14-3-3ϵ and 14-3-3σ Inhibit Toll-like Receptor (TLR)-mediated Proinflammatory Cytokine Induction*

Background: TLRs are key sensors of viral and bacterial components and lead to the production of proinflammatory cytokines. Results: 14-3-3ϵ and 14-3-3σ proteins curtail TLR2-, TLR3-, TLR4-, TLR7/8-, and TLR9-mediated proinflammatory cytokine induction. Conclusion: 14-3-3ϵ and 14-3-3σ play a critical, hitherto underappreciated role in modulating TLR functionality. Significance: Learning how TLRs are modulated is crucial for understanding innate immunity. Toll-like receptors (TLRs) are a group of pattern recognition receptors that play a crucial role in the induction of the innate immune response against bacterial and viral infections. TLR3 has emerged as a key sensor of viral double-stranded RNA. Thus, a clearer understanding of the biological processes that modulate TLR3 signaling is essential. Limited studies have applied proteomics toward understanding the dynamics of TLR signaling. Herein, a proteomics approach identified 14-3-3ϵ and 14-3-3σ proteins as new members of the TLR signaling complex. Toward the functional characterization of 14-3-3ϵ and 14-3-3σ in TLR signaling, we have shown that both of these proteins impair TLR2, TLR3, TLR4, TLR7/8, and TLR9 ligand-induced IL-6, TNFα, and IFN-β production. We also show that 14-3-3ϵ and 14-3-3σ impair TLR2-, TLR3-, TLR4-, TLR7/8-, and TLR9-mediated NF-κB and IFN-β reporter gene activity. Interestingly, although the 14-3-3 proteins inhibit poly(I:C)-mediated RANTES production, 14-3-3 proteins augment Pam3CSK4, LPS, R848, and CpG-mediated production of RANTES (regulated on activation normal T cell expressed and secreted) in a Mal (MyD88 adaptor-like)/MyD88-dependent manner. 14-3-3ϵ and 14-3-3σ also bind to the TLR adaptors and to both TRAF3 and TRAF6. Our study conclusively shows that 14-3-3ϵ and 14-3-3σ play a major regulatory role in balancing the host inflammatory response to viral and bacterial infections through modulation of the TLR signaling pathway. Thus, manipulation of 14-3-3 proteins may represent novel therapeutic targets for inflammatory conditions and infections.

The human innate immune system provides the first line of defense against various infectious agents, such as bacteria, viruses, parasites, and helminths. Toward the effective functioning of innate immunity, various classes of protein-based receptors serve to sense various danger-associated molecular patterns and pathogen-associated molecular patterns (PAMPs) 2 (1). These different classes of pathogen recognition receptors include membrane-bound pathogen recognition receptors, such as Toll-like receptors (TLRs); receptor kinases; mannose receptors; cytoplasmic pathogen recognition receptors, such as nucleotide oligomerization domain receptors, the RIG-I (retinoic acid-inducible gene I)-like receptor (RLR) family, and the recently described AIM2 and DAI cytosolic DNA receptors, the activation of which leads to the production of proinflammatory cytokines and type I interferon (IFN) (2).
TLR3 plays a critical role in innate immunity because it serves to recognize viral dsRNA originating from dsRNA viruses, such as reovirus (4). TLR3 also recognizes dsRNA produced during the replication of many viruses, including ssRNA viruses, such as West Nile virus, respiratory syncytial virus, and encephalomyocarditis virus. In addition, TLR3 also recognizes a synthetic analog of dsRNA known as polyriboinosinic:polyribocytidylic acid (poly(I:C)) (3). It has been shown that activation of TLR3 using poly(I:C) inhibits HIV infection by increasing the expression of type I IFN antiviral factors, thus restricting HIV expression and replication (8). Also, poly(I:C) has proven beneficial as a mucosal adjuvant for an influenza virus vaccine in a murine infection model (4). Activation of TLR3 signaling has also been linked to the inhibition of tumor cell growth (9) and to amplification of inflammation following the sensing of necrosis in viral dsRNA-independent settings, such as experimental polymicrobial septic peritonitis and ischemic gut injury (10). Given the pleiotropic role exerted by TLR3 in innate immunity, it is essential that the molecular mechanisms that serve to modulate its functionality be fully deciphered. To this end, we have shown that Mal and MyD88 have the ability to inhibit TLR3-mediated IFN-␤ production (7,11). Further, we have shown that the transcription factor YinYang1 (YY1), induced by poly(I:C), translocates to the nucleus, where it interacts with the IFN-␤ promoter and inhibits the binding of IRF-7 to the latter (12).
Two-dimensional Gel Electrophoresis-Whole cell lysates were extracted from WT and MAVS Ϫ/Ϫ iBMDMs following stimulation with poly(I:C) for various times (0, 1.5, 8, and 24 h). The proteins were precipitated using the acetone precipitation method followed by incubation in lysis buffer (7 M urea, 4% CHAPS, 2 M thiourea, 100 mM DTT, 5% ampholytes, and one protease inhibitor mixture tablet (PICS)/50 ml of lysis buffer). Protein separation by two-dimensional gel electrophoresis in the first dimension was performed by isoelectric focusing using 24-cm pH 4 -7 IPG strips (GE Healthcare) and in the second dimension by SDS-PAGE. Rehydration of IPG strips, isoelectric focusing, equilibration of focused strips, and SDS-PAGE second dimensional separation was carried out as described previously (22). The separated proteins were visualized by silver staining (23), and high resolution gel images where scanned using a Typhoon Trio variable mode imager from GE Healthcare. Comparative and statistical analysis of two-dimensional gels was performed with the Progenesis software program from Non-Linear Dynamics (Newcastle, Tyne, UK).
Mass Spectrometry Analysis (Progenesis, MS, Mascot)-Differentially expressed proteins were subjected to in-gel trypsin digestion, and the resulting peptides were analyzed by peptide mass fingerprinting using an Ion Trap LC/MS apparatus from Agilent Technologies (model 6430). Excision, washing, destaining, trypsin digestion, and peptide recovery was performed as described previously (22). Peptides were separated using a nanoflow Agilent 1200 series system, equipped with a Zorbax 300SB C18 5-m, 4-mm, 40-nl precolumn and a Zorbax 300SB C18 5-m, 43 mm ϫ 75-m analytical reversed phase column using HPLC-Chip technology, and 0.1% formic acid was used as mobile phase A, and 50% acetonitrile, 0.1% formic acid was used as mobile phase B. Samples were loaded at a flow rate of 4 l/min onto the enrichment column, and the peptide fragments were eluted with a constant nanopump flow rate of 0.6 ml/min. The capillary voltage was set to 1900 V, and the flow and the temperature of the drying gas were 4 liters/min and 300°C, respectively. Database searches were performed using Mascot MS/MS ion search (Matrix Science, London, UK). All pI values and molecular masses of the identified proteins were compared with the relative position of their corresponding two-dimensional spots on analytical slab gels.
First Strand cDNA Synthesis-Total cellular RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Thereafter, 1 g of total RNA was mixed with 1 l of random hexamers (500 g/ml), and water was added to a final volume of 17 l and incubated for 5 min at 70°C. The mixture was then briefly centrifuged and chilled on ice for 2 min. Thereafter, the other reaction components were added in the following order: 5 l of 5ϫ RT buffer, 1.3 l of 10 mM dNTP, 0.7 l of RNasin (Promega), 1 l of Moloney murine leukemia virus reverse transcriptase (Promega), and nucleasefree water to a total volume of 25 l. Reactions were incubated at 37°C for 40 min followed by 42°C for 40 min and heating to 80°C for 5 min. The first strand cDNA was stored at Ϫ20°C for up to 1 month.
Reporter Gene Assays-HEK293 and HEK293-TLR2, -3, -4, -7/8, and -9 cells (5 ϫ 10 4 cells/well; 96-well plate) were transfected with 80 ng/well luciferase reporter gene plasmid for NF-B, IFN-␤, and CCL5 and cotransfected with the expression vectors encoding 14-3-3⑀ or 14-3-3 and MyD88, Mal, TRIF, or TRAM using Lipofectamine as described previously (7). A total of 40 ng/well phRL-TK (TK-Renilla-luciferase) reporter gene was co-transfected simultaneously to normalize data for transfection efficiency. After 24 h, cells were stimulated with ligands as indicated for a further 24 h. Thereafter, cell lysates were prepared and reporter gene activity was measured using the Dual-Luciferase assay system (Promega). Data are expressed as the mean -fold induction relative to control levels, for a representative experiment from a minimum of three separate experiments, each performed in triplicate.
Extraction of Cellular Nuclear Fraction-14-3-3⑀, 14-3-3, and control esiRNA transfected U373-CD14 cells were stimulated with Pam 3 CSK 4 , poly(I:C), LPS, R848, and CpG for the indicated times. After ligand stimulations, the cells were col-lected, and nuclear extracts were prepared using the nuclear extraction kit as described by the manufacturer (Cayman Chemical).
Transfection and Co-immunoprecipitation-HEK293, HEK293-TLR3, and U373-CD14 cells (1 ϫ 10 6 cells/well; 6-well plate) FIGURE 1. 14-3-3 proteins are novel components of TLR signaling pathway. A, experimental workflow. Wild-type and MAVS Ϫ/Ϫ iBMDMs were stimulated with poly(I:C) (20 g/ml) for a range of times (0, 1.5, 8, and 24 h). The proteins from the cells were extracted using acetone precipitation, and samples were subjected to two-dimensional gel electrophoresis as described under "Experimental Procedures." Gel cross-comparison was performed using Progenesis software, and differentially expressed protein spots were picked, digested with trypsin, and identified using nano-LC-MS/MS as described under "Experimental Procedures." The data analysis was performed using the MASCOT database, and biological and cellular characteristics of the proteins were recorded. B and C, average -fold change of the differentially expressed protein spots shown as log normalized volumes comparing the protein spot volume in gels stimulated for 1.5, 8, and 24 h with poly(I:C) as compared with the control gels (0 h). D, RT-PCR was performed to verify the absence of MAVS adaptor in MAVS Ϫ/Ϫ iBMDMs using forward and reverse primers. The samples were diluted 20ϫ and were subjected to DNA gel electrophoresis. GAPDH was used to normalize all samples, and -fold changes were determined relative to control. Wild-type iBMDMs (E-I), U373-CD14 (J-N), and synoviocyte cells (O-S) were stimulated with Pam 3 CSK 4 (1 g/ml), poly(I:C) (20 g/ml), LPS (1 g/ml), R848 (1 g/ml), and CpG (5 g/ml) for a range of times (0, 0.5, 1.5, 3, 4, and 8 h), and total RNA was isolated, converted to first strand DNA, and used as a template for quantitative real-time RT-PCR as described under "Experimental Procedures" to assay the expression levels of 14-3-3⑀ and 14-3-3. The data presented are representative of at least three independent experiments performed in triplicate (mean Ϯ S.E. (error bars)). Statistical analysis was performed using unpaired Student's t test and two-way ANOVA tests comparing the test samples with their respective controls.
were transfected with the indicated plasmids, whereby the total amount of DNA (3 g/well) was kept constant using Lipofectamine 2000 (Invitrogen). After 24 h, cells were stimulated with the indicated ligands followed by cell lysis in 600 l of lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 2 mM EDTA, pH 8.0, 1% Nonidet P-40, 0.5% sodium deoxycholate supplemented with 1 mM PMSF, 1 mM, DDT, 1 mM NaVO 3, 5 mM EGTA, and 1 tablet/10 ml of PICS) and left on ice for 20 min. The lysates were subjected to centrifugation for 10 min at 14,000 rpm to remove cell debris. Next, 2 g of the indicated antibody was incubated with 70 l of A/G Plus-agarose beads (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) overnight at 4°C, washed, and incubated with the cell lysates for 2 h at 4°C. The immune complexes were precipitated, washed, and eluted by the addition of Laemmli loading buffer followed by SDS-PAGE and immunoblotting using the indicated antibodies.
Data Analyses-Unpaired Student's t test and two-way ANOVA tests were used to carry out statistical analysis of data.

14-3-3 Proteins Are Novel Components of the TLR Signaling
Pathway-In order to explore the molecular mechanisms that modulate TLR3 functionality following viral sensing, iBMDMs were stimulated with the synthetic TLR3 ligand, poly(I:C), followed by an assay of changes in protein expression using two-dimensional gel electrophoresis. Although poly(I:C) is sensed by TLR3, it may also be sensed by the RLRs RIG-1 and Mda-5, which utilize the signaling adaptor MAVS (24); thus, MAVS Ϫ/Ϫ iBMDMs were included in the study as a control to preclude the signaling proteins induced through the cytosolic RLRs. Following stimulation of WT and MAVS Ϫ/Ϫ iBMDMs with poly(I:C) for 0 -24 h and two-dimensional gel electrophoresis, differentially expressed proteins were selected using Progenesis software and were then subjected to in-gel trypsin digestion followed by nano-LC-MS/MS (Fig. 1A). A number of proteins were found to be differentially expressed (up-/down-regulated) at all time points following poly(I:C) stimulation of WT iBM-DMs when compared with control (0 h) iBMDMs (Table 1). Importantly, we found that several members of the 14-3-3 protein family (14-3-3 protein , ␦, , ␤, ␣, ⑀, and ) were suppressed upon stimulation with poly(I:C) for 1.5 and 8 h. Similarly, suppression of 14-3-3 expression was also observed in MAVS Ϫ/Ϫ iBMDMs following poly(I:C) stimulation, indicating that poly(I:C)-mediated suppression of 14-3-3 proteins occurs through TLR3 rather than through the RLRs (Table 2). Although a limited number of studies have described a role for 14-3-3 proteins in TLR signaling (18,25), their functional characterization and role in TLR signaling require further clarification. Given this, we opted to validate and functionally characterize the role of two 14-3-3 proteins, 14-3-3⑀ and 14-3-3, in TLR signaling. Statistical comparison of the two-dimensional Mass spectrometry identification of differentially expressed protein spots in wild-type BMDMs stimulated with poly(I:C) (20 g/ml) over a range of times (0, 1.

5, 8, and 24 h)
The -fold change represents an average differential change of the protein spot over time compared with the control (0 h). The experiment and two-dimensional gel electrophoresis for each stimulation time point were performed in triplicate as independent experiments (mean Ϯ S.E.). Ϫ4.8 0.05 gels using Progenesis software showed comparable suppression of 14-3-3⑀ and 14-3-3 protein expression in both WT and MAVS Ϫ/Ϫ BMDMs following stimulation with poly(I:C) for 1.5 and 8 h (Fig. 1, B and C). Thus, the absence of MAVS expression does not significantly affect the modulatory effects of poly(I:C) on 14-3-3⑀ and 14-3-3 mRNA expression and confirms that poly(I:C) mediates its effects primarily through TLR3 in iBMDMs. An absence of MAVS adaptor expression in MAVS Ϫ/Ϫ BMDMs was confirmed using RT-PCR (Fig. 1D).
Common Biological Processes Modulated by 14-3-3⑀ and 14-3-3 in TLR Signaling Networks-Upon identification and validation of MyD88, Mal, TRIF, TRAM, TRAF3, and TRAF6 as interacting partners of 14-3-3⑀ and 14-3-3, we opted to investigate whether 14-3-3⑀ and 14-3-3 and their interacting proteins, MyD88, Mal, TRIF, TRAM, TRAF3, and TRAF6, are involved in the co-regulation of similar biological processes and diseases. To do this, Pathway Studio Software (Ariadne Genomics) was used to investigate and integrate the search for biological processes and diseases co-associated with 14-3-3 proteins and their interacting partners. Assimilated data demonstrated that both 14-3-3⑀ and 14-3-3 and their interacting partners play a regulatory role in biological processes, such as cytokine production and the inflammatory response (Fig. 9A). 5. 14-3-3⑀ and 14-3-3 inhibit TLR adaptor-dependent NF-B and IFN-␤ reporter gene activity but differentially affect CCL5 reporter gene activity. A-L, HEK293 cells were co-transfected with vectors encoding a reporter gene for either the IFN-␤, NF-B, or CCL5 promoter (80 ng) and either empty vector (40 ng), MyD88, MAL, TRIF, or TRAM plasmids (10 ng) and increasing amounts of an expression vector encoding 14-3-3⑀ or 14-3-3 (0, 1, 5, 10, 20, or 30 ng), as indicated. A total of 40 ng/well phRL-TK (TK-Renilla-luciferase) reporter gene was co-transfected simultaneously to normalize data for transfection efficiency After 24 h, cell lysates were harvested and assessed for luciferase reporter gene activity. The data presented are representative of at least three independent experiments performed in triplicate (mean Ϯ S.E. (error bars)). Statistical analysis was performed using unpaired Student's t test and two-way ANOVA tests comparing the test samples with their respective controls.

DISCUSSION
TLRs are critically involved in mediating a normal physiological response to invading pathogens toward their elimination, and it is becoming increasingly appreciated that TLRs are involved in inflammatory pathologies (26 -28). The innate immune system has evolved in such a way to provide many strategies to limit TLR functionality with a view to curtailing unwanted inflammatory responses (7,11). Given the central role played by TLRs toward the sensing of PAMPs and dangerassociated molecular patterns and emanation of the inflammatory response, albeit inappropriate in certain cases, we sought to investigate novel mechanisms that may have evolved toward the modulation of TLR functionality. Given that dsRNA is one of the most important viral PAMPs, we opted to investigate the immune responses that are instigated upon exposure of iBMDMs to synthetic dsRNA, namely poly(I:C). Using an integrated quantitative proteomics approach, we have identified 14-3-3⑀ and 14-3-3 proteins as critical new regulators of the TLR2, TLR3, TLR4, TLR7/8, and TLR9 signal transduction pathways. Briefly, upon stimulation of murine iBMDMs with Pam 3 CSK 4 , poly(I:C), LPS, R848, and CpG, ligand-dependent degradation of 14-3-3⑀ and 14-3-3 at 0.5-3 h was observed, followed by induction at later time points. Similarly, degradation followed by induction of 14-3-3 mRNA was also demonstrated in human U373-CD14 astrocytoma cells and human synoviocytes upon Pam 3 CSK 4 , poly(I:C), LPS, R848, and CpG stimulation. These findings correlate with another study (29), which showed that the increased expression of multiple 14-3-3 proteins was evident upon stimulation of keratinocytes with dsRNA for 18 h.
Toward the functional characterization of 14-3-3⑀ and 14-3-3 in TLR signaling, we have shown that both of these proteins impair TLR2, TLR3, TLR4, TLR7/8, and TLR9 ligandinduced IL-6, TNF␣, and IFN-␤ production. This disagrees, at least in part, with a previous study showing that 14-3-3 enhanced TLR2-and TLR4-mediated TNF␣ and IL-8 production in RAW264.7 cells (19). However, the same study also showed that 14-3-3 suppressed TLR2-, TLR4-, and TLR9-mediated IL-6 production and did not affect IP-10 production (19). We also show that 14-3-3⑀ and 14-3-3 impair TLR2-, TLR3-, TLR4-, TLR7/8-, and TLR9-mediated NF-B and IFN-␤ reporter gene activity, which is in contrast to a previous study showing that 14-3-3 enhanced TLR4-mediated NF-B reporter gene activity (19). Interestingly, the same study showed that 14-3-3 suppressed TLR2-mediated NF-B reporter gene activity (19). However, it must be noted that differences in functionality exist between the various 14-3-3 isoforms and may account for the observed experimental discrepancies (25). Regarding the modulation of TLR2-, TLR3-, TLR4-, TLR7/8-, and TLR9-driven RANTES production, we provide evidence that, similar to NF-B and IFN-␤ induction, 14-3-3 proteins serve to inhibit poly(I:C)-mediated CCL5 mRNA and protein production. Interestingly, 14-3-3 proteins are required for Pam 3 CSK 4 -, LPS-, R848-, and CpG-mediated production of RANTES in a Mal/MyD88-dependent manner. Given these findings, we propose that the differential effects of Pam 3 CSK 4 , poly(I:C), LPS, R848, and CpG on RANTES production may be due to alternative adaptor utilization. More specifically, poly(I: C)-mediated CCL5 induction, facilitated by TRIF, is impaired by 14-3-3. In contrast, Pam 3 CSK 4 -, LPS-, R848-, and CpG-mediated 14-3-3 expression, facilitated by MyD88, is enhanced by 14-3-3. Because studies have shown that the CCL5 promoter contains numerous response elements for the transcriptional activators NF-B, AP-1, SP-1, IRF3, IRF-1-RE, and STAT1 (31)(32)(33), we propose that transcriptional induction of CCL5 mRNA may be differentially regulated by MyD88 and TRIF through the induction/suppression of various transcriptional activators. Herein, we preclude NF-B and IRF3 because they are comparably modulated following stimulation with the various PAMPs. Thus, we propose that the differential effects of TRIF and MyD88 on RANTES production may be facilitated through the differential activation of the other key CCL5 transcriptional activators, such as AP-1, SP-1, IRF-1-RE, and STAT1. Thus, it is plausible to speculate that PAMPs that drive MyD88-dependent TLR signaling may, in conjunction with 14-3-3, induce the expression of specific transcriptional activators to drive CCL5 transcription. In contrast, PAMPs that drive MyD88-independent TRIF signaling, in conjunction with 14-3-3, may induce the expression of inhibitory elements or suppress the expression of transcriptional activators toward the inhibition of CCL5 transcription. Interestingly, we have previously shown U373-CD14 cells were pretreated with either control lamin A/C or 14-3-3⑀ or 14-3-3 esiRNA to target their suppression. After 24 h, cells were stimulated with Pam 3 CSK 4 (1 g/ml; P3C), poly(I:C) (20 g/ml; P(I:C)), LPS (1 g/ml), R848 (1 g/ml), and CpG (5 g/ml) for 0 -120 min as indicated. Next, the cell lysates were harvested, and NF-B (phospho-p65 (A)), p38 (phospho-p38 (C)), and ERK1/2 (phospho-ERK1/2 (D)) signaling was assessed by immunoblot analysis with total protein serving as a loading control. Alternatively, the nuclear protein fraction was isolated, and immunoblot analysis was performed using an anti-IRF3 antibody (B). Equal protein loading was confirmed using an anti-lamin A/C antibody. Next, densitometry was performed to determine the relative activation of the indicated protein, whereby the band intensity of the activated protein was normalized relative to the respective total protein level. The data presented are representative of three independent experiments. that differences exist in terms of the regulation of RANTES production by poly(I:C). Specifically, we have shown that although MyD88 inhibits poly(I:C)-mediated RANTES production, Mal does not (7,11). Therefore, our work correlates with other studies showing that CCL5 induction is a complex cell type-and ligand-dependant process in vivo (32,33).
Recently, the role of 14-3-3␤ in TLR signaling has been investigated (18). Following LPS stimulation of cells, PKC⑀ is recruited to TLR4, followed by phosphorylation of PKC⑀ and subsequent association with 14-3-3␤ in a MyD88-dependent manner (18). Binding of 14-3-3␤ to PKC⑀ has been shown to lock PKC⑀ in an open conformation, thus regulating its lipid binding activity (34). Another study has shown that dsRNA induces 14-3-3-mediated signaling pathways in human kerati-nocytes (29). However, the exact role played by 14-3-3 proteins in TLR signaling requires further clarification. Herein, we employed co-immunoprecipitation experiments and have shown that 14-3-3⑀ and 14-3-3 bind to MyD88, Mal, TRIF, and TRAM. Further, 14-3-3⑀ and 14-3-3 also bind TRAF3 and TRAF6 but not IKK⑀, IRF3, IRF7, or p38. Notably, although the interaction between the 14-3-3 proteins and the TLR signaling molecules is ligand-independent, our data clearly show that stimulation of cells with PAMPs is required to activate TLR signaling and to facilitate the immunomodulatory capacity of the 14-3-3 proteins toward the curtailment of TLR-mediated proinflammatory cytokine production. In support of these findings is the fact that 14-3-3 isoforms have previously been shown to interact with another member of the TRAF family, TRAF7,  . Cellular processes and diseases associated with 14-3-3⑀ and 14-3-3 and interacting partners. The newly identified and verified 14-3-3⑀ and 14-3-3 protein interacting partners, including MyD88, MAL, TRIF, TRAM, TRAF3, TRAF6 were uploaded onto Pathway Studio software (Ariadne Genomics). A and B, 14-3-3⑀ and 14-3-3 and interacting partners were analyzed using Pathway Studio software for the cellular process network (A) and common diseases (B) shared between them. The dark highlighted circular/oval-shaped entities represent the 14-3-3 proteins and interacting partners. The rectangular entities indicate the cellular processes (A) and diseases (B) that are co-regulated by 14-3-3⑀ and 14-3-3 and interacting partners. and components of the TLR signaling pathway, namely TAB2, TAK1, and TBK1 (TRAF family member-associated NF-B activator-binding kinase 1) (1,25,35). Together, these data suggest that 14-3-3⑀ and 14-3-3 mediate their cellular effects through the TLR adaptors and TRAF3 and TRAF6. The 14-3-3 proteins are a family of conserved, ubiquitously expressed, acidic proteins, consisting of seven known mammalian isoforms (␤, ␥, ⑀, , , , and ), which are differentially expressed in mammalian cells and are subject to phosphorylation (13-15). 14-3-3 proteins have at least 200 interacting partners, and in most cases, binding is dependent on the phosphorylation of the target protein at either serine or threonine motifs (15). Binding partners include a multitude of functionally diverse signaling proteins, including kinases, phosphatases, and transmembrane receptors, which allows 14-3-3 proteins to play a vital role in a variety of regulatory processes, such as metabolism, apoptosis, cell proliferation, growth, differentiation, and intracellular signaling (16). It is becoming increasingly appreciated that abnormal 14-3-3 expression or dysregulation of 14-3-3/target protein interactions contribute to many diseases (e.g. cancer, joint inflammation, and bacterial/viral infections) (17). More specifically, 14-3-3 is purported to be a tumor suppressor and is silenced in breast cancers (17). In contrast, 14-3-3⑀ expression is enhanced in lung cancers (36). Because TLR activation and dysregulation have also been linked with such pathologies (1,25,30,37), it is tempting to speculate that coordinated dysregulation of TLRs and 14-3-3 proteins may occur in these diseases. Herein, we propose that upon stimulation of TLR2, TLR3, TLR4, TLR7/8, and TLR9 with their respective ligands, 14-3-3⑀ and 14-3-3 expression is reduced, thus permitting early proinflammatory cytokine production. Later, 14-3-3⑀ and 14-3-3 expression is induced, thereby curtailing proinflammatory cytokine production. Whether 14-3-3 proteins serve to curtail the unwanted inflammatory milieu that exists during chronic inflammatory diseases is certainly worthy of further investigation.
Regarding the role played by 14-3-3 proteins during viral and bacterial infections, we propose that initial sensing of the pathogen by the TLR leads to suppression of 14-3-3⑀ and 14-3-3 expression, thereby permitting the induction of TLRdriven early proinflammatory cytokine and type I IFN production toward the elimination of the infection. Later, following curtailment of the infection, 14-3-3⑀ and 14-3-3 expression is induced, thus leading to the inhibition of proinflammatory cytokines and type I IFN production. Thus, we propose that TLR-mediated cytokine and chemokine production is a selfregulating process that is facilitated, at least in part, by 14-3-3⑀ and 14-3-3.
Thus, by identifying 14-3-3⑀ and 14-3-3 as critical negative regulators of TLR2-, TLR3-, TLR4-, TLR7/8-, and TLR9-dependent proinflammatory and type I IFN production, the present study provides insight into the mechanisms that serve to modulate TLR-dependent signaling. More specifically, 14-3-3⑀ and 14-3-3 inhibit TLR2-, TLR3-, TLR4-, TLR7/8-, and TLR9dependent IL-6, TNF␣, and IFN-␤ induction. Interestingly, 14-3-3⑀ and 14-3-3 augment, rather than impair, TLR2-, TLR4-, TLR7/8-, and TLR9-mediated RANTES production but not TLR3-mediated RANTES production. These effects are mediated through interaction of the 14-3-3 proteins with the TLR adaptors and both TRAF3 and TRAF6. This interaction may serve to recruit auxiliary immunomodulatory molecules to the complex. Alternatively, the interaction may curtail the ability of the TLR adaptors and/or TRAF3 and TRAF6 to engage with downstream signaling proteins, thus impeding cytokine production. Although the precise molecular role for 14-3-3 in TLR signaling requires further investigation, it is clear that the 14-3-3 proteins respond to changes in the phosphorylation status of target proteins within the cell, so they respond to the dynamic changes that occur within a cell upon sensing of invading pathogens.
In conclusion, 14-3-3⑀ and 14-3-3 play a major regulatory role in balancing the host inflammatory response to viral and bacterial infections. Thus, monitoring and manipulation of 14-3-3 proteins may represent novel diagnostic and therapeutic targets for inflammatory conditions and infections.