ROLE OF TLR4 TYROSINE PHOSPHORYLATION IN SIGNAL TRANSDUCTION AND ENDOTOXIN TOLERANCE

Abstract

In this study, we examined whether tyrosine phosphorylation of the Toll-IL-1 resistance (TIR) domain of Toll-like receptor (TLR) 4 is required for signaling and blocked in endotoxin tolerance. Introduction of the P712H mutation, responsible for lipopolysaccharide (LPS) unresponsiveness of C3H/HeJ mice, into the TIR domain of constitutively-active mouse ΔTLR4 and mutation of the homologous P714 in human CD4-TLR4 rendered them signaling-incompetent and blocked TLR4 tyrosine phosphorylation. Mutations of tyrosine residues Y674A and Y680A within the TIR domains of CD4-TLR4 impaired its ability to elicit phosphorylation of p38 and JNK mitogen-activated protein kinases, IκB-α degradation, and activation of NF-κB and RANTES reporters. Likewise, full-length human TLR4 expressing Y674A or Y680A mutations showed suppressed capacities to mediate LPS-inducible cell activation. Signaling deficiencies of the Y674A and Y680A TLR4s correlated with altered MyD88-TLR4 interactions, increased associations with a short IRAK-1 isoform, and decreased amounts of activated IRAK-1 in complex with TLR4. Pretreatment of human embryonic kidney (HEK) 293/TLR4/MD-2 cells with protein tyrosine kinase or Src kinase inhibitors suppressed LPS-driven TLR4 tyrosine phosphorylation, p38 and NF-κB activation. TLR2 and TLR4 agonists induced TLR tyrosine phosphorylation in HEK293 cells overexpressing CD14, MD-2, and TLR4 or TLR2. Induction of endotoxin tolerance in HEK293/TLR4/MD-2 transfectants and in human monocytes markedly suppressed LPS-mediated TLR4 tyrosine phosphorylation and recruitment of Lyn kinase to TLR4, but did not affect TLR4-MD-2 interactions. Thus, our data demonstrate that TLR4 tyrosine phosphorylation is important for signaling and is impaired in endotoxin-tolerant cells, and suggest involvement of Lyn kinase in these processes.

Activation of innate immune responses is critical for the early host defense against microbial infections and for subsequent development of adaptive immunity (1-4). Toll-like receptors (TLRs)³ play a central role in these processes by sensing conserved pathogen-associated molecular patterns (PAMPs) from bacteria (TLR2, TLR4, TLR5, TLR9) (4-10) and viruses (TLR4, TLR3, TLR7−9) (11-15). All mammalian TLRs share a similar structural organization, with an ectodomain containing leucine-rich repeats, a transmembrane domain, and a cytoplasmic domain with an intracellular Toll-IL-1 resistance (TIR) domain essential for signal transduction (1-4,16). TLRs are expressed on a variety of cells, including epithelial and endothelial cells, neutrophils, monocytes, macrophages, and dendritic cells (DC), either at the cell surface (TLR2, TLR4, and TLR5) or intracellularly in endosomes (TLR3, 7, 8, and 9) (1-4,17-20). TLR sensing of certain bacterial structures (e.g., lipopolysaccharide (LPS) is initiated by a co-receptor, CD14, that acts by binding various PAMPs and presenting them to TLRs that trigger signal transduction (21-23). Several other molecules, e.g., CD11b/CD18 (Mac-1), CD36, and Dectin-1, have also identified as coreceptors that facilitate TLR-mediated signaling (24-29). In the case of TLR4, an extracellular protein, MD-2, is necessary for TLR4 responsiveness to LPS (30-32), and is part of a tri-molecular signaling complex comprised of CD14, LPS, and TLR4 (32,34).

Recognition of PAMPS by TLRs leads to TLR oligomerization and recruitment of adapter proteins and kinases to their intracellular TIR signaling domains. These processes trigger activation of transcription factors and expression of cytokines, as well as adhesion and costimulatory molecules via two main signaling pathways. The “MyD88-dependent” pathway is activated by all TLRs except TLR3, engages adapter proteins MyD88 and TIR domain containing adapter protein (TIRAP) also called MyD88-like adapter (Mal) (engaged by TLR2 and TLR4), as well as the kinases IL-1R-associated kinase (IRAK)-4 and IRAK-1. This pathway results in early NF-κB and MAPK activation, production of proinflammatory cytokines, and B cell proliferation (1-4,16,18,35,36). The “MyD88-independent pathway” is triggered by TLR4 and TLR3 only, uses the adapters TIR domain containing adapter-inducing interferon (TRIF) and TRIF-related adapter molecule (TRAM) (TLR4) or TRIF alone (TLR3), and kinases TRAF-associated NF-κB activator (TANK)-binding kinase (TBK)-1 and IκB kinase (IKK)-ε. This pathway mediates DC maturation, activation of interferon regulatory factors (IRFs), delayed NF-κB and MAPK activation, and induction of type I interferons (IFNs) (37-52). Mice deficient for either the MyD88-dependent or MyD88-independent components are resistant to LPS, but more susceptible to bacterial infections, indicating that both pathways play a role in antimicrobial defense (36,40).

Prior exposure to LPS induces a state of cell refractoriness to subsequent LPS challenge known as endotoxin tolerance, which is thought to limit excessive inflammatory responses (rev in 53,54). A subset of septic shock survivors develop a tolerant phenotype manifested by decreased monocytic responses to LPS and increased susceptibility to bacterial infections (55). The molecular mechanisms of tolerance remain poorly understood. Although LPS tolerance was suggested to be the consequence of decreased TLR4/MD2 expression (56), other studies showed unaltered TLR4 expression, but suppressed IRAK-1-MyD88 interactions and IRAK-1 activation in LPS-tolerant cells (57-61). Our previous results also demonstrated comparable TLR4 expression in normal and LPS-tolerant mouse macrophages (62) and human monocytes (63), whereas recruitment of MyD88 to TLR4 and IRAK-1 activation was blunted (63). In this report, we suggest that impaired recruitment of MyD88 to TLR4 and activation of IRAK-1 in endotoxin-tolerant cells is secondary to a decrease in TLR4 tyrosine phosphorylation. Our findings support the hypothesis that tyrosine phosphorylation of the TLR4 TIR domain is required for TLR4 signaling, and is blocked in endotoxin-tolerant cells. Tyrosine-deficient TLR4 species exhibited constitutive associations with MyD88, suppressed LPS-inducible MyD88 recruitment to TLR4, increased interactions with a short isoform of IRAK-1, and impaired activation of IRAK-1 at the receptor. Furthermore, endotoxin-tolerant cells were found to show suppressed LPS-inducible TLR4 tyrosine phosphorylation, which correlates with impaired MyD88 recruitment to TLR4 and inhibited IRAK-1 activation. Moreover, the ability of protein tyrosine kinase and Src kinase inhibitors to block TLR4 tyrosine phosphorylation and signaling, coupled with the observation of LPS-inducible recruitment of Lyn kinase to TLR4 and its impairment in LPS-tolerant cells, suggest involvement of Lyn kinase in TLR4 signaling.

Experimental Procedures

Reagents and Cell Culture

Polyclonal antibody (Ab) to human TLR4 (H80), Abs to MyD88, IκB-α, tubulin, β-actin and IRAK-1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-phospho (p)-p38 and anti-pJNK Abs were from Promega (Madison, WI). Anti-Flag monoclonal Ab (M2), M2-horseradish peroxidase (HRP) conjugate, and anti-HA polyclonal Ab were obtained from Sigma (St. Louis, MO), and anti-phosphotyrosine Ab PY20 was from BD Biosciences (San Jose, CA). Human HEK293T cells were maintained as described previously (64). Protein-free, phenol-water extracted E. coli LPS and mycobacterial soluble tuberculosis factor (STF) were prepared as described (65,66), and the synthetic TLR2 agonist, Pam3Cys (S-[2,3-Bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-Lys4-OH trihydrochloride) was purchased from EMC Microcollections GmbH (Tubingen, Germany). An HEK293 cell line stably transfected with untagged human TLR4 and Flag-tagged human MD-2 (HEK/TLR4/MD-2) was kindly provided by Dr. Douglas Golenbock (University of Massachusetts Medical School, Worchester, MA). Protein tyrosine kinase inhibitors herbimycin A, genistein, and Src kinase inhibitors PP1 and PP2 were purchased from Calbiochem (San Diego, CA). Human monocytes were prepared by counterflow elutriation and resuspended in RPMI 1640 medium (BioWhittaker, Walkersville, MD) supplemented with 5% FBS (HyClone, Logan, UT), 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen, Carlsbad, CA).

Recombinant plasmids and transient transfection

pCDNA3- YFP-human (hu)TLR4, pCDNA3-huCD14, pCMV1-Flag-huTLR2, pCMV-β-galactosidase, and pELAM-luciferase, were obtained from Dr. Douglas T. Golenbock (University of Massachusetts Medical School, Worcester, MA). pEFBOS-His/Flag-huMD-2 was provided by Dr. Kensuke Miyake (Research Institute for Microbial Diseases, Osaka University, Osaka, Japan), and pCDNA3-YFP-MyD88 was obtained from Dr. Katherine A. Fitzgerald (University of Massachusetts Medical School, Worcester, MA). pGL3-RANTES-luciferase reporter plasmid was kindly provided by Dr. John Hiscott (McGill University, Montreal, Canada ). Expression vectors encoding haemagglutinin (HA)-ΔTLR4 WT and HA-ΔTLR4 P712H have been described (67), and expression plasmids pFlag-CMV-1 encoding WT or PGV714−716 CD4-TLR4 were kindly provided by Dr. Stephen T. Smale (Howard Hughes Medical Institute, UCLA, Los Angeles, CA). P714H, Y674A and Y680A mutations were introduced into the TIR domain of CD4-TLR4 or YFP-TLR4 by site-directed mutagenesis, using Quick-Change Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA). HEK293T cells were cultured overnight in 150 mm TC dishes (5×10⁶ cells per dish) and cotransfected for 3 h with expression vectors as described in Figure Legends (25 μg total plasmid DNA per dish) using Superfect transfection reagent (Qiagen, Valencia, CA). After 48 h, cellular extracts were prepared as described (68). For real-time PCR analyses and determination of cytokine levels by ELISA, transfections were carried out in 6-well plates according to manufacturer's protocol.

Isolation of RNA, real-time PCR analysis, and IL-8 secretion

Total RNA was isolated with RNeasy kits (Invitrogen), followed by DNase digestion and re-purification as recommended by the manufacturer. cDNA was prepared from 1 μg RNA using Reverse Transcription System (Promega), and subjected to real-time PCR with gene-specific primers for human hypoxanthine phosphoribosyltransferase (HPRT), IL-8, TNF-α, and IFN-β on a MyIQ minicycler with Optical Detection Module (Bio-Rad, Hercules, CA). Real-time PCR data were processed using −2^ΔΔCT method (69). IL-8 levels in supernatants were measured by ELISA in the Cytokine Core Laboratory (University of Maryland, Baltimore).

Co-immunoprecipitation and immunoblotting

Cell extracts (1−3 mg total protein) were pre-cleared with protein G-agarose beads (20 μl per sample, Roche Applied Science, Indianapolis, IN) and incubated overnight with 1 μg of respective Ab in lysis buffer containing 20 mM HEPES (pH 7.4), 0.5% Triton X-100, 150 mM NaCl, 12.5 mM β-glycerophosphate, 50 mM NaF, 1 mM DTT, 1 mM sodium orthovanadate, 2 mM EDTA, 1 mM PMSF, and protease inhibitor cocktail (Roche). Thereafter, protein G-agarose beads were added (45 μl per sample) and incubation continued for 4 h. Beads were washed 5 times with lysis buffer, and proteins were separated by SDS-PAGE on 4−20% minigels (InVitrogen), transferred to Immobilon-P membranes (Millipore, Billerica, MA), and subjected to Western analysis as described previously (68).

Reporter assays

Expression vectors pELAM-luciferase (NF-κB reporter assays) and pGL3-RANTES-luciferase (RANTES reporter assays) were added (0.3 μg/well each) to corresponding transfection mixtures, pCMV-β-galactosidase reporter (0.2 μg/well) was co-transfected for normalization (NF-κB reporter assays), and total plasmid DNA amount was adjusted to 1.5 μg/well with pcDNA3. Following transfections, cells were recovered for 24 h, lysed in a passive lysis buffer (Promega) and firefly luciferase vs. β-galactosidase activities were measured using Reporter Assay Systems (Promega) and β-galactosidase assay kit (Tropix, Galacto-Light System) on a Berthold LB 9507 luminometer (Berthold Technologies, Bad Wildbad, Germany). For RANTES reporter assays, luciferase activity was normalized to total protein concentrations measured in cell extracts.

Statistical analysis

Statistical analysis was performed using the GraphPad Prism 4 program for Windows. Statistical differences among experimental groups were evaluated by the Student's t-test with the level of significance set at p<0.05. Values are expressed as mean ± SD.

RESULTS

The P712H mutation in the TIR domain of murine HA-ΔTLR4 abolishes TLR4 signaling and tyrosine phosphorylation

Positional cloning of TLR4 identified a P712H mutation within the TIR domain as the molecular basis for the LPS-hyporesponsive phenotype of C3H/HeJ mice (4,5). However, the molecular mechanisms responsible for the signaling deficiency of the mutant P712H TLR4 still remain obscure. In this study, we hypothesized that the P712H mutation alters the secondary structure of the TIR domain, resulting in the failure of TLR4 to undergo agonist-induced post-translational modifications that may be necessary for initiation of downstream signaling. Since an earlier report correlated LPS-induced TLR4 tyrosine phosphorylation with TLR4 signaling (70), we initially sought to determine whether signaling incompetence of P712H TLR4 variants also correlates with changes in TLR4 tyrosine phosphorylation. To this end, we used an expression vector encoding murine TLR4 that has a deletion of a large part of the ectodomain (ΔTLR4), rendering it constitutively active, as evidenced by activation of NF-κB, COX-2, and TLR4-MyD88 association (67). Overexpression of wild-type (WT) HA-ΔTLR4 in HEK293T cells markedly induced IL-8 gene expression and production (Fig. 1A and B), IκB-α degradation, and p38 phosphorylation (Fig. 1C), while the P712H HA-ΔTLR4 mutant failed to activate cells. IRAK-1 phosphorylation was also severely inhibited in cells that overexpress P712H HA-ΔTLR4 versus the response elicited by WT HA-ΔTLR4 (Fig. 1C). Under conditions of equal total HA-ΔTLR4 protein expression, immunoprecipitation and Western blot analysis revealed strong tyrosine phosphorylation of WT, but not mutant (P712H) HA-ΔTLR4 species (Fig. 1D). Collectively, these results indicate that the lack of signaling capacity of the P712H HA-ΔTLR4 mutant correlates with its impaired tyrosine phosphorylation.

Fig. 1. The P712H mutation in the TIR domain of mouse HA-ΔTLR4 abolishes TLR4 signaling and tyrosine phosphorylation.

HEK293T cells were transiently transfected with expression vectors encoding HA-tagged ΔTLR4 WT, P712H mutant, or a control vector pCDNA3. After 48 h, total RNA was isolated to examine IL-8 mRNA levels by real-time PCR (A), cell-free supernatants were collected to determine IL-8 production (B), and cell extracts prepared for Western analysis of IRAK-1, IκB-α, p38 phosphorylation, and tubulin expression (C). TLR4 species were also immunoprecipitated with α-HA Ab and subjected to immunoblotting with α-HA Ab (total TLR4 expression) and α-phosphotyrosine Ab (TLR4 phosphorylation) (D). Results of a representative experiment (n=3) are shown.

The effect of the PGV714−716AAA mutation in the TIR domain of human CD4-TLR4 fusion protein on cell activation and TLR4 tyrosine phosphorylation

We next sought to extend the linkage between signaling competence of mouse TLR4 and its ability to undergo tyrosine phosphorylation to human TLR4. In doing so, we made use of the human P714A mutation (71) that corresponds to the homologous P712H mutation in murine TLR4 (4,5,71) and blocks activation of NF-κB-, AP-1-, and C/EBP-dependent reporter plasmids elicited by constitutively-active WT CD4-TLR4 (71). Extending these results, we observed that overexpression of the PGV714−716AAA CD4-TLR4 (designated P714A) in HEK293T cells failed to activate IL-8 mRNA expression and secretion (Figs. 2A and B), IRAK-1 and p38 MAPK phosphorylation, and IκB-α degradation (Fig. 2C), in contrast to robust responses elicited by WT CD4-TLR4. As shown in Fig. 2D, Western analysis of immunoprecipitated CD4-TLR4 proteins showed comparable expression of WT and PGV714−716AAA mutant. Similar to the results obtained for the P712H HA-ΔTLR4, the PGV714−716AAA CD4-TLR4 failed to undergo tyrosine phosphorylation, whereas tyrosine phosphorylation of WT CD4-TLR4 was clearly observed (Fig. 2D). Taken together, these data indicate that the PGV714−716AAA mutation in the TIR domain of human TLR4 precludes TLR4 tyrosine phosphorylation and renders human TLR4 functionally incompetent.

Fig. 2. The effect of the PGV714−716 mutation in the TIR domain of human CD4-TLR4 on cell activation and TLR4 tyrosine phosphorylation.

CD4-TLR4 WT or PGVP714−716AAA mutant (designated P714A) was overexpressed in HEK293T cells, and transfection with pCDNA3 was used as a control. After 48 h, IL-8 gene expression was examined by real-time PCR (A), IL-8 production was determined in supernatants by ELISA (B), and Western analysis of IRAK-1 phosphorylation, IκB-α degradation, and p38 phosphorylation was performed in cellular extracts. Tubulin expression was assessed as a loading control (C). CD4-TLR4 was immunoprecipitated with α-CD4 Ab and analyzed by immunoblotting with α-Flag Ab (total TLR4 expression) and α-phosphotyrosine Ab (TLR4 phosphorylation) (D). Data from a representative experiment (n=4) are shown.

Tyrosine to alanine substitutions in the TIR domain of TLR4 inhibit activation of NF-κB, RANTES reporters, phosphorylation of JNK and p38 MAPK, and degradation of IκB-α

Substitution of several tyrosine residues within the TIR domain was shown to impair the ability of CD4-TLR4 to activate NF-κB, C/EBP, and AP-1 reporter plasmids (71), suggesting that tyrosine residues in the TIR domain are necessary for TLR4 signal transduction. However, since additional adjacent non-tyrosine residues were also mutated, it is possible that the observed decrement in TLR4 activity might be due to changes in the protein structure caused by multi-amino acid mutations. In addition, only cell activation caused by constitutively-active CD4-TLR4 was studied in this report, and it was not elucidated whether tyrosine deficiencies affect ligand (LPS)-mediated TLR4 signaling. To address these questions, we first examined whether activation of NF-κB and MyD88-independent (41) RANTES reporters by constitutively-active CD4-TLR4 would be impaired by point tyrosine substitutions in the TIR domain. WT CD4-TLR4 and two CD4-TLR4 point mutants, Y674A and Y680A, were overexpressed in HEK293T cells co-transfected with NF-κB or RANTES reporters, and reporter induction was measured compared to a control (transfection with pcDNA3). The Y674A mutation completely abrogated the ability of CD4-TLR4 to activate NF-κB and RANTES reporters, showing signaling deficiency comparable to that of the P714H YFP-TLR4, whereas the Y680A substitution had a less pronounced effect (Fig. 3A). Next, we examined the effect of tyrosine deficiency in the TIR domain of CD4-TLR4 on activation of endogenous signaling intermediates, e.g., JNK and p38 MAPK phosphorylation and IκB-α degradation. Under conditions of comparable CD4-TLR4 protein expression, strong p38 and JNK phosphorylation and IκB-α degradation was elicited by WT CD4-TLR4, while CD4-TLR4 mutants deficient for 674Y or 680Y yielded little or no detectable MAPK phosphorylation and IκB-α degradation (Fig. 3B).

Fig. 3. Tyrosine-alanine substitutions in the TIR domain impair TLR4-induced signaling.

(A) and (B): HEK293T cells were transiently transfected with expression vectors encoding either CD4-TLR4 WT, CD4-TLR4 P714H mutant, or CD4-TLR4 species expressing Y674A and Y680A mutations. As a control, pCDNA3 was transfected. (A): NF-κB (pELAM-lucifearse) and RANTES (pGL3-RANTES-lucifearse) reporters were co-expressed along with pCMV-β-galactosidase reporter, cells were recovered for 24h, and luciferase vs. β-galactosidase activities were measured. *p<0.005, **p<0.05 reflect statistically significant differences in reporter activation between CD4-TLR4 WT and the indicated mutants. (B): TLR4 species were immunoprecipitated with α-CD4 Ab and subjected to immunoblotting with α-Flag Ab (total TLR4 expression) and α-phosphotyrosine Ab (TLR4 phosphorylation). (C) and (D): Full-length WT, P714H, and Y674A TLR4 species were overexpressed in HEK293T cells along with CD14 and MD-2, in control cultures, pcDNA3 transfection was carried out. (C): NF-κB (pELAM-lucifearse) and pCMV-β-galactosidase reporters were co-transfected, and cells were stimulated with medium or 100 ng/ml LPS for 24 h. Luciferase vs. β-galactosidase activities were measured in cell extracts. (D): After recovery for 24 h, YFP-TLR4 proteins were immunoprecipitated with α-GFP Ab, and total TLR4 expression was examined by immunoblotting with α-GFP Ab. (B) and (D): Cell lysates were subjected to Western analysis of JNK and p38 phosphorylation and IκB-α degradation. β-actin immunoblots show a loading control. The middle band in the JNK immunoblot represents an unspecific band. Results of a representative experiment (n=5) are presented.

To demonstrate functional significance of tyrosine deficiencies for LPS-mediated signaling, the Y674A and Y680A point mutations were introduced into the TIR domain of full-length YFP-TLR4. As a reliable means to compare the extent of inhibition, the P714H point mutation (homologous to the murine P712H mutation) was also created in the TIR domain of YFP-TLR4. WT, P714H mutant, or tyrosine-deficient (Y674A, Y680A) species of YFP-TLR4 were overexpressed in HEK293T cells (together with CD14 and MD-2 to enable LPS responsiveness), and LPS-induced NF-κB reporter activation, IκB-α degradation and phosphorylation of JNK and p38 MAPK were examined. Stimulation with LPS strongly induced NF-κB reporter activation (Fig. 3C), phosphorylation of p38 and JNK, and led to degradation of IκB-α (Fig. 3D) in cells overexpresssing WT YFP-TLR4. In contrast, the presence of the P714H and Y674A mutations led to complete inhibition of TLR4 to elicit these LPS-mediated responses, demonstrating that the agonist-mediated signal-transducing functions of TLR4 are also severely compromised in the Y674A tyrosine-deficient mutant. Similar to its lower inhibitory effect on the ability of constitutively active CD4-TLR4 to induce cellular activation, the Y680A YFP-TLR4 mutation also showed weaker suppression of LPS-induced activation of NF-κB reporter (Fig. 3C), and p38/JNK phosphorylation (data not shown). Together, these results show the importance of individual tyrosine residues in the TIR domain for enabling TLR4 signal-transducing capacities.

Mutations of Y674 and Y680 tyrosine residues in the TIR domain of TLR4 alter TLR4-MyD88 interactions, impair activation of IRAK-1 at the receptor and result in interaction with a short form of IRAK-1

In the next series of experiments, we sought to elucidate molecular mechanisms responsible for signaling incompetence of tyrosine-deficient TLR4. To this end, three distinct experimental approaches were employed to study the effect of the Y674A and Y680 mutations on TLR4 interactions with MyD88 and IRAK-1. First, we examined the effect of these mutations on the ability of constitutively-active CD4-TLR4 to interact with co-expressed YFP-MyD88 and endogenous IRAK-1. Since overexpressed MyD88 is known to activate IRAK-1 constitutively (43-46), and would be expected to compromise the ability of IRAK-1 to interact with TLR4, co-transfection with YFP-MyD88 was omitted in the case of IRAK-1-TLR4 interaction studies. In agreement with previously reported data (43-46), concomitant expression of constitutively active WT CD4-TLR4 and YFP-MyD88 in HEK293T cells resulted in ligand-independent TLR4-MyD88 associations (Fig. 4A). Surprisingly, the Y674A and Y680A CD4-TLR4s were also able to interact with YFPMyD88, and the degree of interaction correlated with their relative signaling deficiency (Fig. 4A). Specifically, WT CD4-TLR4 and the Y680A mutant with the weakest signaling deficiency showed the lowest associations, whereas non-signaling Y674A CD4-TLR4 exhibited the strongest interaction with MyD88 (Fig. 4A). Consistent with the ability of MyD88 to associate with TLR4 via their TIR domains and with IRAK-1 via death domains (72,73), we observed higher amounts of unmodified endogenous IRAK-1 in complex with the Y674A mutant compared to that associated with WT CD-TLR4 (Fig. 4B). Of great interest, tyrosine-deficient CD4-TLR4 variants, but not WT CD4-TLR4, interacted with a short form of IRAK-1 (Fig. 4B) that showed electrophoretic mobility similar to that reported for the IRAK-1c isoform, a negative regulator of TLR signaling (74).

Fig. 4. The effect of the Y674A and Y680A mutations on constitutive (CD4-TLR) and LPS-inducible (full-length YFP-TLR4) interactions of TLR4 with MyD88 and IRAK-1 species.

WT, Y674A and Y680A variants of CD4-TLR4 were overexpressed in HEK293T cells with (A) or without (B) YFP-MyD88. CD4-TLR4 species were immunoprecipitated with α-Flag-agarose, and TLR4 interactions with YFP-MyD88 (A) or IRAK-1 (B) were examined using α-GFP or α-IRAK-1 Abs. Total CD4-TLR4 expression was measured by immunoblot analysis of TLR4 immune complexes with α-Flag-HRP, and YFP-MyD88 expression was estimated in immunoprecipitated YFP-MyD88 using α-GFP Ab. (C) and (D): WT, Y674A YFP-TLR4 variants were co-expressed in HEK293T cells along with CD14 and MD-2. LPS stimulation was carried out as indicated (C) or for 5 min (D). YFP-TLR4s or endogenous MyD88 were immunoprecipitated, and TLR4, endogenous MyD88 and IRAK-1 expression was analyzed by immunoblotting with α-GFP, α-MyD88, and α-IRAK-1 Abs, respectively. Results of a representative experiment (n=4) are shown.

Next, we sought to examine the effect of tyrosine mutations in the TLR4 TIR domain on agonist-inducible TLR4-MyD88-IRAK-1 signalosome assembly. For this purpose, we used co-immunoprecipitation to study LPS-mediated recruitment of endogenous MyD88 and IRAK-1 to full-length WT, Y674A or Y680A YFP-TLR4s co-expressed in HEK293T cells along with CD14 and MD-2. The Y674A YFP-TLR4 mutant, but not WT YFP-TLR4, constitutively associated with endogenous MyD88 (Fig. 4C). LPS stimulation failed to increase these amounts that were significantly lower than MyD88 quantities recruited to WT YFP-TLR4 after LPS stimulation for 5 min (Fig. 4C). In striking contrast, LPS induced potent recruitment of endogenous MyD88 to WT YFP-TLR4 within 5 min, followed by rapid dissociation of MyD88 from the TLR4 receptor complex, as opposed to delayed MyD88 dissociation from the Y674A mutant (Fig. 4C). Co-immunoprecipitation analyses further revealed strong LPS-induced association of activated, phosphorylated IRAK-1 species with WT YFPTLR4, whereas significantly lower amounts of activated IRAK-1 interacted with the Y674A mutant (Fig. 4D). Figure 4 D further demonstrates that only the Y674A YFP-TLR4 mutant, but not WT YFP-TLR4, exhibited the ability to interact with a short form of IRAK-1, which corresponds by electrophoretic mobility to a negative regulator of TLR signaling, IRAK-1c. Taken together, these data indicate that tyrosine deficiency in the TIR domain of TLR4 results in constitutive interactions with endogenous MyD88 and a short isoform of IRAK-1 (most likely representing IRAK-1c), impairs LPS-inducible recruitment of MyD88 to TLR4, and suppresses phosphorylation of IRAK-1 at the receptor.

TLR2 and TLR4 agonists induce tyrosine phosphorylation of respective TLRs

Given the correlation between signal-transducing capacities of constitutively-active HA-ΔTLR4 and CD4-TLR4 proteins and their ability to undergo tyrosine phosphorylation, it was important to show agonist-induced tyrosine phosphorylation of unmodified, full-length TLR4. To this end, HEK293T cells were transiently transfected with pCDNA3-huTLR4, along with pCDNA3-huCD14 and pEFBOS-HA-huMD-2 expression vectors to impart LPS sensitivity. In addition to transiently transfected cells, we also used an HEK293 cell line stably expressing untagged human TLR4 and Flag-MD-2 to study TLR4 tyrosine phosphorylation. Since TLR2 has been shown to undergo tyrosine phosphorylation upon stimulation with heat-killed S. aureus (74), HEK293T cells were also transiently transfected with pCMV-1-Flag-TLR2, along with pCDNA3-huCD14 and pEFBOS-HA-huMD-2, and stimulated with two different TLR2 agonists, mycobacterial STF, and synthetic Pam3Cys. Thereafter, Flag-TLR2 and untagged TLR4 were immunoprecipitated from cell extracts with anti-Flag and anti-TLR4 Abs, respectively, and analyzed by immunoblotting for tyrosine phosphorylation of respective TLRs and for total TLR expression. Extending previously published data (74), both STF and Pam3Cys induced tyrosine phosphorylation of TLR2 in a time-dependent manner, with a response peaking within 5 min and declining by 15 min (Figs. 5A and B). TLR4 also exhibited tyrosine phosphorylation within 1−15 min following LPS stimulation in HEK293/TLR4/CD14/MD-2 transient and stable transfectants (Figs. 5C and 7B). These data show that agonist stimulation induced tyrosine phosphorylation of TLR2 and TLR4 in HEK293 cells overexpressing the respective TLRs.

Fig. 5. TLR2 and TLR4 agonists induce tyrosine phosphorylation of respective TLRs.

HEK293T cells were transiently transfected with pCDNA3-huCD14, pEFBOS-HA-huMD-2, along with either pCMV-1-Flag-huTLR2 (A,B) or pCDNA3-huTLR4 (C). Cells were recovered for 24 h, followed by stimulation with TLR2 (STF and Pam3Cys, A and B) and TLR4 (LPS, C) agonists. TLR2 and TLR4 were immunoprecipitated from cell extracts with α-Flag and α-TLR4 H80 Ab, respectively, and subjected to immunoblot analysis for total TLR2 (α-Flag Ab) or TLR4 (H80) expression, as well as TLR tyrosine phosphorylation (α-phosphotyrosine Ab PY20). Shown are data of one out of three experiments.

Fig. 7. Endotoxin tolerance induction inhibits TLR4 tyrosine phosphorylation in HEK293/TLR4/MD-2 stable transfectants but does not affect TLR4-MD-2 interactions.

Cells were pretreated with for 20 h medium or 10 ng/ml LPS, followed by washing and restimulation with 100 ng/ml LPS as shown. (A): RNA was isolated, reverse-transcribed, and analyzed by real-time PCR for IL-8 gene expression. *p<0.05. (B) TLR4 proteins were immunoprecipitated from cell extracts with α-TLR4 Ab H80 (B) or α-phosphotyrosine Ab PY20 (C). TLR4 immune complexes (B) were analyzed with α-phosphotyrosine Ab PY20 (TLR4 phosphorylation), α-TLR4 Ab H80 (TLR4 total expression), or α-Flag Ab to estimate amounts of Flag-MD-2 associated with TLR4. Immunoprecipitated phosphotyrosine proteins (C) were subjected to immunoblot analysis using α-TLR4 Ab H80. Total cellular extracts were analyzed by immunoblotting with α-TLR4 antiserum to measure total TLR4 protein expression (C). Results of a representative experiment (n=4) are depicted.

Protein tyrosine kinase and Src kinase inhibitors suppress LPS-inducible TLR4 tyrosine phosphorylation and signaling

Next, we sought to gain an initial insight into which kinases could be involved in TLR4 tyrosine phosphorylation and signaling. To this end, we used protein tyrosine kinase inhibitors, genistein and herbimycin A, as well as Src kinase inhibitors, PP1 and PP2, to examine whether inhibition of these kinases compromise LPS-mediated responses and/or TLR4 tyrosine phosphorylation. Based on the literature, pretreatment of cells with protein tyrosine kinase and Src kinase inhibitors before LPS stimulation was necessary because simultaneous addition of these inhibitors with LPS often failed to mediate a suppressive effect (70). For this reason, the effects of inhibitor pretreatment on LPS-inducible responses in HEK293/TLR4/MD-2 stable transfectants were examined. LPS stimulation resulted in marked phosphorylation of p38 evident in DMSO (vehicle control)-pretreated cells by 15 min (Fig. 6A), and strongly induced activation of the NF-κB reporter (Fig. 6B). LPS-mediated p38 phosphorylation was abolished by prior exposure of cells to protein tyrosine kinase inhibitors herbimycin A, genistein and Src kinase inhibitor PP1. Herbimycin A and Src kinase inhibitors PP1 and PP2 also exerted a strong inhibitory effect on LPS-induced NF-κB reporter activation. Furthermore, herbimycin A, genistein, and PP1 also completely abrogated LPS-inducible TLR4 tyrosine phosphorylation (Fig. 6A), suggesting an important role of Src kinases in LPS-mediated TLR4 tyrosine phosphorylation and signaling.

Fig. 6. The effect of protein tyrosine kinase and Src kinase inhibitors on TLR4 phosphorylation and signal transduction.

HEK293/TLR4/MD-2 stable transfectants were pretreated for 45 min with genistein (100 μM), herbimycin A (1 μM), PP1 and PP2 (10 μM each), followed by stimulation with LPS for 15 min (A) or 6 h (B). In NF-κB reporter assays (B), NF-κB (pELAM-luciferase) and β-galactosidase (pCMV-β-galactosidase) reporters were co-transfected. (A): TLR4 tyrosine phosphorylation was analyzed in TLR4 immune complexes and total cell lysates vs. TLR4 total expression using α-phosphotyrosine (PY20) and α-TLR4 Abs. LPS-inducible phosphorylation of p38 was measured with α-phosphop38 Ab, and equal protein loading was controlled by running β-actin immunoblot. (B). NF-κB reporter activation was examined in cell extracts by analysis of luciferase vs. β-galactosidase activities. Shown are results of a representative (n=4) experiments.

Induction of endotoxin tolerance correlates with suppressed LPS-inducible tyrosine phosphorylation of TLR4 and recruitment of Lyn kinase to TLR4 but does not affect TLR4-MD-2 interactions

Our previous studies showed that induction of endotoxin tolerance does not change total protein levels of TLR4 and MD-2, but suppresses MyD88 recruitment to TLR4 and IRAK-1 activation (63). In view of the crucial significance of TLR4 tyrosine phosphorylation for signal transduction (Figs. 1-4; 70,71), we next sought to determine whether LPS tolerance alters agonist-induced tyrosine phosphorylation of TLR4. First, we assessed the effect of endotoxin tolerance on tyrosine phosphorylation status of TLR4 in stably transfected HEK293/TLR4/MD-2 cells. To detect these modifications, we assessed both the proportion of tyrosine-phosphorylated species of TLR4 in total immunoprecipitated TLR4 proteins, and, reciprocally, the proportion of TLR4 species among total phosphotyrosine proteins immunoprecipitated with anti-phosphotyrosine Ab. LPS caused a strong increase in steady-state levels of IL-8 mRNA (Fig. 7A) and TLR4 tyrosine phosphorylation, which was detected within 1−5 min in medium-pretreated cells (Figs. 7B and C). Tolerant cells showed reduced LPS-mediated IL-8 mRNA expression (Fig. 7A) and no detectable tyrosine phosphorylation of TLR4 (Figs. 7B and C). Total TLR4 levels were not affected by tolerance induction, confirming our previous findings (62,63) and the results reported by others (57,59).

In our previous study, we showed that endotoxin tolerance does not modulate TLR4 and MD-2 protein levels (63,64). Because of the importance of relative stoichiometry of expressed MD-2 molecules in enabling optimal LPS responsiveness to TLR4 (75), the ability of MD-2 to associate with TLR4 was next examined in medium-pretreated and LPS-tolerized HEK293T/TLR4/MD-2 stable transfectants. Coimmunoprecipitation analysis revealed comparable amounts of MD-2 associated with TLR4 in control and LPS-tolerant cells regardless of whether or not cells were stimulated with LPS (Fig. 7B, third panel). These data indicate that endotoxin tolerance does not alter the ability of MD-2 to interact with TLR4.

To confirm our results obtained in the overexpression system, we examined the effect of endotoxin tolerance on tyrosine phosphorylation of endogenous TLR4 in human monocytes. LPS pretreatment of monocytes significantly inhibited LPS-mediated IκB-α degradation, p38 phosphorylation, and TNF-α gene expression compared to responses observed in medium-pretreated cells (Figs. 8A, B), demonstrating LPS tolerance induction. LPS-tolerant human monocytes showed suppressed LPS-inducible up-regulation of both MyD88-dependent (e.g., TNF-α) and MyD88-independent (e.g., IFN-β) genes (Fig. 8B), extending our previous findings obtained in mouse macrophages (76). Figure 8C shows that LPS failed to elicit tyrosine phosphorylation of endogenous TLR4 in endotoxin-tolerant human monocytes, in contrast to the strong response observed in control cells.

Fig. 8. Suppressed LPS-inducible tyrosine phosphorylation of TLR4 in endotoxin-tolerant human monocytes.

Human monocytes were pretreated for 20 h with medium or 10 ng/ml LPS, washed and restimulated with 100 ng/ml LPS over time courses (A and C), or for 1h (IFN-β mRNA induction) and 3 h (TNF-α gene expression, B). *p<0.005; **p<0.05. Cellular extracts were examined by immunoblotting for IκB-α degradation and p38 phosphorylation (A). B: RNA was isolated and subjected to reverse transcription and real-time PCR analysis of TNF-α and IFN-β mRNA levels. (C) Cellular extracts were subjected to immunoprecipitation with α-TLR4 Ab H80 followed by immunoblotting with α-phosphotyrosine Ab PY20 (TLR4 tyrosine phosphorylation). Total TLR4 expression was analyzed by immunoblotting with α-TLR4 antiserum. Results of a representative experiment (N =7) are shown.

Our data using protein tyrosine kinase and Src kinase inhibitors suggest the role for Src kinases in mediating TLR4 tyrosine phosphorylation and signal transduction. To elucidate further whether Src kinases play a role in TLR4 phosphorylation, we studied whether one of the Src kinase family members, Lyn, is recruited to TLR4 upon LPS stimulation and how induction of LPS tolerance may affect this process. For this purpose, Flag-tagged Lyn was overexpressed in HEK293/TLR4/MD-2 stable transfectants, cells were pretreated with medium or tolerized with LPS, and LPS-inducible recruitment of Lyn to TLR4 was examined by co-immunoprecipitation. LPS stimulation of medium-pretreated cells led to a strong recruitment of Flag-Lyn to TLR4 within 1−5 min, with a maximal response reached by 15 min (Fig. 9). In contrast, very little Lyn was found in association with TLR4 in HEK293/TLR4/MD-2 cells rendered endotoxin-tolerant (Fig. 9). Taken together, these data show that induction of endotoxin tolerance results in suppression of LPS-inducible tyrosine phosphorylation of TLR4 and recruitment of Lyn to TLR4, as well as the failure of LPS to induce MyD88-dependent and MyD88-independent signaling, but does not affect TLR4-MD-2 interactions.

Fig. 9. LPS-induced recruitment of Lyn to TLR4 is suppressed in LPS-tolerant HEK/TLR4/MD-2 cells.

Flag-Lyn was overexpressed in HEK293/TLR4/MD-2 stable transfectants, cells pretreated for 20 h with medium or 10 ng/ml LPS, followed by LPS stimulation as shown. Recruitment of Flag-Lyn to TLR4 was examined in TLR4 immunoprecipitates by immunoblotting with α-Flag-HRP or in Flag-Lyn immunoprecipitates immunoblotted with α-TLR4 antibody. Total expression of Flag-Lyn was measured in Lyn immune complexes with α-Flag-HRP, and total TLR4 expression was determined by immunoblotting of cellular extracts with α-TLR4 antibody. β-actin immunoblot was used to control for protein loading. The results of a representative experiment (n=3) are presented.

DISCUSSION

Tyrosine phosphorylation regulates signal transducing functions of many receptors, including TLR2 and TLR3 (71,74,77). Chen et al. (70) reported that LPS induces tyrosine phosphorylation of endogenous TLR4 in human monocytes, and Ronni et al. (71) showed abolishment of activation of NF-κB-, AP-1-, and C/EBP-dependent reporter plasmids by CD4-TLR4 fusion proteins with several tyrosine-alanine substitutions in their TIR domain. However, the molecular mechanisms by which alterations in tyrosine phosphorylation of the TIR domain of TLR4 affect signaling have not been elucidated. It is also not known whether the endotoxin-tolerant phenotype is secondary to interference with post-translational modifications of TLR4. In this paper, our data support the hypothesis that agonist-induced engagement of TLRs leads to post-translational modifications within the TIR domain that may play an important role in creating docking platforms for the recruitment of downstream signal-competent adapter proteins (e.g., MyD88) and kinases (e.g., IRAK-4 and IRAK-1). Our results also suggest that one mechanism by which endotoxin tolerance inhibits recruitment of MyD88 to TLR4 and IRAK-1 activation could operate by altering TLR4 tyrosine phosphorylation, creating a “signal-incompetent” TLR4 docking platforms. This is, in turn, translated into preferential association between hypophosphorylated TLR4 and a short isoform of IRAK-1 (possibly IRAK-1c), and might result in recruitment of other negative regulators of TLR4 signaling. Finally, the ability of protein tyrosine kinase and Src kinase inhibitors to block TLR4 tyrosine phosphorylation and signal transduction, coupled with the demonstration of LPS-mediated recruitment of Lyn to TLR4 and its inhibition in LPS-tolerant cells, suggest the involvement of Lyn in TLR4 tyrosine phosphorylation, signaling and tolerance.

Several lines of evidence presented in this paper suggest that post-translational modifications regulate the signal-transducing functions of TLR4. First, introduction of the P712H mutation into the TIR domain of a constitutively active mouse ΔTLR4 abrogated receptor tyrosine phosphorylation and blocked its ability to elicit IRAK-1 phosphorylation and activation of p38 and NF-κB (Fig. 1). Secondly, expression of the PGV714−716AAA mutation (encompassing the homologous P712H mouse mutation) in another constitutively active TLR4 construct, human CD4-TLR4, rendered it signal-incompetent and incapable of undergoing tyrosine phosphorylation (Fig. 2). Both the P712H ΔTLR4 and P714A CD4-TLR4 mutants failed to activate IRAK-1 (Figs. 1 and 2), and the mutation of P714 blocked activation of NF-κB and RANTES reporters. Furthermore, the P714H TLR4 mutants were incapable of eliciting constitutive (CD4-TLR4) and LPS-inducible (YFP-TLR4) phosphorylation of p38 and JNK, degradation of IκB-α, and NF-κB and RANTES reporter activation (Figs. 3 and 4). These results demonstrate functional significance of TLR4 tyrosine phosphorylation for activation of both MyD88-dependent and MyD88-independent signaling cascades and indicate that the conserved proline is necessary for this to occur. Third, mutation of two tyrosine residues within the TIR domain of TLR4 led to profound suppression of TLR4-mediated phosphorylation of JNK and p38 MAPK, degradation of IκB-α, and activation of NF-κB and RANTES reporters (Fig. 3). Finally, we demonstrate that TLR2 and TLR4 agonists induce tyrosine phosphorylation of respective TLRs overexpressed in HEK293T cells, as well as endogenous TLR4 in human monocytes (Figs. 5, 7, 8). Our findings indicate that TLR4 tyrosine phosphorylation is an important post-translational modification necessary for LPS signaling.

To the best of our knowledge, this paper demonstrates for the first time that inhibition of LPS-mediated MyD88-dependent and MyD88-independent signaling cascades in endotoxin-tolerant HEK293/TLR4/MD-2 cells and human monocytes correlates with the inability of TLR4 to undergo tyrosine phosphorylation. It is noteworthy that inhibited tyrosine phosphorylation of the P712H ΔTLR4 mutant (Fig. 1D) correlates with its failure to induce p38 phosphorylation, IκB-α degradation(Figs. 1C and D) and NF-κB reporter activation (71). It is also intriguing that the mouse P712H mutation and a functionally homologous P714A substitution both abolished the capacity of constitutively-active mouse ΔTLR4 and human CD4-TLR4 to activate IRAK-1 (Figs. 1 and 2C). These data, coupled with blocked TLR4 tyrosine phosphorylation (Figs. 7 and 8), impaired recruitment of MyD88 to TLR4, and suppressed IRAK-1 activation observed in endotoxin-tolerant human monocytes (63) suggest the existence of a “non-signaling,” nonphosphorylated conformation of TLR4. Since proper TLR4 complex assembly with adapter proteins is important for recruitment and activation of IRAKs (1-3,72,73,78), it was of importance to delineate whether deficient tyrosine phosphorylation of TLR4 alters recruitment of downstream adapter proteins and kinases. Coimmunoprecipitation studies revealed that tyrosine-deficient CD4-TLR4 species were not compromised in their abilities to associate with co-expressed YFP-MyD88, and, in contrast to WT CD4-TLR4, interacted with a short isoform of IRAK-1, most likely representing IRAK-1c (Fig. 4A and B). Because the use of constitutively active CD4-TLR4 and YFP-MyD88 could yield artificial results, our next approach was to employ full-length YFP-TLR4 that are incapable of autoactivation but mediate signal transduction in conjunction with CD14 and MD-2 when activated by LPS (34). This paper shows that the tyrosine-deficient Y674A YFP-TLR4 mutant, but not WT YFP-TLR4, constitutively associated with endogenous MyD88 (Fig. 4C). In contrast to potent LPS-inducible recruitment of endogenous MyD88 to WT YFP-TLR4 with the peak response at 5 min, MyD88 amounts associated with the Y674A mutant remained unchanged and were significantly lower (Fig. 4C). This is reminiscent of the failure of LPS to mediate recruitment of MyD88 to TLR4 observed in endotoxin-tolerant human monocytes (63). It is noteworthy that although the Y674A and Y680A TLR4 species were not compromised in their capacities to interact with unmodified IRAK-1, IRAK-1 activation at the mutant Y674A TLR4 was severely inhibited, as judged by suppressed association of activated, phosphorylated IRAK-1 with tyrosine-deficient TLR4 (Fig. 4D). It is interesting to note that our recent studies demonstrated that truncated, kinase-deficient mutants of IRAK-4 expressed in a patient hyporesponsive to LPS and suffering from recurrent infections also exhibit increased association with MyD88 and inability to interact with activated IRAK-1 (68).

The findings presented herein directly demonstrate increased associations of tyrosine-deficient TLR4 variants with a short isoform of IRAK-1 whose electrophoretic mobility corresponds to IRAK-1c (Fig. 4B and D). IRAK-1c acts as a negative regulator of TLR4 signaling, as evidenced by suppression of IL-1 and LPS-mediated activation of NF-κB and production of TNF-α and IL-6 (79). That IRAK-1c shows impaired interactions with IRAK-4, fails to be phosphorylated by IRAK-4 (79), and exhibits increased associations with tyrosine-deficient TLR4 (Fig. 4) may explain compromised activation of IRAK-1 at the Y674A TLR4 mutant. It is tempting to speculate that preferential accumulation of IRAK-1c within hypophosphorylated TIR domains of tolerized TLR4 may account for impaired IRAK-1 phosphorylation and activation widely observed in tolerance (54,63). In addition, we cannot exclude a possibility of preferential recruitment of another negative regulator of TLR signaling, an alternatively-spliced, short form of MyD88 (MyD88s), to tyrosine-deficient (e.g., Y674A) or tolerized, hypophosphorylated TLR4 variants. While not deficient in its interactions with IL-1R and IRAK-1, MyD88s fails to interact with IRAK-4 because of its lack of the intermediate domain critical for MyD88-IRAK-4 associations, leading to the failure of IRAK-1 phosphorylation and activation upon assembly of IL-1R/MyD88s/IRAK-1 complexes (80,81). Finally, defective tyrosine phosphorylation within the TLR4 TIR domain could alter recruitment and/or activation of IRAK-4. Studies are in progress to address the exact molecular mechanism by which tyrosine deficiencies in the TLR4 TIR domain impair LPS-inducible MyD88 recruitment and IRAK-1 activation at the receptor complex.

It remains to be determined whether inhibition of TLR4 tyrosine phosphorylation occurs due to modulation of expression levels and/or activities of tyrosine kinases or phosphatases. Several tyrosine kinases have been implicated in TLR4-mediated signaling, including Bruton's tyrosine kinase (82,83), as well as p56^lyn, p58^hck, and p59^c-fgr (84,85). However, a possible role for tyrosine kinases for TLR4 signaling was questioned by Delude et al. (86), since inhibition of LPS-induced tyrosine kinase activity with genistein failed to inhibit LPS-mediated NF-κB translocation. Nevertheless, a recently published report on involvement of c-Src tyrosine kinase in TLR3 tyrosine phosphorylation and signaling (87) further reinforces the importance of Src kinases in TLR phosphorylation and activation. In this paper, we demonstrate that protein tyrosine kinase and Src kinase inhibitors blocked LPS-mediated TLR4 tyrosine phosphorylation, activation of p38 MAPK and induction of NF-κB reporter activity (Fig. 6). Furthermore, LPS stimulation of HEK293/TLR4/MD-2 stable transfectants led to recruitment of overexpressed Flag-Lyn to TLR4, and induction of endotoxin tolerance significantly inhibited Lyn-TLR4 association (Fig. 9). Although further experiments using Lyn gene silencing techniques and cells obtained from Lyn^−/− mice are necessary to unambiguously demonstrate whether Lyn is responsible for TLR4 phosphorylation, our data presented herein support the hypothesis that Src tyrosine kinases are involved in LPS-induced TLR4 phosphorylation and its modulation in endotoxin tolerance. In addition to altered expression and/or activities of protein kinases, changes in expression and enzymatic activity of phosphatases may also contribute to suppressed TLR4 tyrosine phosphorylation in tolerant cells. In this regard, Ropert et al. (88) reported that induction of tolerance to LPS coincided with increased phosphatase activity, and treatment of tolerant cells with okadaic acid, an inhibitor of phosphatase activity, restored TLR4-mediated signaling, reversing the tolerant phenotype. Nimah et al. (89) demonstrated increased expression of the dual specificity phosphatase, MAP kinase phosphatase-1 (MKP-1), during tolerance that regulates TNF-α expression by inhibiting p38 activity. In addition, MKP-1^−/− macrophages exhibited a significantly reduced capacity to become tolerant to LPS (89). Since the cytosolic phosphatase SHIP has been shown to be required for the development of an LPS-refractory state (90), these data further support a role for altered phosphatase activity in tolerance.

Overexpression experiments and studies in knockout mice have unambiguously shown that MD-2 is indispensable for conferring LPS sensitivity to TLR4 (30-34). Therefore, it was of importance to elucidate whether endotoxin tolerance affects TLR4-MD-2 interactions that may be a consequence of altered TLR4 tyrosine phosphorylation. However, we observed similar amounts of MD-2 associated with TLR4, regardless of LPS stimulation or endotoxin tolerance induction, indicating that suppressed TLR4-mediated signaling in tolerized cells is not due to an altered TLR4-MD-2 association. Given the role of MD-2 in LPS binding and presentation to TLR4 (33,34), our data are also consistent with the finding that similar amounts of labeled LPS bind to control and endotoxin-tolerant cells (91).

In conclusion, our data demonstrate that tyrosine phosphorylation of TLR4 is required for its ability to signal. We also show a correlation between abolished TLR4 tyrosine phosphorylation and suppressed LPS-mediated p38 MAPK phosphorylation, IκB-α degradation, and expression of MyD88-dependent and MyD88-independent cytokines. Similar suppression of TLR4 signaling could be recapitulated in P712H murine and P714H human TLR4 mutants that also show deficient tyrosine phosphorylation, and, conversely, tyrosine deficiency in the TIR domain of TLR4 is associated with its signaling incompetence. These results suggest an important role for the loss of LPS-induced TLR4 tyrosine phosphorylation in tolerance that is manifested at the molecular level by impaired recruitment of MyD88 to TLR4 and IRAK-1 activation. However, association of an extracellular accessory protein, MD-2, with TLR4 was found to be unaltered in LPS-tolerized cells. Suppressed LPS-inducible recruitment of endogenous MyD88 and increased association of IRAK-1c isoform with tyrosine-deficient TLR4 suggest that changes in TLR4 tyrosine phosphorylation interfere with its ability to provide proper signaling platforms for recruitment of signaling-competent intracellular adapter proteins and kinases, resulting in diminished TLR signaling in tolerized cells.