Published online before print June 4, 2007
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
,1
* Department of Public Health and Cell Biology, University of Rome "Tor Vergata," Rome, Italy;
Fondazione Santa Lucia IRCCS, Laboratory of Neuroembryology, Rome, Italy;
Research Center, Pediatric Hospital "Bambino Gesù," Rome, Italy; and
R&D Sigma-Tau S.p.A., Pomezia (RM), Italy
1 Correspondence: Department of Public Health and Cell Biology, University of Rome "Tor Vergata," Via Montpellier, 1, 00133, Rome, Italy. E-mail: claudio.sette{at}uniroma2.it
ABSTRACT
MyD88 is an adaptor protein, which plays an essential role in the intracellular signaling elicited by IL-1R and several TLRs. Central to its function is the ability of its Toll/IL-1R translation initiation region (TIR) domain to heterodimerize with the receptor and to homodimerize with another MyD88 molecule to favor the recruitment of downstream signaling molecules such as the serine/threonine kinases IL-1R-associated kinase 1 (IRAK1) and IRAK4. Herein, we have synthesized and tested the activity of a synthetic peptido-mimetic compound (ST2825) modeled after the structure of a heptapeptide in the BB-loop of the MyD88-TIR domain, which interferes with MyD88 signaling. ST2825 inhibited MyD88 dimerization in coimmunoprecipitation experiments. This effect was specific for homodimerization of the TIR domains and did not affect homodimerization of the death domains. Moreover, ST2825 interfered with recruitment of IRAK1 and IRAK4 by MyD88, causing inhibition of IL-1ß-mediated activation of NF-
B transcriptional activity. After oral administration, ST2825 dose-dependently inhibited IL-1ß-induced production of IL-6 in treated mice. Finally, we observed that ST2825 suppressed B cell proliferation and differentiation into plasma cells in response to CpG-induced activation of TLR9, a receptor that requires MyD88 for intracellular signaling. Our results indicate that ST2825 blocks IL-1R/TLR signaling by interfering with MyD88 homodimerization and suggest that it may have therapeutic potential in treatment of chronic inflammatory diseases.
Key Words: TLR • IL-1 receptor • inflammation • autoimmune diseases • innate immunity
INTRODUCTION
IL-1 was described originally as a cytokine, which acts as an endogenous pyrogen with potent, proinflammatory activity. It plays a crucial role in a wide range of host responses to infection and inflammation [1
] as well as in the neuronal injury occurring in acute and chronic neurodegenerative disorders [2
]. Two separate genes encode for the IL-1
and IL-1ß isoforms, which share a high sequence homology [3
] and induce similar effects by binding to the same receptor, i.e., IL-1R Type I (IL-1RI). Upon ligand binding, the IL-1RI forms a signaling heterodimer with the IL-1R accessory protein (IL-1RAcP) [4
]. IL-1RI and IL-1RAcP belong to the receptor superfamily referred to as the TLR/IL-1R superfamily. In addition to IL-1RI and IL-1RAcP, mammals express at least 11 members (TLR1–11) of this family [5
, 6
], which are specialized in the recognition of microbial products, such as the lipid A moiety of Gram-negative bacterial LPS, a ligand of TLR4 [7
], or nonmethylated CpG oligodeoxyribonucleotides, deriving from the bacterial DNA, which binds TLR9 [8
]. The highly conserved, cytoplasmic region in the TLR/IL-1R class of receptors, i.e., the Toll/IL-1R translation initiation region (TIR) domain, plays an essential role in signaling downstream of these receptors by recruiting several effectors and favoring their functional interaction. Central to this signaling pathway is the role of adaptor molecules, which bridge the receptors to the protein kinases required to propagate the signal inside the cells [9
]. The IL-1R and all of the TLRs with the sole exception of TLR3 use the adaptor protein MyD88 to initiate their signaling pathway [10
, 11
]. MyD88 is a 33-kDa protein, which contains an N-terminal death domain (DD) and a C-terminal TIR domain separated by a short linker region [12
]. MyD88 is recruited to the receptor complexes as a dimer, which is stabilized by homophylic interactions occurring between the DD and TIR domains [13
]. Once recruited by the receptor TIR domain, MyD88 associates with the IL-1R-associated kinase 4 (IRAK4) through homophylic interaction between the respective DDs. This event leads to recruitment of another kinase, IRAK1, to the complex and to its phosphorylation/activation by IRAK4 [14
]. IRAK1 and IRAK4 then dissociate from the complex, interact with TNF receptor-associated factor 6, another adaptor molecule, and cause the activation of TGF-ß-activated kinase 1. This complex signaling pathway is required to induce the downstream activation of the IB kinase complex and the release of NF-B and for activation of MAPKs p38 and JNK [4
, 15
]. In addition to MyD88, other adaptor proteins containing a TIR domain have been identified [16
]. MyD88 adaptor-like or TIR domain-containing adaptor protein functions downstream of TLR2 and TLR4 [17
, 18
]. TIR-related adaptor protein inducing IFN (Trif) is recruited by TLR3 and TLR4 and is responsible for activation of IFN-induced genes [19
, 20
]. Trif-related adaptor molecule (Tram) acts exclusively in the pathway downstream of TLR4 activation and appears to be required to recruit Trif to the receptor [21
, 22
].
MyD88-deficient mice are defective in the production of IL-6 and TNF in response to stimulation with IL-1 or other microbial components recognized by TLR2, TLR4, TLR5, and TLR9 [11 , 23 ]. Conversely, it has been demonstrated that the response of macrophages to LPS stimulation of TLR4 is only partly dependent on MyD88, whereas IFN-inducible genes such as glucocorticoid attenuated-response gene 16 or immune-responsive gene 1 are still up-regulated, even in the absence of a functional MyD88 [24 ]. Thus, this branch of signaling seems to depend on the other adaptor molecules Trif and Tram [16 ]. Loss of MyD88 expression and/or function has anti-inflammatory effects in several pathological situations such as early artherosclerosis [25 ], arthritis [26 ], and inflammatory diseases of the CNS such as multiple sclerosis [27 ]. For this reason, the development of molecules, which interfere specifically with MyD88-dependent signaling, could be useful in the treatment of several immunological disorders [28 ]. As homodimerization of MyD88 and its interaction with the receptors require the TIR domain, this region is targeted for studies aimed at developing drugs, which can block MyD88 activation [29 , 30 ]. Our laboratory previously identified a heptapeptide, which mimics the BB-loop of the conserved TIR domain of MyD88. This peptide interferes strongly with dimerization of MyD88 and activation of the IL-1 signaling pathway in live cells [31 ]. It is interesting that site-directed mutagenesis of three charged residues within this heptapeptide also caused loss of homodimerization, interaction with the receptor, and IL-1ß signaling [32 ], thus confirming our results through a different approach. Herein, we report the identification and characterization of a novel compound (ST2825), which mimics this MyD88 BB-loop heptapeptide and has biological activity toward this adaptor protein. Our studies suggest that this newly synthesized compound has potential as an anti-inflammatory drug.
MATERIALS AND METHODS
Synthesis of ST2825
The peptidomimetic ST2825 was synthesized as described previously in Patent No. WO 200606709 [33
].
Plasmids
Hemagglutinin (HA)-tagged DD of MyD88-expressing plasmid was a kind gift from Dr. Marta Muzio ("Mario Negri" Institute, Milan, Italy). Expression vectors for Flag-tagged MyD88 or Myc-tagged MyD88 were constructed by inserting PCR-generated cDNA fragments in the mammalian expression vectors p3X-Flag or pCDNA3-N2-Myc, respectively. For the NF-B reporter assays, the NF-B luciferase and Renilla luciferase constructs were used according to the manufacturer's instructions (Promega, Madison, WI, USA).
Cell culture and transfections
The human embryonic kidney (HEK)293T and HeLa cell lines were cultured in DMEM, supplemented with 10% FBS (Invitrogen, Carlsbad, CA, USA), and grown in a 37°C humidified atmosphere of 5% CO2. For coimmunoprecipitation of Flag-MyD88/Myc-MyD88, HEK293T cells were cultured in 10 cm-diameter dishes and transfected by the calcium-phosphate method with 4–5 µg of the appropriate plasmids. The peptidomimetic compound ST2825 was added to the medium 7 h after transfection. To detect Flag-MyD88 associated with HA-MyD88Death, Myc-IRAK1 kinase death (KD), Myc-MyD88TIR, or Myc-IRAK4KD, HEK293T cells were cultured in 6 cm-diameter dishes and transfected by Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The peptidomimetic compound ST2825 was added to the medium 6 h after transfection.
For mitogenic stimulation, Hela cells were starved overnight in medium without serum, pretreated for 15 min with or without various concentrations of ST2825 prior to stimulation in the presence or absence of 100 nM epidermal growth factor (EGF; PeproTech, Rocky Hill, NJ, USA) for various times. At the end of stimulation, cells were washed with 2 ml ice-cold PBS and lysed in 80 µl buffer [50 mM HEPES, pH 7.4, 15 mM MgCl2, 150 mM NaCl, 15 mM EGTA, 10% glycerol, 1% Triton X-100, protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA), 20 mM ß-glycerophosphate, 2 mM DTT, 1 mM Na3VO4]. Cells were pelleted by centrifugation at 10,000 g for 10 min, and the resulting supernatants were diluted in SDS sample buffer for Western blot analysis of levels of phospho (p)ERK1/2.
Cell viability assay
Cell viability was assessed by 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) staining according to the manufacturer's instructions (Roche Applied Science, Indianapolis, IN, USA), measuring the reaction product with a spectrophotometer (absorbance at 450 nm). Briefly, HeLa cells were seeded at 105 cells/ml in a 96-well tissue-culture plate. After incubating overnight, the medium was discarded, and the cells were added with tissue culture medium, 10% FBS, containing ST2825 (previously dissolved in DMSO) at concentrations ranging from 0.1 to 10 µM and DMSO at 0.1% final concentration. The cells were incubated for 6 and 18 h and then added with the yellow XTT (0.3 mg/ml) for further 2 h of incubation. At the end of the incubation periods, reactions were quantified by using a Sirio S Seac microplate reader (Radim S.p.A., Pomezia, Italy).
Coimmunoprecipitation assay
HEK293T cells were collected 20 h after transfection, washed in ice-cold PBS, and lysed in buffer containing 50 mM HEPES, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 20 mM ß-glycerophosphate, 2 mM DTT, 1 mM Na3VO4, and protease inhibitors. After 10 min on ice, cell lysates were centrifuged at 10,000 g for 10 min at 4°C, and cytosolic fractions were collected for immunoprecipitation. Cell extracts (1 mg total proteins) were precleared by incubation for 1 h with protein G-Sepharose beads (Sigma-Aldrich) under constant shaking at 4°C. After preclearing, cell extracts were incubated with 2 µg mouse anti-Flag M2 (Sigma-Aldrich) for 1 h under constant shaking at 4°C. Concurrently, protein G-Sepharose beads were presaturated with 0.1% BSA (Sigma-Aldrich) in PBS in the same conditions for 1 h. After incubation, the beads were washed twice with lysis buffer and then incubated with cell extracts containing the antibody for 1 h further at 4°C under constant shaking. Sepharose bead-bound immunocomplexes were washed three times in lysis buffer and eluted in SDS-PAGE sample buffer for Western blot analysis.
Western blot analysis
Cell extracts or immunoprecipitated proteins were diluted in SDS sample buffer as described above and boiled for 5 min. Proteins were separated on A 8–12% SDS-PAGE gel and transferred to polyvinylidene fluoride Immobilon-P membranes (Millipore, Bedford, MA, USA) using a semidry blotting apparatus (Bio-Rad, Hercules, CA, USA). Membranes were saturated with 5% nonfat dry milk in PBS containing 0.1% Tween 20 for 1 h at room temperature and incubated overnight at 4°C with the following primary antibody (1:1000 dilution): mouse anti-HA (Berkeley Antibody Company, Babco, Richmond, CA, USA), mouse anti-Myc (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-Flag M2 (Sigma-Aldrich), and rabbit anti-pERK1/2 (Cell Signaling Technology, Beverly, MA, USA). Secondary anti-mouse IgGs conjugated to HRP (Amersham Biosciences, Little Chalfont, UK) were incubated with the membranes for 1 h at room temperature at a 1:10,000 dilution in PBS containing 0.1% Tween 20. Immunostained bands were detected by the chemiluminescence method (Santa Cruz Biotechnology).
NF-B reporter assay
HeLa cells (1x105) were cultured in 12-well plates and transfected with 0.5 µg of a NF-B-dependent luciferase reporter gene and R. luciferase reporter gene (4 ng) as an internal control using the FuGENE 6 reagent (Roche Diagnostic, Nutley, NJ, USA) according to the manufacturer's instructions. Twenty-four hours after transfection, cells were placed in medium without serum for 2 h and pretreated with or without various concentrations of ST2825 for 15 min prior to stimulation in the presence or absence of IL-1ß (30 ng/ml; R&D Systems, Minneapolis, MN, USA) or polyinosinic:polycytidylic [poly(I:C); 100 µg/ml; Invivogen, San Diego, CA, USA] for an additional 30 min. After rinsing with PBS, cells were incubated with medium without serum for 3 h, then harvested, lysed, and analyzed using a biocounter luminometer as described elsewhere [31
]. Data were normalized for transfection efficiency, dividing firefly luciferase activity by that of R. luciferase. Data are expressed as mean-fold induction ± SD from a minimum of three separate experiments.
In vivo effect of ST2825 on IL-1ß response
Mice (female C57Bl/6 purchased from Harlan, Indianapolis, IN, USA) were divided into experimental groups of 15 mice. They were injected i.p. with saline (control animals) or recombinant murine IL-1ß (20 µg/Kg). A time-course analysis of IL-6 production established that the peak of cytokine was reached 2 h after IL-1ß injection. ST2825, administered orally as 0.5% suspension in carboxymethylcellulose (CMC) or CMC alone, was supplied to the experimental mice groups. Two hours after IL-1ß injection, the animals were killed, and sera were collected to assay IL-6 levels. Mice, which were treated orally with 100 and 200 mg/Kg ST2825, showed lower levels of IL-6 versus CMC-treated mice (P<0.01; data were evaluated with one-way ANOVA followed by Dunnett's t-test).
PBMC proliferation assay
Venous blood was obtained from healthy donors from the Ospedale Pediatrico Bambino Gesù (Rome, Italy) after informed consensus. Human PBMC were isolated by Ficoll-PaqueTM Plus (Amersham Pharmacia Biotech, Little Chalfont, UK) density-gradient centrifugation. The lymphocyte-enriched buffy coat was removed and washed in PBS. PBMC were labeled with 5-chloromethylfluorescein diacetate (CMFDA) at the final concentration of 0.1 µg/ml (CellTracker CMFDA, Molecular Probes, Eugene, OR, USA), seeded at the concentration of 5 x 105 cells/well in 96-well plates (Becton Dickinson, San Jose, CA, USA), and cultured with 200 µl complete RPMI 1640 (Invivogen), supplemented with 10% FCS (Hyclone Laboratories, Logan, UT, USA). ST2825 was added to the medium at the indicated concentrations in the presence or absence of human CpG oligodeoxynucleotides (ODN 2006, Hycult Biotechnology, The Netherlands), used at the optimal concentration of 2.5 µg/ml. Cell proliferation was measured on Day 7 by FACSCalibur flow cytometer (Becton Dickinson) interfaced to a Macintosh CellQuest computer program. Thirty thousand events were analyzed whenever possible. To identify B cell subsets, antibodies to CD19, CD27, and CD38 (Becton Dickinson) were used.
ELISA immunoassay
Secreted Igs were detected at Day +7 by ELISA assay. Briefly, 96-well plates (Corning Inc., Corning, NY, USA) were coated overnight with purified goat anti-human IgA + IgG + IgM (Jackson ImmunoResearch, West Grove, PA, USA). After washing with PBS containing 0.05% Tween and blocking with PBS containing 1% gelatin (1 h, room temperature), plates were incubated for 1 h at 37°C with the supernatants of the cultured cells. After washing, plates were incubated for 1 h with peroxidase-conjugated fragment goat anti-human IgA,IgG, and IgM antibodies (Jackson ImmunoResearch). O-Phenylendiamine tablets (Sigma-Aldrich) were used as chromogenic substrate. OD was measured at 405 nm, and concentrations were calculated by interpolation from the standard curve.
RESULTS
ST2825 interferes with MyD88 homodimerization in a TIR-dependent manner
We reported previously that a peptide within the BB-loop region of the TIR domain of MyD88 (196–202) inhibits MyD88 homodimerization and downstream activation of NF-B in response to IL-1 stimulation [31
]. On the basis of the structure of this heptapeptide, a series of synthetic molecules was produced and screened for biological activity toward MyD88 signaling [33
]. Among the molecules initially screened, ST2825 (Fig. 1A
) appeared to possess the ability to interfere with IL-1ß-induced activation of NF-B (Fig. 1C)
. By contrast, this molecule did not interfere with activation of NF-B induced by poly(I:C), a ligand that stimulates the MyD88-independent TLR3 (Fig. 1D)
, nor with EGF-mediated activation of the MAPKS ERK1/2 in HeLa cells (Fig. 1E)
. As shown in Figure 2
, incubation of HeLa cells with ST2825 for 6 or 18 h did not strongly affect cell viability (13% decrease at 6 h and 16% at 18 h for the maximal dose used).
 View larger version (36K): [in a new window] |
Figure 1. ST2825 specifically inhibits IL-1 induction of NF-B-regulated reporter gene in HeLa cells. (A) Schematic representation of the structure of ST2825. (B) Superimposition of the structure of ST2825 and the heptapeptide RDLVPGT in the BB-loop of MyD88. (C and D) NF-B activity luciferase reporter assay. HeLa cells were cotransfected with a NF-B-regulated firefly reporter and phRL-TK (constitutively expressed R. luciferase). Cells were allowed to recover overnight, starved in medium without serum for 2 h, and pretreated with or without various concentration of ST2825 for 15 min prior to stimulation in the presence or absence of IL-1 ß (30 ng/ml) or poly(I:C) (100 µg/ml) for 30 min. After two washings with PBS, the cells were incubated with medium without serum for a further 3 h. Cell extracts were prepared and assayed for firefly and R. luciferase (for normalizing transfection efficiency). Data are presented as normalized firefly luciferase activity. (E) Hela cells were pretreated for 15 min with or without various concentrations of ST2825 prior to stimulation in the presence or absence of 100 nM EGF for the indicated time. Whole cell extracts were prepared and analyzed by Western blot for the levels of phosphorylated ERK1/2.
|
 View larger version (18K): [in a new window] |
Figure 2. Effects of ST2825 on HeLa cell viability, which was assessed by XTT as described in Materials and Methods in HeLa cells treated with DMSO of increasing doses of ST2825 for 6 h (A) or 18 h (B). Absorbance at 450 nm was quantified as a measure of cell viability using a microplate reader. Data are represented as mean ± SD of three separate experiments. Ctrl, Control.
|
40% inhibition of dimerization at 5 µM ST2825 and 80% inhibition at 10 µM ST2825 (Fig. 3A
, Lanes 3 and 4). Figure 3B
shows the quantitative data of three experiments.
 View larger version (20K): [in a new window] |
Figure 3. ST2825 interferes with MyD88 dimerization. (A) HEK293T cells were transfected with Myc-MyD88 alone (Lane 1) or in combination with Flag-MyD88 (Lanes 2–4). Seven hours after transfection, cells were incubated for 13 h with DMSO (Lanes 1 and 2) or with 5 (Lane 3) or 10 µM (Lane 4) ST2825. At the end of incubation, cells were collected, and dimerization of MyD88 was assessed by coimmunoprecipitation. Cell extracts were immunoprecipitated (I.P.) with anti-Flag antibody, and immunoprecipitated proteins were analyzed in Western blot with the anti-Flag antibody or the anti-Myc antibody to reveal the association. The effect of ST2825 on MyD88 dimerization is dose-dependent. (B) Densitometric analysis of the results shown in A is depicted.
|
 View larger version (36K): [in a new window] |
Figure 4. ST2825 does not interfere with MyD88 DD dimerization but inhibits MyD88 TIR domain homodimerization. HEK293T cells were transfecteded with HA-DD or Myc-TIR alone (A and B, Lanes 1) or in combination with Flag-MyD88 (A and B, Lanes 2–4). Six hours after transfection, cells were incubated for 13 h with DMSO (A and B, Lanes 1 and 2) or with 5 (A and B, Lanes 3) or 10 µM (A and B, Lanes 4) ST2825. Dimerization of MyD88 DDs was evaluated by coimmunoprecipitation. Cell extracts were immunoprecipitated with anti-Flag antibody, and immunoprecipitated proteins were analyzed by Western blotting with anti-Flag, anti-Myc, or anti-HA antibodies to reveal the dimerization.
|
 View larger version (39K): [in a new window] |
Figure 5. ST2825 inhibits recruitment of IRAK1KD and IRAK4KD by MyD88. HEK293T cells were transfected with Myc-IRAK1KD or Myc-IRAK4KD alone (A and B, Lanes 1) or in combination with Flag-MyD88 (A and B, Lanes 2 and 3). Six hours after transfection, cells were incubated for 13 h with DMSO (A and B, Lanes 1 and 2) or with 10 µM ST2825 (A and B, Lanes 3). Cell extracts were immunoprecipitated with anti-Flag antibody, and immunoprecipitated proteins were analyzed by Western blotting with the anti-Flag antibody or the anti-Myc antibody to reveal the interaction. ST2825 interferes strongly with recruitment of IRAK1 and IRAK4 by MyD88.
|
 View larger version (23K): [in a new window] |
Figure 6. ST2825 attenuates IL-1ß-stimulated IL-6 production in vivo. Two groups of control mice and three groups of treated mice were injected with 20 µg/Kg IL-1ß. Vehicle CMC (control groups) or different doses of ST2825 were administered orally before IL-1ß injection. Two hours after injection, mice were killed, and IL-6 production was measured by ELISA in the collected sera. Statistical analyses of the data were performed using the one-way ANOVA followed by Dunnett's t-test.
|
 View larger version (31K): [in a new window] |
Figure 7. ST2825 blocks the proliferation and differentiation of human B cells activated through TLR9. (A) Representative experiment of proliferation assay with CMFDA. PBMC were labeled with CMFDA and cultured for 7 days under the indicated conditions. Staining with anti-CD19 was performed to identify B cells and with anti-CD27 to identify B memory (me; lower-squared regions) and plasma cells (PC; upper-squared regions). Numbers indicate the percentage of B memory and plasma cells in the B cell population (B cells 100%). (B) The histograms show the absolute number of B cells in each well after 5 days of culture with ST2825. PBMC were cultured with 16, 8, and 4 µM ST2825 in the absence or presence of CpG. ST2825 blocks the increase of B cell number induced by CpG stimulation without affecting cell viability, as demonstrated by the equal number of B cells in the wells containing untreated B cells or B cells treated with 8 µM ST2825 (the effective dose used in all experiments). (C) The panel shows the frequency of CD27bright CD38pos plasma cells in B cells from healthy donors (n=3; indicated by different symbols in the panel) cultured for 7 days with CpG alone or in combination with increasing concentrations of ST2825 (1, 4, 8, 16 µM). Black lines represent the mean of the values obtained from the three different donors. Dots representing identical values overlap. (D) ELISA immunoassay of the supernatants relative to the cells cultured as described in C was recovered to measure antibody secretion. Histograms show the IgM, IgA, and IgG antibody concentration for each different culture condition. Results represent the mean ± SD of the values in the three donors.
|
DISCUSSION
MyD88 is an adaptor protein essential for the intracellular signaling in response to IL-1ß and TLR9 stimulation. Although activation of these receptors is required for an efficient defense response to pathogenic infection, their excessive signaling can lead to clinical syndromes [38 , 39 ]. Hence, limiting MyD88-mediated signaling might be beneficial in the treatment of pathological, inflammatory situations. Herein, we describe a novel, synthetic compound (ST2825), which interferes with MyD88 signaling in vitro and in vivo. Using coimmunoprecipitation experiments, luciferase reporter assays, FACS analysis, and ELISA, we demonstrate that ST2825 has the potential to suppress an inflammatory response to IL-1R and TLR9 stimulation, suggesting that the approach used in our work could be useful to develop compounds that exert anti-inflammatory actions [40 ].
Proper and adequate TLR/IL-1R signaling is essential for microbial recognition, inflammation, microbial clearance, and cell death [41 ]. However, hyperactivation of TLR/IL-1R signaling may cause septic shock and autoimmune disorders, and an ineffective or inappropriate response might favor diseases such as cancer and allergy [42 ]. Recent studies have shown how these signaling pathways are tightly regulated in inflammatory cells. Indeed, a panoply of antagonistic factors exists in inflammatory cells to ensure the appropriate modulation of TLR/IL-1R signaling [43 , 44 ]. For instance, ST2 [45 ], RP105 [46 ], and SIGIRR [47 ] are membrane-bound orphan receptors that function as negative regulators for TIR-mediated signaling. Many physiological antagonists of TLR/IL-1R signaling act at the intracellular level, such as IRAKM [48 ], a splice variant of IRAK2 [49 ], MyD88s, a splice variant of MyD88 [50 ], PI-3K [51 ], suppressor of cytokine signaling 3 [52 ], A20 [53 ], and Triad3A, a member of the Triad3 family of RING finger E3 ubiquitin ligases [54 ]. The abundance of endogenous, negative regulators highlights the necessity to control proper activation of this pathway in inflammatory cells. Nevertheless, uncontrolled activation still occurs in several pathological situations, indicating the requirement for synthetic, therapeutic inhibitors.
The remarkable progress in the elucidation of TLR signaling has drawn a keen interest toward TLR-targeted therapeutics from pharmaceutical companies [55 , 56 ]. Indeed, agonists and antagonists of IL-1R/TLR signaling can find therapeutic application depending on the clinical setting. Activators may have adjuvant roles, thus improving vaccine efficacy and reinforcing the immune response to cancer, and inhibitors may help to dampen inflammation in disorders such as allergy, atherosclerosis, and autoimmune diseases [57 ].
MyD88 is a suitable target for drugs aimed at inhibiting exogenous and endogenous activation of TLR/IL-1R signaling [16 ]. Rebek Jr. and colleagues [29 ] first synthesized and proved the in vivo efficacy of a low molecular weight MyD88 inhibitor by modeling a tripeptide sequence of the BB-loop of the TIR domain and subsequently, described bifunctional TIR mimetic compounds capable of disrupting the interaction of MyD88 with the IL-1R/IL-1RAcP complex [30 ]. In a previous investigation, our group showed that self-association of MyD88 could be interfered specifically by a heptapeptide targeting the BB-loop [31 ]. The synthetic compound ST2825 was modeled after this sequence (RDVLPGT) of MyD88. Herein, by using several approaches, we show that ST2825 interferes efficiently with MyD88 homodimerization and with IL-1ß- and CpG-driven signaling in distinct experimental settings.
The target of ST2825 is likely the interface between the BB-loops of MyD88 TIR domains [31 ]. As MyD88 dimerizes through homophylic interactions between the DD and the TIR domains, it was important to determine whether ST2825 required the TIR domain to exert its effect. The dimerization assays with the isolated TIR or DD domain and full-length MyD88 presented here demonstrate that ST2825 interferes only with the association between full-length MyD88 and the TIR domain, without affecting dimerization with the DD domain of the protein. This result indicates that ST2825 recognizes the target after which it was modeled.
Using the coimmunoprecipitation approach, we also showed that ST2825 prevents the recruitment of IRAK1 and IRAK4 by MyD88. As ST2825 has no effect on dimerization of MyD88 DD domains, these results suggest that MyD88 homodimerization is a prerequisite for the recruitment of IRAKs and that ST2825 interferes with these events by preventing TIR domain homophylic interactions between MyD88 molecules. The addition of ST2825 attenuates the activation of NF-B in HeLa cells stimulated with IL-1ß. It is more important that orally administered ST2825 attenuated the production of IL-6 induced by IL-1ß injection in mice. This result suggests that ST2825 is also effective in an in vivo model of acute inflammation. However, given the moderate effect on IL-6 production, it remains to be evaluated whether ST2825 may confer clinical advantages.
It is remarkable that we found that ST2825 also inhibited signaling effectively from the TLR9 ligand CpG, which operates in the endosomal compartment. This receptor depends strictly on MyD88 for signal transduction [2 ]. CpG-containing DNAs are internalized rapidly by immune cells and then interact with TLR9 present in endocytic vesicles [58 ]. TLR9 activation triggers a MyD88-dependent response in B cells, which undergo proliferation, class switching, and differentiation into antibody-secreting plasma cells [58 ]. In B cells, this activation is enhanced by simultaneous signals delivered through the antigen receptor. It has been shown that coactivation of naive B cells by TLR9 and the B cell antigen receptor drives their differentiation to plasma cells, whereas TLR9 alone is sufficient for memory B cell activation and differentiation into plasma cells [36 ]. In our experimental setting, we observed that CpG induced extensive proliferation of memory B cells, differentiation into plasma cells, and secretion of antibodies of all isotypes in the culture supernatants. It is striking that ST2825 suppressed all these events. The effect was dose-dependent, within the same range of the concentrations required to interfere with MyD88 homodimerization and downstream events, and did not produce toxic effects on cell survival. As TLR9 antagonists have already demonstrated their beneficial effects in murine models of systemic lupus erythematosus and rheumatoid arthritis [59 , 60 ], our studies suggest that ST2825 may be useful in the treatment of these autoimmune diseases. Future experiments will be aimed at testing the efficacy and the side-effects of ST2825 in murine models of immunological disorders.
ACKNOWLEDGEMENTS
This work was supported by a Research Contract (Conto Terzi) from Sigma-Tau Industrie Farmaceutiche Riunite S.p.A. (to C. S.). The authors acknowledge Drs. Daniela F. Angelini and Luca Battistini for their help with FACS analyses.
Received December 21, 2006; revised March 27, 2007; accepted April 10, 2007.
REFERENCES
B involves the Toll adapters TRAM and TRIF J. Exp. Med. 198,1043-1055B J. Biol. Chem. 280,15809-15814Related Article
This article has been cited by other articles:
 |
|
 |
|
  M. P. Paronetto, V. Messina, E. Bianchi, M. Barchi, G. Vogel, C. Moretti, F. Palombi, M. Stefanini, R. Geremia, S. Richard, et al. Sam68 regulates translation of target mRNAs in male germ cells, necessary for mouse spermatogenesis J. Cell Biol., April 20, 2009; 185(2): 235 - 249. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Funami, M. Sasai, H. Oshiumi, T. Seya, and M. Matsumoto Homo-oligomerization Is Essential for Toll/Interleukin-1 Receptor Domain-containing Adaptor Molecule-1-mediated NF-{kappa}B and Interferon Regulatory Factor-3 Activation J. Biol. Chem., June 27, 2008; 283(26): 18283 - 18291. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Nyman, P. Stenmark, S. Flodin, I. Johansson, M. Hammarstrom, and P. Nordlund The Crystal Structure of the Human Toll-like Receptor 10 Cytoplasmic Domain Reveals a Putative Signaling Dimer J. Biol. Chem., May 2, 2008; 283(18): 11861 - 11865. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. A. Frost and C. H. Lang Regulation of muscle growth by pathogen-associated molecules J Anim Sci, April 1, 2008; 86(14_suppl): E84 - E93. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
