Human TLRs 10 and 1 Share Common Mechanisms of Innate Immune Sensing but Not Signaling This information is current as Yue Guan, Diana Rose E. Ranoa, Song Jiang, Sarita K. of September 28, 2021. Mutha, Xinyan Li, Jerome Baudry and Richard I. Tapping J Immunol 2010; 184:5094-5103; Prepublished online 26 March 2010; doi: 10.4049/jimmunol.0901888 http://www.jimmunol.org/content/184/9/5094 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2010/03/25/jimmunol.090188 Material 8.DC1 http://www.jimmunol.org/ References This article cites 74 articles, 29 of which you can access for free at: http://www.jimmunol.org/content/184/9/5094.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 28, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Human TLRs 10 and 1 Share Common Mechanisms of Innate Immune Sensing but Not Signaling Yue Guan,* Diana Rose E. Ranoa,* Song Jiang,* Sarita K. Mutha,† Xinyan Li,* Jerome Baudry,‡ and Richard I. Tapping*,x TLRs are central receptors of the innate immune system that drive host inflammation and adaptive immune responses in response to invading microbes. Among human TLRs, TLR10 is the only family member without a defined agonist or function. Phylogenetic analysis reveals that TLR10 is most related to TLR1 and TLR6, both of which mediate immune responses to a variety of microbial and fungal components in cooperation with TLR2. The generation and analysis of chimeric receptors containing the extracellular recognition domain of TLR10 and the intracellular signaling domain of TLR1, revealed that TLR10 senses triacylated lipopep- tides and a wide variety of other microbial-derived agonists shared by TLR1, but not TLR6. TLR10 requires TLR2 for innate immune recognition, and these receptors colocalize in the phagosome and physically interact in an agonist-dependent fashion. Downloaded from Computational modeling and mutational analysis of TLR10 showed preservation of the essential TLR2 dimer interface and lipopeptide-binding channel found in TLR1. Coimmunoprecipitation experiments indicate that, similar to TLR2/1, TLR2/10 complexes recruit the proximal adaptor MyD88 to the activated receptor complex. However, TLR10, alone or in cooperation with TLR2, fails to activate typical TLR-induced signaling, including NF-kB–, IL-8–, or IFN-b–driven reporters. We conclude that human TLR10 cooperates with TLR2 in the sensing of microbes and fungi but possesses a signaling function distinct from that of other TLR2 subfamily members. The Journal of Immunology, 2010, 184: 5094–5103. http://www.jimmunol.org/ he innate immune system is the first line of defense in fense, monocytes/macrophages express most TLRs (6), whereas response to an invading pathogen. Cellular innate immune the expression of individual TLRs in dendritic cells depends upon T defenses are necessary for the direct killing of pathogens, their subtype (9, 10). The microbial agonists for TLRs include and they also mediate the immediate release of proinflammatory a wide variety of structures. For example, TLRs 3, 7, 8, and 9 mediators that enable immune cells to access the site of infection, collectively respond to nucleic acids from bacteria and viruses, as well as the responses of professional APCs essential for gen- and these intracellular family members induce the expression of erating effective adaptive immunity (1, 2). Primary triggers of type-1 IFNs that possess potent antiviral activities (11, 12). In by guest on September 28, 2021 inflammatory and adaptive responses are the TLRs, a family of contrast, TLRs 1, 2, 4, 5, and 6 sense outer membrane components cell surface innate immune sensors that exist in organisms ranging of bacteria, fungi, and protozoan organisms, and these receptors from lower invertebrates to higher mammals. Mammalian TLRs are expressed on the surface of mammalian cells. Although most alert the host to the presence of infection through direct recog- TLRs signal as homodimers, TLR2 requires TLR1 or TLR6 for nition of conserved structural components of viruses, bacteria, activity (13). TLR1/2 and TLR6/2 heterodimers discriminate fungi, and protozoans (3, 4). In addition to microbial and viral among different microbial products that include bacterial lip- products, TLRs sense molecules associated with damage to self- oproteins, bacterial lipoteichoic acids, nonenteric bacterial LPSs, tissues as well as host products of inflammation (5). fungal phospholipomannans, protozoan GPI anchors, and myco- Humans possess 10 TLR family members (1–10), subsets of bacterial lipomannans and phosphatidylinositols (3). The cell which are expressed in leukocytes and the epithelial cells of surface TLRs engage a core signaling pathway, leading to the mucosal surfaces (6–8). In accordance with their role in host de- activation of NF-kB, AP-1, and other transcription factors that drive the production of proinflammatory chemokines, cytokines, and cell-adhesion molecules (14). x *Department of Microbiology, †Department of Biochemistry, and College of Med- Despite extensive research on the TLRs, human TLR10 has icine, University of Illinois, Urbana, IL 61801; and ‡Center for Molecular Biophys- ics, Oak Ridge National Laboratory, Oak Ridge, TN 37831 remained an orphan receptor without a known agonist or function. TLR10 was initially cloned in 2001 and shares the greatest ho- Received for publication June 15, 2009. Accepted for publication February 18, 2010. mology with TLR1 and TLR6 (15). In mammals, TLR 10, 1, and 6 This work was supported by the National Institutes of Health, National Institute of Allergy and Infectious Diseases Grant AI052344 (to R.I.T.). genes are tandemly arranged and seem to have arisen from du- Address correspondence and reprint requests to Dr. Richard Tapping, Department of plication events. Phylogeny supports the idea that TLR10 arose Microbiology, University of Illinois, B103 CLSL MC110, 601 South Goodwin Av- before the gene duplication that generated TLR1 and TLR6 (16, enue, Urbana, IL 61801. E-mail address: [email protected] 17). Chickens possess two TLRs that share ancestral origins with The online version of this article contains supplemental material. the TLR1-6-10 cluster and two additional TLRs that share origins Abbreviations used in this paper: ECD, extracellular domain; HA, hemagglutinin; with TLR2 (18). The fact that these chicken TLRs mediate re- HKAL, heat-killed Acholeplasma laidlawii; HKSA, heat-killed Staphylococcus sponses to lipopeptides through cooperative interactions indicates aureus; LTA-SA, lipoteichoic acid from S. aureus; LRR, leucine-rich repeat; Malp-2, macrophage-activating lipopeptide-2; MEF, murine embryonic fibroblast; PamCSK4, that this recognition function predates the mammalian expansion S-[2,3-bis(hydroxy)-propyl]-(R)-cysteinyl-(lysyl)3-lysine; Pam3CSK4, N-palmitoyl- of the TLR1-6-10 cluster (19). The TLR2 subfamily is thought to S-[2,3-bis(palmitoyloxy)-propyl]-(R)-cysteinyl-(lysyl)3-lysine; PamCysPamSK4, N- palmitoly-S-[2-hydroxy-3-(palmitoyloxy)propyl]-(R)-cysteinyl-(lysyl)3-lysine; P.G. have evolved under positive and balancing selection and, among LPS, Porphyromonas gingivalis LPS; TIR, Toll/IL-1R; TM, transmembrane. primates, Toll/IL-1R (TIR) domains seem to have undergone www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901888 The Journal of Immunology 5095 purifying selection (20, 21). The independent maintenance of veloped by Jin et al. (25, 26). In short, a truncated portion of the TLR TLR10 and its associated TIR domain suggest a distinct biological ectodomain lacking the C-terminal cap was fused to the highly conserved role for this receptor (16, 19). LRR C-terminal capping module of VLRB.61, a hagfish variable lym- phocyte receptor. With the aid of the computer software DNAWorks TLR10 is an unusual family member in that it is highly expressed (Bethesda, MD) (27), six adjacent sets of ∼60-bp overlapping oligonu- in the B cell lineage, suggesting that it plays a critical role in B cell cleotides were used to synthesize the sequence encoding aa 133–200 of the function. The presence of sequence gaps and retroviral insertions hagfish VLRB.61 clone. Using overlap extension PCR, the VLR coding 9 reveals that mouse TLR10 is a pseudogene, a situation that pre- region was fused to the 3 -end of TLR 1, 2, and 10 ECD, encompassing aa 22–476, 17–508, and 20–474, respectively. The PCR products were cloned cludes the generation and phenotypic assessment of a TLR10 as a BglII/NheI fragment into a modified pDisplay vector containing an knock-out mouse. In this article, we report that human TLR10 HA-tag upstream of the BglII site and an Fc domain of the human IgG1 shares a variety of agonists with TLR1, including a number of cell- downstream of the NheI site (kindly provided by Dr. David M. Kranz, surface components of bacteria and fungi. Similar to TLR1 and Department of Biochemistry, University of Illinois at Urbana Champaign). 9 TLR6, TLR10 requires TLR2 for recognition. However, we found A thrombin cleavage site (LVPRGS) was also added at the 3 -end of the TLRvlr hybrid to allow cleavage of the soluble TLR from the Fc fusion that TLR10 lacks the downstream signaling typically associated protein. A Flag-tagged TLR2vlr-Fc construct was also engineered. with other TLR2 family members. TLR10 mutants. Site-directed mutagenesis of the TLR10 mutants was performed in TLR10 chimeric receptor TLR10-1.