Characterization of SIGIRR/IL-1R8 Homolog from Zebrafish Provides New Insights into Its Inhibitory Role in Hepatic This information is current as of October 3, 2021. Wei Feng, Yi-Feng Gu, Li Nie, Dong-Yang Guo, Li-xin Xiang and Jian-zhong Shao J Immunol 2016; 197:151-167; Prepublished online 20 May 2016;

doi: 10.4049/jimmunol.1502334 Downloaded from http://www.jimmunol.org/content/197/1/151

Supplementary http://www.jimmunol.org/content/suppl/2016/05/20/jimmunol.150233 Material 4.DCSupplemental http://www.jimmunol.org/ References This article cites 69 articles, 28 of which you can access for free at: http://www.jimmunol.org/content/197/1/151.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

Characterization of SIGIRR/IL-1R8 Homolog from Zebrafish Provides New Insights into Its Inhibitory Role in Hepatic Inflammation

Wei Feng,*,† Yi-Feng Gu,*,† Li Nie,*,† Dong-Yang Guo,*,† Li-xin Xiang,*,† and Jian-zhong Shao*,†,‡

Single Ig IL-1R–related molecule (SIGIRR, also called IL-1R8 or Toll/IL-1R [TIR]8), a negative regulator for Toll/IL-1R signaling, plays critical roles in innate immunity and various diseases in mammals. However, the occurrence of this molecule in ancient vertebrates and its function in homeostasis and disorders remain poorly understood. In this study, we identified a SIGIRR homology from zebrafish (Danio rerio [DrSIGIRR]) by using a number of conserved structural and functional hallmarks to its mammalian counterparts. DrSIGIRR was highly expressed in the liver. Ablation of DrSIGIRR by lentivirus-delivered small interfering RNA in the liver significantly enhanced hepatic inflammation in response to polyinosinic-polycytidylic acid [poly(I:C)] Downloaded from stimulation, as shown by the upregulation of inflammatory and increased histological disorders. In contrast, depletion of TIR domain–containing adaptor inducing IFN-b (TRIF) or administration of TRIF signaling inhibitor extremely abrogated the poly(I:C)-induced hepatic inflammation. Aided by the zebrafish embryo model, overexpression of DrSIGIRR in vivo significantly inhibited the poly(I:C)- and TRIF-induced NF-kB activations; however, knockdown of DrSIGIRR promoted such activations. Furthermore, pull-down and Duolink in situ proximity ligation assay assays showed that DrSIGIRR can interact with the TRIF . Results suggest that DrSIGIRR plays an inhibitory role in TRIF-mediated inflammatory reactions by competitive http://www.jimmunol.org/ recruitment of the TRIF adaptor protein from its TLR3/TLR22 receptor. To our knowledge, this study is the first to report a functional SIGIRR homolog that existed in a lower vertebrate. This molecule is essential to establish liver homeostasis under inflammatory stimuli. Overall, the results will enrich the current knowledge about SIGIRR-mediated immunity and disorders in the liver. The Journal of Immunology, 2016, 197: 151–167.

ingle Ig IL-1R–related molecule (SIGIRR), also known as TIR domain with substitutions of the two sites (Ser447 Toll/IL-1R (TIR)8 or IL-1R8, is an important member of and Arg-Tyr536) required for the signaling of IL-1RI, as well as a S the TLR/IL-1R superfamily (1–4). In contrast with other unique 95-aa-long cytoplasmic tail, which differentiates SIGIRR by guest on October 3, 2021 TIR family members, SIGIRR comprises a single extracellular Ig from other TIR-containing family members (4, 5). SIGIRR has domain, a transmembrane region, and a conserved intracellular become increasingly attractive for its critical negative regulatory function on IL-1RI, IL-18R, T1/ST2, and various TLR (TLR-1–5, -7, and -9) signaling pathways (5–10). Dysfunction of SIGIRR results in *College of Life Sciences, Zhejiang University, Hangzhou 310058, People’s Repub- lic of China; †Key Laboratory for Cell and Engineering of Zhejiang Province, various inflammatory diseases, as well as autoimmune-relevant Hangzhou 310058, People’s Republic of China; and ‡Laboratory for Marine Biology disorders and tumors, such as ulcerative colitis, psoriatic inflam- and Biotechnology, Qingdao National Laboratory for Marine Science and Technol- mation, rheumatoid arthritis, lupus nephritis, and chronic lympho- ogy, Qingdao 266200, People’s Republic of China cytic leukemia. These findings suggest the diverse roles of SIGIRR Received for publication November 2, 2015. Accepted for publication May 2, 2016. in immunologic homeostasis and tolerance in a variety of tissues This work was supported by National Basic Research Program of China (973) Grants 2012CB114404 and 2012CB114402; National Natural Science Foundation of China (11–15). Nevertheless, little evidence exists regarding the functions Grants 31172436, 31272691, 31372554, 31472298, and 31572641; Hi-Tech Research of SIGIRR in the liver, except for a recent report showing that and Development Program of China (863) Grant 2012AA092202; and by Scientific Mycobacterium tuberculosis in SIGIRR-deficient mice Research Fund of Zhejiang Provincial Science and Technology Department Grant 2013C12907-9. induced an exaggerated hepatic inflammatory response (16). The sequences presented in this article have been submitted to GenBank (http://www. The liver plays a pivotal role in the of lipids, pro- ncbi.nlm.nih.gov/genbank/) under accession number KJ574205. teins, and carbohydrates, clearance of toxins and pathogens, and Address correspondence and reprint requests to Prof. Jian-zhong Shao and Assoc. regulation of immune responses (17, 18). As a component of the Prof. Li-xin Xiang, Zhejiang University, YuHangTang Road 866, Hangzhou 310058, alimentary system, the liver is continuously exposed to various Zhejiang, People’s Republic of China. E-mail addresses: [email protected] and [email protected] Ags and pathogen-associated molecular patterns of the large The online version of this article contains supplemental material. population of commensal bacteria, such as LPS and bacterial genomic DNA, from the through the portal Abbreviations used in this article: DIGIRR, double Ig IL-1R–related molecule; Drex- SIGIRR, extracellular region of DrSIGIRR; DrSIGIRR, SIGIRR homology from vein; however, no obvious inflammation existed in the healthy zebrafish (Danio rerio); EGFP, enhanced GFP; HEK293T, human embryonic liver, which is partly ascribed to the modulation of the TLR sig- 293T; hpf, hour postfertilization; IRF, IFN regulatory factor; LV, lentivirus; MO, morpho- lino oligonucleotide; PLA, proximity ligation assay; poly(I:C), polyinosinic-polycytidylic naling to normally regulate the innate immune responses, also acid; shRNA, short hairpin RNA; SIGIRR, single Ig IL-1R–related molecule; siRNA, known as liver tolerance (19, 20). Naturally, TLRs are found to be small interfering RNA; TIR, Toll/IL-1R; TRIF, Toll/IL-1R domain–containing adap- widely expressed on parenchymal and nonparenchymal cells in tor inducing IFN-b; UTR, untranslated region. the liver, which are essential for the defense against pathogen Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 invasions (21). However, excessive activation of TLR-medicated www.jimmunol.org/cgi/doi/10.4049/jimmunol.1502334 152 NEGATIVE REGULATION OF SIGIRR/IL-1R8 IN LIVER INFLAMMATION Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 1. Molecular characterization and subcellular distribution of DrSIGIRR. (A) Genomic structure of the zebrafish SIGIRR gene compared with human and mouse SIGIRRs. Rectangles represent the , and lines between the exons indicate the introns. sizes are indicated above each exon; intron sizes are indicated below each intron. (B) Comparative gene location maps of human, mouse, and zebrafish SIGIRRs. The same color represents the same gene in different species. The contig harboring the SIGIRR gene shows the conserved linkage of three in the human 11, mouse chromosome 7, and zebrafish chromosome 25. Arrows indicate the gene orientation. (C) Schematic overview of the domain organization of SIGIRR and DIGIRR . All the proteins have the Ig-like domain, transmembrane domain, and the TIR domain. However, the proteins of zebrafish SIGIRR and Tetraodon DIGIRR have a signal peptide that does not exist in mammalian SIGIRRs. (D) The tertiary TIR domain structure of the SIGIRR proteins was obtained by homology modeling from the SWISS-MODEL program. The chain is colored blue to red from the N terminus to the C terminus. Secondary structures, such as the BB loop and a-helix E, are labeled. (E) Subcellular distribution analysis of DrSIGIRR in HEK293T cells. HEK293T cells were transfected with the plasmid pEGFP-SIGIRR, fixed, and stained with the probe DiI, which showed that DrSIGIRR was mainly localized on the cell membrane with the merged signals of DiI. Scale bar, 10 mm. Values represent the mean 6 SD. signaling pathways may result in almost all the liver diseases mechanisms underlying the TLR-dependent inflammatory re- examined, such as acute hepatitis, alcoholic liver injury, autoim- sponses in the liver remain incompletely understood and are waiting mune hepatitis, and hepatocellular carcinoma (19, 22, 23). For to be determined. example, Con A–induced autoimmune-associated hepatitis can be In the present study, we describe the molecular and functional prevented in TLR3-deficient mice, and the mortality caused by identifications of a SIGIRR homology from zebrafish (Danio acetaminophen-induced hepatotoxicity can be reduced by TLR9 rerio [DrSIGIRR]), an emerging attractive model organism for the antagonists (24, 25). Thus, TLR activation is a double-edged study of human diseases and comparative immunology. DrSIGIRR sword, which must be tightly regulated to keep a check on dereg- exhibits a number of conserved structural and functional hall- ulated activation of TLR-dependent immune responses, thereby marks to its mammalian counterparts, including similar genomic preventing inflammatory disorders. However, the negative regulatory synteny and organization, compositions of Ig-like and TIR domains, The Journal of Immunology 153

can interact with TRIF protein. These observations suggest that DrSIGIRR may play a negative regulatory role in TRIF-mediated inflammatory reactions by competitive recruitment of TRIF adaptor protein from the TLR3/22 receptor, thus providing new insights into the inhibitory role of SIGIRR in hepatic inflammation. To our knowledge, the above findings provide the first evidence that a functional SIGIRR homolog exists in a lower vertebrate, which is essential to establish liver homeostasis. Overall, this study will enrich the current knowledge about SIGIRR-mediated immunity in the liver and provide significant implications for developing a novel zebrafish model to investigate SIGIRR-based liver diseases and clinical therapies.

Materials and Methods Experimental fish and embryo Wild-type AB zebrafish (D. rerio) were maintained in a circulating water bath at 28˚C under standard laboratory conditions (26). Embryos were col- lected at different stages of embryonic development as determined using previous protocols (27). Downloaded from Molecular cloning DrSIGIRR cDNA was generated by RT-PCR using the homologous sequences predicted from the University of California Santa Cruz and National Center for Biotechnology Information genome databases (28). The encoding sequences of zebrafish MyD88 (DrMyD88), TLR3 (DrTLR3), and TLR22 (DrTLR22) (NM_212814, NM_001013269.3, and NM_001128675.1, respectively) were amplified according to their pub- http://www.jimmunol.org/ lished data in the National Center for Biotechnology Information database. The primers used in cloning are listed in Supplemental Table I. PCR prod- ucts were purified using a gel extraction kit (Qiagen), inserted into pGEM-T FIGURE 2. An unrooted phylogenetic tree of the amino acid sequences vector (Promega), and transformed into competent Escherichia coli TOP10 of DrSIGIRR and other IL-1R family members was constructed with the cells (Invitrogen). Plasmid DNAs were purified using a miniprep kit neighbor-joining method by MEGA 5.0. Node values represent the per- (MEGA) and sequenced on an ABI 3730 sequencer (Invitrogen). Gene and centage bootstrap confidence derived from 1000 replicates. The GenBank protein structures, multiple amino acid sequence alignment, and phylo- accession numbers of the sequences are as follows: Homo sapiens IL-18R, genetic analysis were executed using databases and software programs as Q13478.1; Mus musculus IL-18R, Q61098.1; Sus scrofa IL-18R, described previously (29–31).

BAC19817.1; Felis catus IL-18R, AAO19575.1; H. sapiens ST2, by guest on October 3, 2021 BAA02233.1; M. musculus ST2, P14719.2; Gallus gallus ST2, BAB20772.1; Real-time PCR for analysis Salmo salar ST2, Q8AXT5; H. sapiens IL-1RI, AAA59137.1; M. musculus Total RNAs from developing embryos and various tissues (heart, spleen, IL-1RI, AAA39279.1; Rattus norvegicus IL-1RI, Q05KR1; G. gallus IL-1RI, liver, intestine, kidney, gill, brain, skin, and muscle) from healthy fish or AAA48924.1; Oryctolagus cuniculus IL-1RI, XP_002709930.1; Oncorhynchus liver tissues from poly(I:C)-stimulated fish were isolated using TRIzol mykiss IL-1RI, CAC82935.1; Bos taurus IL-1RI, NP_001193664.1; reagent (Invitrogen) and transcribed into first-strand cDNAwith oligo(dT)18 S. salar IL-1RLP, CAC83729.1; Callithrix jacchus IL-1RI, XP_002757499.1; (Takara Bio) to determine the gene expression patterns. Real-time PCR was Tetraodon nigroviridis DIGIRR, ABO15773.1; Takifugu rubripes DIGIRR, performed using a Mastercycler ep realplex instrument (Eppendorf) with a SYBR Premix Ex Taq kit (Takara Bio), following the manufacturers’ in- ACA03786.1; Gasterosteus aculeatus DIGIRR, ACA51853.1; H. sapiens structions as previously described (31, 32). Briefly, the reaction mixtures in a SIGIRR, Q6IA17.3; M. musculus SIGIRR, Q9JLZ8.2; R. norvegicus total volume of 10 ml were incubated for 2 min at 95˚C, followed by 40 SIGIRR, NP_001020058.1; B. taurus SIGIRR, NP_001075912.1; G. gallus cycles of 15 s at 95˚C, 15 s at 60˚C, and 20 s at 68˚C. The relative gene SIGIRR, NP_001186471.1; S. scrofa SIGIRR, BAL43001.1; S. salar expression was calculated using the 22DCT or 22DDCT method with initial SIGIRR, NP_001167309.1; D. rerio, KJ574205; Anas platyrhynchos, normalization of DrSIGIRR or other genes against b-actin. In all cases, each XP_005030227.1; Sarcophilus harrisii, XP_003774280.1. PCR trial was performed with triplicate samples and repeated at least three times. The related primers are listed in Supplemental Table I. substitutions of the two functional amino acid sites required for Plasmid constructions signal transduction in the TIR domain, subcellular localization The sequence encoding the extracellular region of DrSIGIRR (DrexSIGIRR) on the cellular surface membrane, and an inhibitory role in was inserted into pET32b (Invitrogen) between the BamHI and XhoI sites the MyD88-dependent NF-kB signaling pathway. Remarkably, to construct the prokaryotic expression vector pET32b-exSIGIRR. The full coding sequence of DrSIGIRR was subcloned into pEGFP-N1 (Clontech, DrSIGIRR was found to be highly expressed in the liver. Ablation Palo Alto, CA), pFLAG-CMV1 (Sigma-Aldrich), and pcDNA6/myc-HisB of DrSIGIRR by small interfering RNA (siRNA) significantly (Invitrogen) to construct the eukaryotic expression vectors pEGFP-SIGIRR, increased the liver inflammatory responses to polyinosinic- pFLAG-SIGIRR, and pcDNA6-SIGIRR to obtain the recombinant fusion polycytidylic acid [poly(I:C)] stimulation. In contrast, depletion of proteins of DrSIGIRR with enhanced GFP (EGFP), Flag-tag, and Myc-tag, TIR domain–containing adaptor inducing IFN-b (TRIF) or ad- respectively. The encoding sequences of DrMyD88, DrTLR3, and DrTLR22 were subcloned into pCMV-Tag2B (Invitrogen) or pcDNA6/myc-HisB. The ministration of a TRIF signaling inhibitory peptide extremely plasmids of pEGFP-TRIF and pCMV-TRIF were previously constructed in abrogated the poly(I:C)-induced hepatic inflammation. A zebrafish our laboratory (33). The NF-kB luciferase construct was purchased from embryo model was used to demonstrate the overexpression of Clontech, and the pRL-TK (Renilla luciferase reporter plasmid) vector was DrSIGIRR in embryos inhibited in both poly(I:C)- and TRIF- obtained from Promega. The primers used for construct generation are listed in Supplemental Table I. All constructs were sequenced to verify the induced NF-kB activation; in contrast, knockdown of DrSIGIRR correct sequences and orientations. Plasmids for transfection and micro- promoted such activations. Furthermore, pull-down and Duolink in injection were prepared free of endotoxin using an EZNA plasmid mini kit situ proximity ligation assay (PLA) assays showed that DrSIGIRR (Omega Bio-Tek). 154 NEGATIVE REGULATION OF SIGIRR/IL-1R8 IN LIVER INFLAMMATION

FIGURE 3. Inhibition of IL-1b– and CpG-ODN– induced NF-kB activation by overexpressing DrSIGIRR in zebrafish embryos. (A and B) Expression patterns of DrMyD88 and DrSIGIRR at the embryonic stages of 6, 12, 24, 36, 48, and 96 hpf against b-actin. The relative expression value was averaged from three replicates, each containing 30–50 embryos. (C and D) One-cell stage embryos were coinjected with the DrIL-1b (60 pg/embryo) (C) or CpG-ODN 1670 (1.5 ng/embryo) (D) alone or with the plasmid pcDNA6-SIGIRR (60 pg/embryo) plus the NF-kB Downloaded from luciferase reporter and the pRL-TK Renilla lucifer- ase reporter. At 24 h after microinjection, luciferase activity was detected and expressed as the fold in- duction over the control with three replicates as de- scribed in Materials and Methods. Values represent the mean 6 SD. **p , 0.01. http://www.jimmunol.org/ by guest on October 3, 2021

Subcellular localization indicated expression plasmid (pCMV-MyD88 or pEGFP-TRIF) alone or with the DrSIGIRR mRNA (or pcDNA6-SIGIRR) plus NF-kB luciferase Human embryonic kidney 293T (HEK293T) cells were seeded into multiwell reporter plasmid (50 pg/embryo) and pRL-TK Renilla luciferase reporter plates (Corning) and cultured in DMEM (HyClone Laboratories) supple- plasmid (5 pg/embryo, as an internal control) was injected into one-cell mented with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin stage embryos. The empty plasmid was added to ensure that all samples at 37˚C in 5% CO2 to allow growth into 70–90% confluence. These cells received equal amounts of DNA. At 24 h after microinjection, embryos were transfected with pEGFP-SIGIRR plasmid DNA (1–2 mg) using were collected for luciferase activity detection as previously described (32, FuGENE HD transfection reagent (Promega) in accordance with the 33). For IFN production assay, plasmids in various combinations (pEGFP- manufacturer’s instructions (31, 32). At 24 h posttransfection, the cells TRIF alone, pEGFP-TRIF plus DrMO, and pEGFP-TRIF plus DrMO were fixed in 4% paraformaldehyde for 10 min and stained with 10 mM DiI and DrSIGIRR mRNA) were microinjected into one-cell stage embryos. (Beyotime) at 37˚C for 5 min. Fluorescence images were obtained using Empty plasmid (pEGFP-N1) or DrSIGIRR mRNA alone was injected as a laser scanning confocal microscope (Zeiss LSM 710) (31). negative control. At 24 h after microinjection, the embryos with strong and Morpholino oligonucleotide and capped mRNA GFP expression were used for further analysis. Real-time PCR was per- formed to measure the mRNA levels of IFN regulatory factor (IRF)3 and The morpholino oligonucleotide (MO) against DrSIGIRR mRNA (DrMO) IFN1 as described earlier (33, 34). (59-GTGACAGCGCCACATTGCAGCCCTC-39) and the standard control MO (59-CTCTTACCTCAGTTACAATTTATA-39) were designed and Preparation of recombinant protein and polyclonal Ab synthesized using Gene Tools. For DrMO validation, the DrSIGIRR cDNA The pET32b-exSIGIRR was transformed into Escherichia coli BL21(DE3). fragment that encompassed partial 59–untranslated region (UTR) and the The recombinant DrexSIGIRR protein was induced by 0.5 mM isopropyl ATG site were amplified and cloned into pEGFP-N1 to generate a DrSIGIRR- b-D-thiogalactoside and purified through Ni-NTA agarose affinity chro- EGFP vector. This vector (50 pg/embryo) was then injected into one-cell stage matography (Qiagen) in accordance with the manufacturer’s instructions. embryos with control or DrMO (4 ng/embryo). GFP fluorescence was Subsequently, 6-wk-old male New Zealand White rabbits weighing ∼1.5 visualized at 24 h after microinjection via fluorescence microscopy (Zeiss kg each were immunized with 100 mg purified DrexSIGIRR protein in Axiovert 40 CFL; Carl Zeiss, Jena, Germany). The capped mRNA of CFA or IFA four times, followed by collection of anti-serum. The Ab titer was DrSIGIRR was transcribed in vitro using a Message Machine kit (Ambion), determined by microplate-based ELISA, and specificity to the DrSIGIRR according to the user’s manual, and then solubilized in diethyl pyrocarbonate protein was assessed via Western blot analysis (35, 36). water for microinjection. Western blot analysis Evaluation of DrSIGIRR in NF-kB activation and IFN production Tissue samples were treated with a lysis buffer (1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% sodium deoxycholate, 50 mM Tris-HCl The effects of DrSIGIRR on MyD88- and TRIF-induced NF-kB activa- [pH 7.4]) containing protease inhibitor mixture (Roche). The proteins were tion or TRIF-induced IFN production were determined via MO-mediated separated by 12% SDS-PAGE and transferred onto 0.45-mm polyvinylidene knockdown and mRNA rescue assays. For NF-kB activation assay, the difluoride membranes (Bio-Rad Laboratories, Hercules, CA), then blocked The Journal of Immunology 155 Downloaded from http://www.jimmunol.org/

FIGURE 4. DrSIGIRR negatively regulates MyD88-induced NF-kB activation. (A) Validation of DrSIGIRR MO (DrMO). Embryos were injected with the plasmid DrSIGIRR-EGFP (50 pg/embryo) plus DrMO (4 ng/embryo) or the control MO (4 ng/embryo) and examined by fluorescence microscopy at 24 h after microinjection. Micrographs showed that DrMO efficiently blocked the expression of the DrSIGIRR-EGFP vector and embryos were almost without green fluorescence. In contrast, the control MO cannot block the expression and presented strong green fluorescence, thereby indicating that DrMO can efficiently knock down DrSIGIRR expression in zebrafish embryogenesis. Original magnification 350. (B) pCMV-MyD88 (60 pg/embryo) alone, with DrSIGIRR mRNA (100 pg/embryo), with DrMO (4 ng/embryo), or with capped DrSIGIRR mRNA and DrMO plus the NF-kB luciferase reporter and the pRL-TK Renilla luciferase reporter were injected into one-cell stage embryos. The empty plasmid (pCMV-tag 2B) or DrSIGIRR mRNA was injected into the one-cell stage embryos as negative controls. Luciferase activity was assayed at 24 h after microinjection and expressed as the fold induction relative to by guest on October 3, 2021 the control with three replicates as described in Materials and Methods. Values represent the mean 6 SD. **p , 0.01. (C and D) DrSIGIRR coimmu- noprecipitates with DrMyD88. HEK293T cells (C) were transiently transfected with the empty vector or the Flag-tagged DrSIGIRR and the Myc-tagged DrMyD88, followed by immunoprecipitation with rabbit anti-DrSIGIRR or rabbit IgG as the negative control. Western blot analysis was performed with the mouse anti-Myc or anti-Flag Ab against DrMyD88 or DrSIGIRR, respectively. The same lysates were simultaneously immunoblotted as described above to monitor the expression of DrSIGIRR and DrMyD88. One-cell stage zebrafish embryos (D) were microinjected with plasmid pcDNA6-MyD88, harvested 24 h after microinjection, and immunoprecipitated with mouse anti-Myc mAb or mouse IgG as a negative control. Western blot analysis was performed with the mouse anti-Myc or rabbit anti-DrSIGIRR Ab against DrMyD88 or endogenous DrSIGIRR, respectively. with 5% (w/v) nonfat dry milk at 4˚C for overnight. After washing with Duolink in situ PLA TBST three times for 30 min, the blots were incubated with rabbit anti- DrSIGIRR Ab or other mAbs (Abcam), detected with HRP-conjugated Duolink in situ PLA was performed using a Duolink PLA kit (Sigma- goat anti-rabbit/mouse IgG Ab (Abcam), and visualized with ECL re- Aldrich) to further detect the association between DrSIGIRR and agents (GE Healthcare), all in accordance with the manufacturers’ DrTRIF in vivo. One-cell stage zebrafish embryos were coinjected with protocols. pcDNA6-SIGIRR and pCMV-TRIF. After 24 h, the embryos were col- lected and then sliced into 8-mm-thick frozen sections. The sections were Coimmunoprecipitation and pull-down assay fixed in 4% paraformaldehyde for 15 min at room temperature, washed Coimmunoprecipitation was performed in either the HEK293T cell line or with PBS twice for 20 min, permeabilized with 0.2% Triton X-100, the zebrafish embryo model. pcDNA6-MyD88 and pFLAG-SIGIRR were blocked with Duolink blocking buffer for 30 min at 37˚C, and finally in- transfected into HEK293T cells using FuGENE HD, or pcDNA6-MyD88 cubated with mouse anti-Myc mAb and rabbit anti-Flag mAb. PLA de- and pcDNA6-TRIF were microinjected into one-cell stage zebrafish em- tection was subsequently performed in accordance with the manufacturer’s instructions, and the signals were detected using a laser scanning confocal bryos. At 48 or 24 h posttransfection of microinjection, cells or embryos 3 were lysed with cold lysis buffer (1% Triton X-100, 150 mM NaCl, 1 mM microscope (Zeiss LSM 710) with a 63 oil immersion objective. The EDTA, 20 mM Tris-HCl [pH 7.4]) containing protease inhibitor mixture specificity of PLA was identified using negative controls, which consisted of samples incubated with each single species primary Ab but with both (Roche) for 30 min at 4˚C. Lysates were centrifuged for 15 min at 14,000 rpm and supernatants were incubated with rabbit anti-DrSIGIRR Ab, species of secondary Abs. mouse anti-Myc mAb, or rabbit/mouse IgG (negative control) at 4˚C Preparation of lentiviruses overnight, and then incubated with 30 ml protein A–agarose beads (Roche) for 4 h. The beads were washed four times with lysis buffer, and the ob- siRNAs against DrSIGIRR or DrTRIF were designed using related siRNA tained samples were analyzed by 12% SDS-PAGE and Western blot assay. template design tools (31, 37). Short hairpin RNA (shRNA) oligonucleo- For pull-down assay, pcDNA6-TRIF and pFLAG-SIGIRR were respec- tides containing the selected siRNAs (four for DrSIGIRR and five for tively transfected into HEK293T cells. At 24 h posttransfection, cells were DrTRIF) (Supplemental Table I) were synthesized (Invitrogen), annealed, lysed with cold lysis buffer, and the collected supernatants of DrSIGIRR and ligated into the pSUPER.retro.puro vector (Oligoengine, Seattle, WA) and DrTRIF were incubated at 4˚C overnight, followed by immunopre- digested with BglII and HindIII to generate the constructs of si1, si2, si3, cipitation with rabbit anti-DrSIGIRR Ab and Western blot analysis as si4, and TRIF1–5. The most efficient shRNAs and H1 were described above. subcloned into a PLB plasmid containing a CMV-EGFP expression cassette 156 NEGATIVE REGULATION OF SIGIRR/IL-1R8 IN LIVER INFLAMMATION Downloaded from

FIGURE 5. Expression of DrSIGIRR in different tissues and in poly(I:C)-stimulated . (A and B) The expression pattern of DrSIGIRR in different tissues was detected at mRNA levels (normalized to b-actin) via real-time PCR (A) and protein levels via Western blot (B). Values represent the mean 6 SD from three replicates. (C and D) DrSIGIRR mRNA (normalized to b-actin) and protein expression were examined in the liver of adult zebrafish before and after poly(I:C) injection at 50 mg/g body weight once daily for 2 d at various time points by real-time PCR (C) and Western blot (D), correspondingly. mRNA levels were expressed as fold induction relative to the control group. Values represent the mean 6 SD of 10–15 fish at each time point from three independent experiments. *p , 0.05, **p , 0.01. http://www.jimmunol.org/ to produce EGFP-tagged infective viruses (38, 39). Subsequently, the lenti- administration of lentiviruses or 1 h after antagonist peptide pretreatment, viruses were packaged and propagated in HEK293T cells via cotransfection poly(I:C) was injected i.p. into the zebrafish (50 mg/g body weight), and the of PLB, pCMV-VSVG, and pCMV-dR8.2 using FuGENE HD, followed by control groups received mock PBS only. Liver samples were collected at 6 h concentration via ultracentrifugation. The titers of the produced lentiviruses after poly(I:C) challenge (10–15 fish in each group) to evaluate the level of were determined by transduction and flow cytometric analysis of EGFP liver inflammation based on the changes in expression and histology. expression in HEK293T cells (31, 39). The silencing efficacy of the lenti- viruses (LV4 for DrSIGIRR and LVtrif for DrTRIF) was evaluated in the Statistical analysis liver by real-time PCR after the zebrafish were injected with LV4, LVtrif, or 5 Statistical evaluations of the differences between the means of experimental LVc (lentivirus harboring scrambled shRNA as negative control) (2 3 10 by guest on October 3, 2021 transduction units/fish) once a day for 3 d. groups were conducted by multiple Student t tests and the data are expressed as mean 6 SD. A p value ,0.05 was considered statistically significant. All Poly(I:C)-induced liver inflammation experiments were replicated at least three times independently. Poly(I:C)-induced liver inflammation was evaluated in zebrafish using previously reported protocols in mice with slight modifications (40). Each Results zebrafish was i.p. injected with poly(I:C) (Sigma-Aldrich) at a gradient Molecular identification of DrSIGIRR dosage of 25, 50, and 75 mg/g body weight. A moderate concentration (50 mg/g body weight) of poly(I:C) was then administrated into zebrafish The full-length DrSIGIRR cDNA (GenBank accession no. once a day for 2 d, and the liver samples were collected at 3, 6, 12, 24, and KJ574205) comprises a 1281-bp open reading frame that encodes 48 h after the final injection. Liver inflammatory responses were determined 426 aa, an 84-bp 59-UTR, and a 462-bp 39-UTR (Supplemental Fig. 1). by the enhanced histopathological alterations and the upregulated inflam- The DrSIGIRR gene consists of nine exons and eight introns, in matory cytokines (TNF-a,IFN-g,IL-1b, IL-6, and IFN1) and IRF3 via real- time PCR. The control fish received PBS at the same doses and time points. agreement with many other species (Fig. 1A). Genes adjacent to the DrSIGIRR locus on chromosome 25 (such as Ptdss2, Pkp3,and Histological analysis Ano9) show an overall conservation with those of other species Liver samples were harvested at the indicated time points after poly(I:C) (Fig. 1B). The DrSIGIRR protein is predicted to exhibit a molecular stimulation, fixed in 10% paraformaldehyde, and then embedded in paraffin. mass of ∼48 kDa, with a theoretical isoelectric point of ∼5.96. This Sections (∼6 mm) were collected for H&E staining and TUNEL assay using an protein contains a single extracellular Ig-like domain (34–128 aa) in situ cell death detection kit, POD (Roche). Histological changes were ex- amined under a light microscope (Zeiss Axiostar Plus) at 31000 magnification. distinct from double Ig IL-1R–related molecule (DIGIRR; with two) and IL-1R (with three) molecules, a transmembrane domain Evaluation of DrSIGIRR and DrTRIF in liver inflammation (135–157 aa), an intracellular TIR domain (181–327 aa), and a The functions of DrSIGIRR and DrTRIF in poly(I:C)-induced liver in- peculiar terminal intracellular tail with 95 aa, all of which are flammation were examined by lentivirus-based siRNA knockdown and similar to those of the other SIGIRR proteins (Fig. 1C). Multiple antagonist peptide–based inhibition assays. For knockdown assay, zebra- sequence alignment showed that DrSIGIRR shares 58–59% amino fish were injected i.p. with the lentivirus (LV4 or LVtrif or LV4 plus LVtrif) acid similarities to its mammalian counterparts, with the highest once a day for 3 d as described earlier. For antagonist inhibition assay, zebrafish were pretreated 1 h via i.p. with two TRIF antagonist peptides similarities (77–78%) among the TIR domains (Supplemental Fig. (with a penetration sequence at the N terminus) derived from a mice model 2). Similar to the TIR domains in other SIGIRR proteins, the (41, 42). The sequences of the peptides for DrTRIF (DrT1 and DrT2) DrSIGIRR TIR domain is predicted to display three TIR boxes and control are shown as follows: RQIKIWFQNRRMKWKK-CIEDAI with a conserved tertiary structure containing a central five-stranded DNSAFV-NH2 (DrT1), RQIKIWFQNRRMKWKK-FSEDFAQAGRSTLR- NH2 (DrT2), and RQIKIWFQNRRMKWKK-SLHGRGDPMEAFII-NH2 parallel b-sheet (bA–bE) surrounded by a total of five a helices (DrControl). These peptides were synthesized by Leon Chemical (Shanghai) (aA–aE) (Fig. 1D) (33). The b-sheet and a helix structures are with purity of .95% as determined by HPLC. At 3 d after the final connected by a number of loops (such as AB, BB, CD, DD, and EE The Journal of Immunology 157

FIGURE 6. Poly(I:C)-induced liver inflammation. (A) Dosage dependence of poly(I:C)-induced liver in- flammation. Adult zebrafish were injected with PBS or 25, 50, and 75 mg/g body weight of poly(I:C) for 3 h. Real-time PCR measured the mRNA levels of different inflammatory cytokines. (B) Liver sections were prepared for H&E staining or TUNEL assay to detect apoptosis. H&E staining of poly(I:C)-treated livers showed evidently distorted hepatic plates, less Downloaded from compact and homogeneous hepatocytes, edema, and spotty necrosis (indicated by the arrow). TUNEL assay demonstrated that more cells underwent apoptosis as compared with the controls. Scale bars, 5 mm. Values in (A) represent the mean 6 SD of 10–15 fish for each group from three independent experiments. *p , 0.05, , **p 0.01. http://www.jimmunol.org/ by guest on October 3, 2021

loops) based on the strands and helices that they connect. The BB their signaling study. The results showed that DrMyD88 was loop, a prominent feature on the surface of the TIR domain, is constitutively expressed at different developmental stages (Fig. 3A), conserved between zebrafish and mammals (Fig. 1D). The two whereas DrSIGIRR presented a lower expression before 12 h amino acid sites (Ser447 and Arg-Tyr536) in the BC loop and a helix postferilization (hpf) and a higher expression at 24 hpf (Fig. 3B). E, which are functionally crucial for signal transduction of IL-1R/ These findings indicated that both the DrMyD88-dependent sig- TLR family members, are substituted by Cys238 and Ala-Leu322, naling pathway and the DrSIGIRR-mediated regulatory mechanism respectively, and distinguish SIGIRR TIR domains from those of were established in the embryo before 24 hpf, suggesting that the the other family members (Supplemental Fig. 3). Clearly, a high embryo is a promising model for in vivo study of DrMyD88 and structural conservation of TIR domains exists between DrSIGIRR DrSIGIRR. Nevertheless, the supposed biological significance of and other SIGIRRs. Phylogenetic analysis revealed that fish the expressed DrMyD88 and DrSIGIRR in the early defense against SIGIRRs initially cluster together with fish DIGIRRs, the ortholog pathogen invasion or in the regulation of embryonic development of SIGIRR recently identified in fish, then merge with the avian and remains to be further clarified. As expected, the administration of mammalian SIGIRRs into a large group with high bootstrap prob- zebrafish embryos with IL-1R/TLR stimuli (IL-1b and CpG- ability (Fig. 2). Finally, DrSIGIRR is found to be predominantly oligodeoxynucleotides [ODNs]) significantly (p , 0.01) induced distributed on the cell surface membrane, with structural localiza- NF-kB activation at 24 h after microinjection. In contrast, over- tion similar to that of mammalian SIGIRRs (Fig. 1E) (7, 12). expression of DrSIGIRR in the embryos substantially (p , 0.01) inhibited such activations (Fig. 3C, 3D). To evaluate whether Functional identification of DrSIGIRR DrSIGIRR plays an inhibitory role in a MyD88-dependent man- The classical function of SIGIRR proteins in mammals is its ner, pCMV-MyD88 or pCMV-MyD88 combined with DrSIGIRR exertion as a negative regulator of MyD88-dependent NF-kB mRNA was injected into the embryos. The results showed that activation in IL-1R/TLR signaling pathways. Therefore, such overexpression of DrMyD88significantly(p , 0.01) induced NF-kB characteristic of DrSIGIRR was examined using NF-kB Dual- activation at 24 h after microinjection, whereas coexpression of Luciferase reporter system in a zebrafish embryo model for DrSIGIRR significantly (p , 0.01) suppressed such activation in vivo functional identification purpose. The expression patterns (Fig. 4B). For further clarification, MO-mediated DrSIGIRR of zebrafish MyD88 (DrMyD88) and DrSIGIRR during embryo- knockdown and mRNA-based rescue experiments were performed at genesis were initially analyzed to optimize the time points for 24 h after microinjection. Accordingly, a SIGIRR-EGFP expression 158 NEGATIVE REGULATION OF SIGIRR/IL-1R8 IN LIVER INFLAMMATION

FIGURE 7. Time dependence of poly(I:C)-induced liver inflammation. Adult zebrafish were injected with

poly(I:C) at 50 mg/g body weight once daily for 2 d. Downloaded from Liver samples were collected at 3, 6, 12, 24, and 48 h after the final injection. The mRNA levels of cytokines were measured by real-time PCR. Values represent the mean 6 SD of 10–15 fish at each time point from three independent experiments. *p , 0.05, **p , 0.01. http://www.jimmunol.org/ by guest on October 3, 2021

vector containing the DrMO target sequence was first generated to which provides initial insights into the involvement of DrSIGIRR validate the efficacy of DrMO against DrSIGIRR. The SIGIRR- in hepatic biology (Fig. 5A, 5B). To investigate the role of DrSIGIRR EGFP vector was coinjected into one-cell stage embryos with in liver inflammation, we performed a poly(I:C)-induced hepatic control MO or DrMO. The data revealed that DrMO can effi- inflammatory assay by administering the adult zebrafish with var- ciently suppress the expression of DrSIGIRR in zebrafish embryos ious concentrations of poly(I:C) at different times. The results (Fig. 4A). Correspondingly, DrMyD88-induced NF-kB activation showed that poly(I:C) induced a moderate inflammation in the liver in DrSIGIRR morphants was significantly (p , 0.01) upregulated at the dosage of ∼50 mg/g body weight during a 3–12 h time period, compared with that in wild-type embryos, and such upregulation as determined by the upregulated inflammatory cytokines (TNF-a, can be remarkably (p , 0.01) inhibited by the administration of IFN-g,IL-1b, IL-6, IFN1) and IRF3 (Figs. 6A, 7), as well as the DrSIGIRR mRNAs (Fig. 4B). Moreover, coimmunoprecipitation appearance of histological disorders, including distorted hepatic assayshowedthatDrMyD88 can interact with DrSIGIRR in plates, less compact and homogeneous hepatocytes, edema, spotty HEK293T cells (Fig. 4C) and bind to the endogenous DrSIGIRR necrosis via H&E staining, and increased apoptosis, as detected by in zebrafish embryo cells (Fig. 4D). The above observations TUNEL staining (Fig. 6B). Notably, the expression of DrSIGIRR in demonstrate that DrSIGIRR plays a conserved negative regulatory the liver was found to be significantly declined (p , 0.01 or p , role in MyD88-dependent NF-kB signaling pathways. Thus, 0.05) at mRNA and protein levels during the poly(I:C) stimulation, SIGIRR is structurally and functionally conserved from fish to implying that DrSIGIRR plays a profound role in poly(I:C)-induced mammals throughout the vertebrate evolution, making zebrafish hepatic inflammation (Fig. 5C, 5D). To provide evidence for this an attractive model for the study of SIGIRR biology. notion, we performed an in vivo DrSIGIRR knockdown assay fol- lowed by a lentivirus-based siRNA delivery protocol. Among the Inhibitory function of DrSIGIRR in hepatic inflammation four generated constructs harboring four candidate siRNAs targeted Tissue expression analysis showed that DrSIGIRR yielded the to different regions of DrSIGIRR, pSUPER-DrSIGIRR-4 (si4) was highest expression in the liver at both mRNA and protein levels, identified to be the most effective (.70%) in inducing DrSIGIRR The Journal of Immunology 159 Downloaded from http://www.jimmunol.org/

FIGURE 8. Silencing of DrSIGIRR gene by the lentivirus-based siRNA delivery system. (A) Screening of effective siRNAs against DrSIGIRR. Four designed siRNAs targeting different regions of DrSIGIRR mRNA were respectively inserted into the pSUPER vector (si1–4). The HEK293T cells were cotransfected with si1–4 or the control plasmid (pSUPER vector harboring the scrambled siRNA) with the overexpression plasmid pcDNA6-SIGIRR. The efficacy of siRNAs was measured by real-time PCR. (B and C) The silencing efficiency of lentivirus-delivered siRNA (si4) against the DrSIGIRR gene by guest on October 3, 2021 in vivo was analyzed by real-time PCR (B) and Western blot analysis (C). (D) The efficacy of lentivirus infection was detected in HEK293T cells by GFP fluorescence under a fluorescence microscope (Zeiss Axiovert 40 CFL). After 3 d of infection with the constructed lentiviruses (LVc or LV4), most HEK293T cells strongly expressed GFP, thereby demonstrating that both viruses have high infective efficacy. Scale bars, 50 mm. (E) Lentivirus titers were assessed according to the percentage of GFP+ HEK293T cells after exposure to different dilutions of lentiviruses by flow cytometry. The shaded graphs indicate the fluorescence of control cells without lentivirus infection. The number above the bracketed line shows the percentage of GFP+ cells in each group from three independent experiments. The upper and lower panels indicate the LVc and LV4 groups. Values in (A) and (B) represent the mean 6 SD of three independent experiments. **p , 0.01. mRNA degradation (Fig. 8A). This siRNA encoding sequence was 44). TLR22 is also found to be another functional homolog to TLR3 then used to produce the recombinant lentivirus. The generated in teleost fish (45–47). These observations suggest that the TLR3/22- lentivirus (LV4) after concentration showed a highly infectious ef- mediated TRIF signaling pathway may participate in poly(I:C)- ficacy, as determined by most of the HEK293T cells with strongly elicited hepatic inflammation in zebrafish. Therefore, the expression expressed GFP (Fig. 8D), and reached a titer above 1 3 106 levels of TLR3 and TLR22 were detected to be substantially ele- transduction units/ml, as shown by flow cytometry (Fig. 8E). The vated in the liver after poly(I:C) stimulation (Fig. 10A, 10B). To LV4 also exhibited a high efficiency (.60%) for DrSIGIRR in- provide further clarification, two TRIF signaling inhibitory peptides terference in zebrafish liver via real-time PCR and Western blot derived from the sequences of the BB loop and the helix B in analysis (Fig. 8B, 8C). The LV4 was then administered into the fish DrTRIF TIR domain (which were homologous to those in mouse once a day for 3 d before stimulation with poly(I:C). As expected, TRIF) were used in the experiment. The results showed that pre- knockdown of DrSIGIRR considerably increased the inflammatory treatment of zebrafish with one of these inhibitory peptides (DrT1, responses of liver to poly(I:C) challenge, as shown by the significant not DrT2; data not shown) at 2.5 nmol/g body weight for 1 h upregulation (p , 0.01 or p , 0.05) of TNF-a,IFN-g,IL-1b,IL-6, considerably inhibited the poly(I:C)-driven hepatic inflammation IFN1, and IRF3 (Fig. 9A), as well as enhanced histological defects, compared with that of the control group, as determined by the including massive liver degeneration, necrosis, and apoptosis, com- significant decline (p , 0.01 or p , 0.05) of the exaggerated ex- pared with those in the control group that received the lentivirus pression of inflammatory cytokines (TNF-a,IFN-g,IL-1b,IL-6, harboring scrambled siRNA (LVc) (Fig. 9B) at 6 h. and IFN1) and IRF3 (Fig. 10C). Furthermore, an in vivo TRIF knockdown assay was performed using the lentivirus-based siRNA TRIF signaling participates in poly(I:C)-induced hepatic delivery protocol as described above. A recombinant lentivirus inflammation (LVtrif) harboring an encoding sequence for siRNA with the most TLR3 is known as the receptor for poly(I:C) that activates down- efficient silencing activity for DrTRIF (.70%) was constructed for stream signaling through the adaptor protein TRIF in mammals (43, this purpose (Fig. 11). This LVtrif was administered into fish once a 160 NEGATIVE REGULATION OF SIGIRR/IL-1R8 IN LIVER INFLAMMATION Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 9. Knockdown of DrSIGIRR exhibits stronger liver inflammation after poly(I:C) stimulation. (A) Expression levels of cytokine mRNAs (normalized to b-actin) were detected by real-time PCR. Values represent mean 6 SD of 10–15 fish in each group from three independent experiments. *p , 0.05, **p , 0.01. (B) Histological images of liver tissues from DrSIGIRR normal (LVc) and DrSIGIRR knockdown (LV4) adult zebrafish after injection with PBS or poly(I:C). H&E staining of liver sections showed severe liver necrosis and TUNEL assay revealed that more apoptotic hepatocytes were present in the LV4 group compared with the LVc group. The arrow indicates the site of spotty necrosis. Scale bars, 5 mm. day for 3 d before poly(I:C) stimulation. As expected, knockdown and DrTRIF are involved in the TRIF-mediated inflammatory of DrTRIF significantly abated the poly(I:C)-induced hepatic in- reactions. Both molecules play opposite roles in these processes. flammation, as shown by the remarkable (p , 0.01 or p , 0.05) For further evaluation, the involvement of DrSIGIRR in the TRIF decreases in the excessive production of TNF-a,IFN-g,IL-1b,IL-6, signaling pathway was examined in poly(I:C)-induced hepatic IFN1, and IRF3 (Fig. 12), as well as the improvement of the his- inflammation by a lentivirus-based double-knockdown assay. As tological damages, including reduced degeneration and apoptosis expected, the simultaneous knockdown of DrSIGIRR and DrTRIF of the hepatocytes (Fig. 13). Clearly, the above observations sug- in the liver significantly impaired the enhanced inflammatory re- gest that TLR3/22-mediated TRIF signaling plays a major role in sponses to poly(I:C) occurring in the DrSIGIRR single-deficient poly(I:C)-induced liver inflammation. livers (p , 0.01 or p , 0.05), as determined by changes in the cytokines (TNF-a,IFN-g,IL-1b, IL-6, IFN1, and IRF3) and the Association of DrSIGIRR with TRIF-mediated inflammatory histological damages between the two silenced livers (Figs. 12, 13). reactions These results provide preliminary information that DrSIGIRR plays The above-mentioned DrSIGIRR and DrTRIF knockdown/ an inhibitory role in the TRIF signaling pathway. To clarify this inhibitory assays, which were conducted independently in the observation, the zebrafish embryo model was used again to deter- liver under poly(I:C) stimulation, suggested that both DrSIGIRR mine the effect of DrSIGIRR on the poly(I:C)- and TRIF-induced The Journal of Immunology 161

FIGURE 10. TLR3/22-mediated TRIF signaling involves poly(I:C)-induced hepatic inflammation. (A and B) DrTLR3 and DrTLR22 mRNA expression levels (normalized to b-actin) in the liver from adult zebrafish were detected before and after poly(I:C) injection at 50 mg/g body weight once daily for 2 d at various time points by real-time PCR. (C) The Downloaded from DrCorDrT1 peptide was injected i.p. at 1 h before the poly(I:C) challenge as described in Materials and Methods. The mRNA expression of cytokines (shown in each panel) was measured at 3 h after poly(I:C) injection. Values represent mean 6 SD of 10–15 fish from three independent experiments. *p , 0.05, **p , 0.01. http://www.jimmunol.org/ by guest on October 3, 2021

NF-kB activations. As expected, poly(I:C) administration of the Overall, DrSIGIRR plays a negative regulatory role in the DrTRIF- embryos significantly induced the NF-kB activation at 24 h after mediated inflammatory reactions, probably via the competitive re- microinjection (p , 0.01), whereas overexpression of DrSIGIRR cruitment of the DrTRIF adaptor protein from the TLR3/22-TRIF in the embryos significantly inhibited such activation (p , 0.01; signaling pathway. Fig. 14A). Similarly, the overexpression of DrTRIF in the em- bryos at 24 h after microinjection induced significant NF-kB ac- Discussion tivation in a dose-dependent manner (p , 0.01; Fig. 14B). In SIGIRR is an important member of the IL-1R subgroup of the TIR contrast, the overexpression of DrTRIF with DrSIGIRR during superfamily, which encompasses a group of structurally homolo- that stage substantially suppressed the NF-kB activation (p , gous proteins essential for the modulation of host defenses against 0.01; Fig. 14C). Then, MO-mediated DrSIGIRR knockdown assay infection, inflammation, injury, and stress in mammals (48–50). was performed in the embryos at 24 h after microinjection. The The IL-1R subgroup members are distinguished by the presence of results showed that overexpression of DrTRIF induced signifi- extracellular Ig-like domains and a conserved intracellular TIR cantly higher NF-kB activation in DrSIGIRR morphants than in domain activating the NF-kB, MAPK, and JNK pathways (50, DrSIGIRR-expressing wild-type embryos (p , 0.01). However, 51). The IL-1R family members, including IL-1RI, IL-1RI ac- the rescue experiment revealed that administration of DrSIGIRR cessory protein, IL-18 receptors (IL-18Ra and IL-18Rb), and IL- mRNA into the morphants severely reduced the induction of NF- 1R–related protein 2, usually contain three Ig-like domains in their kB activation (p , 0.01; Fig. 14D). Additionally, DrSIGIRR extracellular regions and serve as positive regulatory receptors in demonstrated its inhibitory role in DrTRIF-induced IFN production the IL-1R signaling pathways (1, 52). However, SIGIRR contains (Fig. 15A). The above data provide functional suggestions that only one Ig-like domain and functions as a negative regulator to DrSIGIRR negatively regulates DrTRIF-mediated signaling. Fur- suppress the Toll/IL-1R (such as TLR4 and IL-1R) signaling ther examinations through pull-down and Duolink in situ PLA as- pathways (5, 6, 50). Evolutionally, SIGIRR is supposed to origi- says revealed that DrSIGIRR can interact with the DrTRIF protein nate from a common IL-1R–like ancestor with the loss of distinct in vitro (Fig. 15B) and in the zebrafish embryo cells (Fig. 15C). functional elements, including two extracellular Ig-like domains 162 NEGATIVE REGULATION OF SIGIRR/IL-1R8 IN LIVER INFLAMMATION Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 11. Silencing of DrTRIF gene by the lentivirus-based siRNA delivery system. (A) Screening of the effective siRNAs against DrTRIF. Five designed siRNAs targeting different regions of DrTRIF mRNA were respectively inserted into the pSUPER vector (TRIF1–5). The HEK293T cells were cotransfected with TRIF1–5 or the control plasmid with the overexpression plasmid pCMV-TRIF. The efficacy of the siRNAs was measured by real-time PCR. (B) The in vivo silencing efficiency of the lentivirus-delivered siRNA (TRIF3) was analyzed by real-time PCR. (C) The infectious efficacy of the lentivirus (LVtrif) was detected in HEK293T cells by GFP fluorescence under a fluorescence microscope. After 3 d of infection with LVtrif, most HEK293T cells strongly expressed GFP, thereby demonstrating the high infective ability of LVtrif. Scale bar, 50 mm. (D) Lentivirus titer was assessed according to the percentage of GFP+ HEK293T cells after exposure to different dilutions of the lentivirus by flow cytometry. The shaded graphs indicate the fluorescence of control cells without lentivirus infection. The number above the bracketed line shows the percentage of GFP+ cells in each group from three independent experiments. Values in (A) and (B) represent the mean 6 SD of three independent experiments. *p , 0.05, **p , 0.01. essential for IL-agonist binding, and the substitution of two con- MyD88-dependent NF-kB activation. To our knowledge, this served amino acid sites (Ser447 and Arg-Tyr536) in the TIR do- work is the first to identify a SIGIRR homolog from teleost fish. main, which are required for signal transduction (31). Thus, SIGIRR originated at least as early as in the fish species, with SIGIRR has long been considered as an orphan receptor with conserved structures and functions throughout vertebrate evolu- unknown ligand molecules. Recently, human SIGIRR was found tion. Therefore, our study may contribute to the definition of the to act as a coreceptor with IL-18Ra for the recognition of IL-37, evolutionary history of the SIGIRR family and its associated uncovering the existence of a previously unknown mechanism signaling pathways, as well as the development of a zebrafish underlying the SIGIRR-mediated inflammatory regulation (3, 53). model for research on SIGIRR biology. Although well identified in mammals and predicted to be con- Functionally, SIGIRR has become increasingly attractive be- served from chicken to humans on the basis of sequence and cause of its critical negative regulation of various Toll/IL-1R– expression patterns (54), the occurrence and existence of the mediated mechanisms of immune homeostasis and tolerance. SIGIRR homolog in lower vertebrates, including teleost fish, are SIGIRR dysfunction results in various inflammatory diseases, poorly understood. In the present study, we identified a SIGIRR such as lupus nephritis, rheumatoid arthritis, brain or psoriatic homolog (DrSIGIRR) from zebrafish. This DrSIGIRR shares a inflammation, and disorders of colonic epithelial homeostasis, the number of conserved structural and functional features with its latter of which may lead to colonic inflammation and tumori- mammalian counterparts, as shown by the similar genome synteny, genesis (11–14, 55, 56). However, limited evidence shows the gene organization, tertiary structure of the TIR domain, existence functions of SIGIRR in the liver. Previous tissue expression of one Ig-like domain, two substitutions of the key functional analyses showed that SIGIRR widely distributes in various tissues amino acid sites, and the negative regulatory function of the examined (2, 50). However, the expression levels in tissues differ The Journal of Immunology 163 Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 12. Functional evaluation of DrTRIF and DrSIGIRR in liver inflammation by knockdown assays. The mRNA expression levels of the inflammatory cytokines and IRF3 (shown in each panel) in response to poly(I:C) stimulation were measured by real-time PCR. Values are shown in means 6 SD from three independent experiments. *p , 0.05, **p , 0.01. among various species. For instance, mice showed a higher ex- FIGURE 13. Histological changes and apoptotic alterations in DrTRIF pression of SIGIRR in the kidney, gastrointestinal tract, and , and DrSIGIRR knockdown livers under poly(I:C) stimulation. The his- swine demonstrated a stronger expression in the kidney and lymph tological images of liver tissues were obtained from the normal (NM), nodes, and chicken exhibited a higher expression in the kidney, negative control (LVc), DrTRIF knockdown (LVtrif), DrSIGIRR gastrointestinal tract, and liver (54, 57). This phenomenon implies knockdown (LV4), and the DrTRIF and DrSIGIRR double knockdown the functional diversity of SIGIRR among cellular activities, (LV4+LVtrif) adult zebrafish after injection with PBS (for the NM group) which was supposed to be the adaptation partly ascribed to the or poly(I:C) (for the LVc, LVtrif, LV4, and LV4+LVtrif groups). H&E evolutionary pressure (54). In the present study, we showed that staining and TUNEL assay of liver sections showed the following: DrSIGIRR was highly expressed in the liver and kidney but healthy tissue structures with little damages and apoptosis (in NM); the appearance of histological disorders with less compact, edema, spotty moderately expressed in the spleen, gill, intestine, and skin, with necrosis, as indicated by the arrow, and apoptotic hepatocytes (in LVc); expression patterns similar to those of several other species, es- the improvement of the histological damages with less degeneration and pecially humans (58). These observations indicate the important apoptosis of the hepatocytes (in LVtrif); enhanced histological defects functional involvement of SIGIRR in the liver. To evaluate this with massive liver necrosis and more apoptotic hepatocytes (in LV4); and notion, we initially performed a dynamic expression analysis of decreased histological damages with less degeneration and apoptosis of DrSIGIRR in response to poly(I:C) stimulation in the liver. The the hepatocytes (in LV4+LVtrif) as compared with the LV4 group. Scale results showed that DrSIGIRR underwent a significant decline bars, 5 mm. at 6 h after poly(I:C) treatment, which was accompanied by the 164 NEGATIVE REGULATION OF SIGIRR/IL-1R8 IN LIVER INFLAMMATION

DrSIGIRR plays an inhibitory role in liver inflammation. Poly(I:C) is a synthetic analog of viral RNA widely used to mimic virus infection to activate the TLR3 signaling pathway (43). TLR3 is a well-recognized receptor for poly(I:C) that activates downstream signaling via the TRIF adaptor protein, which is conserved be- tween teleost fish and mammals (45, 47, 63). TLR22 is a func- tional homolog of TLR3 in teleost fish (46). Therefore, the TLR3/22-mediated TRIF signaling pathway may participate in poly(I:C)-induced hepatic inflammation in zebrafish, and DrSIGIRR may serve as a negative regulator during the inflam- matory responses. Our study showed that the siRNA depletion of DrTRIF or the administration of a TLR3/22-TRIF signaling in- hibitory peptide in the liver significantly inhibited inflammation, whereas the double deficiency of DrTRIF and DrSIGIRR im- paired the enhanced inflammation in the livers with single defi- ciency of DrSIGIRR. With the zebrafish embryo model, the inhibitory effect of DrSIGIRR on the TLR3/22-TRIF signaling pathway was further determined by the downstream NF-kB activation. As expected, the results displayed that overexpression of DrTRIF in DrSIGIRR morphants significantly induced NF-kB Downloaded from activation, whereas such activation can be significantly hampered by DrSIGIRR administration. Detailed mechanisms of SIGIRR in the negative regulation of Toll/IL-1R signaling were well addressed by the MyD88- dependent signaling pathways. For example, SIGIRR inhibits IL-1R signaling by disturbing the heterodimerization of IL-1RI and http://www.jimmunol.org/ IL-1R accessory protein through binding to these molecules with its extracellular Ig domain. The mutated cytoplasmic TIR domain inhibited signaling by attenuating the recruitment of receptor- proximal proteins, such as MyD88, TRAF6, and IRAK to the receptor (6). In contrast, only the TIR domain of SIGIRR was necessary to inhibit TLR4 signaling, probably through its inter- action with the receptor via the BB loop region in the TIR domain to prevent the dimerization of the TLR4 and MyD88 complexes by guest on October 3, 2021 for signal transduction (14). However, the mechanism by which SIGIRR participates in the TRIF-mediated signaling pathways FIGURE 14. DrSIGIRR negatively regulates the poly(I:C)- and TRIF- remains to be elucidated. In the present study, we explored the induced NF-kB activation. (A) One-cell stage zebrafish embryos were possible inhibitory mechanism of DrSIGIRR in the TLR3/22- coinjected with poly(I:C) alone or with the plasmid pcDNA6-SIGIRR (60 TRIF signaling pathway. Pull-down binding assay results pg/embryo) plus the NF-kB luciferase reporter and the pRL-TK Renilla revealed that the recombinant DrSIGIRR protein can interact with luciferase reporter (as the internal control). (B) One-cell stage embryos the DrTRIF protein but not obviously with the TLR3 or TLR22 were coinjected with different concentrations of pEGFP-TRIF (0, 30, 60, protein (data not shown). Moreover, Duolink in situ PLA assay and 120 pg/embryo) plus the NF-kB luciferase reporter and the pRL-TK results showed that the DrSIGIRR protein can bind with DrTRIF C Renilla luciferase reporter. ( ) One-cell stage embryos were coinjected in zebrafish embryos. Many previous studies have shown that with plasmids pEGFP-TRIF (60 pg/embryo) alone or with the different teleosts TLR3, TLR22, and TRIF share conserved functional do- concentrations of pcDNA6-SIGIRR (0, 30, and 60 pg/embryo) plus the mains with their mammalian homologs; zebrafish TRIF can as- NF-kB luciferase reporter and pRL-TK Renilla luciferase reporter. (D) One-cell stage embryos were coinjected with pEGFP-TRIF (60 pg/em- sociate with TLR3, RIP1, and TBK1 to initiate NF-kB activation bryo) alone, with DrMO (4 ng/embryo), or with DrMO (4 ng/embryo) and and IFN production (45, 46, 64). Moreover, the TIR domain of the capped mRNA of DrSIGIRR (100 pg/embryo) plus the NF-kB lucif- TLR3 is responsible for its interaction with TRIF (45). These erase reporter and the pRL-TK Renilla luciferase reporter. Luciferase ac- observations imply that DrSIGIRR may play its negative regula- tivity was assayed at 24 h after microinjection and expressed as the fold tory role in the TLR3/22-TRIF signaling pathway by competitive induction over the control with three replicates, as described in Materials recruitment of DrTRIF from the TLR3/22 receptor based on the and Methods. Values represent the mean 6 SD. **p , 0.01. TIR–TIR interaction. However, further investigations are still needed to clarify the precise molecular mechanisms underlying DrSIGIRR activity in the TLR3/22-TRIF signaling pathway. upregulation of proinflammatory cytokines. As a negative regu- Additionally, the cellular mechanism of poly(I:C)-induced liver lator in inflammatory responses, the decreased expression of inflammation in mice models is characterized to be NK-mediated, DrSIGIRR might be the prerequisite for the induction of inflam- which is dependent on Kupffer cell activation (40). The Kupffer matory reactions. In support, a similar result was found in the cells activated by poly(I:C) via TLR3 are supposed to express expression pattern of SIGIRR after LPS or infection stimulation in surface Rae-1 and produce soluble IL-12, IL-18, and TNF-a.Rae-l several mouse models (59–62). Consequently, we developed an expression on Kupffer cells directly activated NK cells by its in- siRNA-mediated DrSIGIRR-deficient liver model in zebrafish and teraction with NKG2D. IL-12, IL-18, and TNF-a indirectly stim- found that the ablation of DrSIGIRR significantly enhanced the ulated NK cells to produce IFN-g. Finally, the NK cell–derived hepatic inflammatory responses to poly(I:C) stimulation. Therefore, IFN-g and the Kupffer cell–derived TNF-a synergistically induced The Journal of Immunology 165 Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 15. Detection of the negative regulation of DrSIGIRR in TRIF-mediated IFN production and interaction between DrSIGIRR and DrTRIF. (A) DrSIGIRR negatively regulates TRIF-mediated IFN production. pEGFP-TRIF (60 pg/embryo) alone, with DrMO (4 ng/embryo), or with capped DrSIGIRR mRNA (100 pg/embryo) and DrMO was injected into zebrafish embryos in the one-cell stage. Empty plasmid (pEGFP-N1) or DrSIGIRR mRNA alone was injected into respective one-cell stage embryos as negative controls. Real-time PCR was performed to detect the expression levels of IRF3 and IFN1 at 24 h after microinjection with three replicates (each containing 30–50 embryos). Values represent the mean 6 SD. *p , 0.05, **p , 0.01. (B) Association of DrSIGIRR with DrTRIF detected by pull-down assay. Myc-tagged DrTRIF and Flag-tagged DrSIGIRR were transiently transfected into respective HEK293T cells. At 24 h posttransfection, cells were lysed and supernatants of Flag-SIGIRR and Myc-TRIF were incubated at 4˚C overnight. The proteins were immunoprecipitated with rabbit anti-DrSIGIRR Ab or rabbit IgG as a negative control for 6 h at 4˚C, followed by Western blot analysis with mouse anti-Myc or anti-Flag Ab against DrTRIF or DrSIGIRR, respectively. (C) The association of DrSIGIRR with DrTRIF was detected by Duolink in situ PLA. One-cell stage embryos were coinjected with plasmids pcDNA6-SIGIRR and pCMV-TRIF and then collected to slice into 8-mm-thick frozen sections at 24 h after microinjection. The sections underwent PLA to detect the interaction, as described in Materials and Methods. The control groups (a and b) were incubated with mouse anti-Myc mAb (Myc) and rabbit anti-Flag mAb (Flag). The experimental groups (c and d) were incubated with mouse anti-Myc mAb and rabbit anti-Flag mAb (Myc+Flag) simultaneously. Red fluorescence was shown in groups (c) and (d), which indicated the protein interaction signal. The nuclei were stained with DAPI in blue. Scale bar, 5 mm. hepatocyte damage (65, 66). However, the cellular mechanisms distribution of SIGIRR in Kupffer cells, NK cells, or other cell underlying the influence of SIGIRR on poly(I:C)-triggered hepatic types in the liver. inflammation remain to be clarified. These mechanisms may Animal models are powerful tools for exploring major issues in largely depend on completely understanding the precise cellular immunology and the disease mechanisms that are difficult to address 166 NEGATIVE REGULATION OF SIGIRR/IL-1R8 IN LIVER INFLAMMATION in humans. Aside from the widely used mouse models, the zebrafish 19. Seki, E., and D. A. Brenner. 2008. Toll-like receptors and adaptor molecules in liver disease: update. Hepatology 48: 322–335. model has attracted considerable interest from researchers because of 20. Nakamoto, N., and T. Kanai. 2014. 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Primer Name Sequence (5’→ 3’ ) Use Accession Number

DrSIGIRR-F KJ574205 CCGCCGAAGAGAGTCTGTCAGCTTC Gene cloning

DrSIGIRR-R KJ574205 GTCGTCTTCCAGGTTGTATCTCGGC Gene cloning

DrSIGIRR-QF KJ574205 AAGCAAATAAGCCAGGACATCAG Real-time PCR

DrSIGIRR-QR KJ574205 TGGTGGGAAGGGTCTTGTAG Real-time PCR

3 ’-Adapter CTGATCTAGAGGTACCGGATCC Gene cloning

GATCCCCGCATGGAAAGCAGTGAATTTTCA RNAi DrSIGIRR-siRNA1-F AGAGA AATTCACTGCTTTCCATGCTTTTTA

AGCTTAAAAAGCATGGAAAGCAGTGAATTT RNAi DrSIGIRR-siRNA1-R CTCTTGAAAATTCACTGCTTTCCATGCGGG

GATCCCCGCCGATGTCGAAGGTTGATTTCA RNAi DrSIGIRR-siRNA2-F AGAGAATCAACCTTCGACATCGGCTTTTTA

AGCTTAAAAAGCCGATGTCGAAGGTTGATT RNAi DrSIGIRR-siRNA2-R CTCTTGAAATCAACCTTCGACATCGGCGGG

GATCCCCGGAGAACAAGTACAGTCATTTCA RNAi DrSIGIRR-siRNA3-F AGAGAATGACTGTACTTGTTCTCCTTTTTA

AGCTTAAAAAGGAGAACAAGTACAGTCATT RNAi DrSIGIRR-siRNA3-R CTCTTGAAATGACTGTACTTGTTCTCCGGG

GATCCCCGCTCACAAACCTCACAATTTTCA RNAi DrSIGIRR-siRNA4-F AGAGA AATTGTGAGGTTTGTGAGCTTTTTA

AGCTTAAAAAGCTCACAAACCTCACAATTT RNAi DrSIGIRR-siRNA4-R CTCTTGAAAATTGTGAGGTTTGTGAGCGGG

DrSIGIRR-pc6-EcoRI Eukaryotic TTTGAATTCGCCACCATGTGGCGCTGT KJ574205 expression

DrSIGIRR-pc6-XhoI Eukaryotic TTTCTCGAGCGTAAATCATCATCCGTG KJ574205 expression

DrSIGIRR-pFlag-EcoRI Eukaryotic CCGGAATTCATGGCGCTGTCAC KJ574205 expression

DrSIGIRR-pFlag-XbaI Eukaryotic TGCTCTAGATCATAAATCATC KJ574205 expression

DrSIGIRR-pET32b- Prokaryotic TTTGGATCCGTCCCTTTGTGGC BanHI KJ574205 expression DrSIGIRR-pET32b-XhoI Prokaryotic TTTCTCGAGTGAAGCCACCAC KJ574205 expression

DrSIGIRR-pEGFP-EcoRI Localization GCCGAATTCGTAAATCATCATCCGTG KJ574205

DrSIGIRR-pEGFP-XhoI Localization TTTCTCGAGGCCACCATGTGGCGCTGT KJ574205

DrMyD88-F NM_212814 ATGGCATCAAAGTTAAGTATAG Gene cloning

DrMyd88-R NM_212814 TTAGGGCAGTGAAAGTGCTTTG Gene cloning

DrTLR22-F Gene cloning ATGAAAAGGAAATCAAGAGAAAGC NM_001128675

DrTLR22-R Gene cloning CTACAAAATACAGTTCTCTGATTTATGG NM_001128675

DrTLR3-F Gene cloning ATGGATCTAATGAAACTCATATTATTG NM_001013269

DrTLR3-R Gene cloning CTAGTTGACCTTGTTTGTAGAGGC NM_001013269

DrMyD88-pCMV-EcoRI Eukaryotic TTTGAATTCATGGCATCAAAGTTAAGT NM_212814 expression

DrMyD88-pCMV-XhoI Eukaryotic TTTCTCGAGTTAGGGCAGTGAAAGTGC NM_212814 expression

DrMyD88-pc6-BamHI CGGGATCCGCCACCATGGCATCAAAGTTAA Eukaryotic NM_212814 GT expression

DrMyD88-pc6-XhoI Eukaryotic CCCTCGAGCGGGGCAGTGAAAGTGCTTTGG NM_212814 expression

GATCCCCGCAGCACATGCTAAGCTAATTCA RNAi DrTRIF-siRNA1-F AGAGATTAGCTTAGCATGTGCTGCTTTTTA

AGCTTAAAAAGCAGCACATGCTAAGCTAAT RNAi DrTRIF -siRNA1-R CTCTTGAA TTAGCTTAGCATGTGCTGCGGG

GATCCCCGGATGTTAGTTCCTATTATTTCAA RNAi DrTRIF -siRNA2-F GAGA ATAATAGGAACTAACATCCTTTTTA

AGCTTAAAAAGGATGTTAGTTCCTATTATTC RNAi DrTRIF -siRNA2-R TCTTGAA ATAATAGGAACTAACATCCGGG

GATCCCCCAACTTACGAAGTTGTCATTTCAA RNAi DrTRIF -siRNA3-F GAGA ATGACAACTTCGTAAGTTGTTTTTA

AGCTTAAAAACAACTTACGAAGTTGTCATT RNAi DrTRIF -siRNA3-R CTCTTGAA ATGACAACTTCGTAAGTTGGGG

DrTRIF -siRNA4-F GATCCCCGGGAATCATTTCCGCAAATTTCA RNAi AGAGAATTTGCGGAAATGATTCCCTTTTTA

AGCTTAAAAAGGGAATCATTTCCGCAAATT RNAi DrTRIF -siRNA4-R CTCTTGAA ATTTGCGGAAATGATTCCCGGG

GATCCCCGGAAGATCGACGTTAAGATTTCA RNAi DrTRIF –siRNA5-F AGAGAATCTTAACGTCGATCTTCCTTTTTA

AGCTTAAAAAGGAAGATCGACGTTAAGATT RNAi DrTRIF –siRNA5-R CTCTTGAA ATCTTAACGTCGATCTTCCGGG

β-actin-QF AF057040 AGGTCATCACCATCGGCAAT Real-time PCR

β-actin-QR AF057040 GATGTCCACGTCGCACTTCA Real-time PCR

IL-1β-QF NM_212844 TGGACTTCGCAGCACAAAATG Real-time PCR

IL-1β-QR NM_212844 GTTCACTTCACGCTCTTGGATG Real-time PCR

IL-6-QF NM_001261449 TGCAAGTCAAATTCAGAGCATAC Real-time PCR

IL-6-QR NM_001261449 ACCCTTACAGCCATGTGGCGAAC Real-time PCR

IRF3-QF NM_001143904 AGAAACATGGATGAGTCAG Real-time PCR

IRF3-QR NM_001143904 ATTAGTAAGCCTGGTCTG Real-time PCR

IFN1-QF NM_207640 TGGAGGACCAGGTGAAGTT Real-time PCR

IFN1-QR NM_207640 ATTGACCCTTGCGTTGCTT Real-time PCR

TNFα-QF NM_212859 GCTGGATCTTCAAAGTCGGGTGTA Real-time PCR

TNFα-QR NM_212859 TGTGAGTCTCAGCACACTTCCATC Real-time PCR

IFNγ-QF NM_212864 GACGTATGCAGAAACGCTATGG Real-time PCR

IFNγ-QR NM_212864 ATGCTTTAGCCTGCCGTCTCT Real-time PCR

TLR3-QF Real-time PCR TCTCTGGAGCATAACACAA NM_001013269

TLR3-QR Real-time PCR CTCTCGGAAGGCAGTATT NM_001013269

TLR22-QF Real-time PCR ACACCTTACCAACCACAT NM_001128675

TLR22-QR Real-time PCR TGAATCCTCTGAACATACTCT NM_001128675

TRIF-QF EF204937 CAATGACACAGCCAGTTC Real-time PCR

TRIF-QR EF204937 CTTGAGTTGGTGGTTTGC Real-time PCR

MyD88-QF NM_212814 CGAAAAAAGGTGTAAGAGGATGG Real-time PCR MyD88-QR NM_212814 TGGATTTGTAGACGACAGGGATT Real-time PCR

F, forward primer; R, reverse primer.

SUPPLEMENTAL FIGURE S1. sequence of the DrSIGIRR cDNA with the deduced amino acid sequence below it. The signal peptide is underlined; the transmembrane region is shown in dark gray. The start and stop codons are denoted by bold and italic.

SUPPLEMENTAL FIGURE S2. Multiple alignment of the DrSIGIRR protein with its homologues from other species. Residues shaded in black are completely conserved across all species aligned, and those in gray have similar side chains. Dashes in the amino acid sequences indicate gaps that were introduced to maximize alignment. The conserved domains (Ig domain, transmembrane domain, and TIR domain) are indicated above the alignment. Conserved motifs of TIR box are boxed out in solid lines. Triangles indicate the two important residues that are predicted to form the disulfide bonds of an

Ig-like domain fold. Gene accession numbers are noted in the legend of FIGURE 2.

SUPPLEMENTAL FIGURE S3. Alignment of the substituted amino acids in the cytoplasmic TIR regions of representative IL-1R family members. Residues shaded in black are critical for signal transduction. Residues shaded in gray indicate the substitution of amino acids. Gene accession numbers are noted in the legend of FIGURE 2.