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Toll-like receptor (TLR) stimulation triggers a signaling pathway via MyD88 and IRAK-4 that is essential for proinflammatory cytokine induction. Although NF-κB has been shown to be one of the key transcriptional regulators of these cytokines, evidence suggests that other factors may also be important. Here we showed that MyD88-deficient macrophages have defective c-Rel activation, which has been linked to IL-12 p40 induction, but not IL-6 or TNFα. We also investigated other transcription factors and showed that C/EBPβ and C/EBPδ expression was limited in MyD88- or IRAK-4-deficient macrophages treated with LPS. Importantly, the absence of both C/EBPβ and C/EBPδ resulted in the impaired induction of proinflammatory cytokines stimulated by several TLR ligands. Our results identify c-Rel and C/EBPβ/δ as important transcription factors in a MyD88-dependent pathway that regulate the induction of proinflammatory cytokines.
Toll-like receptors (TLRs) are germline-encoded receptors that engage and initiate a response to pathogen-associated molecular patterns (PAMPs) (1–3). Each member of the Toll-like receptor family interacts with a different profile of pathogen components (2). For example, lipopolysaccharide (LPS), derived from the outer membrane of Gram-negative bacteria, stimulates TLR4. This triggers the release of proinflammatory cytokines and initiates innate and adaptive immune responses (4, 5). Importantly, improper regulation of this pathway can lead to the massive release of proinflammatory cytokines and result in acute sepsis or chronic inflammatory disorders (6, 7).
MyD88 and IRAK-47 define an important signaling pathway downstream of TLR stimuli that is essential for the production of proinflammatory cytokines such as IL-6, IL-12 p40, and TNFα (2, 5, 8, 9). MyD88- and IRAK-4-deficient macrophages show severe defects in proinflammatory cytokine expression upon TLR4 stimulation (8, 9). MyD88 and IRAK-4 are also critical for TLR2, TLR7, TLR9 and IL-1R-induced cytokine expression (2, 9). NF-κB has been shown to play an essential role in the induction of proinflammatory cytokines (10, 11). Accordingly, IL-6, IL-12 p40, and TNFα (12–15) promoters contain critical NF-κB binding sites. However, LPS/TLR4-induced activation of NF-κB occurs in MyD88- or IRAK-4-deficient macrophages, because the MyD88-independent pathway can also activate NF-κB (8, 9, 16–18). Therefore TLR4 signaling provides a unique opportunity to study whether other transcription factors can regulate the expression of proinflammatory cytokines downstream of MyD88.
Five different members are included in the NF-κB family of transcription factors, p65 (RelA), RelB, c-Rel, NF-κB1 (p50) and NF-κB2 (p52) (19, 20). These factors form homo or heterodimers, and regulate the transcription of many immune related genes. c-Rel was originally identified as the cellular homolog of v-Rel, the oncogene of avian reticuloendotheliosis virus strain T and is preferentially expressed in the hematopoietic system (21, 22). Defective lymphocyte proliferation and humoral immunity were found in c-Rel-deficient mice (23). c-Rel has also been shown to play an important role in the induction of cytokines, including the production of IL-2 from T cells, as well as IL-12 and IL-23 production in macrophages and DCs (23–28). Studies have shown that c-Rel plays an important role in collagen-induced arthritis, experimental autoimmune encephalomyelitis (EAE) and several infectious diseases (29–33). However, studies examining NF-κB induction downstream of TLR stimuli, generally evaluate the induction of the predominant heterodimer composed of p65 (RelA) and p50 (NF-κB1) (8, 11, 34, 35). Therefore we examined whether c-Rel was involved in the TLR-MyD88 pathway.
To further investigate whether transcription factors other than NF-κB play a critical role in TLR-induced cytokine production, we examined a potential role for CCAAT/enhancer binding proteins (C/EBP). There are six members of the C/EBP family. C/EBPs are auto-regulatory transcription factors and belong to the basic-leucine zipper (bZIP) family of transcription factors (36–38). C/EBPβ, also known as NF-IL6, CRP2, IL-6DBP, LAP, NF-M, AGP/EBP, or ApC/EBP, were identified as sequence-specific transcription factors by various approaches (39–43).
C/EBPβ has been shown to be involved in multiple biological functions including a role in adipocyte differentiation, proliferation, tumor progression and immune function (36, 42, 44–48). C/EBPβ-deficient mice show a profound susceptibility to infection with Listeria monocytogenes, Salmonella typhimurium and Candida albicans (44, 45). C/EBPβ-deficient mice also mount a Th2 biased response upon challenge with C. albicans (45). Curiously, C/EBPβ deficient mice had high levels of IL-6 in the serum, which may be responsible for the lymphoproliferative disorder observed in older mice (45). The high circulating levels of IL-6 was unexpected since previous studies have suggested that C/EBPβ is involved in the expression of IL-6 (39, 41).
C/EBPδ (NF-IL6β, CRP3, CELF, RcC/EBP2) was also independently cloned by several groups (42, 43, 49). Notably, one approach utilized an LPS-activated human monocyte cDNA library to clone a transcription factor that specifically bound to a probe derived from the IL-1-responsive element of the IL-6 promoter (49). Like C/EBPβ, C/EBPδ also plays a role in adipocyte differentiation (42, 48) and is found to be induced by various inflammatory stimuli (36, 49–51). However, the function of C/EBPδ in inflammation has not been fully elucidated.
Evidence suggests that there may be potential overlapping or synergistic roles for C/EBPβ and C/EBPδ. Initial studies showed that C/EBPδ not only bound to the same DNA sequence as C/EBPβ, but was shown to act as a homodimer or heterodimer with C/EBPβ (49). Furthermore, both NF-κB and C/EBP binding sites in the promoter have been shown to be critical for transcriptional activation of IL-6 and TNFα (39, 52–54). The dependence of IL-6 expression upon C/EBPβ or C/EBPδ has previously been reported (55, 56). In addition, the induction of membrane-bound PGE2 synthase (mPGES) mRNA in response to LPS was impaired in the absence of either MyD88 or NF-IL6 (C/EBPβ), suggesting that MyD88 might be the upstream of C/EBPβ (57). Therefore, to explain the defective cytokine expression observed in MyD88 KO macrophages, we hypothesized that both C/EBPβ/δ may be activated downstream of MyD88.
The goal of this study was to evaluate the contribution of various transcription factors to the induction of proinflammatory cytokines downstream of MyD88. We found that both c-Rel and C/EBPβ/δ are important components of the MyD88-dependent pathway and differentially contribute to the optimal induction of proinflammatory cytokines.
MyD88, IRAK-4, c-Rel, C/EBPβ and C/EBPδ mice in C57BL/6 background were generated as described (8, 9, 23, 58–60). C57BL/6J mice were obtained from The Jackson Laboratory. Gender-matched littermates were used as control animals, and at least three pairs were compared in each experiment. In this study, no significant difference was observed between MyD88 +/+ and +/− macrophages. All mice were maintained in a specific pathogen-free environment at the Ontario Cancer Institute according to institutional guidelines.
Bone marrow mononuclear cells were obtained from femurs and tibias, and plated at 5×106 cells per 10cm plate with 6 mL of RPMI 1640 medium containing 10% FCS and 25 ng/mL M-CSF. On Day 1 and Day 3, 3 mL of the above medium/M-CSF (R&D systems) was added to the culture plates. On Day 5, floating cells were discarded and adherent bone marrow-derived macrophages were cultured in 10 mL fresh RPMI 1640 medium with 10% FCS before stimulation. The following TLR ligands were used to stimulate macrophages: LPS from Escherichia coli 0111:B4 (Sigma or InvivoGen), purified lipoteichoic acid (LTA) from S. aureus (InvivoGen), Imiquimod (R837) and CpG (ODN 1826) (InvivoGen).
2×105 MEF cells were cultured in 2mL α-MEM medium with 10% FCS in 6-well for overnight before stimulation with LPS or IL-1β (R&D systems). Due to the fact that C/EBPβ KO females are infertile, C/EBPβ/δ DKO E16.5 embryos were obtained by the intercrosses between [C/EBPβ +/−] / [C/EBPδ −/−] males and females. Fetal livers from E16.5 embryos were digested by collagenase for 30 min. Fetal liver cells were plated at 5×105 cells per 6-well with 1.5 mL of RPMI 1640 medium containing 10% FCS and 25 ng/mL murine M-CSF. On Day 1 and Day 3, 750 μL of the above medium/M-CSF was added to the culture plates. On Day 5, floating cells were discarded and adherent fetal liver-derived macrophages were cultured in 2 mL RPMI 1640 medium with 10% FCS before stimulation. 24 hr after stimulation, the concentration of cytokines was determined by ELISA (BD Biosciences), according to the manufacturer' instructions.
After stimulation with LPS, macrophages from each 10-cm plates were lysed in 200 μL of Buffer A (0.65% NP-40, 10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, pH = 7.4, supplemented with protease inhibitors) on ice for 15 minutes. The nuclei were obtained by centrifugation (10,000 g) at 4 °C for 1 min. Then these nuclei were washed twice in Buffer A to remove cytoplasmic protein contamination. The nuclei were incubated in 20 μL of Buffer C (20 mM HEPES, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, pH = 7.9, supplemented with protease inhibitors) on ice for 30 min. These tubes were centrifuged (14,000 g) at 4 °C for 10 min to obtain the supernatant (nuclear extract).
NF-κB probe (5'-ATC AGG GAC TTT CCG CTG GGG ACT TTC CG-3' and 5'-CGG AAA GTC CCC AGC GGA AAG TCC CTG AT-3') and C/EBPβ probe (5'-GAT CAT TTT GTG TAA GAC-3' and 5'-GAT CGT CTT ACA CAA AAT-3') were synthesized and annealed. The probe was labelled with 32P and incubated with nuclear extract (10 μg), poly dI-dC (3 μg), in 15μL binding buffer (20 mM HEPES, 1 mM EDTA, 0.1 mM KCl, 10% glycerol, pH = 7.9) for 15 min. In super-shift assays, 2 μg of specific antibody was added and incubated for another 15 min. The protein-DNA complex and free probes were separated by PAGE in 0.5X Tris-borate-EDTA buffer.
After LPS treatment, total RNA was extracted from macrophages using TRIzol reagent (Invitrogen), according to the manufacturers' instructions. 5 μg of each RNA sample was electrophoresed through 1% denaturing formaldehyde-agarose gel, then transferred to a nylon membrane (Hybond-N Membrane, Amersham) and hybridized to 32P-labeled IκBα, IL-6 or GAPDH cDNA.
The following primary antibodies were used in this study: Anti-C/EBPβ (C-19, sc-150), C/EBPδ (C-22, sc-151), USF-2 (C-20, sc-862), NF-κB p65 (C-20, sc-372), c-Rel (C, sc-71) and RelB (C-19, sc-226) antibodies from Santa Cruz Biotechnology; Anti-NF-κB p50 (06–886) antibody from Upstate; Anti-NF-κB2 p100/p52 (#4882), C/EBPβ (LAP, #3087) and C/EBPδ (#2318) from Cell Signaling Technology. Secondary horseradish peroxidase (HRP)-conjugated antibodies and the ECL Plus Western blotting detection system were purchased from Amersham Pharmacia.
Previous studies have indicated that NF-κB induction is relatively normal in MyD88 deficient macrophages, while cytokine induction is severely impaired (8). It is possible that TRIF provides an important alternate pathway to MyD88-independent NF-κB induction and that other transcription factors contribute to MyD88-mediated cytokine induction. To evaluate this possibility we re-examined the induction of NF-κB using primary bone marrow-derived macrophages (BMM). By electrophoretic mobility shift assay (EMSA), slightly delayed NF-κB activation was observed in bone marrow-derived macrophages from MyD88-deficient mice (Fig. 1A). Similar findings were observed using thioglycollate-activated peritoneal macrophages (8). As sustained activation of NF-κB is also important for cytokine production, EMSA were done at longer time points (up to 6 hr). The absence of MyD88 only slightly reduced the later phase of LPS-induced NF-κB activation (Fig. 1B). This was consistent with the published results using MEF (mouse embryonic fibroblast) cells (18). Furthermore, the DNA-binding complexes from both wild type control and MyD88-deficient macrophages contained both p65 and p50 subunits, the two major components of active NF-κB heterodimers (Fig. 1C).
To further evaluate NF-κB transcriptional activity, we demonstrated that induction of IκBα transcripts, a typical NF-κB target gene, was not defective in MyD88-deficient macrophages (Fig. 1D). In contrast, the induction of proinflammatory cytokines such as TNFα and IL-6 was severely impaired (Fig. 1D). It is unlikely that the minor alterations in NF-κB activity accounts for the profound reduction of proinflammatory cytokine expression in MyD88-deficient cells.
To examine whether other Rel family members contribute to the impaired cytokine expression, Western blot analyses were done using total cell lysates or nuclear protein extracts from MyD88-deficient macrophages (Fig. 1, E and F). The expression of NF-κB subunits, including c-Rel, p65, RelB and p100, was slightly reduced in MyD88-deficient macrophages (Fig. 1E). Only minimal differences in nuclear expression of p65, p50 and RelB were observed when comparing LPS-stimulated MyD88 +/− and −/− macrophages (Fig. 1F). No significant differences in the processing of p100, and the phosphorylation of p65 (Ser536) and IKKα/β (Ser180/Ser181) were observed (data not shown). Interestingly, we found that the nuclear translocation of c-Rel in response to LPS treatment was severely impaired in MyD88-deficient macrophages (Fig. 1F).
Since c-Rel has been previously shown to play a selective role in the induction of IL-12p40, we compared the role of c-Rel and MyD88 in the induction of various proinflammatory cytokines (25, 27). c-Rel −/− and MyD88 −/− macrophages showed clearly reduced IL-12p40 production upon stimulation with a variety of TLR ligands (Fig. 2A) while the induction of IL-6 or TNFα was similar in WT and c-Rel deficient macrophages (Fig. 2, B and C). Therefore c-Rel plays a selective role in the induction of IL-12 p40, but not IL-6 or TNFα downstream of MyD88.
We next investigated another transcription factor that may play a role in inflammatory responses. Activation protein-1 (AP-1), mainly controlled by MAP kinase pathways (61), is capable of contributing to proinflammatory cytokine expression. However, consistent with previously reports (8), we found that the absence of MyD88 resulted in the delayed activation of AP-1 but played a minor role in the sustained activation of AP-1 (data not shown). Similar to NF-κB, it is unlikely that the delayed induction of AP-1 could explain the profound defect in proinflammatory cytokine production in MyD88 deficient cells.
Next we performed experiments to determine whether C/EBPβ was induced downstream of MyD88. LPS stimulation led to the induction of C/EBPβ activity in wild type macrophages measured by EMSA (Fig. 3A). Interestingly, in MyD88 KO macrophages the LPS-induced C/EBPβ expression was clearly impaired (Fig. 3A). In addition, a super-shift assay was performed and the result indicates that this C/EBPβ probe bound to C/EBPβ, but not C/EBPδ (Fig. 3A).
C/EBPβ is an auto-regulatory transcription factor that is mainly regulated at the transcriptional level (36). C/EBPβ contains three isoforms, LAP*, LAP, and LIP, which are generated from different in-frame initiation codons. Both LAP* and LAP are fully active, as they contain a complete transcription activation domain and a bZIP DNA binding domain, while LIP contains a bZIP domain and can serve as a negative regulator (36). In wild type macrophages, the expression of all C/EBPβ isoforms were strongly induced upon LPS stimulation. In contrast, C/EBPβ induction by LPS was dramatically decreased in macrophages from MyD88-deficient mice (Fig. 3B). It is possible that the absence of C/EBPβ can possibly be rescued by C/EBPδ due to functional redundancy. Interestingly, the expression of C/EBPδ was undetectable in MyD88-deficient macrophages (Fig. 3C). The impaired induction of both C/EBPβ/δ expression was further confirmed using antibodies from another commercial source (data not shown). To demonstrate that C/EBPβ and C/EBPδ are indeed regulated at the transcriptional level, the induction of C/EBPβ and C/EBPδ mRNA was examined. Northern blot analysis showed that the LPS-mediated induction of C/EBPβ was dramatically decreased in MyD88-deficient macrophages, while the induction of C/EBPδ mRNA was also dependent upon MyD88 (Fig. 3D).
The induction of C/EBPβ/δ was not limited to LPS purified from E. coli 0111:B4. LPS purified from Salmonella minnesota and synthetic Lipid A both induced the expression of C/EBPβ/δ via a MyD88-dependent pathway (Fig. 3E). Therefore, in the absence of MyD88, LPS-induced expression of both C/EBPβ and C/EBPδ is impaired. To examine whether C/EBPβ/δ is important in other TLR signaling pathways, macrophages were co-cultured with a variety of TLR stimuli. LTA/TLR2, Imiquimod/TLR7, and CpG/TLR9 induced C/EBPβ/δ expression in macrophages from control mice, whereas the induction of C/EBPβ and C/EBPδ by these TLR ligands were impaired in the absence of MyD88 (Fig. 3F). Recently, a study showed that BCG (Mycobacterium bovis bacillus Calmette-Guerin) infected macrophages strongly induced the transcription of C/EBPβ, demonstrated by real-time PCR. In addition, the induction of C/EBPβ mRNA was impaired in MyD88 −/− macrophages (62). Collectively, these data demonstrate that various TLR stimuli can induce C/EBPβ and C/EBPδ via the MyD88-dependent pathway.
Since IRAK-4 is downstream of MyD88, the induction of C/EBPβ and C/EBPδ was also examined in IRAK-4-deficient macrophages. Figure 4A showed that LPS stimulation of IRAK4−/− macrophages did not induce C/EBPβ and C/EBPδ to similar levels as in control macrophages. We also examined whether the kinase activity of IRAK-4 was necessary for C/EBPβ/δ induction, using mice with a knock-in mutation that inactivates the kinase activity of IRAK-4. The induction of LIP and C/EBPδ was moderately impaired in the IRAK-4 kinase-dead macrophages, but the induction of LAP* and LAP forms of C/EBPβ was not impaired (Fig. 4B). Therefore, the induction of C/EBPβ and C/EBPδ is downstream of IRAK-4, but differentially dependent upon the kinase activity of IRAK-4.
To compare the kinetics of C/EBPβ, C/EBPδ, IL-6 and TNFα induction at the mRNA level, RNA was isolated from WT bone marrow-derived macrophages stimulated with LPS for various time points and subjected to Northern blot analysis. These results show that C/EBPβ/δ mRNA is induced earlier than IL-6, but not TNFα (Fig. 4C).
According to previous reports, neither C/EBPβ or LAP-deficient macrophages exhibited a significant decrease in the induction of IL-6 and TNFα, while G-CSF and PGE2 induction was impaired (44, 57, 63, 64). Since C/EBPβ/δ double deficiency causes early neonatal lethality, this suggests that C/EBPβ and C/EBPδ may compensate for each other in certain signaling pathways (65). To further explore potential overlapping functions, we examined the induction of C/EBP family members and cytokine production in C/EBPβ or C/EBPδ deficient macrophages. Upon LPS stimulation, C/EBPδ induction was similar in C/EBPβ-deficient macrophages (Fig. 5A), and the expression of C/EBPβ was also normally induced in C/EBPδ-deficient macrophages (Fig. 5B). Furthermore, we found that C/EBPδ-deficient macrophages did not show significant defects in IL-6 and TNFα secretion in response to LPS/TLR4 or CpG/TLR9 stimulation (Fig. 5, C and D). Therefore, it is possible that either C/EBPβ or C/EBPδ is sufficient to support the induction of proinflammatory cytokines.
Because C/EBPβ/δ double deficiency causes early neonatal lethality, we decided to utilize MEFs to further study the function of C/EBPβ/δ. Similar to primary macrophages, LPS could induce the expression of C/EBPβ and C/EBPδ in MEFs. In addition, the induction of C/EBPβ/δ was impaired in either MyD88 −/− or IRAK-4 −/− MEFs (Fig. 6, A and B). To examine whether C/EBPβ and C/EBPδ play a key role in the LPS-induction of proinflammatory cytokines, C/EBPβ/δ double knock out (DKO) MEFs were analyzed. Interestingly, IL-6 production was severely impaired in DKO cells following LPS stimulation (Fig. 6C). In addition, IL-1-induced IL-6 production was also severely defective in cells lacking C/EBPβ/δ (Fig. 6C). Importantly, the induction of IκBα transcripts, an NF-κB target gene, was not affected in C/EBPβ/δ DKO MEFs stimulated with LPS or IL-1β (Fig. 6D). This demonstrates that the signal transduction from receptor to NF-κB activation was not altered in these DKO cells, and that blockade of C/EBPβ/δ alone led to a specific defect in cytokine induction. Therefore, these experiments suggest that either C/EBPβ or C/EBPδ is capable of supporting IL-6 production.
To further confirm whether the absence of both C/EBPβ/δ also have an impact on cytokine production in macrophages, we utilized fetal liver-derived macrophages from DKO embryos, since C/EBPβ/δ double deficiency causes early neonatal lethality. C/EBPβ/δ deficient fetal liver-derived macrophages showed a marked decrease in IL-6 production after stimulation with various TLRs (Fig. 7A). The absence of both C/EBPβ/δ was also important for TNFα secretion in macrophages (Fig. 7B). In addition, LPS stimulated C/EBPβ/δ DKO MEFs also have defective TNFα induction, determined by real-time PCR (data not shown). Similar to results in Figure 6D, the induction of IκBα transcripts was not affected in C/EBPβ/δ DKO macrophages stimulated with LPS, indicating that it is unlikely that NF-κB activation was dramatically altered (Fig. 7C). Collectively, our results demonstrated that C/EBPβ/δ are important transcriptional regulators of TLR induced IL-6 and TNFα production.
As LPS-induced NF-κB and AP-1 activities are relatively preserved in MyD88-deficient cells, the key transcription factors that account for the severe cytokine defects in these knockout cells remained largely undefined. In addition, identification of key transcription factors that regulate the expression of proinflammatory cytokines other than NF-κB will provide a complete understanding for the precise control of the inflammatory signaling network. In this study, we examined various NF-κB family members and identified a specific defect in nuclear translocation of c-Rel, but not other NF-κB subunits, which accounted for the well-preserved NF-κB activity in MyD88 knockout cells. In addition, we found that the induction and activation of two C/EBP members C/EBPβ and δ were severely impaired in MyD88-deficient macrophages stimulated by LPS. The importance and potential specificity of c-Rel and C/EBPβ/δ activities in TLR signals is focus of our discussion in the transcriptional regulation of the inflammatory signaling network.
This study, as well as reports from other groups, demonstrated that the deficiency of MyD88-dependent pathway had a minimal effect on the classical NF-κB pathway, where p65 and p50 subunits play the major role (8, 18). We demonstrated that the induction of RelB and p52 subunits, which are the main components in the alternative NF-κB pathway (11), was also normal in MyD88 KO macrophages. Interestingly, c-Rel was the only subunit of NF-κB that failed to be activated in the absence of MyD88. Unlike p65 and p50 subunits, the importance and unique role of c-Rel in TLR signaling has not been fully appreciated so far.
NF-κB p65 and p50 subunits play a general role in the induction of many proinflammatory cytokines, including IL-1β, IL-6 and TNFα (11). This study and other studies show that NF-κB c-Rel is the main subunit which regulates IL-12/IL-23 p40 expression in macrophages in response to a variety of TLR ligands (25, 27). Other reports also suggest c-Rel specifically regulate IL-12 p35 and IL-23 p19 transcription in dendritic cells (26, 28, 30). This unique specificity of c-Rel suggests that c-Rel plays an important role in programming the adaptive immune response, since IL-12 and IL-23 mediated interferon-γ production favors T helper 1 responses (66). Accordingly, experiments suggest that c-Rel expression by antigen presenting cells is required for the development of Th1 responses and T cell proliferation (30, 33, 67). Importantly, the new finding that c-Rel is regulated in a MyD88-dependent fashion provides a deeper understanding in linking the coordinate activation of innate and adaptive immunity.
Defective c-Rel activation cannot explain why the induction of other cytokines, such as IL-6 and TNFα, is severely abrogated in the absence of MyD88. Our study demonstrates that C/EBPβ and C/EBPδ are key transcription factors mediating MyD88-dependent cytokine production in macrophages. Other models have shown that both C/EBPβ and C/EBPδ are involved in adipocyte differentiation and the early neonatal lethality of C/EBPβ/δ DKO mice is potentially due to defective adipocyte maturation (42, 48, 65). C/EBPβ also plays an essential role in female reproduction and skin tumorigenesis, whereas C/EBPδ involved in chromosomal stability and certain pro-apoptotic gene expression (47, 58, 68, 69). In addition, C/EBPβ can regulate HIV gene expression by binding to the long terminal repeat (LTR) (70, 71).
It is probably not surprising that C/EBP family of transcription factors can participate in regulating cytokine expression, consistent with many previous studies (15, 39, 49, 52–56, 72, 73). However, in previous studies and this study, neither C/EBPβ nor C/EBPδ single knock-out cells show impaired proinflammatory cytokine expression, such as IL-6 (Fig. 5C) (44, 63, 64). It is interesting that the expression of both C/EBPβ and C/EBPδ are abolished in MyD88- and IRAK-4-deficient macrophages stimulated with LPS, unlike NF-κB and AP-1. This highlights a unique function of C/EBPβ/δ in the MyD88-mediated pathway that controls initiation of many inflammatory responses and innate immunity (2, 5).
Our current study suggests that C/EBPβ/δ plays a specific role in the differential regulation of LPS induced cytokines at the transcriptional level. Notably, the kinetics of the induction of C/EBPβ/δ in LPS-treated wild type cells precedes the induction of IL-6, but is delayed compared to TNFα (Fig. 4C). Cells lacking C/EBPβ/δ showed clear defects in IL-6 induction and TNFα production, but not in IκBα expression induced by LPS (Fig. 6 and and7).7). These results parallel those observed in MyD88-deficient cells. As shown in Fig. 1D, the initial phase of TNFα induction was not impaired in MyD88-deficient macrophages, but the sustained TNFα production was impaired. This is consistent with the observation in MEFs in a previous report (18). Together, these findings suggest that MyD88- C/EBPβ/δ signaling is not critical for the initial phase of TNFα induction. Furthermore, for the initial phase of TNF induction, we cannot exclude the possibility that C/EBPβ/δ may also play a role together with other transcription factors (Fig. 4 and and7)7) (18, 72). Another possible explanation is that MyD88-deficient macrophages may have less C/EBPβ expression the resting state. However, the C/EBPβ expression levels in resting and activated macrophages has been evaluated recently by real-time PCR (62). The results indicate that the C/EBPβ expression level in resting MyD88-deficient macrophages is comparable to wild type macrophages.
How exactly MyD88-mediated LPS signals controls induction of C/EBPβ and δ expression in a precise but delayed kinetics remains an open question (Fig. 3, A–C) (36). The mechanisms underlying this issue may be complicated and likely involves multiple signaling systems. Downstream of LPS many cytokines are produced, including TNF, IL-1, IL-6, IL-12, various chemokines, and the recently reported 4–1BBL, which in turn trigger sequential signaling pathways that may significantly affect the initial LPS stimuli (74–76). Sequential signaling events are also documented in signals triggered by TNF (77). It is possible that C/EBPβ/δ induction is controlled by one primary signaling pathway initiated by LPS/TLR4 signaling itself and various downstream cytokine signals. Alternatively, C/EBPβ/δ may be controlled by the collective output from multiple pathways, which may culminate in a certain “activation threshold” of the cells some time (1~2 hours in macrophages) following LPS stimulation. Accordingly, it has been reported that transcriptional regulation of C/EBPβ and δ genes can be activated by multiple cytokines, growth factors, and various transcription factors (36).
Evidence has shown that the expression of the C/EBP family of transcription factors are sensitive to regulation when cells undergo major changes, particularly during cell differentiation and proliferation (78). C/EBPβ/δ, as well as C/EBPα, play a role in transcriptional control of genes specific for lineage differentiation and cell proliferation. One example is that during adipocyte differentiation, C/EBPβ/δ are turned on during the proliferative phase of pre-adipocytes, which are activated by insulin, cAMP, glucocorticoids, and other growth factors (48). The expression of C/EBPβ and C/EBPδ is differentially regulated by cAMP and glucocorticoids, respectively (42). But together these two C/EBP proteins control a specific set of genes that are critical for the initial phase of adipocyte transformation (48, 79). Similarly, in this study on LPS-stimulated macrophages, C/EBPβ and δ can be differentially regulated by the requirement of IRAK-4 kinase activity (Fig. 4B) and together play a role in the induction and maintenance of proinflammatory cytokines in a timely manner. In future studies, it will be interesting to further dissect signaling pathways downstream of MyD88 and IRAK-4 that lead to the activation of C/EBPδ and different forms of C/EBPβ.
It is also interesting to note that in C/EBPβ and C/EBPδ double-deficient macrophages, LPS-induced cytokine induction is strikingly reduced but not completely impaired (Fig. 7, A and B). It is likely that some other transcription factors, including NF-κB, also contribute to the cytokine expression mediated by MyD88-dependent pathway. Indeed, in addition to LPS/TLR4, the function of C/EBPβ/δ is extended to other TLR pathways (Fig. 3E) in which NF-κB activity is severely impaired in MyD88-deficient cells (2). The recently discovered IRF5, may be another candidate transcription factor as it is activated by MyD88 and translocated into the nucleus upon TLR activation (80). Intriguingly, IRF5-deficient macrophages and mice also exhibited a partial defect in IL-6 and TNFα induction when challenged with LPS in vitro and in vivo, respectively. Exactly how C/EBPs, IRFs, and NF-κB divide their work on cytokine induction and potentially coordinate with each other in a timely, regulated manner in response to TLR signals will also be an exciting topic for future studies.
In this study, both macrophages and MEFs were utilized to study the transcriptional regulation of proinflammatory cytokines downstream of MyD88. It has been shown that human synovial fibroblasts, umbilical vein endothelial cells and mouse endothelial cells only utilize the MyD88-dependent pathway, but not the TRIF-dependent pathway, to activate NF-κB in response to LPS (81, 82). This raises the possibility that TLR signaling might be different between macrophages and MEFs. Similar to macrophages, it has been shown that NF-κB induction was slightly delayed in MyD88- or IRAK-4-deficient MEFs (9, 18). The nuclear translocation of c-Rel in response to LPS treatment was also severely impaired in MyD88 KO MEFs (data not shown). In addition, the induction of C/EBPβ/δ was also impaired in MyD88- or IRAK-4-deficient MEFs (Fig. 6, A and B). These results indicate that macrophages and MEFs utilize the same signaling mediators under the conditions used in this study. However, it is notable that C/EBPβ/δ DKO MEFs have a more severe defect in IL-6 expression, compared to fetal liver-derived macrophages. Therefore, MEFs appear to be more dependent on C/EBPβ/δ for IL-6 transcription than macrophages.
TLR mediated signals are intricately regulated and a full understanding of the signaling pathways is required to properly control immune and inflammatory responses (6). Our study contributes to the understanding of how MyD88 and IRAK-4 pathway activate downstream transcription factors and proinflammatory cytokines in two major areas. We show that MyD88 not only activates AP-1 and NF-κB subunit p65 and p50, but also specifically activates c-Rel, C/EBPβ and C/EBPδ. Particularly in LPS signalling, where NF-κB and AP-1 activities are relatively preserved in MyD88-deficient macrophages, the specific defect in c-Rel and the profound defect in C/EBPβ/δ activation likely accounts for the reduction of IL-12 p40, IL-6 and TNFα. The absence of both C/EBPβ/δ specifically in TLR signaling impairs key proinflammatory cytokines without affecting other NF-κB-dependent genes such as IκBα. Further understanding of these pathways and key molecules could provide critical, novel drug targets in order to combat various inflammatory and immune disorders (7, 83–85).
We gratefully acknowledge E. Sterneck for helpful discussion and generously providing the C/EBPβ and C/EBPδ mice. We also thank S. Kasayama and H. Yamamoto for the C/EBPβ/δ DKO cells. We are indebted to C. Mirtsos, P. Y. B. Au, H. Chau, N. J. Chen, T. Collins, E.K. Deenick, S. Katz, P.A. Lang, W. J. Lin, J. D. Watson, Y.W. Su and J. Xu for technical support and assistance. We are also grateful to L. Z. Penn, D. L. Barber, P. Cheung, J. Gommerman, and H. Okada for suggestions and discussions.
This work was supported by Canadian Institutes of Health Research Operating grant MOP-57734. F. Shen and S. L. Gaffen were supported by the NIH grants AR050458 and AR054389. P.S. Ohashi holds a Canada Research Chair in Autoimmunity and Tumor Immunity.
7Abbreviations used in this paper: IRAK-4, IL-1R-associated kinase 4; TRIF, Toll/IL-1R domain-containing adaptor-inducing IFN-β, bZIP, basic leucine zipper; KO, knockout; DKO, double KO; LTA, lipoteichoic acid; MEF, mouse embryonic fibroblast; WT, wild type; IRF, IFN regulatory factor.
Disclosures The authors have no financial conflict of interest.