021 was measured by RT-qPCR. The gene expression is normalized against rps11. The results are shown as the mean 6 SEM of three replicates and are representative of three independent experiments. **p , 0.01, ***p , 0.001. (C) Md1, Md1_tv1, and Md1_tv2 FLAG-tagged proteins were expressed in HEK293 cells, and their stability was analyzed for 24 h in the presence of actinomycin D (Act D). Blots were probed with Abs to FLAG and histone 3 and then developed with ECL reagents. ND, not detected; ns, not significant. 1384 IDENTIFICATION OF NOVEL Md1 SPLICE VARIANTS of Md1 by RT-PCR, resulting in the detection of two cDNA viral resistance (Fig. 5A). These results were further confirmed in products (Fig. 2A). Sequencing of these amplicons revealed that rescue experiments after knocking down Md1 with a previously the larger cDNA corresponded to the previously known full-length validated MO that targets the exon 1/intron 1 boundary and, mouse Md1 (19), whereas the smaller cDNA fragment corre- therefore, impairs the generation of the three variants for up to 7 d sponded to a novel Md1 splice variant lacking the region encoded postfertilization (23). Md1-deficient larvae showed higher sus- by exons 2 and 3. This mouse Md1 splice variant lacking both ceptibility to SVCV infection (Fig. 5B), confirming previous re- exons 2 and 3, subsequently referred to as Md1 transcript variant 2 sults (23). Strikingly, whereas forced expression of either md1 or (Md1_tv2) (gene accession number LN875561), had a single md1_tv1 fully restored the viral resistance of Md1-deficient larvae, (ORF) encoding a putative polypeptide of 90 aa, with a predicted md1_tv2 overexpression resulted in increased viral resistance of molecular mass of 9.6 kDa (Fig. 2B–D). Notably, mouse Md1_tv2 Md1-deficient animals compared with their wild-type siblings. lacks three of the cysteine residues that are crucial for the protein md1_tv2 induces the expression of ifnphi1 in challenged folding (positions 58, 102, and 112), as well as the 13 aa evolu- zebrafish larvae tionarily conserved in all the analyzed MD1 sequences from different species (Fig. 2B, Supplemental Fig. 1). As occurred To clarify the strong antiviral effect of Md1_tv2 while bearing in in zebrafish Md1_tv2, the three-dimensional structure was mind that Md1 regulates the proinflammatory and antiviral re- strongly altered and the ML domain was truncated in this case sponses (23), we next explored whether the novel variants also (Supplemental Figs. 2, 3). Although no evidence suggesting the existence of a mouse MD1 splice variant lacking only exon 2 was found (Fig. 2A), its generation in other cell populations or after Downloaded from appropriate stimulation cannot be ruled out. Expression patterns of md1, md1_tv1, and md1_tv2 genes in zebrafish Suspecting that the newly identified zebrafish Md1 splice variants,

especially Md1_tv2, may play roles in the immune response other http://www.jimmunol.org/ than those previously described for the full-length Md1 (23), we addressed this question by analyzing the md1, md1_tv1, and md1_tv2 expression patterns. For this, their mRNA levels were measured by RT-qPCR in zebrafish embryos during the first stages of development (Fig. 3A) as well as in different adult tissues and organs (Fig. 3B). We found that the three variants were maternally transferred, and that, whereas md1_tv1 mRNA levels were always lower than those of md1, md1_tv2 was not detected in any case after fertilization (Fig. 3A). However, the three transcript variants by guest on September 26, 2021 were present in all of the studied adult tissues. The md1_tv1 and md1_tv2 mRNA levels were significantly lower than the md1 mRNA levels in all adult tissues examined, but, curiously, showed similar expression levels in gut, spleen, kidney, gill, and skin, which play a key role in the immune response of fish (Fig. 3B). We next analyzed the stability of the three tagged Md1 variants ectopically expressed in HEK293 cells, because no Abs are available to zebrafish Md1. The results showed that all variants were expressed at similar levels, and, although their stability was found to be similar, Md1-tv1 splice variant appeared slightly more stable (Fig. 3C). md1, md1_tv1, and md1_tv2 are differentially regulated in challenged fish We then turned our attention to clarifying whether these variants responded to infection and stimulation in a distinct manner de- pending on the developmental stage. To evaluate this, the expression of md1, md1_tv1, and md1_tv2 was assayed by RT-qPCR in zebrafish larvae and adults infected with SVCV. It was found that the md1_tv2 transcript was not expressed in larvae (Fig. 4A), but only in adult fish (Fig. 4B), corroborating the above observations. Notably, SVCV resulted in increased transcript levels of full- FIGURE 4. mRNA levels of md1, md1_tv1, and md1_tv2 in zebrafish length md1 in larvae (Fig. 4A) and of md1_tv1 and md1_tv2 in larvae and adults infected with SVCV. (A) Zebrafish larvae were infected 3 7 8 adult fish (Fig. 4B). with 2.5 10 –10 TCID50/ml SVCV at 3 d postfertilization by immersion, and whole larvae were collected at 48 h postinfection and pooled for md1_tv2 shows a strong antiviral effect in zebrafish larvae gene expression analysis. (B) Adult zebrafish were infected i.p. with TCID50/ml SVCV, and gene expression analysis was performed in pooled As zebrafish Md1 regulates the antiviral response of zebrafish head at 48 h postinfection. md1, md1_tv1, and md1_tv2 mRNA levels were (23), we sought to determine the antiviral role played by the novel measured by RT-qPCR. The gene expression is normalized against rps11. Md1 transcript variants. Although forced expression of md1 and The results are shown as the mean 6 SEM of three replicates and are md1_tv1 did not affect the resistance of zebrafish larvae to SVCV representative of three independent experiments. ***p , 0.001. ND, not infection, md1_tv2 overexpression was able to significantly increase detected; ns, not significant. The Journal of Immunology 1385 Downloaded from http://www.jimmunol.org/

FIGURE 5. Md1_tv2 shows greater ability to promote the clearing of viral infection than full-length Md1 and Md1_tv1. Survival of zebrafish larvae injected at the one-cell stage with antisense (As), md1, md1_tv1,ormd1_tv2 mRNAs alone (A) or in combination with standard control (Std) or Md1 MOs 7 8 (B). In both cases, larvae were challenged by immersion with 2.5 3 10 –10 TCID50/ml SVCV at 3 d postfertilization (light gray lines) or left uninfected by guest on September 26, 2021 (black lines). The infected controls (injected with As mRNA) are shown with dashed lines. The number of samples (n) is indicated. **p , 0.01, ***p , 0.001. modulate such responses. For this, the mRNA levels of the genes (injected with antisense RNA) in response to VaDNA, whereas encoding proinflammatory IL-1b (Il1b) and antiviral IFNphi1 md1_tv2 failed to affect the NF-кB activation levels. (Ifnphi1) were measured in zebrafish larvae upon SVCV infection or bacterial DNA (VaDNA, TLR9 agonist) injection. In all cases, Discussion including the controls, an induction of il1b and ifnphi1 was found Although several splice variants of TLR4 and its accessory mol- in response to viral infection and stimulation with VaDNA, as ecules have already been identified and characterized in mammals, expected, and no effect of forced expression of any of the Md1 for example, soluble mouse TLR4 (38, 39), MyD88(S) (40), transcript variants in nonchallenged fish was observed (Fig. 6). MD2B (41), and MD2 short (42), to the best of our knowledge, the However, md1 and md1_tv1 overexpression resulted in increased current study reports for the first time the existence of MD1 splice mRNA levels of il1b and ifnphi1 compared with controls (injected variants. The conservation in mouse macrophages of the most with antisense RNA) in challenged larvae (Fig. 6). Interestingly, interesting MD1 splice variant from both a structural and func- although the forced expression of md1_tv2 failed to affect il1b tional point of view points to the need to extend our functional transcript levels in any case (Fig. 6A, 6C), it promoted the highest studies to the mouse and to review previous works in mammals mRNA levels of ifnphi1 in challenged larvae (Fig. 6B, 6D). that did not take into account the existence of MD1 splice variants. In addition, further studies would be of interest using human к md1 and md1_tv1, but not md1_tv2, regulate NF- B activation samples because it is likely that the human MD1 also undergoes In light of these intriguing results, we delved further into the alternative splicing, which may have functional consequences. signaling pathways regulated by the zebrafish Md1 variants and, One interesting observation of this study is the expression profile more specifically, into the different activity of Md1_tv2 versus Md1 of the zebrafish Md1 splice variants during development, in par- and Md1_tv1. As the master regulator of inflammation, NF-кBis ticular the maternal transfer of md1_tv2, which, curiously, was not also crucial for the induction of il1b gene in fish (59), and, because expressed by the embryo itself during the first days of development, a NF-kB reporter line was available (44, 60–62), we visualized the but was expressed at similar levels to full-length md1 in the adult dynamics of NF-кB in real time in zebrafish larvae forced to ex- lymphomyeloid organs, namely spleen and kidney, and mucosal/ press the different Md1 variants after being stimulated with pathogen portal of entry, namely gut, skin, and gill, suggesting that VaDNA (Fig. 7). The results showed a drastic activation of NF-кB it is produced by immune cells other than the oocyte. As forced in the whole larvae in response to bacterial DNA in all cases, as expression of md1_tv2 splice variant was the most powerful on expected from previous results (25, 60, 63). md1 and md1_tv1 inducing the antiviral response and, at the same time, shows were able to induce NF-кB to a greater extent than in the controls reduced proinflammatory activity, it is tempting to speculate its 1386 IDENTIFICATION OF NOVEL Md1 SPLICE VARIANTS Downloaded from http://www.jimmunol.org/

FIGURE 6. Md1_tv2 shows greater ability to induce antiviral ifnphi1 than full-length Md1 and Md1_tv1, and impaired ability to induce proinflammatory il1b. by guest on September 26, 2021 (A and C) Zebrafish one-cell embryos were injected with antisense (As), md1, md1_tv1,ormd1_tv2 mRNAs alone or in combination with 6.5 pg/egg VaDNA, and whole embryos were collected for gene expression analysis at 24 h postfertilization. (B and D) Zebrafish one-cell embryos were injected with antisense (As), 7 8 md1, md1_tv1,ormd1_tv2 mRNAs, infected with 2.5 3 10 –10 TCID50/ml SVCV by immersion at 3 d postfertilization, and collected for gene expression analysis 48 h later. il1b (A and B)andifnphi1 (C and D) mRNA levels were measured by RT-qPCR. The gene expression is normalized against rps11.Theresults are shown as the mean 6 SEM of three replicates and are representative of three independent experiments. **p , 0.01, ***p , 0.001. ns, not significant. relevance in the protection of newly hatched embryos through the The relevance of zebrafish Md1 splice variants is also highlighted induction of IFNs, while preventing excessive inflammation, which by their drastic induction upon viral infection of adult fish, whereas may be deleterious during early developmental stages (64). full-length md1 transcript levels were unaffected. Curiously, this

FIGURE 7. Md1 and Md1_tv1, but not Md1_tv2, prime the NF-kB activation triggered by bacterial DNA. Zebrafish one-cell NF-kB:eGFP embryos were injected with antisense (As), md1, md1_tv1,ormd1_tv2 mRNAs in the presence of PBS (negative control) or 6.5 ng VaDNA. (A) The mean GFP fluorescence was quantified in whole larvae at 3 d postfertilization. Each dot represents the mean GFP fluorescence per single larva. The mean 6 SEM of the whole larval GFP fluorescence for each group is also shown. The number of samples (n)is indicated. **p , 0.01, ***p , 0.001. (B) Representative images showing the different NF-kB activation levels in response to VaDNA and the different splice variants. ns, not significant. The Journal of Immunology 1387 induction parallels that of IFNs and IFN-stimulated genes following Disclosures SVCV infection (54) further suggesting a role for the alternative The authors have no financial conflicts of interest. splicing of md1 gene in the regulation of the antiviral response of this species. This relevance was definitively confirmed using gain- and loss-of-function strategies in zebrafish larvae, where Md1_tv2 References 1. Akira, S., S. Uematsu, and O. Takeuchi. 2006. Pathogen recognition and innate was found to show higher antiviral activity compared with full- immunity. Cell 124: 783–801. length Md1 and Md1_tv1, but no proinflammatory activity at all. 2. Janeway, C. A., Jr., and R. Medzhitov. 2002. Innate immune recognition. Annu. Strikingly, all Md1 splice variants primed innate immunity, because Rev. Immunol. 20: 197–216. 3. Aderem, A., and R. J. Ulevitch. 2000. Toll-like receptors in the induction of the they failed to induce the expression of proinflammatory and anti- innate immune response. Nature 406: 782–787. viral genes in the absence of a second stimulus provided by a 4. Medzhitov, R., and C. Janeway, Jr. 2000. Innate immune recognition: mecha- PAMP or by an infection. Furthermore, whereas Md1 and Md1-tv1 nisms and pathways. Immunol. Rev. 173: 89–97. 5. Xu, Y., X. Tao, B. Shen, T. Horng, R. Medzhitov, J. L. Manley, and L. Tong. fully restored the viral resistance of Md1-deficient zebrafish larvae, 2000. Structural basis for signal transduction by the Toll/interleukin-1 receptor Md1_tv2 not only was able to rescue their viral resistance, but also domains. Nature 408: 111–115. 6. Medzhitov, R., P. Preston-Hurlburt, and C. A. Janeway, Jr. 1997. A human ho- to further increase it, confirming the stronger ability of this splice mologue of the Drosophila Toll protein signals activation of adaptive immunity. variant to induce IFNphi1, the most powerful antiviral IFN in Nature 388: 394–397. zebrafish (54, 65). The three-dimensional structure prediction of the 7. Rock, F. L., G. Hardiman, J. C. Timans, R. A. Kastelein, and J. F. Bazan. 1998. A family of human receptors structurally related to Drosophila Toll. Proc. Natl. zebrafish and mouse Md1 splice variants may explain these func- Acad. Sci. USA 95: 588–593. tional differences. Although zebrafish Md1_tv1 retained five of 8. Poltorak, A., X. He, I. Smirnova, M. Y. Liu, C. Van Huffel, X. Du, D. Birdwell, six conserved cysteine residues, which have been shown to form E. Alejos, M. Silva, C. Galanos, et al. 1998. Defective LPS signaling in C3H/HeJ Downloaded from and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282: 2085–2088. disulfide bonds crucial for the three-dimensional structure of the 9. Qureshi, S. T., L. Larivie`re, G. Leveque, S. Clermont, K. J. Moore, P. Gros, and ML domain of Md1 and responsible for binding LPS and other D. Malo. 1999. Endotoxin-tolerant mice have mutations in Toll-like receptor 4 phospholipids (18, 58, 66), the zebrafish Md1_tv2 lacked these (Tlr4) [Published erratum appears in 1999 J. Exp. Med. 189: 1518.]. J. Exp. Med. 189: 615–625. cysteine residues, resulting in an altered structure and disorgani- 10. Ohto, U., K. Fukase, K. Miyake, and Y. Satow. 2007. Crystal structures of human zation of the two antiparallel b-sheets that form a b-cup fold re- MD-2 and its complex with antiendotoxic lipid IVa. Science 316: 1632–1634. 11. Nagai, Y., S. Akashi, M. Nagafuku, M. Ogata, Y. Iwakura, S. Akira, T. Kitamura, quired for lipid ligand binding (18, 58). Therefore, it is likely that A. Kosugi, M. Kimoto, and K. Miyake. 2002. Essential role of MD-2 in LPS http://www.jimmunol.org/ the antiviral activity of Md1_tv2 does not depend on the binding of responsiveness and TLR4 distribution. Nat. Immunol. 3: 667–672. LPS or an endogenous lipid ligand, such as the major constituents 12. Shimazu, R., S. Akashi, H. Ogata, Y. Nagai, K. Fukudome, K. Miyake, and M. Kimoto. 1999. MD-2, a molecule that confers lipopolysaccharide respon- of phospholipid in eukaryotic cells, phosphatidylcholine and siveness on Toll-like receptor 4. J. Exp. Med. 189: 1777–1782. phosphatidylethanolamine, which were found to bind mouse Md1 13. Akashi-Takamura, S., and K. Miyake. 2008. TLR accessory molecules. Curr. and have been suggested to play a role in retaining the Md1 structure Opin. Immunol. 20: 420–425. 14. Lee, C. C., A. M. Avalos, and H. L. Ploegh. 2012. Accessory molecules for Toll- (58). Taking into account the expected inability of Md1_tv2 to bind like receptors and their function. Nat. Rev. Immunol. 12: 168–179. any ligand, it is tempting to speculate that Md1_tv2 acts as an en- 15. Divanovic, S., A. Trompette, S. F. Atabani, R. Madan, D. T. Golenbock, A. Visintin, R. W. Finberg, A. Tarakhovsky, S. N. Vogel, Y. Belkaid, et al. 2005. dogenous danger signal that primes antiviral immunity (67). Negative regulation of Toll-like receptor 4 signaling by the Toll-like receptor Although further investigation is required to elucidate the homolog RP105. Nat. Immunol. 6: 571–578. by guest on September 26, 2021 mechanism involved in the regulation of the inflammatory and 16. Divanovic, S., A. Trompette, S. F. Atabani, R. Madan, D. T. Golenbock, A. Visintin, R. W. Finberg, A. Tarakhovsky, S. N. Vogel, Y. Belkaid, et al. 2005. antiviral responses in zebrafish by the Rp105/Md1 complex and the Inhibition of TLR-4/MD-2 signaling by RP105/MD-1. J. Endotoxin Res. 11: novel Md1 splice variants, our in vivo analysis of NF-kB activation 363–368. in challenged larvae confirms the functional differences of full- 17. Kimoto, M., K. Nagasawa, and K. Miyake. 2003. Role of TLR4/MD-2 and RP105/MD-1 in innate recognition of lipopolysaccharide. Scand. J. Infect. Dis. length Md1 and Md1_tv1 with Md1_tv2. Thus, Md1 and Md1_tv1 35: 568–572. were both able to potentiate the activation of NF-kB triggered by 18. Yoon, S. I., M. Hong, G. W. Han, and I. A. Wilson. 2010. Crystal structure of the bacterial DNA/TLR9 signaling pathway, whereas Md1_tv2 soluble MD-1 and its interaction with lipid IVa. Proc. Natl. Acad. Sci. USA 107: 10990–10995. failed to do so. Importantly, none of the Md1 variants was able to 19. Miyake, K., R. Shimazu, J. Kondo, T. Niki, S. Akashi, H. Ogata, Y. Yamashita, activate NF-kB in nonchallenged fish. These results are not com- Y. Miura, and M. Kimoto. 1998. Mouse MD-1, a molecule that is physically associated with RP105 and positively regulates its expression. J. Immunol. 161: pletely unexpected because exogenous expression of either mouse 1348–1353. or human RP105/MD1 complex in Ba/F3 cells potentiated LPS- 20. Miyake, K., Y. Yamashita, M. Ogata, T. Sudo, and M. Kimoto. 1995. RP105, a induced NF-kB activation via TLR4/MD2 (68). In sharp contrast, novel B cell surface molecule implicated in B cell activation, is a member of the leucine-rich repeat protein family. J. Immunol. 154: 3333–3340. however, similar experiments in HEK293 have shown that the 21. Jault, C., L. Pichon, and J. Chluba. 2004. Toll-like receptor gene family and TIR- RP105/MD1 complex acts as a negative regulator or LPS signaling domain adapters in Danio rerio. Mol. Immunol. 40: 759–771. via TLR4/MD2 (15), further highlighting the complexity of the 22. Meijer, A. H., S. F. Gabby Krens, I. A. Medina Rodriguez, S. He, W. Bitter, B. Ewa Snaar-Jagalska, and H. P. Spaink. 2004. Expression analysis of the Toll-like re- interactions between these two complexes and emphasizing the ceptor and TIR domain adaptor families of zebrafish. Mol. Immunol. 40: 773–783. need to re-examine the role of RP105 and MD1 in vivo by taking 23. Candel, S., M. P. Sepulcre, R. Espıń-Palazoń, S. D. Tyrkalska, S. de Oliveira, J. Meseguer, and V. Mulero. 2015. Md1 and Rp105 regulate innate immunity and into account the presence and relevance of different splice variants. viral resistance in zebrafish. Dev. Comp. Immunol. 50: 155–165. In conclusion, we have identified that an evolutionarily con- 24. Pietretti, D., H. P. Spaink, A. Falco, M. Forlenza, and G. F. Wiegertjes. 2013. served alternative splicing mechanism operates in the MD1 gene Accessory molecules for Toll-like receptors in Teleost fish: identification of TLR4 interactor with leucine-rich repeats (TRIL). Mol. Immunol. 56: 745–756. with important functional consequences for the regulation of innate 25. Sepulcre, M. P., F. Alcaraz-Pe´rez, A. Lo´pez-Munõz, F. J. Roca, J. Meseguer, immunity and the antiviral response. Our results pave the way for M. L. Cayuela, and V. Mulero. 2009. Evolution of lipopolysaccharide (LPS) future studies aimed at clarifying the role of MD1 and its splice recognition and signaling: fish TLR4 does not recognize LPS and negatively regulates NF-kappaB activation. J. Immunol. 182: 1836–1845. variants in the regulation of innate and adaptive immune responses 26. Sullivan, C., J. Charette, J. Catchen, C. R. Lage, G. Giasson, J. H. Postlethwait, from fish to mammals. P. J. Millard, and C. H. Kim. 2009. The gene history of zebrafish tlr4a and tlr4b is predictive of their divergent functions. J. Immunol. 183: 5896–5908. 27. Hughes, A. L. 1994. The evolution of functionally novel proteins after gene Acknowledgments duplication. Proc. Biol. Sci. 256: 119–124. We thank I. Fuentes and P. Martıńez for excellent technical assistance 28. Reams, A. B., and J. R. Roth. 2015. Mechanisms of gene duplication and amplification. Cold Spring Harb. Perspect. Biol. 7: a016592. and Dr. P. Fernańdez-Somalo (Laboratorio Central de Veterinaria, Spanish 29. Lynch, M., and J. S. Conery. 2000. The evolutionary fate and consequences of Ministry of Agriculture) for providing the SVCV isolate 56/70. duplicate genes. Science 290: 1151–1155. 1388 IDENTIFICATION OF NOVEL Md1 SPLICE VARIANTS

30. Brett, D., H. Pospisil, J. Valca´rcel, J. Reich, and P. Bork. 2002. Alternative 50. Kelley, L. A., and M. J. Sternberg. 2009. Protein structure prediction on the Web: splicing and genome complexity. Nat. Genet. 30: 29–30. a case study using the Phyre server. Nat. Protoc. 4: 363–371. 31. Kopelman, N. M., D. Lancet, and I. Yanai. 2005. Alternative splicing and gene 51. Kelley, L. A., S. Mezulis, C. M. Yates, M. N. Wass, and M. J. Sternberg. 2015. duplication are inversely correlated evolutionary mechanisms. Nat. Genet. 37: The Phyre2 web portal for protein modeling, prediction and analysis. Nat. 588–589. Protoc. 10: 845–858. 32. Black, D. L. 2003. Mechanisms of alternative pre-messenger RNA splicing. 52. McGuffin, L. J., M. T. Buenavista, and D. B. Roche. 2013. The ModFOLD4 Annu. Rev. Biochem. 72: 291–336. server for the quality assessment of 3D protein models. Nucleic Acids Res. 41: 33. Graveley, B. R. 2001. Alternative splicing: increasing diversity in the proteomic W368–W372. world. Trends Genet. 17: 100–107. 53. Reed, L. J., and H. Muench. 1938. A simple method of estimating fifty percent 34. Matlin, A. J., F. Clark, and C. W. Smith. 2005. Understanding alternative end points. Am. J. Hyg. 27: 493–497. splicing: towards a cellular code. Nat. Rev. Mol. Cell Biol. 6: 386–398. 54. Lo´pez-Munõz, A., F. J. Roca, J. Meseguer, and V. Mulero. 2009. New insights 35. Wang, B. B., M. O’Toole, V. Brendel, and N. D. Young. 2008. Cross-species EST into the evolution of IFNs: zebrafish group II IFNs induce a rapid and transient alignments reveal novel and conserved alternative splicing events in legumes. expression of IFN-dependent genes and display powerful antiviral activities. J. BMC Plant Biol. 8: 17. Immunol. 182: 3440–3449. 36. Gupta, S., D. Zink, B. Korn, M. Vingron, and S. A. Haas. 2004. Genome wide 55. Lo´pez-Munõz, A., F. J. Roca, M. P. Sepulcre, J. Meseguer, and V. Mulero. 2010. identification and classification of alternative splicing based on EST data. Bio- Zebrafish larvae are unable to mount a protective antiviral response against water- informatics 20: 2579–2585. borne infection by spring viremia of carp virus. Dev. Comp. Immunol. 34: 546–552. 37. Lu, J., E. Peatman, W. Wang, Q. Yang, J. Abernathy, S. Wang, H. Kucuktas, and 56. Pfaffl, M. W. 2001. A new mathematical model for relative quantification in real- Z. Liu. 2010. Alternative splicing in teleost fish genomes: same-species and time RT-PCR. Nucleic Acids Res. 29: e45. cross-species analysis and comparisons. Mol. Genet. Genomics 283: 531–539. 57. de Oliveira, S., C. C. Reyes-Aldasoro, S. Candel, S. A. Renshaw, V. Mulero, and 38. Iwami, K. I., T. Matsuguchi, A. Masuda, T. Kikuchi, T. Musikacharoen, and A. Calado. 2013. Cxcl8 (IL-8) mediates neutrophil recruitment and behavior in Y. Yoshikai. 2000. Cutting edge: naturally occurring soluble form of mouse Toll- the zebrafish inflammatory response. J. Immunol. 190: 4349–4359. like receptor 4 inhibits lipopolysaccharide signaling. J. Immunol. 165: 6682– 58. Harada, H., U. Ohto, and Y. Satow. 2010. Crystal structure of mouse MD-1 with 6686. endogenous phospholipid bound in its cavity. J. Mol. Biol. 400: 838–846. 39. Jaresova´, I., D. Rozkova´,R.Spı´sek, A. Janda, J. Bra´zova´, and A. Sediva´. 2007. 59. Olavarrıá, V. H., M. P. Sepulcre, J. E. Figueroa, and V. Mulero. 2010. Prolactin- Kinetics of Toll-like receptor-4 splice variants expression in lipopolysaccharide- induced production of reactive oxygen species and IL-1b in leukocytes from the Downloaded from stimulated antigen presenting cells of healthy donors and patients with cystic bony fish gilthead seabream involves Jak/Stat and NF-kB signaling pathways. J. fibrosis. Microbes Infect. 9: 1359–1367. Immunol. 185: 3873–3883. 40. Janssens, S., K. Burns, J. Tschopp, and R. Beyaert. 2002. Regulation of 60. Candel, S., S. de Oliveira, A. Lo´pez-Munõz, D. Garcıá-Moreno, R. Espıń- interleukin-1- and lipopolysaccharide-induced NF-kappaB activation by alter- Palazoń, S. D. Tyrkalska, M. L. Cayuela, S. A. Renshaw, R. Corbalań-Ve´lez, native splicing of MyD88. Curr. Biol. 12: 467–471. I. Vidal-Abarca, et al. 2014. Tnfa signaling through tnfr2 protects skin against 41. Ohta, S., U. Bahrun, M. Tanaka, and M. Kimoto. 2004. Identification of a novel oxidative stress-induced inflammation. PLoS Biol. 12: e1001855. isoform of MD-2 that downregulates lipopolysaccharide signaling. Biochem. 61. de Oliveira, S., P. Boudinot, Aˆ . Calado, and V. Mulero. 2015. Duox1-derived

Biophys. Res. Commun. 323: 1103–1108. H2O2 modulates Cxcl8 expression and neutrophil recruitment via JNK/c-JUN/ http://www.jimmunol.org/ 42. Gray, P., K. S. Michelsen, C. M. Sirois, E. Lowe, K. Shimada, T. R. Crother, AP-1 signaling and chromatin modifications. J. Immunol. 194: 1523–1533. S. Chen, C. Brikos, Y. Bulut, E. Latz, et al. 2010. Identification of a novel human 62. de Oliveira, S., A. Lo´pez-Munõz, S. Candel, P. Pelegrıń, Aˆ . Calado, and MD-2 splice variant that negatively regulates lipopolysaccharide-induced TLR4 V. Mulero. 2014. ATP modulates acute inflammation in vivo through dual oxi- signaling. J. Immunol. 184: 6359–6366. dase 1-derived H2O2 production and NF-kB activation. J. Immunol. 192: 5710– 43. Westerfield, M. 1993. The Zebrafish Book. A Guide for the Laboratory Use of 5719. Zebrafish Danio (Brachydanio) rerio. Institute of Neuroscience, University of 63. Alcaraz-Pe´rez, F., V. Mulero, and M. L. Cayuela. 2008. Application of the dual- Oregon, Eugene, OR. luciferase reporter assay to the analysis of promoter activity in zebrafish em- 44. Ogryzko, N. V., E. E. Hoggett, S. Solaymani-Kohal, S. Tazzyman, T. J. Chico, bryos. BMC Biotechnol. 8: 81. S. A. Renshaw, and H. L. Wilson. 2014. Zebrafish tissue injury causes upregu- 64. Galindo-Villegas, J., D. Garcıá-Moreno, S. de Oliveira, J. Meseguer, and lation of interleukin-1 and caspase-dependent amplification of the inflammatory V. Mulero. 2012. Regulation of immunity and disease resistance by commensal response. Dis. Model. Mech. 7: 259–264. microbes and chromatin modifications during zebrafish development. Proc. Natl.

45. Larkin, M. A., G. Blackshields, N. P. Brown, R. Chenna, P. A. McGettigan, Acad. Sci. USA 109: E2605–E2614. by guest on September 26, 2021 H. McWilliam, F. Valentin, I. M. Wallace, A. Wilm, R. Lopez, et al. 2007. 65. Aggad, D., M. Mazel, P. Boudinot, K. E. Mogensen, O. J. Hamming, Clustal W and Clustal X version 2.0. Bioinformatics 23: 2947–2948. R. Hartmann, S. Kotenko, P. Herbomel, G. Lutfalla, and J. P. Levraud. 2009. The 46. Schultz, J., F. Milpetz, P. Bork, and C. P. Ponting. 1998. SMART, a simple two groups of zebrafish virus-induced interferons signal via distinct receptors modular architecture research tool: identification of signaling domains. Proc. with specific and shared chains. J. Immunol. 183: 3924–3931. Natl. Acad. Sci. USA 95: 5857–5864. 66. Inohara, N., and G. Nun˜ez. 2002. ML: a conserved domain involved in innate 47. Letunic, I., T. Doerks, and P. Bork. 2012. SMART 7: recent updates to the immunity and lipid metabolism. Trends Biochem. Sci. 27: 219–221. protein domain annotation resource. Nucleic Acids Res. 40: D302–D305. 67. Collins, S. E., and K. L. Mossman. 2014. Danger, diversity and priming in innate 48. Buchan, D. W., F. Minneci, T. C. Nugent, K. Bryson, and D. T. Jones. 2013. antiviral immunity. Cytokine Growth Factor Rev. 25: 525–531. Scalable web services for the PSIPRED protein analysis workbench. Nucleic 68. Ogata, H., I. Su, K. Miyake, Y. Nagai, S. Akashi, I. Mecklenbraüker, Acids Res. 41: W349–357. K. Rajewsky, M. Kimoto, and A. Tarakhovsky. 2000. The Toll-like receptor 49. Jones, D. T. 1999. Protein secondary structure prediction based on position- protein RP105 regulates lipopolysaccharide signaling in B cells. J. Exp. Med. specific scoring matrices. J. Mol. Biol. 292: 195–202. 192: 23–29.