TLRs are a group of pattern-recognition receptors that play a crucial role in danger recognition and induction of the innate immune response against bacterial and viral infections. The TLR adaptor molecule, Toll/IL-1R domain-containing adaptor inducing IFN (TRIF), facilitates TLR3 and TLR4 signaling and concomitant activation of the transcription factors, NF-κB and IFN regulatory factor 3, leading to proinflammatory cytokine production. Whereas numerous studies have been undertaken toward understanding the role of TRIF in TLR signaling, little is known about the signaling components that regulate TRIF-dependent TLR signaling. To this end, TRIF-interacting partners were identified by immunoprecipitation of the TRIF signaling complex, followed by protein identification using liquid chromatography mass spectrometry. Following stimulation of cells with a TLR3 or TLR4 ligand, we identified a disintegrin and metalloprotease (ADAM)15 as a novel TRIF-interacting partner. Toward the functional characterization of the TRIF:ADAM15 interaction, we show that ADAM15 acts as a negative regulator of TRIF-mediated NF-κB and IFN-β reporter gene activity. Also, suppression of ADAM15 expression enhanced polyriboinosinic polyribocytidylic acid and LPS-mediated proinflammatory cytokine production via TRIF. In addition, suppression of ADAM15 expression enhanced rhinovirus 16 and vesicular stomatitis virus–mediated proinflammatory cytokine production. Interestingly, ADAM15 mediated the proteolytic cleavage of TRIF. Thus, ADAM15 serves to curtail TRIF-dependent TLR3 and TLR4 signaling and, in doing so, protects the host from excessive production of proinflammatory cytokines and matrix metalloproteinases. In conclusion, to our knowledge, our study clearly shows for the first time that ADAM15 plays an unexpected role in TLR signaling, acting as an anti-inflammatory molecule through impairment of TRIF-mediated TLR signaling.

The innate immune response is highly conserved and represents the first line of defense against microbial pathogens (1). The TLR family is a conserved component of the innate immune system that responds to specific pathogen-associated molecular pattern from viruses, bacteria, fungi, and parasites (2, 3). Signaling through TLRs can be broadly divided into two pathways that are linked to downstream activation of NF-κB, MAPKs, and IFN regulatory factors (IRFs) (4). All TLRs have a Toll/IL-1R domain that facilitates their interaction with TLR adaptor proteins, five of which have been identified to date, namely MyD88, MyD88 adaptor-like, Toll/IL-1R domain-containing adaptor inducing IFN (TRIF), TRIF-related adaptor molecule (TRAM), and Sterile-alpha and Armadillo motif-containing protein (SARM) (4); these adaptors facilitate activation of the MyD88-dependent and TRIF-dependent pathways. Whereas MyD88 is used by TLR2, TLR5, TLR7, TLR8, and TLR9, TRIF is used by TLR3. Both the MyD88 and TRIF-dependent pathways are used by TLR4 toward the induction of proinflammatory cytokines and IFN-stimulated genes (5). Additionally, activation of the TLR pathway also leads to the induction of matrix metalloproteinases (MMPs), including MMP-9 (gelatinase B) and MMP-10 (stromelysin 2) (6, 7). Activation of MMPs has been associated with the maintenance of normal homeostasis, and the upregulation of MMPs has been linked with inflammatory responses, cancer, and arthritis (7).

During TLR3 and TLR4 signaling, TRIF, in association with TRAM in the case of TLR4, initiates a signaling pathway through TRAF3, TANK-binding kinase 1 (TBK1), and inhibitor of nuclear factor-κB (IκB)–kinase complex [IKKε]), which mediates the direct phosphorylation of IRF3 and IRF7 and the formation of IRF3 and IRF7 hetero- or homodimers. The IRF3/7 dimers then translocate to the nucleus, in association with transcriptional coactivators, such as CBP and p300, and bind to target sequences in DNA, such as IFN-stimulated response elements (8, 9). Also, the IRFs, together with the transcription factors ATF-2/c-Jun and NF-κB from the IFN-β enhanceosome, bind to a nucleosome-free enhancer region within the IFN-β promoter upstream of the transcription start site, leading to transcriptional activation of the IFN-β gene (10). The enhancer itself is divided into four positive regulatory domains (PRD) whereby NF-κB binds to the PRDII element within the IFN-β enhancer region, ATF-2/c-Jun binds to the PRDIV element and is activated by JNK, and IRF3 and IRF7 bind to the PRDI-III element (8). Also, TRIF-dependent activation of NF-κB occurs through binding of TRAF6 to TRIF and subsequent ubiquitination-dependent recruitment and activation of TAK1 (11). Despite the numerous studies that have been undertaken toward understanding the role of TRIF in TLR signaling, little is known about the signaling components that regulate the dependent TLR signaling pathway. In this study, we sought to identify novel components of the TRIF signaling pathway. To this end, TRIF-interacting partners were identified by immunoprecipitation of the TRIF signaling complex, followed by protein identification using liquid chromatography mass spectrometry (LC-MS). Following stimulation of cells with a TLR3 or TLR4 ligand, we identified a disintegrin and metalloprotease (ADAM)15 as a novel TRIF-interacting partner. ADAM15, also known as metalloprotease–arginine-glycine-aspartic acid (RGD)–disintegrin protein (Metargidin), is a type I transmembrane glycoprotein belonging to the ADAMs protein family and is widely expressed in different tissues and cell types (12).

ADAMs are membrane-anchored glycoproteins that facilitate a broad range of biological functions, including proteolysis, cell adhesion, cell fusion, inflammation, angiogenesis, and intracellular signaling. Several ADAM family members have been found to facilitate the release of cytokines, growth factors, receptors, adhesion molecules, and other membrane-bound proteins from the cell surface, a process termed ectodomain shedding (13, 14). Human ADAM15, but not murine ADAM15, is the only known ADAM to contain an integrin-binding motif RGD peptide in the disintegrin domain, which binds integrins αvβ3 and α5β1 in a RGD-dependent manner (14), suggesting that ADAM15 may play a role in cell-to-cell interaction and cell adhesion (13). Regarding subcellular localization, most of the mature form of ADAM15 is localized intracellularly (15). Known ADAM15 substrates include FGFR2iiib and N- and E-cadherin, the shedding of which has been implicated in prostate and breast cancer pathogenesis (12, 16). The cytosolic part of ADAM15 is rich in proline-rich consensus binding sites that facilitate its interaction with Src homology 3 domain-containing proteins (17). Altered expression of ADAM15 has been associated with inflammation in osteoarthritic cartilage and rheumatoid synovium, and within the human papillomavirus-infected gastric mucosa (13, 17).

In this study, we investigated the role played by ADAM15 in TLR3 and TLR4 signaling. We found that ADAM15 interacts with the TLR adaptor TRIF following stimulation of cells with the TLR3 and TLR4 ligands, polyriboinosinic polyribocytidylic acid [poly(I:C)] and LPS, respectively. Toward the functional characterization of the TRIF:ADAM15 interaction, we show that ADAM15 acts as a negative regulator of TRIF-mediated NF-κB and IFN-β reporter gene activation. Also, suppression of ADAM15 expression enhanced poly(I:C) and LPS-mediated proinflammatory cytokine production. Supporting the physiological relevance of our findings, suppression of ADAM15 expression enhanced rhinovirus (RV)16 and vesicular stomatitis virus (VSV)-mediated proinflammatory cytokine production, and ADAM15−/− murine embryonic fibroblasts (MEFs) secreted increased levels of proinflammatory cytokines when compared with wild-type (WT) MEFs upon stimulation with VSV and RV16. Interestingly, ADAM15 mediated the proteolytic cleavage of TRIF. In conclusion, we provide ADAM15 as a novel TRIF-interacting partner and show that ADAM15 binds to TRIF, thereby facilitating the proteolytic cleavage of TRIF and subsequent curtailment of TRIF-dependent TLR3 and TLR4 signaling.

HEK293-TLR3 and HEK293-TLR4 were gifts of K. Fitzgerald (University of Massachusetts Medical School). MEFs WT and ADAM15 knockout were gifts of C. Blobel (Novartis Institutes for Biomedical Research, Basel, Switzerland). HEK293-Blue IFN-αβ were purchased from InvivoGen. Cells were cultured in DMEM with GlutaMAX (Life Technologies-BRL); supplemented with 10% FBS, 1% penicillin-streptomycin, and 1% fungizone; and maintained at 37°C in a humidified atmosphere of 5% CO2. The HEK293-TLR4 culture medium was also supplemented with blasticidin (100 mg/ml) and hygrogold (100 mg/ml). The HEK293-TLR3 culture medium was supplemented with blasticidin (100 mg/ml). The HEK293-Blue IFN-αβ culture medium was supplemented with zeocin (100 mg/ml) and blasticidin (10 mg/ml). HEK293-TLR9 was as described (18). U373-CD14 cells were cultured in RPMI 1640 with GlutaMAX (Life Technologies-BRL) supplemented with 10% FBS, 1% penicillin-streptomycin, 1% fungizone, and 250 μg/ml gentamicin (G418; Sigma-Aldrich). Highly purified protein-free LPS derived from Escherichia coli strain 011:B4 (Alexis) and naked poly(I:C) (InvivoGen) were used for all treatments. ADAM15 small interfering RNA (siRNA) was purchased from Sigma-Aldrich (catalog EHU076821) and siRNA control from Ambion (catalog AM16106). RV16 was from S. Goodbourn (University of London). VSV was from American Type Culture Collection.

The ADAM15-V5 was a gift of E. Dylan (University of East Angelia). The plasmid pcDNA3:MyD88-cmyc was a gift of L. O’Neill (Trinity College Dublin). The plasmid pcDNA3:TRIF-hemagglutinin (HA) was from S. Akira (Osaka University). The IFN-β and NF-κB reporter gene plasmids were as described (9). Melanoma differentiation–associated protein 5 (MDA5) was previously described (19).

HEK293-TLR3 and HEK293-TLR4 cells (1 × 106 cells/well; six-well plate) were transfected with the indicated plasmids using Lipofectamine 2000 (Invitrogen), and the total amount of DNA (3 μg/well) was kept constant. After 24 h, cells were stimulated with the indicated ligands, followed by cell lysis in 600 μl lysis buffer (50 mM HEPES [pH 7.5], 150 mM NaCl, 2 mM EDTA [pH 8.0], 1% Nonidet P-40, 0.5% sodium deoxycholate supplemented with 1 mM PMSF, 1 mM, DDT, 1 mM NaVO3, 5 mM EGTA, and 1 tablet/10 ml protein inhibitory cocktail solution [PICS]), and left on ice for 20 min. The lysates were subjected to centrifugation for 10 min at 14,000 rpm to remove cell debris. Next, 2 μg of the indicated Ab was incubated with 70 μl A/G Plus–agarose beads (Santa Cruz Biotechnology) overnight at 4°C, washed, and incubated with the cell lysates for 2 h at 4°C. The immune complexes were precipitated, washed, and eluted by the addition of Laemmli loading buffer, followed by SDS-PAGE and immunoblotting using the indicated Abs.

U373-CD14 cells were seeded into T175 flask and were stimulated with poly(I:C) and LPS, as indicated, followed by cell lysis in 600 μl lysis buffer (50 mM HEPES [pH 7.5], 150 mM NaCl, 2 mM EDTA [pH 8.0], 1% Nonidet P-40, 0.5% sodium deoxycholate supplemented with 1 mM PMSF, 1 mM DDT, 1 mM NaVO3, 5 mM EGTA, and protease inhibitor mixture), and left on ice for 20 min. The lysates were subjected to centrifugation for 10 min at 14,000 rpm to remove cell debris. Next, 2 μg anti-TRIF polyclonal Ab (Exalpha) was incubated with 50 μl A/G Plus–agarose beads (Santa Cruz Biotechnology) overnight at 4°C, washed, and incubated with the cell lysates for 2 h at 4°C. The immune complexes were precipitated, washed, and subjected to immunoblot analysis using an anti-TRIF polyclonal Ab (Exalpha) and an anti-ADAM15 Ab (from R&D Systems).

Following one-dimensional PAGE of TRIF immunocomplexes, the gel section (10 sections; 25–250 kDa molecular mass) were subjected to in-gel trypsin digestion, and the resulting peptides were analyzed by peptide-mass fingerprinting using an Ion Trap LC-MS apparatus from Agilent Technologies (model 6430). Excision, washing, destaining, trypsin digestion, and peptide recovery were performed, as previously described (20). Peptides were separated using a nanoflow Agilent 1200 series system, equipped with a Zorbax 300SB C18 5-μm, 4-mm, 40-nl precolumn and a Zorbax 300SB C18 5-μm, 43 mm × 75-μm analytical reversed-phase column using HPLC-chip technology, and 0.1% formic acid was used as mobile phase A, and 50% acetonitrile, 0.1% formic acid was used as mobile phase B. Samples were loaded at a flow rate of 4 μl/min onto the enrichment column, and the peptide fragments were eluted with a constant nano pump flow rate of 0.6 ml/min. The capillary voltage was set to 1900 V, and the flow and the temperature of the drying gas were 4 L/min and 300°C, respectively. Database searches were performed using Mascot MS/MS Ion search (Matrix Science, London, U.K.).

HEK293, HEK293-TLR3, HEK293-TLR4, and HEK293-TLR9 cells (5 × 104 cells/well; 96-well plate) were transfected with 80 ng/well luciferase reporter gene plasmid for NF-κB and IFN-β and cotransfected with the expression vectors encoding ADAM15, TRIF, MyD88, and MDA5, as indicated, using lipofectamine, as previously described (8). A total of 40 ng/well phRL-TK (TK-Renilla-luciferase) reporter gene was cotransfected simultaneously to normalize data for transfection efficiency. After 24 h, cells were stimulated with ligands, as indicated, for an additional 24 h. Thereafter, cell lysates were prepared and reporter gene activity was measured using the Dual-Luciferase Assay system (Promega). Data were expressed as the mean fold induction relative to control levels, for a representative experiment from a minimum of three separate experiments, each performed in triplicate.

Human U373-CD14 cells were transfected with ADAM15 and control endoribonuclease-prepared siRNA (esiRNA) to target the suppression of ADAM15 and negative control. In a six-well plate, 20 nM esiRNA was transfected into cells using Dreamfect Gold (OZ Biosciences), as described by the manufacturer. After 24 and 48 h, efficiency of ADAM15 knockdown was assessed by RT-PCR using ADAM15 forward and reverse primers.

Human U373-CD14 cells were transfected with esiRNA to target the suppression of ADAM15 or control esiRNA as a negative control. After 24 h, cells were infected with VSV or RV16 (1:1000) for an additional 24 or 48 h, followed by measurement levels of cytokine mRNA and protein, respectively.

Total cellular RNA was isolated from cells using TRIzol reagent (Invitrogen), according to the manufacturer’s instructions. Thereafter, 1 μg total RNA was mixed with 1 μl random hexamers (500 μg/ml), and water was added to a final volume of 17 μl and incubated for 5 min at 70°C. The mixture was then briefly centrifuged and chilled on ice for 2 min. Thereafter, the other reaction components were added in the following order: 5 μl 5× reverse-transcriptase buffer, 1.3 μl 10 mM dNTP, 0.7 μl RNasin (Promega), 1 μl Moloney murine leukemia virus reverse transcriptase (Promega), and nuclease-free water to a total volume of 25 μl. Reactions were incubated at 37°C for 40 min, followed by 42°C for 40 min and heating to 80°C for 5 min. The first-strand cDNA was stored at −20°C for up to 1 mo.

Total cDNA was used as template for real-time RT-PCR quantitation with Maxima SYBR Green/ROX quantitative PCR master mix (2×) (Fermentas) on a real-time PCR system (DNA Engine Opticon system; MJ Research). For amplification of specific genes, the following primers were used: hIFNβ forward, 5′-AACTGCAACCTTTCGAAGCC-3′ and reverse, 5′-TGTCGCCTACTACCTGTTGTGC-3′; hTNFα forward, 5′-CACCACTTCGAAACCTGGGA-3′ and reverse, 5′-CACTTCACTGTGCAGGCCAC-3′; hCCL5 forward, 5′-TGCCTGTTTCTGCTTGCTCTTGTC-3′ and reverse, 5′-TGTGGTAGAATCTGGGCCCTTCAA-3′; hMMP-9 forward, 5′-TACCACCTCGAACTTTGACAGCGA-3′ and reverse, 5′-GCCATTCACGTCGTCCTTATGCAA-3′; hMMP-10 forward, 5′-AATGGATTGTGGCTCATTGGTGGG-3′ and reverse, 5′-TGGAAGTGGTTTAGGAGGAGGCAA-3′. For each mRNA quantification, the housekeeping gene GAPDH was used as a reference point using the following primers: hGAPDH forward, 5′-TTCGACAGTCAGCCGCATCTTCTT-3′ and reverse, 5′-GCCCAATACGACCAAATCCGTTGA-3′; hTRIF forward, 5′-ACGCCATAGACCACTCAGCTTTCA-3′ and reverse, 5′-AGGTTGCTCATCATGGCTTGGTTC-3′.

Cell-free supernatants were used for measuring IFN-γ, IL-10, IL-12 p70, IL-1β, IL-6, IL-8, and TNF-α levels using a Meso Scale 96-well–plate human 7-plex ultrasensitive assay kit. Levels of MMP-1, MMP-3, and MMP-9 were assessed using a Meso Scale 96-well MMP 3-plex ultrasensitive assay kit. Levels of IFN-β and RANTES were assessed using single-plex assay kits (Meso Scale Discovery), as indicated by the manufacturer. Following viral infection, levels of IL-6 were measured by ELISA (R&D Systems), and levels of TNF-α and CCL5 were also measured by ELISA (PeproTech).

Levels of RV16- and VSV-induced bioactive human type I IFN were assessed using HEK-Blue IFN-αβ cells, essentially as described by the manufacturer (InvivoGen).

The unpaired Student t test and two-way ANOVA tests were used to carry out statistical analysis of data. The p values <0.05 were considered to indicate a statistically significant difference (*p < 0.05, **p < 0.01, and ***p < 0.001).

To explore the molecular mechanisms that modulate TLR3 functionality postviral sensing, TRIF was overexpressed in HEK293-TLR3 cells, followed by stimulation of cells with the synthetic TLR3 ligand, poly(I:C), and SDS-PAGE of the TRIF immunocomplex. The gel was silver stained, and in-gel trypsin digestion followed by Nano-LC-MS/MS was performed. Interestingly, ADAM15 was identified as a TRIF interactor, with a two-peptide match, mascot score 55, and 2% sequence coverage.

To confirm that TRIF and ADAM15 are binding partners, coimmunoprecipitation experiments were performed. Using HEK293-TLR3 cells, it was found that HA-tagged TRIF coimmunoprecipitated with V5-tagged ADAM15 following stimulation with poly(I:C) for 60 min (Fig. 1A). Notably, these experiments were performed in the presence of the metal chelators, EGTA and EDTA, to impair the catalytic activity of ADAM15, a Zn-dependent proteinase (21). The ability of endogenous TRIF to interact with endogenous ADAM15 was also examined using human U373-CD14 cells. Correlating with the overexpression data, it was found that endogenous ADAM15 coimmunoprecipitated with endogenous TRIF following stimulation of cells with poly(I:C) for 60 min (Fig. 1B), but not 80 or 100 min (data not shown). Equal TRIF expression was confirmed by immunoblot analysis of the whole-cell lysates. Regarding TLR4, endogenous ADAM15 coimmunoprecipitated with endogenous TRIF following stimulation of cells with LPS for 20 min. Notably, the coimmunoprecipitation of TRIF with ADAM15 upon stimulation with LPS for 20 min corroborates our proteomic data showing that ADAM15 is present in the endogenous TRIF immunocomplex (data not shown).

FIGURE 1.

TRIF binds to ADAM15. (A) HEK293-TLR3 cells were cotransfected with HA-TRIF, V5-ADAM15, or empty vector (EV). After 20 h, cells were stimulated with 20 μg/ml poly(I:C) for 60 min. Thereafter, immunoprecipitation (IP) was performed using an anti-HA mAb, as described in 2Materials and Methods. (B) U373-CD14 cells were stimulated with either 20 μg/ml poly(I:C) or 1 μg/ml LPS for the indicated times. Thereafter, IP of endogenous TRIF was performed using an anti-human TRIF polyclonal Ab, as described in 2Materials and Methods. Immunoblot analysis was performed using either an anti-human TRIF or an anti-human ADAM15 Ab. The black rectangle indicates the location of the 100-kDa molecular mass marker. Results represent at least three independent experiments.

FIGURE 1.

TRIF binds to ADAM15. (A) HEK293-TLR3 cells were cotransfected with HA-TRIF, V5-ADAM15, or empty vector (EV). After 20 h, cells were stimulated with 20 μg/ml poly(I:C) for 60 min. Thereafter, immunoprecipitation (IP) was performed using an anti-HA mAb, as described in 2Materials and Methods. (B) U373-CD14 cells were stimulated with either 20 μg/ml poly(I:C) or 1 μg/ml LPS for the indicated times. Thereafter, IP of endogenous TRIF was performed using an anti-human TRIF polyclonal Ab, as described in 2Materials and Methods. Immunoblot analysis was performed using either an anti-human TRIF or an anti-human ADAM15 Ab. The black rectangle indicates the location of the 100-kDa molecular mass marker. Results represent at least three independent experiments.

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Given that ADAM15 interacts with TRIF in a ligand-dependent manner, we sought to examine the ability of ADAM15 to modulate TRIF functionality. To this end, HEK293 cells were transiently transfected with the NF-κB reporter gene construct. Regarding the transcriptional activation of the IFN-β gene, assembly of a multiprotein complex encompassing the transcription factors ATF-2/c-Jun, IRF3, IRF7, and NF-κB to form the IFN-β enhanceosome is required. The enhanceosome elements bind to the enhancer region within the IFN-β promoter upstream of the transcription start site. The enhancer is divided into four PRDs, NF-κB binds to the PRDII element, ATF-2/c-Jun binds to the PRDIV element and is activated by JNK, and IRF3 and IRF7 bind to the PRDIII-I element (10). In this study, to decipher the ability of ADAM15 to modulate TRIF-mediated induction of IFN-β, HEK293 cells were transiently transfected with the IFN-β p125, PRDII, and PRDIII reporter gene constructs and increasing amounts of ADAM15 in the presence of a constant concentration of TRIF or, as controls, MyD88 and MDA5. We found that ADAM15 inhibited TRIF-mediated activation of NF-κB, IFN-β p125, PRDII, and PRDIII reporter gene activity (Fig. 2A–D). As controls, the effect of ADAM15 on MDA5, a retinoic acid–inducible gene 1 (RIG-I)–like receptor activated by poly(I:C) in a cell-dependent manner (19), and MyD88-driven reporter gene activity were also tested. At high concentrations of ADAM15 DNA, MyD88-dependent activation of NF-κB reporter gene activity was impaired (Fig. 2E). However, overexpression of low amounts of ADAM15 (5–20 ng) enhanced MyD88-dependent activation of the IFN-β reporter gene activity and did not affect MyD88-dependent NF-κB reporter gene activity (Fig. 2E, 2F). Importantly, we found that overexpression of ADAM15 did not affect MDA5-dependent NF-κB reporter gene activity (Fig. 2G).

FIGURE 2.

ADAM15 inhibits TRIF-mediated reporter gene activity. HEK293T cells were transfected with expression vectors encoding the luciferase reporter genes NF-κB (A, E, G), IFN-β promoter p125 (B, F), IFN-β PRDII (C), or IFN-β PRDIII-I (D) and cotransfected with either empty vectors (EV) or an expression vector encoding the full-length human HA-TRIF (20 ng; A–D), MyD88 (20 ng; E, F), and MDA5 (20 ng; G), and increasing amounts of the expression vector encoding full-length human V5-ADAM15 (5, 20, and 40 ng), as indicated. After 24 h, cell lysates were harvested and assessed for luciferase reporter gene activity (Promega). The data presented are representative of at least three independent experiments performed in triplicate (mean ± SE). *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 2.

ADAM15 inhibits TRIF-mediated reporter gene activity. HEK293T cells were transfected with expression vectors encoding the luciferase reporter genes NF-κB (A, E, G), IFN-β promoter p125 (B, F), IFN-β PRDII (C), or IFN-β PRDIII-I (D) and cotransfected with either empty vectors (EV) or an expression vector encoding the full-length human HA-TRIF (20 ng; A–D), MyD88 (20 ng; E, F), and MDA5 (20 ng; G), and increasing amounts of the expression vector encoding full-length human V5-ADAM15 (5, 20, and 40 ng), as indicated. After 24 h, cell lysates were harvested and assessed for luciferase reporter gene activity (Promega). The data presented are representative of at least three independent experiments performed in triplicate (mean ± SE). *p < 0.05, **p < 0.01, ***p < 0.001.

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As TRIF mediates TLR3 and TLR4 signaling, the effect of ADAM15 on TLR3- and TLR4-dependent NF-κB and IFN-β reporter gene activation was investigated using HEK293 cells stably expressing either TLR3 or TLR4. In HEK293-TLR3 cells, overexpression of ADAM15 significantly inhibited TLR3-dependent activation of NF-κB and IFN-β reporter genes (Fig. 3A, 3B). Furthermore, in HEK293-TLR4 cells, overexpression of ADAM15 significantly reduced TLR4-dependent NF-κB and IFN-β reporter gene activation (Fig. 3C, 3D). As a control, we tested whether ADAM15 affected MyD88-dependent, TRIF-independent TLR9 signaling using HEK293 stably expressing TLR9. We found that overexpression of ADAM15 did not affect TLR9-dependent NF-κB and IFN-β reporter gene activity (Fig. 3E, 3F). These results demonstrate that ADAM15 negatively regulates TRIF-dependent TLR3 and TLR4 signaling, but not TRIF-independent TLR9 signaling. To confirm that ADAM15 inhibits TLR4-mediated NF-κB activation, ADAM15 expression was suppressed, followed by examination of the phosphorylation status of the NF-κB subunit, p65, following LPS stimulation. To this end, ADAM15 expression was suppressed in U373-CD14 cells using esiRNA directed against ADAM15 or control lamin A/C. Selective and specific suppression of endogenous ADAM15 mRNA and protein expression was confirmed by semiquantitative RT-PCR and immunoblot analysis, respectively (Fig. 4A). Following suppression of ADAM15 expression and stimulation of cells with LPS, it was found that, although phosphorylation of p65 at 30 min was suppressed, p65 phosphorylation at 120 min was enhanced following suppression of ADAM15 expression (Fig. 4B). These data indicate that whereas late-stage NF-κB activation, mediated by TRIF (22), is suppressed by ADAM15, early-stage MyD88-dependent NF-κB activation is enhanced by ADAM15.

FIGURE 3.

ADAM15 inhibits TLR-mediated reporter gene activity. HEK293-TLR3 (A, B), HEK293-TLR4 (C, D), and HEK293-TLR9 (E, F) cells were cotransfected with vectors encoding either a luciferase reporter for the NF-κB or IFN-β reporter genes (80 ng) and either empty vector (EV; 40 ng) or with increasing amounts of an expression vector encoding ADAM15 (0, 5, 20, 40 ng), as indicated. A total of 40 ng/well phRL-TK (TK-Renilla-luciferase) reporter gene was cotransfected simultaneously to normalize data for transfection efficiency. After 24 h, cells were stimulated with poly(I:C) (20 μg/ml) (A, B), LPS (1 μg/ml) (C, D), or CpG (3 μg/ml) (E, F). After 24 h, cell lysates were harvested and assessed for luciferase reporter gene activity using the dual luciferase system (Promega). The data presented are representative of at least three independent experiments performed in triplicate (mean ± SE). ***p < 0.001.

FIGURE 3.

ADAM15 inhibits TLR-mediated reporter gene activity. HEK293-TLR3 (A, B), HEK293-TLR4 (C, D), and HEK293-TLR9 (E, F) cells were cotransfected with vectors encoding either a luciferase reporter for the NF-κB or IFN-β reporter genes (80 ng) and either empty vector (EV; 40 ng) or with increasing amounts of an expression vector encoding ADAM15 (0, 5, 20, 40 ng), as indicated. A total of 40 ng/well phRL-TK (TK-Renilla-luciferase) reporter gene was cotransfected simultaneously to normalize data for transfection efficiency. After 24 h, cells were stimulated with poly(I:C) (20 μg/ml) (A, B), LPS (1 μg/ml) (C, D), or CpG (3 μg/ml) (E, F). After 24 h, cell lysates were harvested and assessed for luciferase reporter gene activity using the dual luciferase system (Promega). The data presented are representative of at least three independent experiments performed in triplicate (mean ± SE). ***p < 0.001.

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FIGURE 4.

Suppression of ADAM15 expression enhances proinflammatory cytokine and MMP mRNA expression. (A) Upper, U373-CD14 cells transfected with 20 nM control or ADAM15 esiRNAs. Cells were harvested 24 h after transfection, and total RNA was isolated and reverse transcribed into cDNA. The cDNA template was diluted 25 times, and ADAM15 mRNA was measured by quantitative RT-PCR using primers specific to human ADAM15. GAPDH was used as housekeeping gene. Lower, Whole-cell lysates were subjected to immunoblot analysis using an anti-human ADAM15 mAb (R&D Systems), and β-actin served as a loading control. (B) U373-CD14 cells were pretreated with ADAM15 or control esiRNAs for 24 h. Next, cells were stimulated with 1 μg/ml LPS for the indicated times. Thereafter, cell lysates were harvested and subjected to immunoblot analysis using anti-pp65 and anti–β-actin Abs. (CG) U373-CD14 cells were pretreated with ADAM15 or control esiRNAs. After 48 h, cells were stimulated with either 20 μg/ml poly(I:C) or 1 μg/ml LPS for 3 h to measure cytokine levels. Alternatively, cells were stimulated with either 20 μg/ml poly(I:C) or 1 μg/ml LPS for 24 h to measure MMP levels. Thereafter, total RNA was isolated and used as template to measure levels of IFN-β, TNF-α, RANTES, MMP-9, and MMP-10 mRNAs with GAPDH serving as a housekeeping gene. Data are representative of two independent experiments; each experiment was performed in duplicate (mean ± SE). *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 4.

Suppression of ADAM15 expression enhances proinflammatory cytokine and MMP mRNA expression. (A) Upper, U373-CD14 cells transfected with 20 nM control or ADAM15 esiRNAs. Cells were harvested 24 h after transfection, and total RNA was isolated and reverse transcribed into cDNA. The cDNA template was diluted 25 times, and ADAM15 mRNA was measured by quantitative RT-PCR using primers specific to human ADAM15. GAPDH was used as housekeeping gene. Lower, Whole-cell lysates were subjected to immunoblot analysis using an anti-human ADAM15 mAb (R&D Systems), and β-actin served as a loading control. (B) U373-CD14 cells were pretreated with ADAM15 or control esiRNAs for 24 h. Next, cells were stimulated with 1 μg/ml LPS for the indicated times. Thereafter, cell lysates were harvested and subjected to immunoblot analysis using anti-pp65 and anti–β-actin Abs. (CG) U373-CD14 cells were pretreated with ADAM15 or control esiRNAs. After 48 h, cells were stimulated with either 20 μg/ml poly(I:C) or 1 μg/ml LPS for 3 h to measure cytokine levels. Alternatively, cells were stimulated with either 20 μg/ml poly(I:C) or 1 μg/ml LPS for 24 h to measure MMP levels. Thereafter, total RNA was isolated and used as template to measure levels of IFN-β, TNF-α, RANTES, MMP-9, and MMP-10 mRNAs with GAPDH serving as a housekeeping gene. Data are representative of two independent experiments; each experiment was performed in duplicate (mean ± SE). *p < 0.05, **p < 0.01, ***p < 0.001.

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Given that our data indicate that ADAM15 suppresses TLR3- and TLR4-mediated reporter gene activity and late-stage NF-κB activation through a mechanism involving TRIF, we sought to establish whether ADAM15 affected TLR3- and TLR4-mediated signaling downstream of their respective transcription factors. To this end, we examined the transcriptional expression of proinflammatory cytokines following TLR3 and TLR4 engagement in the absence and presence of ADAM15. We found that silencing of ADAM15 significantly enhanced poly(I:C) and LPS induced IFN-β, TNF-α, and RANTES gene induction, respectively, in human cells (Fig. 4C–E). It has been reported that MMP-9 secretion and activity were abolished in the metastatic prostate cancer PC-3 cell line following suppression of ADAM15 expression (23). Furthermore, ADAM15 has been shown to cleave the ectodomain of MMP-10 (24). Interestingly, it has been proposed that activation of MMP-9 activity may be initiated upon TLR ligand binding (25). Furthermore, TLR3 and TLR2 engagement has been shown to enhance MMP-9 and MMP-10 expression (6, 26). Given the link among TLRs, ADAM15, and MMPs, we opted to investigate the role of ADAM15 in TLR-mediated induction of MMP-9 and MMP-10. Correlating with previous studies (23), it was found that suppression of ADAM15 expression decreased LPS-mediated induction of MMP-9 and MMP-10 mRNA when compared with control cells. Surprisingly, suppression of ADAM15 significantly enhanced poly(I:C)-mediated MMP-9 and MMP-10 mRNA expression when compared with control cells (Fig. 4F, 4G). These data indicate that, whereas ADAM15 is required for LPS-driven MMP-9 and MMP-10 mRNA induction, it impairs poly(I:C)-driven MMP-9 and MMP-10 induction.

Next, the effect of ADAM15 on TLR3- and TLR4-mediated cytokine, chemokine, and MMP induction was assessed. To this end, U373-CD14 cells were transfected with esiRNA-control or esiRNA against ADAM15 for 24 h, followed by stimulation with either poly(I:C) or LPS for an additional 24 h. Following assessment of cytokine and chemokine levels in the cell supernatant, it was found that suppression of ADAM15 expression consistently and significantly increased poly(I:C)-induced IFN-β, IFN-γ, IL-12p70, CCL5, and TNF-α levels compared with the control unstimulated cells (Fig. 5A–E). Furthermore, suppression of ADAM15 enhanced LPS-induced IFN-β, IFN-γ, IL-12p70, and CCL5 levels compared with the control unstimulated cells (Fig. 5I–L). Following LPS stimulation, increases in the levels of TNF-α were also observed following ADAM15 suppression, although the levels were not significant (Fig. 5M). Levels of IL-6, IL-8, and IL-1β were not affected (data not shown). The transcription of many MMPs is promoted by TLR ligands, inflammatory cytokines, and chemokines (27). Thus, the levels of MMP-1, MMP-3, and MMP-9 in cell-free supernatants were also measured following suppression of ADAM15 and subsequent stimulation of cells with poly(I:C) and LPS. Suppression of ADAM15 significantly enhanced poly(I:C)-induced MMP-1, MMP-3, and MMP-9 levels compared with the control (Fig. 5F–H). Suppression of ADAM15 also significantly enhanced LPS-induced MMP-3 and MMP-1 levels compared with the control (Fig. 5O, 5P). Interestingly, suppression of ADAM15 significantly decreased LPS-induced MMP-9 secretion compared with the control (Fig. 5N). These data suggest that ADAM15 plays a role in the negative regulation of poly(I:C) and LPS-mediated cytokine and chemokine induction. Thus, whereas ADAM15 is required for poly(I:C) and LPS-driven MMP-1 and MMP-3 production, ADAM15 differentially modulates poly(I:C) and LPS-mediated MMP-9 production.

FIGURE 5.

Suppression of ADAM15 enhances TLR3- and TLR4-mediated proinflammatory cytokine and chemokine induction. (AP) U373-CD14 cells were pretreated with control or ADAM15 esiRNAs. After 24 h, cells were stimulated with either 20 μg/ml poly(I:C) (A–H) or 1 μg/ml LPS (I–P) for an additional 24 h. Thereafter, levels of IFN-β, IL-12p70, and TNF-α were measured in the cell-free supernatants using the human proinflammatory multiplex assay kit. Levels of RANTES and IFN-β were measured using the human single-plex assay kit (Meso Scale Discovery). MMP-1, MMP-3, and MMP-9 were measured using the human 3-plex meso kit (Meso Scale Discovery). Data are representative of three independent experiments performed in duplicate (mean ± SE). *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 5.

Suppression of ADAM15 enhances TLR3- and TLR4-mediated proinflammatory cytokine and chemokine induction. (AP) U373-CD14 cells were pretreated with control or ADAM15 esiRNAs. After 24 h, cells were stimulated with either 20 μg/ml poly(I:C) (A–H) or 1 μg/ml LPS (I–P) for an additional 24 h. Thereafter, levels of IFN-β, IL-12p70, and TNF-α were measured in the cell-free supernatants using the human proinflammatory multiplex assay kit. Levels of RANTES and IFN-β were measured using the human single-plex assay kit (Meso Scale Discovery). MMP-1, MMP-3, and MMP-9 were measured using the human 3-plex meso kit (Meso Scale Discovery). Data are representative of three independent experiments performed in duplicate (mean ± SE). *p < 0.05, **p < 0.01, ***p < 0.001.

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Next, we sought to investigate the physiological role of ADAM15 in the modulation of virally induced cytokine and chemokine induction. To do this, we examined proinflammatory cytokine levels in the absence and presence of ADAM15 following infection of cells with RV16 and VSV. Whereas RV16 is known to be sensed by TLR3 (28), VSV is known to be sensed by TLR4 through a mechanism requiring TRAM and TRIF, but not MyD88 (29). It was found that suppression of ADAM15 significantly induced RV16- and VSV-mediated IFN-β, TNF-α, IL-6, and CCL5 mRNA levels compared with noninfected control cells (Fig. 6A–D). Interestingly, we found that, whereas suppression of ADAM15 expression increased RV16-mediated MMP-9 and MMP-10 mRNA levels, VSV-mediated MMP-9 and MMP-10 transcription were decreased when compared with noninfected control cells (Fig. 6E, 6F). Moreover, suppression of ADAM15 significantly increased RV16- and VSV-induced type I IFNs, IL-6, TNF-α, and RANTES when compared with noninfected cells (Fig. 6G–J). To support the hypothesis that ADAM15 is a negative regulator of TRIF-mediated TLR3 and TLR4 signaling, we performed comparative analysis of TLR signaling in ADAM15−/− MEFs and WT MEFs. Initially, TLR3, TLR4, and control, TLR2-dependent NF-κB and IFN-β reporter gene activation was investigated using ADAM15−/− MEFs and WT MEFs. Significantly enhanced TLR3- and TLR4-, but not TLR2-mediated NF-κB and IFN-β reporter gene activation was observed in ADAM15−/− MEFs when compared with WT cells (Fig. 7A, 7B).

FIGURE 6.

Suppression of ADAM15 enhances RV16- and VSV-mediated proinflammatory cytokine and chemokine induction. (AF) U373-CD14 cells were pretreated with control or ADAM15 esiRNAs. After 24 h, cells were infected with either RV16 or VSV with noninfected cells serving as a control. After 24 h, cells were collected and total RNA was isolated and used as template to measure levels of IFN-β, TNF-α, IL-6, RANTES, MMP-9, and MMP-10 mRNAs with GAPDH serving as a housekeeping gene. (GJ) U373-CD14 cells were pretreated with control or ADAM15 esiRNAs. After 24 h, cells were infected with either RV16 or VSV. After 48 h, cell-free supernatants were collected, and levels of type I IFN were measured using HEK293-Blue IFN-αβ. Levels of IL-6, TNF-α, and RANTES were measured by ELISA. Data are representative of three independent experiments performed in duplicate (mean ± SE). *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 6.

Suppression of ADAM15 enhances RV16- and VSV-mediated proinflammatory cytokine and chemokine induction. (AF) U373-CD14 cells were pretreated with control or ADAM15 esiRNAs. After 24 h, cells were infected with either RV16 or VSV with noninfected cells serving as a control. After 24 h, cells were collected and total RNA was isolated and used as template to measure levels of IFN-β, TNF-α, IL-6, RANTES, MMP-9, and MMP-10 mRNAs with GAPDH serving as a housekeeping gene. (GJ) U373-CD14 cells were pretreated with control or ADAM15 esiRNAs. After 24 h, cells were infected with either RV16 or VSV. After 48 h, cell-free supernatants were collected, and levels of type I IFN were measured using HEK293-Blue IFN-αβ. Levels of IL-6, TNF-α, and RANTES were measured by ELISA. Data are representative of three independent experiments performed in duplicate (mean ± SE). *p < 0.05, **p < 0.01, ***p < 0.001.

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FIGURE 7.

ADAM15 suppresses TLR-mediated reporter gene activity and VSV-mediated cytokine induction. (A and B) WT and ADAM15−/− MEFs were seeded into six-well plates. After 24 h, cells were transfected with vectors encoding either a luciferase reporter gene for NF-κB or IFN-β (500 ng). A total of 250 ng/well phRL-TK reporter gene was cotransfected simultaneously to normalize data for transfection efficiency. After 24 h, cells were stimulated with Pam2Cys (1 μg/ml), poly(I:C) (20 μg/ml), and LPS (1 μg/ml), as indicated, for 24 h. Thereafter, cell lysates were harvested and assessed for luciferase reporter gene activity using the dual luciferase system (Promega). (CF) WT and ADAM15−/− MEFs were seeded into six-well plates. After 24 h, cells were infected with VSV for 24 h. Thereafter, cells were collected and total RNA was isolated and used as template to measure levels of IFN-β, TNF-α, CCL5, and IFN-α mRNAs with GAPDH serving as a housekeeping gene. The data presented are representative of at least two independent experiments performed in duplicates (mean ± SE). *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 7.

ADAM15 suppresses TLR-mediated reporter gene activity and VSV-mediated cytokine induction. (A and B) WT and ADAM15−/− MEFs were seeded into six-well plates. After 24 h, cells were transfected with vectors encoding either a luciferase reporter gene for NF-κB or IFN-β (500 ng). A total of 250 ng/well phRL-TK reporter gene was cotransfected simultaneously to normalize data for transfection efficiency. After 24 h, cells were stimulated with Pam2Cys (1 μg/ml), poly(I:C) (20 μg/ml), and LPS (1 μg/ml), as indicated, for 24 h. Thereafter, cell lysates were harvested and assessed for luciferase reporter gene activity using the dual luciferase system (Promega). (CF) WT and ADAM15−/− MEFs were seeded into six-well plates. After 24 h, cells were infected with VSV for 24 h. Thereafter, cells were collected and total RNA was isolated and used as template to measure levels of IFN-β, TNF-α, CCL5, and IFN-α mRNAs with GAPDH serving as a housekeeping gene. The data presented are representative of at least two independent experiments performed in duplicates (mean ± SE). *p < 0.05, **p < 0.01, ***p < 0.001.

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Furthermore, WT and ADAM15−/− MEFs were infected with VSV for 24 h, followed by analysis of cytokine/chemokine mRNA levels. We demonstrate that, whereas infection of WT MEFs with VSV resulted in IFN-β, TNF-α, CCL5, and IFN-α induction, a significantly greater induction of these cytokines was evident in ADAM15−/− MEFs (Fig. 7C–F).

To explore the mechanism by which ADAM15 negatively regulates TRIF-mediated TLR3 and TLR4 signaling, we sought to investigate whether ADAM15, a known protease (30, 31), has the ability to mediate the degradation of TRIF and so lead to the curtailment of TRIF-dependent signaling. To this end, HEK293-TLR3 were transfected with TRIF in the absence and presence of ADAM15. Thereafter, cells were left untreated (control) or stimulated with poly(I:C), as indicated, in the absence of EDTA and EGTA, known to partially inhibit the protease activity of ADAM15 (21). Overexpression of ADAM15 facilitated the degradation of TRIF, but not MyD88 (Fig. 8A, 8B). Notably, coexpression of TRIF with catalytically active ADAM15 (16) led to the degradation of TRIF (Fig. 8A; compare TRIF expression in the absence and presence of ADAM15). It has been reported that some ADAMs may be cleaved and subsequently translocated to the nucleus (32, 33). Therefore, we investigated the subcellular distribution of ADAM15 following stimulation of cells with poly(I:C). To this end, ADAM15 was overexpressed in HEK293-TLR3. Thereafter, cells were stimulated with poly(I:C), and the subcellular localization of ADAM15 was investigated by confocal microscopy. To avoid potential artifacts due to high levels of overexpression, the amount of plasmid DNA transfected into the cells was maintained at low levels (200 ng/well; six-well plate). We found that ADAM15 was localized to both the plasma membrane and cytosol and that stimulation with poly(I:C) for 40 or 90 min resulted in a slight increase in the levels of ADAM15 in the cytosol, most likely reflecting its transportation from the plasma membrane to the nucleus (Supplemental Fig. 1). These data indicate that ADAM15 and TRIF may colocalize in the cytosol (32, 33). Furthermore, to support the hypothesis that ADAM15 mediates the proteolytic cleavage of TRIF, ADAM15 expression was suppressed in U373-CD14 cells, followed by stimulation of cells with poly(I:C). Increased levels of endogenous TRIF protein were detected in cells following the suppression of ADAM15 expression when compared with control cells and levels decreased upon stimulation of cells with poly(I:C) (Fig. 7C).

FIGURE 8.

ADAM15 mediates the degradation of TRIF. HEK293-TLR3 (A) and HEK293-TLR4 (B) cells were cotransfected with plasmids encoding TRIF or MyD88 and ADAM15 or empty vector (EV), as indicated. After 20 h, cells were stimulated with 20 μg/ml poly(I:C) or 1 μg/ml LPS, as indicated. Thereafter, cell lysates were harvested and immunoblot analysis was performed to assess TRIF and MyD88 expression levels using anti-HA and anti-myc, and β-actin served as a loading control. (C) U373-CD14 cells were transfected with ADAM15 and control esiRNAs. After 24 h, cells were stimulated with 20 μg/ml poly(I:C) for 24 h, as indicated. Thereafter, cell lysates were subjected to immunoblot analysis using a rabbit anti-TRIF polyclonal Ab (Exalpha), and β-actin served as a loading control. Results are representative of two independent experiments.

FIGURE 8.

ADAM15 mediates the degradation of TRIF. HEK293-TLR3 (A) and HEK293-TLR4 (B) cells were cotransfected with plasmids encoding TRIF or MyD88 and ADAM15 or empty vector (EV), as indicated. After 20 h, cells were stimulated with 20 μg/ml poly(I:C) or 1 μg/ml LPS, as indicated. Thereafter, cell lysates were harvested and immunoblot analysis was performed to assess TRIF and MyD88 expression levels using anti-HA and anti-myc, and β-actin served as a loading control. (C) U373-CD14 cells were transfected with ADAM15 and control esiRNAs. After 24 h, cells were stimulated with 20 μg/ml poly(I:C) for 24 h, as indicated. Thereafter, cell lysates were subjected to immunoblot analysis using a rabbit anti-TRIF polyclonal Ab (Exalpha), and β-actin served as a loading control. Results are representative of two independent experiments.

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To investigate whether ADAM15 and TRIF are involved in the coregulation of similar proteins, share common molecular targets, or are coinvolved in the progression of certain diseases, an interaction map for common regulators/targets, diseases, and cellular processes for TRIF and ADAM15 was generated using Pathway Studio Software (Ariadne). Integration of the data demonstrated that, whereas TRIF regulates IFN-γ expression, ADAM15 indirectly regulates IFN-γ expression (Fig. 9). Also, whereas TNF-α expression is directly regulated by both TRIF and ADAM15, MAPK1 is indirectly regulated by both of these molecules. Moreover, both TRIF and ADAM15 are involved in the regulation of proinflammatory cytokines and cellular processes, such as the inflammatory response, and diseases such as neoplasia and neutrophil infiltration. Interestingly, upregulation of ADAM15 expression has previously been reported in cells treated with proinflammatory cytokines and in tissues of inflammatory diseases (34). Overexpression of ADAM15 has also been shown to increase ERK1/2 activation in endothelial cells (35). Activation of TRIF leads to proinflammatory cytokine induction, which is dependent on NF-κB and MAPK activation (22). Moreover, it has been reported that ADAM15 mRNA and protein levels are increased in prostate cancer and during metastatic progression (36, 37). ADAM15 overexpression has also been reported to promote neutrophil transendothelial migration by a mechanism involving Src/ERK1/2 signaling (35). Interestingly, mice lacking TRIF fail to suppress implanted B16 tumor growth in response to poly(I:C) administration (38, 39). These data demonstrate that both ADAM15 and TRIF are involved in the coregulation of many different signaling events and support the hypothesis that ADAM15 modulates TRIF-dependent signaling.

FIGURE 9.

Cellular processes and diseases associated with TRIF and ADAM15. HEK293-TLR3 cells were cotransfected with HA-TRIF, V5-ADAM15, or empty vector. After 20 h, cells were stimulated with 20 μg/ml poly(I:C) for 60 min. Thereafter, immunoprecipitation of the TRIF immunocomplex was performed using an anti-HA mAb, as described in 2Materials and Methods. Following LC-MS, ADAM15 was identified as a TRIF interactor. Next, the interacting partners were analyzed using Pathway Studio software (Ariadne Genomics) for the cellular process network and common diseases shared between them. Gray dotted line indicates a regulatory role; dark gray solid line indicates direct regulation; and a light gray solid line indicates expression.

FIGURE 9.

Cellular processes and diseases associated with TRIF and ADAM15. HEK293-TLR3 cells were cotransfected with HA-TRIF, V5-ADAM15, or empty vector. After 20 h, cells were stimulated with 20 μg/ml poly(I:C) for 60 min. Thereafter, immunoprecipitation of the TRIF immunocomplex was performed using an anti-HA mAb, as described in 2Materials and Methods. Following LC-MS, ADAM15 was identified as a TRIF interactor. Next, the interacting partners were analyzed using Pathway Studio software (Ariadne Genomics) for the cellular process network and common diseases shared between them. Gray dotted line indicates a regulatory role; dark gray solid line indicates direct regulation; and a light gray solid line indicates expression.

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TRIF is the critical adaptor molecule that facilitates the activation of TLR3, TLR4, and TLR5 signaling pathways (4042). Interestingly, the TRIF signaling pathway is negatively regulated by a number of molecules; however, in most cases, they do not target TRIF specifically, but affect downstream components of the TRIF pathway such as TBK1 or IRFs (4). For example, Src homology 2 domain containing protein tyrosine phosphatase 2 has been shown to negatively regulate TLR3- and TLR4-mediated IFN-β production as well as proinflammatory cytokine production by binding to the kinase domain of TBK1, thereby preventing its activation (43). The fifth TLR adaptor, SARM, has also been shown to negatively regulate the TRIF, but not the MyD88, signaling pathway (44). Also, TRAM adaptor with GOLD domain (TAG), a splice variant of TRAM, has been shown to inhibit LPS-mediated IRF3 activation by displacing TRIF from TRAM (45).

Interestingly, virally encoded proteases have been shown to directly target components of the innate immune system toward the abolition of antiviral signaling. In fact, targeted proteolysis of adaptor molecules, including TRIF, serves to suppress key antiviral signaling pathways (46). For example, the hepatitis C virus serine protease NS3-4A facilitates the proteolysis of TRIF (47) and thus inhibits TLR3-mediated activation of NF-κB and IRF-3. Also, the coxsackievirus B3 virally encoded protein, 3Cpro, mediates TRIF cleavage and thereby inhibits TRIF-mediated type I IFN and apoptotic signaling. Recently, the 3CD protease-polymerase from hepatitis A virus was shown to disrupt TLR3-mediated activation of IRF3 and IFN-β promoter activation by targeting TRIF for degradation (48). Thus, it is clear that targeted degradation of TRIF, certainly by viruses, serves as a strategy to curtail antiviral immune signaling and as a more global mechanism to control inflammatory responses to pathogens.

Given the critical role played by TRIF in innate immune signaling and the strategic importance of TRIF as a molecular target in the context of viral evasion strategies, we opted to explore the pathways and molecules that are modulated by TRIF toward further deciphering its fundamental role in innate immunity. To this end, the TRIF interactome was characterized in response to TRIF-mediated TLR3 and TLR4 ligands. In this study, to our knowledge, we demonstrate for the first time that TRIF interacts with ADAM15 in a TLR3 and TLR4 ligand-dependent manner. We cannot preclude the possibility that ADAM15 may interact with TRIF indirectly through intermediate proteins. Notably, the cytoplasmic domain of ADAM15 contains proline-rich regions that facilitate its interaction with adaptor proteins such as GRB2 (49). Also, both ADAM15 and TRIF have been shown to interact with the p85 subunit of PI3K (13, 50). Whether auxiliary molecules are required to facilitate the interaction of TRIF with ADAM15 requires further investigation. Toward deciphering the functional role of ADAM15 in TRIF signaling, we found that overexpression of ADAM15 inhibited TRIF- mediated NF-κB and IFN-β reporter gene activity. Given that poly(I:C) may be sensed by the RIG-I–like receptor (RLR), MDA5 (8), we investigated whether ADAM15 modulated MDA5-mediated NF-κB activity. We show that ADAM15 did not affect MDA5-mediated NF-κB reporter gene activity. Furthermore, we show that ADAM15 serves to negatively regulate TLR3, TLR4, and TRIF-dependent, but not TLR9-dependent, activation of the NF-κB and IFN-β reporter genes. These findings demonstrate that the inhibitory activity of ADAM15 is dependent on the signaling pathway that is instigated. Also, suppression of ADAM15 enhanced LPS-induced activation of the delayed NF-κB response, mediated through TRIF. Furthermore, suppression of ADAM15 led to enhanced TLR3- and TLR4-mediated production of proinflammatory cytokines and chemokines. Several MMPs, including MMP-3 and MMP-9, contain NF-κB and AP2 transcription factor binding sites within their promoters, indicating that they may be induced during inflammatory episodes, and studies have shown that they may be modulated by ADAMs (23, 51). Moreover, poly(I:C) and LPS have been shown to induce MMP-1, MMP-3, MMP-10, and MMP-13 expression in lung epithelial cells and chondrocytes (6, 52, 53). Thus, we explored the ability of ADAM15 to modulate TLR ligand-mediated MMP induction. It was found that suppression of ADAM15 significantly enhanced poly(I:C)-induced MMP-1, MMP-3, MMP-9, and MMP-10 levels. Also, whereas LPS-mediated induction of MMP-1 and MMP-3 was enhanced upon suppression of ADAM15 expression, MMP-9 levels were inhibited. These data indicate that although ADAM15 impairs TLR3-mediated MMP induction, it is required for LPS-mediated MMP-9 induction. The differential effects of ADAM15 in the context of TLR3 and TLR4 signaling are supported by our data showing that ADAM15 drives MyD88-mediated NF-κB and IFN-β reporter gene activity and that impairment of LPS-mediated early NF-κB activation is evident upon suppression of ADAM15 expression. It has previously been reported that LPS induces MMP-9 expression in macrophages through a reactive oxygen species–p38 kinase-dependent pathway (54). Also, MMP-9 expression appears to be regulated by a number of different signaling pathways in different cell types, for example, protein kinases, MAPKs, and transcription factors such as NF-κB, and AP-1 (51). Thus, the differences in the comparative role played by ADAM15 in TLR3 and TLR4 signaling may reflect differences in the signaling pathways that are instigated, for example, MyD88-dependent versus MyD88-independent signaling. Interestingly, we found that suppression of ADAM15 also enhanced RV16- and VSV-induced cytokine/chemokine transcription and secretion when compared with control cells. We also found that levels of cytokine and chemokine mRNA and protein were enhanced following infection of ADAM15−/− MEFs with VSV when compared with WT cells. As murine ADAM15 lacks the RGD domain, these data suggest that the domain is dispensable in the context of ADAM15 as a regulator of antiviral signaling. Given that ADAM15 exhibits protease activity, we explored the possibility that ADAM15 may facilitate the proteolytic degradation of TRIF, a known target for proteolysis. Our data demonstrate that TRIF, but not MyD88, is subject to proteolytic degradation by ADAM15. We propose that ligand-mediated activation of TRIF signaling enhances the association of TRIF with ADAM15, and, at later time points, TRIF is degraded through an ADAM15-dependent mechanism leading to the negative regulation of TLR3- and TLR4-mediated proinflammatory cytokine production. Regarding the consensus sequence that is recognized by ADAM15, the target peptides of ADAM15 on binding substrates currently remain unknown (55). However, it is known that the cytoplasmic domain of ADAM15 contains proline-rich regions that facilitate its interaction with adaptor proteins such as GRB2 (49). Also, both ADAM15 and TRIF have been shown to interact with the p85 subunit of PI3K (13, 50). Whether auxiliary molecules are required to facilitate the interaction of TRIF with ADAM15 requires further investigation. In conclusion, ADAM15 facilitates the proteolytic cleavage of TRIF and thus impairs TLR3- and TLR4-dependent TRIF signaling events. We cannot preclude the possibility that ADAM15 may activate other MMPs that may facilitate the degradation of TRIF. Regarding the exact mechanism of TRIF degradation, it has recently been demonstrated that, upon TLR3 activation, TRIF downregulation occurs through a lysosomal- rather than a proteasomal- or caspase-mediated process (56). More recently, it has been shown that TRIM38 targets TRIF for degradation via a ubiquitin/proteosome pathway and subsequently leads to the negative regulation of TLR3-mediated IFN-β production (57). Thus, further studies are required to fully decipher the mechanism(s) that facilitates the degradation of TRIF.

Despite the potentially important role that ADAM15 plays in inflammation, cancer, and atherosclerosis, little is known about the regulation of ADAM15 activity (14, 31). Regarding ADAM15 expression patterns, it is found to be expressed only in synoviocyte lining layer in normal synovial tissue, and its expression is strongly increased in the highly hyperplastic lining of individuals with rheumatoid arthritis (14). It has also been speculated that, in rheumatoid arthritis, ADAM15 may degrade the extracellular matrix directly through its metalloprotease activity or indirectly through proteolytic activation of MMPs (14). Upregulation of ADAM15 expression during intestinal bowel disease has also been reported, suggesting its involvement in intestinal inflammation (14). Whereas most of the published data suggest that ADAM15 is a mediator of inflammation, it has also been proposed that ADAM15 may have both a homeostatic and a pathological role depending on the cell and context in which it is expressed (14). For example, ADAM15-deficient mice present with accelerated development of osteoarthritic lesions compared with WT mice, suggesting that ADAM15 has a protective role in the maintenance of joint integrity (58). In this study, we propose that, under normal physiological conditions, ADAM15 serves to curtail TRIF-mediated signaling, thus curbing the production of associated inflammatory mediators. Notably, several splice variants of ADAM15 have been described and are purported to be differentially expressed toward mediating functionally disparate effects (12, 13). In this study, we have shown that WT full-length ADAM15A binds to TRIF. However, the ability of the other ADAM15 splice variants to bind TRIF and to modulate TLR signaling remains to be investigated. In conclusion, to our knowledge, this study shows for the first time that ADAM15 serves to modulate TLR3 and TLR4 signaling events. More specifically, ADAM15 inhibits antiviral immune responses that are mediated through the TLR adaptor, TRIF, and so ADAM15 functions as a negative regulator of TLR3 and TLR4 signaling by a mechanism involving TRIF degradation.

This work was supported by the Science Foundation of Ireland.

The online version of this article contains supplemental material.

Abbreviations used in this article:

ADAM

a disintegrin and metalloprotease

esiRNA

endoribonuclease-prepared siRNA

HA

hemagglutinin

IRF

IFN regulatory factor

LC-MS

liquid chromatography mass spectrometry

MEF

murine embryonic fibroblast

MDA5

melanoma differentiation–associated protein 5

MMP

matrix metalloproteinase

poly(I:C)

polyriboinosinic polyribocytidylic acid

PRD

positive regulatory domain

RGD

arginine-glycine-aspartic acid

RLR

RIG-I–like receptor

RV

rhinovirus

siRNA

small interfering RNA

TBK1

TANK-binding kinase 1

TRAM

Toll/IL-1R domain-containing adaptor inducing IFN–related adaptor molecule

TRIF

Toll/IL-1R domain-containing adaptor inducing IFN

VSV

vesicular stomatitis virus

WT

wild-type.

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The authors have no financial conflicts of interest.