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Previous Article | Next Article 
Molecular and Cellular Biology, February 2008, p. 1338-1347, Vol. 28, No. 4
0270-7306/08/$08.00+0 doi:10.1128/MCB.01412-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Department of Oral Disease Research, National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, 36-3, Gengo, Morioka, Obu, Aichi 474-8522, Japan,1 Laboratory of Oral Molecular Microbiology, Department of Oral Pathobiological Science, Hokkaido University Graduate School of Dental Medicine, Sapporo 060-8586, Japan,2 Department of Infectious Diseases, St. Jude Children's Research Hospital, 332 North Lauderdale Street, Memphis, Tennessee 381053
Received 7 August 2007/ Returned for modification 23 September 2007/ Accepted 28 November 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B. However, regulatory effects of NO on innate immune responses are unclear. Here, we report that NO has a capability to control Toll-like receptor-mediated signaling through S nitrosylation. We found that the adaptor protein MyD88 was primarily S nitrosylated, depending on the presence of endothelial NO synthase (eNOS). S nitrosylation at a particular cysteine residue within the TIR domain of MyD88 resulted in slight reduction of the NF-B-activating property. This modification could be restored by the antioxidant glutathione. Through S nitrosylation, NO could negatively regulate the multiple steps of MyD88 functioning, including translocation to the cell membrane after LPS stimulation, interaction with TIRAP, binding to TRAF6, and induction of IB
phosphorylation. Interestingly, glutathione could reversely neutralize such NO-derived effects. We also found that an acute febrile response to LPS was precipitated in eNOS-deficient mice, indicating that eNOS-derived NO exerts an initial suppressive effect on inflammatory processes. Thus, NO has a potential to retard induction of MyD88-dependent signaling events through the reversible and oxidative modification by NO, by which precipitous signaling reactions are relieved. Such an effect may reflect appropriate regulation of the acute-phase inflammatory responses in living organisms. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B (NF-B) (25, 32, 33, 39), the major transcriptional factor family deeply associated with regulation of the immune system through transcription of a wide range of genes, including cytokines, adhesion molecules, antimicrobial molecules, and antiapoptotic molecules (10, 13, 24). S nitrosylation of NF-B inhibits its DNA binding, promoter activity, and subsequent transcription (25, 33). It has been known that S nitrosylation targets a particular cysteine residue of the NF-B p50 and p65 subunits located in the N-terminal DNA binding loop within the Rel homology domain (25, 32, 33). This residue is conserved in other NF-B subunits, including p52, p100, p105, and c-Rel, and other Rel homology domain-containing molecules. Upstream of NF-B, IB kinase β (IKKβ), a catalytic subunit of the IB (inhibitor of NF-B) kinase complex, also undergoes S nitrosylation, resulting in reduction of its kinase function on phosphorylation of IB (39). Such reduction of the IKKβ function leads to reduced IB ubiquitinylation and proteasomal degradation, resulting in NF-B inhibition (14, 32, 39).
Toll-like receptors (TLRs) are the central innate immune sensors for a broad array of pathogen-associated molecular patterns, ranging from bacterial constituents to viral genomes (2, 35). TLRs initiate early processes of proinflammatory immune responses that help to strengthen the processes of innate and adaptive immunity (2, 20), in which NF-B plays many important roles (13, 24). TLRs utilize MyD88, a Toll/interleukin-1 receptor (IL-1R) homology (TIR) domain-containing adaptor molecule, to activate the NF-B pathway through IL-1R-associated kinases (IRAKs) and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) (1). It has been thought that TLR agonistic molecules, such as lipopolysaccharide (LPS), can regulate NO generation through upregulation of expression of all NO synthase (NOS) isoforms through NF-B activation (4, 9, 32). TLR stimulation can directly activate an antimicrobial property through inducible NOS (iNOS) expression and NO generation in macrophages (46). NO generation is a general feature of immune cells, including neutrophils, monocytes, macrophages, dendritic cells, and NK cells, as well as other cells, including endothelial cells, epithelial cells, and fibroblasts (4), all of which express multiple members of the TLR family. However, it has remained obscure whether generated NO exerts any regulatory effects on TLR signaling or subsequent processes of innate immune responses.
There has been an accumulation of biochemical evidence indicating that TLR signaling components, including IKKβ and NF-B, might be regulated by S nitrosylation. S nitrosylation inhibits the kinase activity of apoptosis signal regulation kinase 1 (ASK1) through inhibition of its binding to substrates (38). ASK1 is known as an important regulator of the TRAF6-p38 mitogen-activated protein kinase (MAPK) pathway downstream of TLR4 and is also involved in modulation of both the NF-B and apoptotic pathways downstream of TLR2 (19, 34). Caspse-1 was recently found to be involved in TLR2- and TLR4-mediated signal transduction of the MyD88-dependent pathway through the cleavage of the TIR domain-containing adaptor protein TIRAP (also known as Mal) (37). Caspase-1 also undergoes S nitrosylation at a cysteine residue within the enzymatic active site, suppressing its proteolytic activity (6, 31). Thus, it is possible that NO provides regulatory effects on the multiple steps of TLR-mediated innate immune signaling through S nitrosylation. In this study, we therefore designed experiments to determine the effect of S nitrosylation on TLR signaling. We further investigated how S nitrosylation affects TLR-initiated immune responses in vivo. We report here that S nitrosylation controls TLR signaling through redox-sensitive and reversible suppression of the MyD88 pathway, which facilitates appropriate control of acute-phase inflammatory responses in vivo.
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MATERIALS AND METHODS |
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Mice. iNOS-deficient (iNOS–/–) mice and endothelial-NOS (eNOS)-deficient (eNOS–/–) mice were from The Jackson Laboratories. C57BL/6J control (wild-type) mice were obtained from Japan SLC. All mice were kept under specific pathogen-free conditions. Male mice between 6 and 10 weeks of age were used for all of experiments. All animal protocols were approved by the National Institute for Longevity Sciences Animal Experimentation Committee at the National Center for Geriatrics and Gerontology (Aichi, Japan).
For LPS-induced acute lung injury, anesthetized mice received Escherichia coli LPS dissolved in pyrogen-free phosphate-buffered saline (PBS) containing 1 mg/ml Evans Blue intratracheally immediately after mechanical ventilation. After 30 min of administration, lung was excised and then lysed for immunoblot analysis. The febrile responses in mice treated with E. coli LPS were tested according to a protocol described previously (45, 49). Mice (n = 6) were maintained at a neutral ambient temperature of 31°C and challenged by intraperitoneal (i.p.) injection of 5 mg LPS/kg of body weight dissolved in pyrogen-free PBS. A high dose of LPS (more than 50 mg/kg) was fatal within 90 min in eNOS–/– mice. A colonic thermocouple was inserted and fixed to the base of the tail with adhesive tape. The change in temperature was monitored at 5-min intervals during a period of 120 min after LPS administration. All of the tests were performed at the temperature of 31°C. After 2 h or 12 h of LPS administration, 2 ml of PBS was injected into the abdominal cavity of each mouse. Then, fluids were collected and centrifuged for assessment of cytokine production by an enzyme-linked immunosorbent assay (ELISA). Preparation of peritoneal macrophages was as described previously (28).
Plasmids. The DNA construct encoding 3x Flag-tagged MyD88 fused to the B subunit of the bacterial DNA gyrase (MyD88-GyrB) was as described previously (11). Plasmids encoding human MyD88 and TIRAP were kind gifts from Margaret K. Offermann (Emory University School of Medicine). The cDNAs of N-terminal Flag-tagged and Myc-tagged MyD88, Myc-tagged TIRAP, and IRAK-1 were amplified by PCR and cloned into the pcDNA3.1 vector (Invitrogen). The construct encoding human TLR2 was as described previously (17). Constructs encoding mutated Flag-MyD88 were obtained using a QuikChange II site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions.
Protein purification. Recombinant Flag-MyD88 proteins were prepared using a FLAG M purification kit (Sigma-Aldrich) from HEK293 cells stably expressing Flag-MyD88 constructs, according to the manufacturer's instructions. Purity of recombinant proteins was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by silver staining and immunoblotting with anti-Flag antibody.
Detection of S-nitrosylated proteins. To detect S-nitrosylated MyD88 from lung lysates from wild-type mice and eNOS–/– mice, we referred to the protocol described by Jaffrey et al. (21). Several experiments were performed using a NitroGlo nitrosylation detection kit (PerkinElmer) according to the manufacturer's instructions. Lung lysates from wild-type and eNOS–/– mice were subjected to the biotin switching S-nitrosylation assay, and then biotinylated proteins were purified on streptavidin-agarose. Purified proteins eluted by 2-mercaptoethanol were detected by immunoblotting with anti-MyD88 antibody.
The quantitative measurement of S-nitrosylated recombinant MyD88 by ELISA was performed as follows. Briefly, recombinant Flag-MyD88 (150 µg) was treated with or without SNAP for 30 min at 37°C in the dark. Then, the free sulfides of Flag-MyD88 were blocked with 4 mM methylmethanethionsulfonate for 15 min. After purification by using Micro Bio-Spin chromatography columns (Bio-Rad Laboratories), Flag-MyD88 was reacted with 25 mM ascorbate to be completely denitrosylated. Free sulfides were then labeled with a biotin-conjugated maleimide, using a biotin labeling kit (SH; Dojindo Laboratories) according to the manufacturer's instructions. The diluents of biotinylated Flag-MyD88 proteins dissolved in Tris-buffered saline (pH 7.2) were stabilized in the wells of immobilizer streptavidin plates (Nunc). Flag-MyD88 proteins in the wells were detected by using anti-Flag antibody and a secondary antibody conjugated with horseradish peroxidase. Colorimetric reaction was detected by absorbance on a spectrophotometer at 450 nm. Results were expressed as means ± standard deviations (SD) of three determinations.
Photolysis of S-nitrosylated proteins. Mouse lung lysates were exposed for 3 min to a UV-visible light mercury vapor lamp according to a protocol recently described (8). The samples were then subjected to the biotin switch technique as described above.
Luciferase reporter assay.
293-TLR2 cells were transiently transfected with wild-type MyD88-GyrB or MyD88-GyrB mutants, each with a cysteine residue replaced with a serine residue, together with 50 ng of an NF-B (5x) luciferase reporter plasmid (pNF-B-Luc; Stratagene) and 5 ng of an internal control luciferase reporter plasmid (pRL-TK; Promega) and incubated for 16 h. At 6 h before the end of incubation, cells were treated with or without 250 µM SNAP. Cells were then stimulated with 100 ng/ml Pam3CSK4 for 6 h. HEK293 cells stably expressing MyD88-GyrB were transfected with pNF-B-Luc and pRL-TK. After 24 h of incubation, cells were stimulated with coumermycin A in the presence and absence of 250 µM SNAP. The dual luciferase activity was measured as described previously (19).
Immunoblot analysis of IRAK-1 and IB.
HAECs were stimulated with 10 ng/ml of LPS for 0 to 90 min. HEK293 cells stably expressing MyD88-GyrB were stimulated with 1 µM coumermycin for 20 min. Cells were lysed in the presence of protease inhibitor and phosphatase inhibitor cocktails (Roche) at 4°C. Cell lysates or lysates from the mouse lungs were separated by SDS-PAGE, followed by immunoblot analyses using anti-IRAK-1, anti-IB, and phosphorylation-specific anti-IB (Ser32/Ser36) antibodies (Cell Signaling Technology).
RNA extraction and reverse transcription-PCR. Total RNA was isolated from mouse peritoneal macrophages stimulated with 100 ng/ml LPS and 10 ng/ml gamma interferon, and transcripts were quantified by real-time quantitative reverse transcription-PCR on a LightCycler ST300 system (Roche). All values were normalized to the level of β-actin mRNA. The primer sets used are as follows: for mouse macrophage inflammatory protein 2 (MIP-2), 5'-ATCCAGAGCTTGAGTGTGACGC-3' (sense) and 5'-AAGGCAAACTTTTTGACCGAA-3' (antisense); for mouse IL-6, 5'-CCACGGCCTTCCCTAC-3' (sense) and 5'-AGTGCATCATCGTTGTTC-3' (antisense); and for mouse β-actin, 5'-AAATCGTGCGTGACATCAAA-3' (sense) and 5'-AAGGAAGGCTGGAAAAGAGC-3' (antisense).
Cytokine ELISA. Concentrations of human IL-8, mouse MIP-2, and mouse IL-6 were determined using a Cytoset ELISA kit (Biosource) according to the manufacturer's instructions.
Subcellular fractionation. Subcellular fractionation of HEK293 cells stably expressing Flag-MyD88 and 293-TLR4 cells stably expressing Flag-MyD88-GyrB was performed using a ProteoExtract subcellular proteome extraction kit (Calbiochem) according to the manufacturer's instructions. This kit enables extraction of different subcellular fractions of the cytoplasm, plasma membrane, nuclei, and cytoskeleton from mammalian cells. 293-TLR4 cells stably expressing Flag-MyD88-GyrB were maintained in serum-free Dulbecco's modified Eagle's medium containing 5% PANEXIN H cell growth supplement (PAN Biotech GmbH) to avoid nonspecific cell activation by animal serum components. The whole-cell lysate was obtained using NuPAGE LDS sample buffer containing 2-mercaptoethanol. Each fraction was mixed with NuPAGE LDS sample buffer and boiled for 5 min, followed by SDS-PAGE and immunoblot analyses using anti-Flag (Sigma), anti-MyD88 (Santa Cruz Biotechnology), anti-IRAK-1, and anti-vimentin (BD Biosciences) antibodies.
Blue native PAGE and immunoprecipitation. HEK293 cells stably expressing Flag-MyD88 were treated with SNAP for 1 h, washed twice with PBS, and lysed with HEPES buffer (pH 7.2) containing 1% Triton X-100, 1% Nonidet-P40, and proteinase inhibitor cocktail (Roche). Cell lysates were separated by blue native PAGE according to the protocol provided by Invitrogen and then immunoblotted with anti-Flag antibody. HEK293 cells transiently transfected combinatorially with Flag-MyD88 and Myc-TIRAP or Flag-MyD88 and IRAK-1 were treated with GSNO or GSH for 1 h. HEK293 cells stably expressing 3x Flag-MyD88-GyrB were stimulated with 1 µM coumermycin A for 20 min. Cells were lysed with HEPES buffer (pH 7.2) containing 1% Triton X-100 and proteinase inhibitor cocktail (Roche). Clarified cell lysates were immunoprecipitated with anti-Flag antibody, followed by immunoblot analysis using anti-Flag, anti-Myc, and anti-TRAF6 (StressGen) antibodies. All experiments were performed at least three times, and representative results are shown.
Immunofluorescent cell staining. HAECs were fixed at –20°C with methanol, and double immunostaining was then carried out with anti-β-actin monoclonal antibody (Santa Cruz Biotechnology) and Alexa 488-conjugated immunoglobulin G secondary antibody (Invitrogen) and then with anti-MyD88 rabbit polyclonal antibody (Santa Cruz Biotechnology) and Alexa 564-conjugated immunoglobulin G secondary antibody (Invitrogen). Cell nuclei were also stained with 2.5 µg/ml of Hoechst 33342 for 30 min.
Statistical analysis. Probability (P) values were calculated by Student's t test and analysis of variance and were considered significant at 0.05 or 0.01.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Bs, ASK1, and caspase-1.
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To determine the requirement of cysteine residues for functioning of MyD88, we utilized MyD88 fused to the B subunit of the bacterial DNA gyrase (MyD88-GyrB). The Streptomyces-derived bivalent antibiotic coumermycin binds GyrB with a stoichiometry of 1:2, acting as a natural dimerizer of GyrB (7). Although overexpressed MyD88 is known to reveal TLR stimulation-independent nonspecific activation of downstream signaling through self-dimerization (11, 36), MyD88-GyrB does not reveal such nonspecific activation unless cells are exposed to TLR stimulation or coumermycin treatment (11). We prepared GyrB-fused wild-type MyD88 and MyD88 mutants, each with one of the nine cysteine residues replaced with a serine residue, and examined the NF-B-activating properties in the TLR2 ligand Pam3CSK4-stimulated HEK293 cells stably expressing TLR2. None of the cysteine replacement mutants abrogated the NF-B-activating property of MyD88 (Fig. 1H). However, the Cys216Ser mutant significantly increased the activity compared with that of wild-type MyD88 (Fig. 1H). Additionally, similar results were found in the cells treated with SNAP (Fig. 1H). Thus, it is possible that Cys216 of MyD88 mediates the suppressive effect of NO.
S nitrosylation alters MyD88-mediated signaling events.
We next explored how S nitrosylation of signaling components alters TLR signaling events. To examine this in vivo, we utilized an animal model of acute lung injury induced by intratracheal administration of LPS. We investigated degradation of IRAK-1 and IB, hallmarks of MyD88-dependent and IKKβ-dependent signaling events, in the lungs 30 min after LPS administration. Interestingly, degradation of IRAK-1 and IB was apparently promoted in eNOS–/– mice compared with that in wild-type mice (Fig. 2A). We also examined whether NO alters degradation of IRAK-1 and IB in cultivated vascular endothelial cells. In HAECs, LPS induced degradation of IRAK-1 and IB within 30 min after stimulation (Fig. 2B). Degradation of IRAK-1 and IB was promoted and occurred within 15 min after stimulation when endogenous NO was predepleted by the L-arginine analog L-NMMA (Fig. 2B). In addition, IRAK-1 degradation was also promoted when confluent HAECs were maintained in culture media without the eNOS activator vascular endothelial growth factor or the phosphatidylinositol 3-kinase inhibitor LY294002 (data not shown). In contrast to these results, degradation was delayed and residual proteins were observed even at 45 min after stimulation when cells were pretreated with SNAP (Fig. 2B). The effect of NO was not altered in the presence of the guanylate cyclase inhibitor ODQ or the cyclic-GMP-dependent protein kinase inhibitor KT5823 (data not shown). Notably, LPS-induced degradation of IRAK-1 and IB in HAECs was prevented by the irreversible thiol modification by N-ethylmaleimide (Fig. 2C), implying that the effect of NO on the signaling events depends on modification of cysteine residues.
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B activation in HEK293 cells was suppressed by pretreatment with cells with NO donors (Fig. 3A). Coumermycin could induce phosphorylation of IB at Ser32 and Ser36, the target residues of IKKβ involved in degradation of IB (51), but SNAP pretreatment could suppress induction of the response (Fig. 3B). SNAP also suppressed coumermycin-induced phosphorylation of MAPKs (data not shown). The coumermycin-dependent dimerization of MyD88-GyrB induced interaction with TRAF6, consistent with a previous study (10), and we found that this interaction was reduced by SNAP treatment (Fig. 3C).
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In HAECs, MyD88 was enriched with filamentous cytoskeletal structures and partly colocalized with β-actin (Fig. 4A). Additionally, a large part of MyD88 stably expressed in HEK293 cells was found in the cytoskeletal fraction (Fig. 4B). These findings are consistent with results of a previous study showing that MyD88 associates with β-actin in HeLa cells (22). Cytoskeletal MyD88 was separated from IRAK-1, which was found only in the cytoplasm (Fig. 4B), suggesting that MyD88 is maintained as an inactive state in cytoskeleton. We found that SNAP treatment altered such cytoskeletal localization of MyD88 into the cytoplasm (Fig. 4C). We further investigated the subcellular localization of MyD88 after TLR4 stimulation in 293-TLR4/MD2-CD14 cells stably expressing Flag-MyD88-GyrB. After LPS treatment, a part of MyD88 was transported to the cytoplasmic membrane from the cytoskeleton (Fig. 4D). However, SNAP treatment retarded such LPS-induced transportation of MyD88 (Fig. 4D). Although native PAGE analysis revealed that MyD88 formed a protein complex (more than 480 kDa) in HEK293 cells, SNAP treatment resulted in reduction in the size of the complex to approximately 450 kDa or 250 kDa, accompanied by an increase in concentration (Fig. 4E). Moreover, higher concentrations of SNAP further altered the complex to render a monomer (approximately 35 kDa) (Fig. 4E), implying disruption of functional MyD88 protein complex by NO.
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NO reversibly suppresses the MyD88 signaling events. S-nitrosylated proteins are known to undergo denitrosylation, by which regulatory effects of NO are conferred to control protein functions. We therefore investigated how S nitrosylation and denitrosylation affect MyD88-mediated signaling events. For this purpose, we utilized GSH because GSH had a capability to denitrosylate MyD88 (Fig. 1C). We found that GSH restored NO-induced impaired interaction of MyD88 with TIRAP (Fig. 4F). Furthermore, the NO-induced cytoplasmic localization of cytoskeletal MyD88 was restored by treatment of the cells with GSH (Fig. 4G). Thus, these results suggest that S nitrosylation alters the MyD88 pathway, and antioxidants or oxidoreductases restore such NO-derived actions, probably through denitrosylation.
NO reversibly suppresses TLR-mediated cellular responses.
HAECs responded to multiple bacterial TLR agonistic molecules, Pam3CSK4 (for TLR1/TLR2), MALP-2 (for TLR2/TLR6), LPS (for TLR4), flagellin (for TLR5), and IL-1β, all of which are known to activate MyD88-dependnet signaling to induce production of the NF-B-driven chemokine IL-8 after stimulation for 3 h (Fig. 5A). Predepletion of endogenous NO by L-NMMA resulted in a significant increase in IL-8 production induced by each stimulator (Fig. 5A), indicating that endogenous NO has a suppressive effect on the TLR-mediated cellular response. Furthermore, IL-8 production by each stimulator was suppressed in the presence of SNAP (Fig. 5A). The effect of NO donors was not altered in the presence of ODQ or KT5823 (data not shown). We investigated whether such a suppressive effect of NO can be restored because S nitrosylation is a reversible protein modification. However, it is difficult to examine the effects of antioxidants or oxidoreductases because the TLR signaling pathway is greatly affected by reactive oxygen species generated from NADPH oxidases (27, 34, 48). Indeed, treatment of cells with ascorbic acid, GSH, NAC, or the NADPH oxidase inhibitor DPI greatly impaired LPS-induced IL-8 production in HAECs (Fig. 5B). We therefore attempted to address whether the effect of NO is transient or persistent. For this purpose, HAECs were pretreated with SNAP for 1 h and washed two times to remove the NO donor. Then, at various times afterwards, the cells were stimulated with LPS and IL-8 production was measured. NO suppression of IL-8 production was gradually neutralized or restored in a time-dependent manner, although the restrictive effect continues for several hours (Fig. 5C).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We found that the suppressive effect of NO on TLR-mediated cellular responses was transient and degraded in a time-dependent manner (Fig. 5C). The specificity of NO regulation may be conferred by the spatial regulation of S nitrosylation within or between proteins and the stimulus-coupled temporal regulation through denitrosylation (14). Signal transduction by ligand-receptor interactions is thought to trigger denitrosylation, restoring substantial protein functions. For example, reduction of the functions of caspase-3 and IKKβ by S nitrosylation is restored by FasL-Fas interaction and TNF--TNFR interaction, respectively (30, 39). Thus, it is possible that TLR ligation-dependent protein denitrosylation also facilitates the restoration of NO suppression although the mechanism of denitrosylation has been poorly studied. Protein denitrosylation is catalyzed by antioxidants or oxidoreductases, including ascorbic acid, thioredoxin-thioredoxin reductase, superoxide dismutase GSH, and GSNO reductase (14, 15, 50, 53). S-nitrosylated MyD88 can be denitrosylated in the presence of ascorbic acid and GSH in vitro (Fig. 1C). Although it is still unclear how TLR ligation activates cellular redox activity, LPS has a potential to activate cellular redox activity and transition of GSH into GSNO (41). More details of TLR-mediated protein S nitrosylation and denitrosylation should be investigated in future studies.
TLR ligation can initiate recruitment of MyD88 to the receptor complex through TIR-TIR interaction. In the case of TLR2 and TLR4, the sorting adaptor TIRAP is essentially required to recruit MyD88 (23). MyD88 then dissociates from the receptor complex and recruits IRAK-1 (and IRAK-4) through death domain (DD)-DD interaction, inducing TRAF6-mediated signaling events and ubiquitin ligation to IRAK-1 or IB, followed by proteasomal degradation (1). Nevertheless, how MyD88 can be initially controlled to be recruited to TLRs has remained unclear. We found that a large part of cellular MyD88 existed in the cytoskeleton and associated with β-actin (Fig. 4A and B), wherein MyD88 formed a complex dissociated from IRAK-1 (Fig. 4B). Our finding suggests that MyD88 preferentially interacts with the cytoskeleton as an inactive form, followed by release into the cytoplasm and recruitment to TLRs after ligation-dependent actin rearrangement. Indeed, inhibition of actin rearrangement by cytochalasin D suppresses LPS-induced signal transduction and cytokine production (5). In addition, cytochalasin D also alters TIRAP recruitment to the cytoplasmic membrane (23). NO restriction of MyD88 function may be achieved through disruption of the protein complex and dissociation of MyD88 from the actin cytoskeleton to the cytoplasm (Fig. 4A to E). NO also reduces the interaction of MyD88 with TIRAP (Fig. 3). These effects may ultimately result in mitigated potential for the ligation-dependent recruitment of MyD88 to TLRs.
We found that S nitrosylation of MyD88 plays some roles in NO modulation of TLR signal transduction. Interestingly, eight of the nine cysteine residues of MyD88 are concentrated in the TIR domain, but the C-terminal DD contains no cysteine residue, suggesting that cysteine modification affects TIR-TIR interaction but not DD-DD interaction. Indeed, NO attenuated the interaction of MyD88 with TIRAP but not that with IRAK-1 (Fig. 3). This result is supported by the result found by Xiong et al. (52) showing that SNAP treatment did not affect the interaction of MyD88 with IRAK-1 in mouse macrophages. Although S nitrosylation of the Cys216 residue of MyD88 may participate in the NO regulation of TLR signal transduction, it is likely that this modification does not have a dominant effect, because Cys216 was not essential for activation of downstream signaling (Fig. 1G). NO modification of Cys216 may yield a slight structural change in the base of the TIR domain, resulting in slightly reduced interaction with a counterpart TIR domain of TIRAP. Alternatively, NO may antagonize other reversible modifications, such as palmitoylation or disulfide bonding to a counterpart molecule, leading to transient impairment of MyD88 functioning. Also, other unknown mechanisms for MyD88 regulation may be negatively influenced by S nitrosylation.
We found that eNOS and iNOS differentially regulate LPS-induced acute-phase immune responses in vivo (Fig. 6). Although the amount of NO derived from eNOS is comparatively small, NO is steadily generated from endothelial cells as a vasodilatory gas that continually maintains an antiproliferative and antiapoptotic environment in vasculatures (16). Simultaneously, eNOS increases the amounts of cellular S-nitrosylated proteins and circulating NO donors by nitrosylating GSH and albumin (40). Such functions of NO from eNOS may systemically reduce cellular reactivity to a TLR stimulus to maintain a weak tolerance, which may lead to prevention of a rapid rise of inflammation. In contrast, NO from iNOS is generated in large quantities and exerts a strong antimicrobial action, although NO from eNOS also has an antimicrobial property (4, 29). The large amount of NO derived from iNOS is thought to disrupt cellular signaling cascades, resulting in anti-inflammatory or immunosuppressive effects. Such functions of NO from iNOS may regionally reduce cellular reactivity to TLR recognition of pathogens to initiate an inducible tolerance, which may transiently prevent promotion of excess inflammatory responses, although excess NO production ultimately results in nitrosative stress and apoptotic cell death (12, 26).
Our study proposes that TLR signal transduction involves an oxidative protein modification by NO and its redox regulation. NO may exert other effects, such as activation of cyclic-GMP-dependent signaling, on TLR signaling events, but such effects may not be dominant, at least in the acute-phase innate immune responses. Further investigations will be necessary to clarify more details about the relationship between such NO regulation and physiological or pathophysiological innate immune responses.
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ACKNOWLEDGMENTS |
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This work was supported by grants-in-aid for Scientific Research on Priority Areas (19041079 to T. Into) and for Young Scientists (B:18791363 to T. Into), provided by the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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FOOTNOTES |
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Published ahead of print on 17 December 2007.
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