TRIL, a Functional Component of the TLR4 Signaling Complex, Highly Expressed in Brain
This information is current as Susan Carpenter, Thaddeus Carlson, Jerome of September 29, 2021. Dellacasagrande, Amaya Garcia, Sharon Gibbons, Paul Hertzog, Anthony Lyons, Lih-Ling Lin, Marina Lynch, Tom Monie, Caroline Murphy, Katherine J. Seidl, Christine Wells, Aisling Dunne and Luke A. J. O'Neill
J Immunol 2009; 183:3989-3995; Prepublished online 26 Downloaded from August 2009; doi: 10.4049/jimmunol.0901518 http://www.jimmunol.org/content/183/6/3989 http://www.jimmunol.org/ References This article cites 30 articles, 8 of which you can access for free at: http://www.jimmunol.org/content/183/6/3989.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 © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology
TRIL, a Functional Component of the TLR4 Signaling Complex, Highly Expressed in Brain1
Susan Carpenter,*‡ Thaddeus Carlson,§ Jerome Dellacasagrande,‡ Amaya Garcia,‡¶ Sharon Gibbons,‡ Paul Hertzog,ʈ Anthony Lyons,† Lih-Ling Lin,§ Marina Lynch,† Tom Monie,# Caroline Murphy,*‡ Katherine J. Seidl,§ Christine Wells,** Aisling Dunne,2* and Luke A. J. O’Neill2,3*
TLR4 is the primary sensor of LPS. In this study, we describe for the first time TLR4 interactor with leucine-rich repeats (TRIL), which is a novel component of the TLR4 complex. TRIL is expressed in a number of tissues, most prominently in the brain but also in the spinal cord, lung, kidney, and ovary. TRIL is composed of a signal sequence, 13 leucine-rich repeats, a fibronectin
domain, and a single transmembrane spanning region. TRIL is induced by LPS in the human astrocytoma cell line U373, in Downloaded from murine brain following i.p. injection, and in human PBMC. Endogenous TRIL interacts with TLR4 and this interaction is greatly enhanced following LPS stimulation. TRIL also interacts with the TLR4 ligand LPS. Furthermore, U373 cells stably overex- pressing TRIL display enhanced cytokine production in response to LPS. Finally, knockdown of TRIL using small interfering RNA attenuates LPS signaling and cytokine production in cell lines, human PBMC, and primary murine mixed glial cells. These results demonstrate that TRIL is a novel component of the TLR4 complex which may have particular relevance for the functional
role of TLR4 in the brain. The Journal of Immunology, 2009, 183: 3989–3995. http://www.jimmunol.org/
oll-like receptors are germline-encoded type 1 transmem- ulating the cell surface expression of TLRs while others are in- brane receptors involved in the innate recognition of mi- volved in ligand delivery and binding. A number of accessory crobial products (1). TLRs possess large leucine-rich re- molecules have been implicated in the TLR4 signaling pathway. T 4 peats (LRRs) within their extracellular domain that serve to MD2, for example, binds LPS and acts as the endotoxin recogni- function in ligand recognition. The cytoplasmic region of the TLRs tion molecule allowing TLR4 to signal (3). MD2 is indispensable is made up of a Toll/IL-1R homology domain, which is involved for TLR4 signaling since MD2-deficient mice are unresponsive to in the downstream signaling cascades initiated following receptor LPS (3, 4). CD14 is a LRR-containing glycoprotein that can be engagement (2). Localization of TLRs is essential to their function. secreted into the serum or expressed as a GPI-linked protein on the by guest on September 29, 2021 TLRs 1, 2, 4, 5, and 6 are localized to the cell surface where they surface of cells (5). The function of CD14 is to bind LPS and directly sense microbial products such as LPS in the case of TLR4 transfer LPS monomers to TLR4/MD2 (6). PRAT4A was origi- and lipopeptides in the case of TLR2. TLRs 3, 7, 8, and 9 are all nally identified as a TLR4-specific chaperone protein (7); however, localized to endosomal compartments where they are responsible it now appears that PRAT4A also plays a role in TLR1 and TLR9 for the recognition of microbial and possibly host-derived nucleic signaling (8). Gp96, an endoplasmic reticulum chaperone protein, acids. was recently shown to be required not only for TLR4 signaling but Accessory molecules are required for signal activation by a also for TLR3, TLR5, TLR7, and TLR9 signaling. Gp96 appears number of TLRs. Some accessory molecules are involved in reg- to be required for posttranslational maturation of TLR proteins (9, 10). Some accessory molecules play roles that are cell-type spe- cific, such as RP105, which is required for TLR2- and TLR4- *School of Biochemistry and Immunology and †Trinity Institute of Neuroscience, Trinity College Dublin, Dublin, Ireland; ‡Opsona Therapeutics, Institute for Molec- mediated responses in B cells (3). In macrophages and dendritic ular Medicine, St. James’s Hospital, Dublin, Ireland; §Inflammation Discovery Re- cells however, RP105 acts as a negative regulator of TLR4 sig- search, Wyeth Research, Cambridge, MA; ¶Dublin Institute of Technology, Dublin, naling (11). Ireland; ʈCentre for Functional Genomics and Human Disease, Monash Institute of Medical Research, Victoria, Australia; #Department of Biochemistry, University of Accessory molecules are clearly vital components of TLR sig- Cambridge, United Kingdom; and **Eskitis Institute for Cell and Molecular Thera- naling complexes. In this study, we describe a previously uniden- pies, Griffith University, Queensland, Australia tified and highly conserved novel protein, TRIL (TLR4 interactor Received for publication May 14, 2009. Accepted for publication July 7, 2009. with leucine-rich repeats; GenBank accession no. NM_014817), The costs of publication of this article were defrayed in part by the payment of page which plays an important and functional role in the TLR4 com- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. plex. It contains 13 LRRs, a signal sequence, a fibronectin domain, 1 This work was supported by the Science Foundation of Ireland. and a single transmembrane spanning region. The LRR motifs are similar to those found in other LRR proteins such as TLRs, 2 A.D. and L.A.J.O. contributed equally to this study. NOGO, and LINGO receptors. TRIL is highly expressed in a num- 3 Address correspondence and reprint requests to Prof. Luke A. J. O’Neill, School of Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland. E-mail ber of tissues, notably in brain, and is up-regulated by LPS both in address: [email protected] vitro and in vivo. We show that TRIL can interact with TLR4 and 4 Abbreviations used in this paper: LRR, leucine-rich repeat; TRIL, TLR4 interactor this interaction is greatly enhanced upon LPS stimulation. We also with leucine rich repeats; CHO, Chinese hamster ovary; siRNA, small interfering demonstrate that TRIL can bind to LPS. Silencing of TRIL atten- RNA; BMDM, bone marrow-derived macrophage. uates the TLR4 signaling pathway in cell lines, human PBMC, and Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 murine mixed glial cells. This study therefore demonstrates that www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901518 3990 TRIL: A NOVEL COMPONENT OF THE TLR4 RECEPTOR COMPLEX
TRIL is a functional protein in the TLR4 complex that may have Stable Chines hamster ovary (CHO) cells were generated using the particular relevance for TLR4-mediated responses in brain. PCDNA5 vector from Invitrogen (V652020). Positive clones were selected using hygromycin antibiotics. Materials and Methods RT-PCR Reagents For quantitative real-time PCR, cDNA was transcribed using a High Anti-Flag Ab and anti-Flag-agarose were purchased from Sigma-Aldrich. Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Prim- Anti--actin Ab was purchased from Sigma-Aldrich. The IB-␣ Ab was a ers and probes for human and mouse TRIL were purchased from gift from Prof. R. Hay (University of Dundee, Dundee, U.K.). The poly- Applied Biosystems (assay identifications Hs00274460_s1 and clonal phospho-p38 Ab was obtained from Cell Signaling Technology. The Mm00503080_s1, respectively). anti-TLR4 Ab was purchased from Imgenex. The anti-mouse IgG (whole molecule) peroxidase conjugate and anti-rabbit IgG (whole molecule) per- Small interfering RNA (siRNA) oxidase conjugate Abs were all purchased from Jackson ImmunoResearch To test the biological function of TRIL, we purchased a RNA interference Laboratories. LPS (TLR4 grade) from Escherichia coli serotype EH100 duplex (Qiagen). Oligonucleotide 1 sequence for human TRIL was was obtained from Alexis. rTNF-␣ as well as IL-6, IL-8, IL-1, TNF-␣, GGCUGGAUCUAGACGGCAA (sense strand sequence for human and RANTES ELISAs were obtained from R&D Systems. Human and TRIL). The additional oligonucleotide tested was CGUAGGAAACUGC murine cDNA panels were obtained from BD Clontech. Human protein GGGCUAUU (Dharmacon). Sequence scrambled negative control oligo- tissue samples were obtained from BD Clontech. The human macro- nucleotide for TRIL was CGUCACUGGGUGAGCAAGA. siRNAs were phage THP1 cell line, HEK293 cells, and U373 parental cells were tested at a number of concentrations. Following optimization, 50 nM of obtained from the European Collection of Animal Cell Cultures. U373/ siRNA oligonucleotide 1 (Qiagen) showed the most consistent knockdown CD14 cells were a gift from Prof. Doug Golenboch (University of Mas- in all cell types. U373/CD14 cells were seeded at 5 ϫ 105cells/ml in 6-well sachusetts, Boston, MA). plates. Oligonucleotides were transfected using Oligofectamine (Invitro- Downloaded from Homology modeling studies gen) in serum-free medium. After 6 h, 1 ml of medium containing 20% FCS and 2ϫ L-glutamine was added to the cells. Cells were harvested after Predicted structural domains in TRIL were identified using the Meta Server 48–72 h and used for further analysis. Human PBMC, THP1 cells, and 3D-Jury. Alignments were manually adjusted and used as templates to murine mixed glial cells were transfected with siRNA using an Amaxa generate models using Modeler 9v3 (12). The Lingo-1 ectodomain (pdb electroportator and a Cell Line Nucleofector Kit V, program V-01 2id5) was the template for the LRR domain. (PBMC), S-019 (THP1), and T-020 (mixed glial cells), according to the manufacturer’s instructions. One ϫ 106cells/ml PBMC or THP1 cells were Animals used per point for nucleofection. Cells were harvested after 72 h and used http://www.jimmunol.org/ for further analysis. C57BL/6 mice (3–4 mo old) were purchased from Harlan. Animals had free access to food and water, were housed in groups of three to six in a Immunoprecipitation, glycosidase treatment, and LPS- binding controlled environment (temperature: 20–22°C; 12:12-h light/dark cycle), assays and were maintained under veterinary supervision for the duration of the experiment. All experiments were conducted under a license from the De- HEK293 cells were seeded at 2 ϫ 105cells/ml in 10- cm dishes. Flag-TLR4 partment of Health and Children (Ireland) and with ethical approval from (pcDNA3.1) was transfected using Genejuice Transfection Reagent (No- Trinity College Ethical Committee. For LPS induction experiments, vagen). Cells were treated as outlined in the figure legends. Cells were C57BL/6 mice were injected with either saline or LPS (100 g/kg E. coli washed in ice-cold PBS and lysed in 500 l of high-stringency lysis buffer serotype 0111.B4; Sigma-Aldrich) i.p. and 3 h later mice were killed by (50 mM HEPES (pH 7.5), 100 mM NaCl, 1 mM EDTA, 10% glycerol, and cervical dislocation and the brain was rapidly removed and stored snap 1% Nonidet P-40). Supernatants were removed and added to the relevant by guest on September 29, 2021 frozen until required for analysis. precoupled Ab. Fifty microliters of each lysate was retained to confirm expression of TRIL and TLR4. Samples were incubated overnight at 4°C. Ab characterization Following incubation, the immune complexes were washed twice with 1 ml Two rabbit polyclonal anti-TRIL Abs were raised against a peptide se- of lysis buffer and once with ice-cold PBS. All supernatants were removed ϫ quence in the C terminus of the protein, aa 797–811-SLRREDRLLQR and beads were resuspended in 30 lof5 sample buffer. The samples FAD (Eurogentic/21Century Biochemicals), one of which was affinity pu- were boiled for 5 min and SDS-PAGE analysis was performed as described rified. Ab specificity was confirmed by peptide competition analysis. previously. ϫ 5 TRIL was immunoprecipitated from TRIL-Flp-In CHO cells (ϳ1–2 ϫ HEK293 cells were seeded at 1 10 cells/ml into 6-well plates. Cells 6 were either mock transfected or transfected with 3 g of TRIL. Samples 10 cells/point) using anti-TRIL-coupled protein A-Sepharose beads. The were harvested and electrophoresed on 10% SDS gels, transferred to poly- complex was washed twice with high-stringency lysis buffer and once with vinylidene fluoride membrane, and blocked in 5% (w/v) dried milk in PBS before resuspending the beads in glycosidase reaction buffer. Beads TBS-Tween 20 buffer. Samples were either probed directly using anti- were incubated with 2.5 U of PNGase F (PP0261–1KT; Sigma-Aldrich) for TRIL Ab or the primary Ab was incubated at 30oC for 30 min with 20 40 min at 37°C. SDS-PAGE sample buffer was added and the samples were g/ml of its corresponding competing peptide. analyzed by Western blotting. HEK293 cells were seeded at 2 ϫ 105cells/ml in 10-cm dishes. Cells Cell culture and stable cell line generation were transfected with 4 g of pcDNA-TRIL plasmid. After 24 h, lysates containing overexpressed TRIL were incubated with 2 g/ml biotinylated HEK293 cells and U373 parental cells were maintained in DMEM and LPS (InvivoGen) or 3 g/ml biotinylated CpG (MWG) before precipitation THP1 cells were maintained in RPMI 1640. All medium used was sup- on avidin beads (Pierce). The complexes were washed in high-stringency plemented with 10% FCS and 1% penicillin-streptomycin solution (v/v). buffer and analyzed by SDS-PAGE. LPS pull-down experiments were also Cerebellar granule neurons were isolated from the dissociated cerebella conducted in the presence and absence of nonbiotinylated LPS for2hat from 5-day-old rats. Cells were gently dissociated with a Pasteur pipette room temperature before precipitation with avidin-agarose for a further 2 h. using 0.4% trypsin and 0.1% DNase I at room temperature. Cells were maintained in DMEM/F12. Cells were also maintained in 10 M cytosine- Results 1--D-arabinofuranoside solution. Cells were maintained on poly-L-orni- thine-coated plates. Cortical astrocytes were isolated from the cortex of Identification and characterization of TRIL 5-day-old rats and maintained in DMEM for 10 days before treatment. A microarray was conducted on embryonic stem cells to identify Human PBMC were isolated from human blood and maintained in IMDM. novel LPS-inducible genes. A previously uncharacterized gene Primary murine mixed glia cells were obtained from 1-day-old neonatal brains. Cells were cultured in DMEM for 10 days before treatment. U373 was identified, which we named TRIL (GenBank accession no. parental cells were used for stable cell line generation. U373 cells were NM_014817). The open reading frame of TRIL comprises 2436 bp transfected by nucleofection (Cell Line Nucleofector Kit V, program and is predicted to encode a protein of 811 aa with a calculated T-020; Amaxa). Cells were maintained in DMEM supplemented with 10% molecular mass of 83 kDa. The chromosomal location has been FCS and 1% penicillin-streptomycin solution (v/v). Cells stably transfected with pcDNA3.1 TRIL were selected and cloned using 300 g/ml neomycin mapped to chromosome 7q15.1 and the coding region consists of analog G418 (InvivoGen) with or without 10 g/ml blasticidin. Overex- one exon. A number of LRR glycoproteins have evolved to encode pression of TRIL was confirmed by immunoblotting and/or FACS analysis. their entire LRR cassette within one exon such as synleurin (13), The Journal of Immunology 3991
protein contains a 23-aa signal sequence as predicted by signal P, suggesting that the protein is directed to the endoplasmic reticulum and from there it could go to several types of vesicles or to the plasma membrane (16). It has a predicted isoelectric point of 9.7, which we have confirmed by two-dimensional electrophoresis (data not shown). A homology model of the TRIL LRR region was generated using Modeler 9v3 (12) and has the characteristic curved solenoid of other LRR domains as shown in Fig. 2B. This model is based on LINGO-1 and is also the common structural element found in the TLRs and CD14. Altogether there are 13 LRR motifs and N- and C-terminal capping structures typical of an extracellular LRR. Each separate TRIL LRR is 24 residues long except for LRR7 which contains two single residue insertions (Fig. 2C). The aspar- agine ladder is strictly conserved throughout.
TRIL expression profiling Downloaded from FIGURE 1. Sequence analysis of TRIL. The amino acid sequence of We conducted quantitative RT-PCR to assess the expression of human and mouse TRIL. Regions with identical amino acids are high- TRIL. Results indicate that TRIL is most highly expressed in mu- lighted in black. rine brain, spinal cord, day 11 embryo, and lung (Fig. 3A). All samples were normalized against GAPDH and expressed relative to the lowest detectable sample. Results from a human cDNA chondroadherin (14), and 5T4 oncofetal leucine-rich glycoprotein panel are consistent with the murine results and also indicate high http://www.jimmunol.org/ (15) in addition to TLR1, TLR2, and TLRs 5–10. The TRIL pro- levels of TRIL in the brain, ovary, small intestine, and spleen (Fig. tein sequence is highly conserved across species and is shown for 3B). Since high expression levels were found in brain, we inves- humans and mice in Fig. 1. Analysis of the TRIL sequence using tigated this further and found that TRIL is highly expressed on rat Meta Server 3D-Jury structural homology software identified two cortical astrocytes and on day 5 cerebellar granule neurons (Fig. distinct structured domains: an N-terminal LRR domain followed 3C). Expression of TRIL was also observed throughout the brain by a type III fibronectin domain before the putative transmembrane in the hippocampus, cortex, and cerebellum at the protein level and region (Fig. 2A). There was no predicted structural domain in the also at the RNA level using laser capture microdissection (data not proposed intracellular portion of TRIL. The N terminus of the shown). by guest on September 29, 2021
FIGURE 2. The TRIL LRR domain. A, Schematic depicting the domain topology of TRIL. The extracellular region (residues 1–697) contains both a LRR domain and type III fibronectin domain. The predicted transmembrane domain is shaded black. The intracellular section (residues 718–811; light gray) is predicted to lack specific structured domains. B, Homology model of the TRIL LRR domain using the LINGO-1 ectodomain (pdb 2id5) as a template. The LRR is shown as a cartoon representation inside the molecular surface. LRR1 is located at the top of the image and the insert containing LRR7 is highlighted in purple. C, Human TRIL LRR sequences. In the consensus sequence, L ϭ leucine, isoleucine, valine, or phenylalanine; N ϭ asparagine, cysteine, serine, or threonine; F ϭ phenylalanine or leucine. For each TRIL LRR consensus sequence, residues are in bold and deviations from the consensus are highlighted. The high levels of deviation from the consensus in the latter half of LRR13 reflect the movement of the structure into the C-terminal cap. 3992 TRIL: A NOVEL COMPONENT OF THE TLR4 RECEPTOR COMPLEX
FIGURE 4. TRIL is a LPS-inducible gene. A, HEK293 cells were trans- Downloaded from fected with 3 g of TRIL (lanes 1 and 3) or mock transfected (lanes 2 and 4). Samples were either probed directly using anti-TRIL Ab or the primary Ab was incubated at 30oC for 30 min with the corresponding competing peptide (20 g/ml) before detection. B, TRIL was immunoprecipitated from CHO cells stably overexpressing TRIL. The complex was resus- pended in glycosidase reaction buffer before incubation with 2.5 U of PNGase F. Samples were then probed using anti-TRIL Ab. C, Wild-type http://www.jimmunol.org/ (WT) U373 and U373 cells stably overexpressing TRIL were treated as described in A. U373/CD14 cells (D) and BMDM (E) were stimulated with 100 ng/ml LPS for the indicated time points and expression of TRIL or -actin was assessed by Western blot. F, C57BL/6 mice were injected i.p. with LPS or water for 3 h. Brain extracts were immunoblotted for TRIL and -actin expression. G, Human PBMC were stimulated with LPS for the FIGURE 3. Expression profile of TRIL. TaqMan quantitative RT-PCR indicated time points. Protein lysates were immunoblotted for TRIL and was conducted on a murine (A) and human (B) cDNA panel to assess TRIL -actin expression as indicated.
expression. TRIL mRNA levels were normalized against GAPDH and ex- by guest on September 29, 2021 pressed relative to the lowest detectable sample. C, Cerebellar granule neurons and cortical astrocytes were prepared from rat brain samples. TRIL the absence of CD14; therefore, we used a cell line stably express- mRNA levels were normalized against GAPDH and expressed relative to total brain. CG, Cerebellar granule. ing CD14 to confer increased LPS responsiveness when analyzing TRIL expression. TRIL was expressed basally and induced by LPS at 2 and 4 h (Fig. 4D). TRIL was also induced in murine bone marrow- derived macrophages (BMDM) after 2 and 4 h and was TRIL is a LPS-inducible protein sustained at 24 h following treatment with LPS (Fig. 4E). In vivo Abs to TRIL were generated to measure endogenous protein lev- expression of TRIL was then assessed in C57BL/6 mice that were els. The peptide sequence to which the Abs were raised is highly exposed to i.p. injection of LPS for 3 h before sacrifice. TRIL is conserved across a number of species. The specificity of the Ab clearly induced in the brain following LPS injection as shown in was confirmed by peptide competition as shown in Fig. 4A for both Fig. 4F. LPS treatment also induced TRIL in primary human overexpressed (lane 1 vs lane 3) and endogenous (lane 2 vs lane PBMC and a marked induction over basal was seen at 60 and 120 4). As can be seen from this figure when TRIL is transiently over- min (Fig. 4G). expressed in 293 cells, it migrates as a doublet. A number of pu- tative N-linked glycosylation sites can be identified in the TRIL TRIL is a component of the TLR4 signaling complex sequence that may explain the different banding patterns observed We next sought to determine whether TRIL plays any role in LPS on electrophoresis. A faster migrating form of the protein was signaling given the nature of the extracellular domain and en- indeed detected following treatment with glycosidase as shown in hanced expression following LPS stimulation. As mentioned Fig. 4B (middle lane), suggesting that the protein does undergo this above, wild-type U373 cells respond poorly to LPS in the absence form of posttranslational modification. Although TRIL expression of CD14 although they express basal levels of both TLR4 and occurs at basal levels in some cell types, we have also observed MD2. We compared LPS-induced cytokine production in wild- enhanced expression of TRIL following treatment with LPS. Since type cells compared with cells stably overexpressing TRIL. As can TRIL is highly expressed in the brain and particularly on astro- be seen in Fig. 5, stable overexpression of TRIL markedly in- cytes, initial studies were conducted in the U373 human astrocy- creased the induction of IL-6, IL-8, and RANTES in response to toma cell line. Once again Ab specificity was confirmed using LPS (Fig. 5, A–C, respectively). Given this ability of TRIL to peptide competition assays. We generated a U373 cell line stably markedly enhance LPS signaling, we next sought to address overexpressing TRIL. Similar to the HEK293 cells, overexpressed whether TRIL can interact with components of the TLR4 receptor TRIL migrates as a doublet and both this band and the band cor- complex. HEK293 cells were transfected with Flag-tagged TLR4 responding to endogenous TRIL were competed out in this exper- and stimulated with LPS for various times (Fig. 6A). TLR4 was iment (Fig. 4C). Wild-type U373 cells respond poorly to LPS in found to interact with endogenous TRIL (Fig. 6, lane 1) and the The Journal of Immunology 3993
FIGURE 5. TRIL modulates responses to LPS. A, U373 cell lines stably expressing TRIL were generated. Cells were maintained in complete me- dium before stimulation with the indicated concentration of LPS for 24 h. FIGURE 7. Knockdown of TRIL affects the LPS but not the TNF-␣ IL-6 (A), IL-8 (B), and RANTES (C) levels were measured by ELISA. signaling pathway. A, U373/CD14 cells were transfected with an siRNA Downloaded from Results are expressed as a mean Ϯ S.D for triplicate determinations. WT, oligonucleotide specific to TRIL (50 nM) or an equivalent concentration of Wild type. scrambled control (Neg. Ctl.) oligonucleotide. Each oligonucleotide was transfected for 72 h before stimulation with LPS (100 ng/ml). Cells were harvested and immunoblotted for TRIL, IB␣, phospho-p38 (pp38), and -actin. B, Cells were stimulated with 20 ng/ml TNF-␣ and immunoblotted strength of this interaction was found to increase on LPS stimu- for TRIL, IB␣, and -actin. C and D, THP1 cells were transfected with lation, being optimal from 1 h (Fig. 6, lane 3). control and TRIL-specific oligonucleotides. Knockdown or control cells http://www.jimmunol.org/ Having established that TRIL was capable of interacting with were stimulated with LPS (100 ng/ml) for 24 h. IL-6 (C) and TNF-␣ (D) TLR4, we next investigated whether TRIL can bind LPS. HEK293 levels were measured by ELISA. Results are expressed as mean Ϯ SD for p Ͻ 0.001. All results are representative of ,ءءء .cells were transfected with full-length TRIL and lysates were in- triplicate determinations cubated with biotinylated LPS or biotinylated CpG as a control for three separate experiments. Neg. Ctl, Negative control. 2 h before precipitation with avidin beads. As shown in Fig. 6B, TRIL could clearly be detected following incubation with LPS; however, no interaction was observed with CpG (Fig. 6B, top). We Knockdown of TRIL affects LPS signaling also found that this interaction could be competed out using un- by guest on September 29, 2021 labeled LPS (Fig. 6B, bottom). This standard assay strongly indi- We next performed siRNA knockdown experiments to determine cates that TRIL can interact with components of the TLR4 receptor whether TRIL functions in the TLR4 signaling pathway. TRIL complex and LPS itself. knockdown was confirmed at the protein level in U373/CD14 cells (Fig. 7A, gel 1, lanes 4–6). This result also further confirms the specificity of the TRIL Ab. Silencing of TRIL affected the LPS signaling pathway by delaying the degradation of IB␣ in re- sponse to LPS (Fig. 7A, gel 2, lanes 4–6). This result was con- firmed with an additional siRNA oligonucleotide. We also found that TRIL knockdown inhibited LPS-induced phosphorylation of p38 (Fig. 7A, gel 3, lanes 4–6). Transfection with a scrambled version of the oligonucleotide (Fig. 7B, Neg. Ctl, gel 1, lanes 1–3) did not affect endogenous levels of TRIL nor did it ablate LPS- induced readouts. Importantly, silencing of TRIL did not affect the TNF-␣ signaling pathway and IB␣ degradation occurred as nor- mal in all samples in response to TNF-␣ (Fig. 7B, gel 2, lanes 4–6). TRIL expression was also knocked down in the human monocytic cell line THP1. This was confirmed at the protein level (Fig. 7C, inset) and cytokine production was monitored. Silencing of TRIL in these cells led to a decrease in IL-6 and TNF-␣ pro- FIGURE 6. TRIL associates with TLR4 and binds LPS. A, HEK293 duction following LPS stimulation (Fig. 7, C and D). To ensure cells were transfected with 4 g of Flag-TLR4 for 24 h before stimulation that there were no off-target affects, endogenous levels of TLR4 with 100 ng/ml LPS for the indicated time points. Interaction with endog- were tested and found to be unaffected by knockdown of TRIL enous TRIL was assessed following immunoprecipitation (IP; top gel) with (data not shown). anti-Flag beads. TRIL and TLR4 expression levels were examined in cell We also knocked down TRIL expression in human PBMC. De- lysates (middle and bottom gels, respectively). B, HEK293 cells were trans- livery of a FITC-tagged TRIL siRNA oligonucleotide was con- fected with 4 g of TRIL for 24 h. Cell lysates were incubated with bio- firmed by FACS analysis and transfection efficiency as achieved tinylated LPS or biotinylated CpG for 2 h before precipitation on avidin beads (top gel). Lysates were assessed for TRIL expression (middle gel). by Amaxa electroporation was found to be close to 100% (data not Cell lysates containing overexpressed TRIL were incubated with biotinyl- shown). Knockdown was confirmed at the protein level (Fig. 8A, ated LPS in the presence or absence of nonbiotinylated LPS (bottom gel) inset). In agreement with results mentioned above, silencing of for 2 h before precipitation on avidin beads. Lysates were analyzed by TRIL affected LPS signaling by delaying IB␣ degradation and SDS-PAGE and immunoblotted (IB) with anti-TRIL. Ctl, Control. inhibiting the phosphorylation of p38 (data not shown). At the 3994 TRIL: A NOVEL COMPONENT OF THE TLR4 RECEPTOR COMPLEX
to as KIAA0644) with the TLRs following sequence analysis of a number of LRR-containing proteins. Furthermore, the consensus sequence of the TRIL LRRs broadly matches that of the TLR family assigned by Matsushima et al. (18) comprising LxxLxLxx- NxLxxLxxxxF/LxxLxx. Consistent with other LRR proteins, TRIL can be modeled with the characteristic curved solenoid shape, contains N- and C-terminal cysteine caps and a conserved asparagine backbone. We have found that TRIL is expressed at high levels in the brain and particularly in astrocytes. Although the protein is expressed basally, we have also found that levels of TRIL increase upon LPS stimulation as shown for U373s, murine BMDM, and human PBMC. TRIL was also induced by LPS in vivo in mouse brain possibly by LPS penetrating the blood-brain barrier or via induc- tion of cytokines such as IL-1. TRIL induction may therefore help prolong LPS signaling. An immediate question that arises relates to the role of TRIL in relation to CD14, given that both proteins possess LRR domains and can bind LPS. Indeed, there is signifi- cant structural similarity between the LRR regions of TRIL and Downloaded from CD14; however, at the amino acid level, the sequence identity is 17%. In addition, both proteins can enhance responses to LPS FIGURE 8. Knockdown of TRIL in primary cells affects LPS re- when stably expressed in U373 cells. TRIL may therefore be a sponses. A and B, PBMC were isolated and transfected with control (Ctl, functional homolog of CD14. CD14 exists as a GPI-anchored pro- CTL) or TRIL-specific siRNA. Cells were stimulated with 100 ng/ml LPS tein and also as soluble factor which functions to transfer LPS to
for 24 h before being harvested and immunoblotted for TRIL (inset). Su- http://www.jimmunol.org/ pernatants were analyzed for IL-6 (A) and TNF-␣ (B) production. C, the TLR4 receptor complex. Interestingly, we have observed a PBMC were stimulated with 500 nM CpGB for 24 h. Supernatants were soluble form of TRIL in FCS. This form was identified by mass analyzed for IL-6. Results are expressed as mean Ϯ SD for triplicate de- spectroscopic analysis; however, this is preliminary data and we p Ͻ 0.001) Similar results were ob- are currently investigating this further. One possibility is that TRIL ,ءءء p Ͻ 0.01 and ,ءء) terminations tained in three additional experiments. D, PBMC were isolated and trans- acts a substitute for CD14 in cells where the latter may be ex- fected with control or TRIL-specific siRNA. Cells were stimulated with 1 pressed at low levels. Alternatively, TRIL may be acting as part of g/ml Pam3Cys4 for 24 h before being harvested. Supernatants were an- the LPS transfer mechanism that exists between accessory mole- ␣ Ϯ alyzed for TNF- production. Results are expressed as mean SD for cules and TLR4. In our studies, LPS treatment of cells increased p Ͻ 0.05).E, Mixed glial cells were isolated ,ء) triplicate determinations
the association of TRIL with TLR4. In addition, we observed that by guest on September 29, 2021 from 1-day-old neonatal mice and cultured for 10 days. Cells were then transfected with control or TRIL-specific siRNA and stimulated with 100 TRIL could specifically associate with LPS. Additional experi- ng/ml LPS for 24 h before being harvested and immunoblotted for TRIL ments will determine whether both TRIL and CD14 are required (inset). Supernatants were analyzed for RANTES production. Neg Ctl, for optimal TLR4 responses. Negative control. TRIL could also have a role similar to RP105, an LRR- con- taining protein with no apparent signaling domain. RP105 binds to MD1 to positively or negatively regulate TLR4 signaling in a cell- cytokine level, knockdown of TRIL led to a decrease in the type-dependent manner (3, 11). We are currently exploring production of TNF-␣ and IL-6 in response to LPS (Fig. 8, A and whether TRIL can bind to MD1 and/or MD2 as well as examining B). We were unable to test the effect of knockdown of TRIL on the the effects of TRIL in additional cell types. production of cytokines by TNF as a control, as TNF could only A number of accessory proteins have also been shown to be induce minimal responses in PBMC; however, knockdown of required for TLR2 signaling. CD14 and vitronectin facilitate the TRIL did not affect CpG-induced IL-6 production (Fig. 8C). In- transfer of lipopeptides to the TLR2 receptor complex (19, 20). It terestingly, knockdown of TRIL also inhibited the induction of therefore appears that multiple accessory proteins are required by TNF-␣ by Pam3Cys4 as shown in Fig. 8D. Finally, we also looked both TLR2 and TLR4 for optimum responses to lipid-based ago- at an additional readout in murine mixed glial cells that produce nists. We have preliminary data to suggest that TRIL could also be RANTES in response to LPS stimulation. Approximately 50% involved in TLR2 signaling since knockdown of TRIL affected knockdown was achieved as shown at the protein level in Fig. 8E Pam3Cys4-induced TNF-␣ production. Further work will enable (inset). Silencing of TRIL in these cells led to a decrease in RAN- us to determine whether TRIL influences additional TLR signaling TES production following LPS stimulation (Fig. 8E). Taken to- pathways. gether, these results indicate that TRIL is a key part of the TLR4 The relatively high expression of TRIL in the brain and the in complex and is important for LPS responses. vivo induction of TRIL by LPS in this tissue is also of interest. It appears that inflammation following brain injury can have a pro- Discussion tective or destructive role depending on which cytokines are pro- TLR signaling in response to LPS requires a number of proteins duced (21, 22). TLR4-deficient mice have been examined follow- that function together as part of a complex to optimally induce ing experimentally induced stroke and were found to have lower proinflammatory responses. We have identified a novel component infarct volumes and lower expression of proteins associated with of this complex, which we have named TRIL. This protein con- brain damage such as IRF-1, NO synthase, cyclooxygenase 2, and tains 13 LRRs, a fibronectin domain, and a putative transmem- matrix metalloproteinase 9 (23, 24). TLR4 has also been shown to brane domain. The predicted domain organization of TRIL is con- mediate innate immunity to bacterial infection in the CNS along sistent with that of Dolan et al. (17) who grouped TRIL (referred with TLR2 (25). Furthermore, TLR4 has been shown to modulate The Journal of Immunology 3995 hippocampal neurogenesis with TLR4-defective mice showing en- 10. Harding, C. V. 2007. gp96 leads the way for toll-like receptors. Immunity 26: hanced neuronal proliferation and differentiation (26). TRIL may 141–143. 11. Divanovic, S., A. Trompette, S. F. Atabani, R. Madan, D. T. Golenbock, participate with TLR4 in these various responses. Given their A. Visintin, R. W. Finberg, A. Tarakhovsky, S. N. Vogel, Y. Belkaid, et al. 2005. structural similarity, there may also be a relationship between Negative regulation of Toll-like receptor 4 signaling by the Toll-like receptor homolog RP105. Nat. Immunol. 6: 571–578. TRIL and the NOGO and LINGO family of proteins, which similar 12. Sali, A., and T. L. Blundell. 1993. Comparative protein modelling by satisfaction to TLR8 are inhibitors of neurite outgrowth (27, 28). Neuroinflam- of spatial restraints. J. Mol. Biol. 234: 779–815. mation has also been associated with neurodegenerative diseases 13. Wang, W., Y. Yang, L. Li, and Y. Shi. 2003. Synleurin, a novel leucine-rich repeat protein that increases the intensity of pleiotropic cytokine responses. Bio- such as Alzheimer’s disease (29, 30). Interestingly, we have pre- chem. Biophys. Res. Commun. 305: 981–988. liminary data which suggest that TRIL expression is increased in 14. Landgren, C., D. R. Beier, R. Fassler, D. Heinegard, and Y. Sommarin. 1998. The the brains of patients with Alzheimer’s disease. Further studies mouse chondroadherin gene: characterization and chromosomal localization. Genomics 47: 84–91. will need to be conducted to determine whether TRIL plays a role 15. King, K. W., F. C. Sheppard, C. Westwater, P. L. Stern, and K. A. Myers. 1999. in the pathogenesis of this condition. Organisation of the mouse and human 5T4 oncofoetal leucine-rich glycoprotein genes and expression in foetal and adult murine tissues. Biochim. Biophys. Acta In conclusion, we have identified a novel component of the 1445: 257–270. TLR4 receptor complex which could represent a new therapeutic 16. Bendtsen, J. D., H. Nielsen, G. von Heijne, and S. Brunak. 2004. Improved target to limit dysregulated TLR4 signaling and may have partic- prediction of signal peptides: SignalP 3.0. J. Mol. Biol. 340: 783–795. 17. Dolan, J., K. Walshe, S. Alsbury, K. Hokamp, S. O’Keeffe, T. Okafuji, ular relevance in the context of neuroinflammation. S. F. Miller, G. Tear, and K. J. Mitchell. 2007. The extracellular leucine-rich repeat superfamily; a comparative survey and analysis of evolutionary relation- ships and expression patterns. BMC Genomics 8: 320. Acknowledgments 18. Matsushima, N., T. Tanaka, P. Enkhbayar, T. Mikami, M. Taga, K. Yamada, and We thank Andrew Hill and Padma Reddy of Biological Technologies Y. Kuroki. 2007. Comparative sequence analysis of leucine-rich repeats (LRRs) Downloaded from (Wyeth Research) for contributions on bioinformatic analysis of TRIL, within vertebrate toll-like receptors. BMC Genomics 8: 124. Mark Stahl (Structural Biology and Chemical Screening Sciences, Wyeth 19. Nakata, T., M. Yasuda, M. Fujita, H. Kataoka, K. Kiura, H. Sano, and K. Shibata. 2006. CD14 directly binds to triacylated lipopeptides and facilitates recognition Research) for helpful discussions. of the lipopeptides by the receptor complex of Toll-like receptors 2 and 1 without binding to the complex. Cell Microbiol. 8: 1899–1909. 20. Gerold, G., K. A. Ajaj, M. Bienert, H. J. Laws, A. Zychlinsky, and J. L. de Diego. Disclosures  2008. A Toll-like receptor 2-integrin 3 complex senses bacterial lipopeptides via
The authors have no financial conflict of interest. vitronectin. Nat. Immunol. 9: 761–768. http://www.jimmunol.org/ 21. Bruce, A. J., W. Boling, M. S. Kindy, J. Peschon, P. J. Kraemer, M. K. Carpenter, F. W. Holtsberg, and M. P. Mattson. 1996. Altered neuronal and microglial References responses to excitotoxic and ischemic brain injury in mice lacking TNF receptors. 1. Janeway, C. A., Jr. 1992. The immune system evolved to discriminate infectious Nat. Med. 2: 788–794. nonself from noninfectious self. Immunol. Today 13: 11–16. 22. Clark, W. M., L. G. Rinker, N. S. Lessov, K. Hazel, J. K. Hill, M. Stenzel-Poore, 2. Medzhitov, R., P. Preston-Hurlburt, and C. A. Janeway, Jr. 1997. A human ho- and F. Eckenstein. 2000. Lack of interleukin-6 expression is not protective mologue of the Drosophila Toll protein signals activation of adaptive immunity. against focal central nervous system ischemia. Stroke 31: 1715–1720. Nature 388: 394–397. 23. Caso, J. R., J. M. Pradillo, O. Hurtado, P. Lorenzo, M. A. Moro, and I. Lizasoain. 3. Nagai, Y., S. Akashi, M. Nagafuku, M. Ogata, Y. Iwakura, S. Akira, T. Kitamura, 2007. Toll-like receptor 4 is involved in brain damage and inflammation after A. Kosugi, M. Kimoto, and K. Miyake. 2002. Essential role of MD-2 in LPS experimental stroke. Circulation 115: 1599–1608. responsiveness and TLR4 distribution. Nat. Immunol. 3: 667–672. 24. Caso, J. R., J. M. Pradillo, O. Hurtado, J. C. Leza, M. A. Moro, and I. Lizasoain.
4. Kim, H. M., B. S. Park, J. I. Kim, S. E. Kim, J. Lee, S. C. Oh, P. Enkhbayar, 2008. Toll-like receptor 4 is involved in subacute stress-induced neuroinflamma- by guest on September 29, 2021 N. Matsushima, H. Lee, O. J. Yoo, and J. O. Lee. 2007. Crystal structure of the tion and in the worsening of experimental stroke. Stroke 39: 1314–1320. TLR4-MD-2 complex with bound endotoxin antagonist Eritoran. Cell 130: 25. Klein, M., B. Obermaier, B. Angele, H. W. Pfister, H. Wagner, U. Koedel, and 906–917. C. J. Kirschning. 2008. Innate immunity to pneumococcal infection of the central 5. Wright, S. D., R. A. Ramos, P. S. Tobias, R. J. Ulevitch, and J. C. Mathison. nervous system depends on toll-like receptor (TLR) 2 and TLR4. J. Infect. Dis. 1990. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS 198: 1028–1036. binding protein. Science 249: 1431–1433. 26. Rolls, A., R. Shechter, A. London, Y. Ziv, A. Ronen, R. Levy, and M. Schwartz. 6. Latz, E., A. Visintin, E. Lien, K. A. Fitzgerald, B. G. Monks, E. A. Kurt-Jones, 2007. Toll-like receptors modulate adult hippocampal neurogenesis. Nat. Cell D. T. Golenbock, and T. Espevik. 2002. Lipopolysaccharide rapidly traffics to Biol. 9: 1081–1088. and from the Golgi apparatus with the toll-like receptor 4-MD-2-CD14 complex 27. Chen, Y., S. Aulia, L. Li, and B. L. Tang. 2006. AMIGO and friends: an emerging in a process that is distinct from the initiation of signal transduction. J. Biol. family of brain-enriched, neuronal growth modulating, type I transmembrane Chem. 277: 47834–47843. proteins with leucine-rich repeats (LRR) and cell adhesion molecule motifs. 7. Wakabayashi, Y., M. Kobayashi, S. Akashi-Takamura, N. Tanimura, K. Konno, Brain. Res. Rev. 51: 265–274. K. Takahashi, T. Ishii, T. Mizutani, H. Iba, T. Kouro, et al. 2006. A protein 28. Ma, Y., J. Li, I. Chiu, Y. Wang, J. A. Sloane, J. Lu, B. Kosaras, R. L. Sidman, associated with toll-like receptor 4 (PRAT4A) regulates cell surface expression of J. J. Volpe, and T. Vartanian. 2006. Toll-like receptor 8 functions as a negative TLR4. J. Immunol. 177: 1772–1779. regulator of neurite outgrowth and inducer of neuronal apoptosis. J. Cell Biol. 8. Takahashi, K., T. Shibata, S. Akashi-Takamura, T. Kiyokawa, Y. Wakabayashi, 175: 209–215. N. Tanimura, T. Kobayashi, F. Matsumoto, R. Fukui, T. Kouro, et al. 2007. A 29. Akiyama, H., S. Barger, S. Barnum, B. Bradt, J. Bauer, G. M. Cole, N. R. Cooper, protein associated with Toll-like receptor (TLR) 4 (PRAT4A) is required for P. Eikelenboom, M. Emmerling, B. L. Fiebich, et al. 2000. Inflammation and TLR-dependent immune responses. J. Exp. Med. 204: 2963–2976. Alzheimer’s disease. Neurobiol. Aging 21: 383–421. 9. Yang, Y., B. Liu, J. Dai, P. K. Srivastava, D. J. Zammit, L. Lefrancois, and Z. Li. 30. Rojo, L. E., J. A. Fernandez, A. A. Maccioni, J. M. Jimenez, and R. B. Maccioni. 2007. Heat shock protein gp96 is a master chaperone for Toll-like receptors and 2008. Neuroinflammation: implications for the pathogenesis and molecular di- is important in the innate function of macrophages. Immunity 26: 215–226. agnosis of Alzheimer’s disease. Arch. Med. Res. 39: 1–16.