Toll-like receptors (TLRs) are germline-encoded receptors that engage and initiate a response to pathogen-associated molecular patterns (PAMPs) (1
). 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
). 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
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
). MyD88- and IRAK-4-deficient macrophages show severe defects in proinflammatory cytokine expression upon TLR4 stimulation (8
). MyD88 and IRAK-4 are also critical for TLR2, TLR7, TLR9 and IL-1R-induced cytokine expression (2
). NF-κB has been shown to play an essential role in the induction of proinflammatory cytokines (10
). Accordingly, IL-6, IL-12 p40, and TNFα (12
) 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
). 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
). 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
). 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
). Studies have shown that c-Rel plays an important role in collagen-induced arthritis, experimental autoimmune encephalomyelitis (EAE) and several infectious diseases (29
). 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
). 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
). 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
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
). C/EBPβ-deficient mice show a profound susceptibility to infection with Listeria monocytogenes
, Salmonella typhimurium
and Candida albicans
). C/EBPβ-deficient mice also mount a Th2 biased response upon challenge with C. albicans
). 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
C/EBPδ (NF-IL6β, CRP3, CELF, RcC/EBP2) was also independently cloned by several groups (42
). 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
) and is found to be induced by various inflammatory stimuli (36
). 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
). The dependence of IL-6 expression upon C/EBPβ or C/EBPδ has previously been reported (55
). 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.