Characterization of SIGIRR/IL-1R8 Homolog from Zebrafish Provides New Insights into Its Inhibitory Role in Hepatic Inflammation 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 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on October 3, 2021 • No Triage! 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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 liver 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 cytokines 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 protein. 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 amino acid 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 Gene 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 infection 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 metabolism 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 gastrointestinal tract 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 kidney 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 exons, and lines between the exons indicate the introns. Exon 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 genes in the human chromosome 11, mouse chromosome 7, and zebrafish chromosome 25. Arrows indicate the gene orientation. (C) Schematic overview of the domain organization of SIGIRR and DIGIRR proteins. 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