Host immune responses are finely regulated by the opposing effects of Th17 and T regulatory (Treg) cells. Treg cells help to dampen inflammatory processes and Th17 cells facilitate various aspects of immune activation. The differentiation of Th cells depends on a unique combination of stimulants and subsequent activation of diverse transcription factors. In particular, cooperative activation of NFAT and Smad3 leads to the induction of Treg cells, and cooperation among STAT3 and Smad3 switches to the induction of Th17 cells. We have previously shown that the IL-1 receptor associated kinase 1 (IRAK-1) selectively activates STAT3 and inactivates NFAT. Physiological studies have shown that IRAK-1−/− mice are protected from developing various inflammatory diseases, including experimental autoimmune encephalomyelitis and atherosclerosis with unknown mechanism. In this study, we demonstrate that IRAK-1 plays a critical modulatory role in the differentiation of Th17 and Treg cells. Following stimulation with TCR agonists and TGFβ, IRAK-1−/− CD4 Th cells display elevated nuclear NFATc2 levels and increased interaction of NFATc2 and Smad3, resulting in increased expression of Foxp3, a key marker for Treg cells. IRAK-1−/− mice have constitutively higher populations of Treg cells. In contrast, when stimulated with TCR agonists together with IL-6 and TGF-β, IRAK-1−/− CD4 Th cells exhibit attenuated STAT3 Ser727 phosphorylation and reduced expression of IL-17 and RORγt compared with wild-type cells. Correspondingly, IRAK-1 deletion results in decreased IL-17 expression and dampened inflammatory responses in acute and chronic inflammatory mice models. Our data provides mechanistic explanation for the anti-inflammatory phenotypes of IRAK-1−/− mice.
CD4+ Th cells play a crucial role in mediating host inflammatory responses. Naive CD4+ T cells can differentiate into Th1, Th2, Th17 effector cells, or the Foxp3 expressing inhibitory T regulatory (Treg)3 cells (1, 2, 3, 4). Th1 cells are responsible for the cell-mediated elimination of intracellular pathogens, whereas promotion of humoral and allergic response is largely dependent on Th2 responses. The significance of Th17 cells is increasingly recognized due to their involvement in organ-specific autoimmunity and numerous inflammatory complications. Elevated levels of IL-17 are associated with the pathogenesis of multiple sclerosis, atherosclerosis, and diabetes (5). In contrast, Treg cells play a central role in dampening various inflammatory effects of effector Th cells and are beneficial for the resolution of inflammatory processes (6, 7).
The counteractions of Th17 and Treg cells are not only reflected in their opposing immune modulatory functions, but also during their differentiation processes. Recent studies have identified retinoic acid-related orphan receptor γt (RORγt), a member of the nuclear hormone receptor super family, as a key transcription factor driving the Th17 differentiation program (8). In contrast, the Treg cells are defined by the expression of the forkhead family transcription factor, Foxp3. TGFβ is involved in the differentiation of both Th17 and Treg cells (9, 10). However, in the case of Treg differentiation, TGFβ-induced transcription factor Smad3 cooperates with NFAT to induce the expression of Foxp3 (11). In contrast, TGFβ and IL-6 trigger the coordinated activation of Smad3 and STAT3 to induce the transcription factor RORγt necessary for Th17 differentiation (4).
The activation statuses of NFATc2 and STAT3 are controlled by signal-dependent phosphorylations mediated by upstream kinases and phosphatases (12, 13). Maximal activation of STAT3 requires phosphorylation at both Tyr705 and Ser727 sites (14). Although JAK2 kinase is shown to be responsible for phosphorylating STAT3 at Tyr705, we have shown that IL-1 receptor associated kinase 1 (IRAK-1) is capable of selectively phosphorylating STAT3 at Ser727 (15). Regarding NFAT, calcium-dependent phosphatase calcineurin dephosphorylates and activates NFAT. We have recently demonstrated that IRAK-1 is responsible for phosphorylating NFAT and maintaining NFAT in an inactive state (16). We therefore hypothesize that IRAK-1 may facilitate the differentiation of Th17 and dampen the induction of Treg cells by affecting the activation status of STAT3 and NFAT. Our hypothesis is further supported by previous functional studies using IRAK-1−/− mice, which are protected from diverse inflammatory diseases such as experimental autoimmune encephalomyelitis (EAE), atherosclerosis, and septic shock (16, 17, 18, 19).
In this study, we examined the expression of Foxp3, IL-17A, and RORγt in wild-type and IRAK-1−/− CD4 T cells. Mechanistically, the activation status of STAT3 and NFATc2 following TGF-β or IL-6 plus TGF-β were evaluated. We found that IRAK-1−/− CD4 T cells have decreased STAT3 phosphorylation at Ser727 and decreased expression of RORγt and IL-17A following TGFβ1 and IL-6 treatment. In contrast, we observed that IRAK-1−/− CD4 T cells have elevated levels of nuclear NFATc2 and increased expression of Foxp3 upon TGF-β treatment. IRAK-1−/− mice also exhibit constitutively elevated levels of CD4+CD25+Foxp3+ Treg cell population. In contrast, IRAK-1−/− mice challenged with various inflammatory agents exhibit reduced IL-17 production, leading to alleviated inflammatory symptoms. Our study reveals a novel contribution of IRAK-1 to the differentiation of Th17 and Treg cells, with significant implications in the pathogenesis of diverse inflammatory diseases.
Materials and Methods
The Ab against Smads and STAT3 (total and phosphorylated) was obtained from Cell Signaling Technology. Anti-laminB, β-actin, and NFAT-c2 Abs were purchased from Santa Cruz Biotechnology. Recombinant TGFβ-1 was purchased from R&D Systems and IL-6 was obtained from BD Biosciences. The TCR agonists, anti-mouse CD3 and CD28, were purchased from BD Biosciences. LPS (E. coli O11:B4) was obtained from Sigma-Aldrich.
Wild-type C57BL/6 mice were obtained from Charles River Laboratories. IRAK-1−/− mice on C57BL/6 background were provided by Dr. James Thomas (University of Texas Southwestern Medical School, Dallas, TX). ApoE−/−/IRAK-1−/− mice were obtained by breeding ApoE−/− mice (The Jackson Laboratory) with IRAK-1−/− mice. All mice were bred and housed at Derring Hall Animal Facility at Virginia Polytechnic Institute and State University, in compliance with approved Animal Care and Use Committee protocols.
Western blot analysis and immunoprecipitation assays
Isolation of whole cell lysates and cytoplasmic and nuclear extracts were performed as described previously (20). Briefly, cells were rinsed in PBS and subsequently lysed on ice in the lysis buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, 1 μg/ml leupeptin, 1 μg/ml pepstatin) for 30 min, followed by addition of 10% Triton X-100. The samples were centrifuged for 10 min at 5000 rpm and the supernatant fractions were transferred and saved as cytoplasmic extracts. Pellets containing the intact nuclei were lysed and solubilized with the high salt buffer (20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 0.4 M NaCl, 0.2 mM EDTA, 0.5 mM DTT, 1 mM PMSF) for 30 min on ice followed by centrifugation at maximum speed for 20 min. The supernatant was saved as the nuclear extract. Western blotting analysis of the protein samples were performed as described previously (21). Immunoblots were developed by using the Amersham ECL Plus Western Blotting Chemiluminescent Detection System (GE Healthcare).
For immunoprecipitations, the cell lysates were treated with 2 μg primary Ab or normal IgG in the presence of 35 μl of protein AG plus agarose (Santa Cruz Biotechnology) and incubated on a rotating wheel at 4°C overnight. The samples were centrifuged briefly and the pellets were washed three times in immunoprecipitation buffer containing protease inhibitors. The bound proteins were eluted from the agarose beads by boiling with 2× Laemmli sample buffer for 5 min. The eluted proteins were loaded on a 7.5% SDS-polyacrylamide gel for electrophoresis followed by Western blot analysis and immunoblotting with the indicated Abs.
Splenocytes were harvested from wild-type and IRAK-1−/− mice as previously described (20). Harvested splenocytes were stained with PE-rat anti-mouse CD4 and allophycocyanin-rat anti-mouse CD25. Cells were subsequently fixed and permeabilized with Cytofix/Cytosper solution from BD Biosciences, and stained with PE-conjugated anti-mouse Foxp3 Ab according to the manufacturer’s protocol. Flow cytometry analyses were performed on a FACSCanto cytometer and data were analyzed using the FACSDiva software (both from BD Biosciences).
In vitro CD4 T cell differentiation
Naive CD4 T cells were isolated from the spleen of wild-type and IRAK-1−/− mice using the MACS CD4 Microbeads (Miltenyi Biotec), according to the manufacturer’s protocol, and differentiated in vitro. For Th17 differentiation, the cells were incubated with plate-bound anti-CD3 (5 μg/ml) mAbs and soluble anti-CD28 (5 μg/ml) in the presence of TGFβ1 (5 ng/ml) and IL-6 (20 ng/ml) at 37°C for 3 days. For Treg differentiation, the naive CD4 T cells were incubated with TGFβ1 (5 ng/ml) in the presence of plate-bound anti-CD3 mAbs and anti-CD28 at 37°C for 3 days. Following differentiation the cells were washed with PBS and used for subsequent experiments as indicated.
Real time RT-PCR
Total RNAs were prepared from either untreated or treated CD4 Th17 or Treg cells using Trizol (Invitrogen) according to the manufacturer’s protocol. Reverse transcription was conducted using the High-Capacity cDNA reverse transcription kit (Applied Biosystems) and subsequent real-time PCR analyses were performed using the SYBR green supermix on an IQ5 thermocycler (Bio-Rad). The relative levels of Foxp3, RORγt, and IL17A transcripts were calculated using the ΔΔCt method after normalizing with GAPDH as the internal control. The relative levels of mRNAs in untreated wild-type cells were adjusted to 1 and served as the basal reference value.
Chromatin immunoprecipitation (ChIP) assays
The isolated splenocytes from wild-type and IRAK-1−/− mice were either untreated or treated with TGFβ (5 ng/ml) alone or in combination with IL-6 (20 ng/ml), followed by cross-linking with 1% formaldehyde in RPMI 1640 complete medium for 15 min with gentle rocking at room temperature. Cells were then washed twice with ice-cold PBS and treated with glycine solution for 5 min to stop the cross-linking reaction. Cells were then lysed in buffer containing SDS and protease inhibitor mixture. Samples were sonicated six times with 30-s pulses at 4°C followed by centrifugation to collect the sheared chromatin. The sheared chromatin was used to set up immunoprecipitation reactions with the indicated Abs using the CHIP-IT Express kit (Active Motif) as per manufacturer’s recommendations. The immunoprecipitated DNA fragments were analyzed by PCR using primers spanning the IL-17A or Foxp3 promoter regions.
Acute and chronic inflammatory treatments
Wild-type and IRAK-1−/− mice of matched gender and age were injected with LPS (E. coli O11:B4; Sigma-Aldrich) (25 mg/kg) or PBS i.p. Total blood was drawn 6 hours after the injection. Plasma was collected and diluted 1/5 in sample diluent (Bio-Plex Diluent kit no. 171-305-008; Bio-Rad). Cytokine levels were assayed using a multiplex bead-based immunoassay as described by the manufacturer’s protocol (Bio-Rad).
High-fat diet feeding and analyses.
ApoE−/− and ApoE−/−/IRAK-1−/− mice of matched gender and age were fed with a Western Diet (TD.94059; Harlan Laboratories) for 3 mo. Plasma levels of IL-17 were measured as described above.
Statistical significance was determined using the unpaired two-tailed Student’s t test. Values of p less than 0.05 were considered statistically significant.
Elevated induction of Foxp3 in IRAK-1−/− CD4+ T cells following stimulation with TGFβ
To test whether IRAK-1 is involved in the induction of Foxp3 Treg cells in vitro, we treated CD4+ T cells harvested from wild-type and IRAK-1−/− splenocytes with plate-bound anti-CD3 Ab and soluble anti-CD28 Ab in the presence or absence of TGFβ. Three days after stimulation, total RNAs were harvested and the relative levels of Foxp3 expression were determined by real-time RT-PCR. As shown in Fig. 1 A, the levels of expressed Foxp3 message were three times higher in IRAK-1−/− CD4+ T cells compared with wild-type CD4+ T cells.
We further studied the CD4+CD25+Foxp3+ T regulatory cell populations in vivo. Splenocytes were harvested from age and gender equivalent wild-type and IRAK-1−/− mice and used to perform flow cytometry analyses. As shown in Fig. 1 B, IRAK-1−/− mice have significantly higher levels of CD4+CD25+Foxp3+ T cells when compared with wild-type mice (p < 0.05). There was no significant difference regarding the frequencies of CD4+CD25+ T cells between wild-type and IRAK-1−/− mice (data not shown). This is consistent with the established notion that CD25 expression is not the definitive marker for Treg cells in both mice and humans. Instead, Foxp3 is the most pertinent indicator for Treg cells. Our data explain previous findings demonstrating that IRAK-1−/− mice have attenuated inflammatory symptoms, including EAE and atherosclerosis (17, 18, 19, 22). Therefore, the higher percentage of Foxp3-expressing Treg cells might explain the protective effect observed in IRAK-1-deficient mice.
Elevated NFATc2 levels contribute to increased Foxp3 induction in IRAK-1−/− CD4+ T cells
The transcription of Foxp3 is induced by the cooperative action of Smad3 and NFAT transcription factors in response to TGFβ (11). We have previously shown that IRAK-1 attenuates the transcriptional activity of NFAT in both macrophages and fibroblasts (16). IRAK-1 phosphorylates NFAT at its Ser-Pro-rich motifs and helps to maintain NFAT in an inactive form (16). To further examine the molecular mechanism underlying the elevated Foxp3 gene expression in IRAK-1−/− CD4+ T cells, we examined the nuclear levels of NFATc2 in wild-type and IRAK-1−/− CD4+ T cells. As shown in Fig. 2,A, TCR activation plus TGFβ treatment induced significantly higher nuclear levels of NFATc2 in IRAK-1−/− CD4+ T cells compared with the wild-type cells. The elevated nuclear NFATc2 levels in IRAK-1−/− cells can also be observed with TCR activation alone (Fig. 2 B).
We further examined the nuclear levels of Smad2 and Smad3 in wild-type and IRAK-1−/− CD4+ T cells. TGFβ treatment equally induced the nuclear accumulation of Smad2 and Smad3 in wild-type and IRAK-1−/− cells (Fig. 2 C). Our data demonstrate that IRAK-1 is preferentially involved in modulating TCR-induced NFATc2 levels, instead of TGFβ-induced Smad3.
Because NFAT cooperates with Smad3 in promoting the expression of Foxp3 (11), we subsequently examined the interaction between NFATc2 and Smad3 in wild-type and IRAK-1−/− cells. As shown in Fig. 2 D, TCR ligation and TGFβ treatment induced significantly elevated interaction between Smad3 and NFATc2 in IRAK-1−/− cells as compared with the wild-type cells.
We also examined the association of Smad3 with the endogenous Foxp3 promoter region using the ChIP assay. As shown in Fig. 2 E, TCR ligation and TGFβ treatment induced significantly higher levels of Smad3 association with the Foxp3 promoter region in IRAK-1−/− CD4+ T cells. Taken together, although IRAK-1 is not affecting the total nuclear levels of Smad2/3 proteins, IRAK-1 deletion indirectly contributes to increased Smad3 binding with Foxp3 promoter through increasing the nuclear levels of NFATc2.
Decreased expression of IL-17 and RORγt in IRAK-1−/− CD4+ T cells following stimulation with TGFβ and IL-6
To determine the role of IRAK-1 in the induction of Th17 cells, we stimulated CD4+ T cells harvested from wild-type and IRAK-1−/− splenocytes with plate-bound anti-CD3 Ab and soluble anti-CD28 in the presence or absence of TGFβ plus IL-6. Three days after stimulation, total RNAs were harvested and the relative levels of IL-17 and RORγt were determined by real-time RT-PCR. As shown in Fig. 3, the levels of induced IL-17A and RORγt were significantly higher in wild-type cells compared with IRAK-1−/− cells (p < 0.05).
IRAK-1−/− T cells have decreased IL-6-stimulated STAT3 phosphorylation
Th17 cells are generated by the cooperative action of the cytokines TGFβ and IL-6 (4, 23, 24). IL-6 acts through the activation of STAT3 to induce the expression of IL-17A as well as the transcription factor RORγt (8). RORγt further contributes to the differentiation and stable maintenance of Th17 cells. We have previously shown that IRAK-1 phosphorylates STAT3 at its Ser727 site and contributes to the full activation of STAT3 (15). To elucidate further the role of STAT3, we determined the levels of activated STAT3 in CD4 T cells isolated from wild-type and IRAK-1−/− mice in response to IL-6 and TGFβ. As shown in Fig. 4,A, STAT3 Ser727 phosphorylation induced by IL6 and TGFβ was significantly attenuated in IRAK-1−/− cells. Furthermore, consistent with our previous finding, IRAK-1 deletion did not affect STAT3 Tyr705 phosphorylation induced by IL-6 (Fig. 4 A).
To define the functional implication for decreased STAT3 Ser727 phosphorylation in IRAK-1−/− cells, we examined the binding of STAT3 with the endogenous IL-17 promoter region through ChIP assay. As shown in Fig. 4 B, compared with wild-type cells, IRAK-1−/− cells have significantly decreased levels of STAT3 bound with the promoter region of IL-17.
Decreased IL-17 expression and attenuated inflammatory responses in IRAK-1−/− mice
IL-17 plays a key role in exacerbating various forms of inflammatory responses (5). Although previous studies have demonstrated that IRAK-1 is associated with the pathogenesis of various inflammatory diseases, including septic shock, EAE, and atherosclerosis, there has been no report regarding the induced levels of IL-17 in IRAK-1−/− mice under various inflammatory conditions (18, 19, 22). Based on our mechanistic studies and other phenotypic findings, we hypothesize that IRAK-1 deletion would attenuate IL-17 expression in vivo. To test this, we set up several acute and chronic inflammatory animal studies. As shown in Fig. 5 A, acute LPS injection capable of inducing septic shock significantly induced IL-17 plasma levels in wild-type mice. In sharp contrast, the levels of IL-17 in IRAK-1−/− mice following the same dose of LPS injection were significantly lower (p < 0.05). This correlated with higher survival rate in IRAK-1−/− mice following LPS inoculation (data not shown).
Furthermore, we evaluated the plasma levels of IL-17 in ApoE−/− and ApoE−/−/IRAK-1−/− mice fed with high-fat diet for 3 months. As we have previously reported, ApoE−/− mice fed with high-fat diet developed severe atherosclerosis whereas ApoE−/−/IRAK-1−/− mice had significantly lower atherosclerotic plaques (16). In our current study, we found that the levels of plasma IL-17 in ApoE−/− mice were significantly elevated following high-fat diet feeding (Fig. 5 B). In contrast, the levels of IL-17 in ApoE−/−/IRAK-1−/− mice were significantly lower (p < 0.01). These results suggest that IRAK-1 plays a major role in promoting inflammation in diverse disease models through the expression of IL-17.
We have herein demonstrated that IRAK-1 plays an important role in modulating the balance between the differentiation of Treg and Th17 cells. IRAK-1 fulfills this function by attenuating TCR-mediated activation of NFATc2, while facilitating the IL-6-mediated activation of STAT3 (Fig. 6).
Our data provides a mechanistic explanation for several previous reports demonstrating that IRAK-1 deletion can provide protection from various inflammatory diseases. Deng et al. initially reported that IRAK-1−/− mice are protected from developing EAE (19). At the time of their discovery, neither Treg cells nor Th17 cells were fully defined. Instead, elevated Th1 response was thought to be responsible for the development of various inflammatory diseases. Deng et al. examined in detail the respective Th1 response and did not observe significant difference among wild-type and IRAK-1−/− mice. Besides autoimmune diseases, studies done by others and us indicate that IRAK-1 is closely associated with the pathogenesis of diverse chronic inflammatory diseases, including atherosclerosis (17, 18, 19, 25). A study using human patient samples has shown elevated IRAK-1 levels in human atherosclerotic plaques (26). Our study using ApoE−/−/IRAK-1−/− mice demonstrated that deletion of IRAK-1 renders protection form high fat diet-induced atherosclerosis (16). In addition, IRAK-1 has also been associated with the risk of lupus and kidney inflammation in humans (26, 27). Our unpublished observation indicated that IRAK-1−/− mice are protected from experimental kidney inflammation using the mouse model of anti-glomerular basement membrane and LPS injection. Mechanistically, this is the first study to define that IRAK-1 deletion contributes to attenuated IL-17 expression and Th17 differentiation, while facilitating Foxp3 expression and Treg differentiation. Recently, Treg cells have been increasingly recognized to be essential in attenuating various inflammatory processes and lessening the progression of inflammatory diseases (28). In contrast, IL-17-producing Th17 cells play a critical role in propagating inflammatory processes that were previously thought to be mediated by the Th1 cells (3). Our data presented in this report provides mechanistic explanation of the protective phenotype of IRAK-1−/− mice and defines a significant connection between IRAK-1 and the balance of Treg cells and Th17 cells.
Excessive expression of IL-17 cytokine is not only associated with chronic inflammatory diseases, but also with acute injury and inflammation. Several recent reports indicate that IL-17 is highly induced following acute endotoxemia in laboratory animals (29, 30, 31), and may contribute to septic shock and multiorgan failure. IRAK-1−/− mice are known to have alleviated symptoms from LPS-induced septic shock (32, 33). Furthermore, humans carrying variant IRAK-1 genes are at a higher risk of getting septic shock (22). Intriguingly, we found that compared with wild-type mice, IRAK-1−/− mice have significantly lower plasma levels of IL-17 following acute lethal dose of LPS injection. The mechanism leading to the rapid expression of IL-17 with septic shock is not known. Besides CD4 Th cells, many other cell types, such as NK cells, αβT cells, lymphoid tissue inducer-like cells, neutrophils, paneth cells, and epithelial cells are also capable of producing IL-17 (34, 35, 36, 37). LPS is a potent inducer of acute phase proteins, including IL-6 during septic shock (38). IL-6 produced may further induce the activation of STAT3 and subsequent transcription of IL-17 in these cells. We cannot rule out the possibility that LPS alone or in combination with other stimulatory factors may directly contribute to STAT3 activation and IL-17 expression. Previous studies done by others and us have shown that LPS can induce STAT3 phosphorylation at both tyrosine and serine residue (15, 39), and LPS-mediated STAT3 serine phosphorylation is dependent on IRAK-1. Future studies are needed to clarify the source of IL-17 expression as well as the molecular mechanisms involved.
IRAK-1-mediated STAT3 and NFATc2 phosphorylation may differentially modulate the activities of STAT3 and NFATc2. As demonstrated previously, the kinase domain of IRAK-1 may adopt a tertiary structure that resembles the cyclin-dependent-kinase (40, 41). Consequently, IRAK-1 preferentially phosphorylates substrates with serine/threonine-proline rich motifs. To date, all of the well-defined IRAK-1 substrates, including IRAK-1 itself, STAT3, NFATc2/c4, and IRF5/7, contain such a motif (15, 42, 43). It has been reported that STAT3 serine phosphorylation is required for the maximum activation of STAT3 transcriptional activity (14). In contrast, NFAT phosphorylation has been shown to facilitate nuclear export of NFAT and attenuate NFAT transcriptional activity (13). Consistent with previous findings, our current study clearly indicates that IL6-mediated STAT3 Ser727 phosphorylation is greatly reduced in IRAK-1−/− cells. This translates into decreased binding of STAT3 to the endogenous promoter of IL-17 and reduced IL-17 expression in IRAK-1−/− CD4 T cells. On the contrary, we found that NFATc2 nuclear levels are significantly higher in TCR-ligated IRAK-1−/− CD4 T cells as compared with the wild-type cells. This phenomenon closely correlates with elevated interaction between NFATc2 and Smad3 in the nucleus of IRAK-1−/− CD4 T cells following TCR ligation and TGFβ stimulation. The induced Foxp3 expression levels are significantly higher in IRAK-1−/− cells compared with wild-type cells. Consequently, IRAK-1 contributes to the exacerbation of inflammation by inducing the expression of IL-17 and decreasing the expression of Foxp3.
Our study expands the repertoire of diverse signaling networks involving IRAK-1. Initially defined as a downstream component of the IL-1 receptor complex (44), IRAK-1 was later recognized as a key molecule in other cellular signaling networks downstream of multiple receptors and coreceptors such as TLRs, CD26, neurotrophin nerve growth factor, and insulin (19, 25, 45, 46, 47, 48). Its close homologue IRAK-4 was also implicated in the TCR-mediated signaling process (48). Our present data demonstrate that IRAK-1 is involved in the signaling process downstream of both IL-6 and TCR agonists. However, it is not clear how IRAK-1 can be recruited in response to IL-6 and TCR agonists. One of the potential connectors could be protein kinase C (PKC). In fact, we and others have reported that several isoforms of PKC can interact, phosphorylate, and activate IRAK-1 (49, 50, 51). Both IL-6 and TCR agonists are well-known activators of PKC, which may subsequently activate IRAK-1. Future studies are needed to further determine the molecular basis for this connection.
Taken together, we have revealed a novel role of IRAK-1 during the induction of RORγt, IL-17, and Foxp3. IRAK-1 serves as a critical link modulating the delicate balance between the differentiation of Th17 and Treg cells (Fig. 6). In combination with functional studies, we posit that IRAK-1 may serve as a viable target for future development of therapeutic interventions of diverse inflammatory diseases ranging from acute septic shock to chronic inflammatory diseases including atherosclerosis and autoimmune complications.
We thank Lin Zhang from our laboratory for assistance with flow cytometry analyses and Samantha Baglin for critical reading of the manuscript.
The authors have no financial conflict of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work was supported by a research grant from the National Institutes of Health AI064414 (to L.L.).
Abbreviations used in this paper: Treg cells, T regulatory cells; ChIP, chromatin immunoprecipitation; EAE, experimental autoimmune encephalomyelitis; IRAK-1, interleukin-1 receptor associated kinase 1; PKC, protein kinase C; RORγt, retinoic acid-related orphan receptor γt.