Differential role for c-Rel and C/EBPbeta/delta in TLR-mediated induction of proinflammatory cytokines

Abstract

TLR stimulation triggers a signaling pathway via MyD88 and IL-1R-associated kinase 4 that is essential for proinflammatory cytokine induction. Although NF-kappaB has been shown to be one of the key transcriptional regulators of these cytokines, evidence suggests that other factors may also be important. In this study, we showed that MyD88-deficient macrophages have defective c-Rel activation, which has been linked to IL-12p40 induction, but not IL-6 or TNF-alpha. We also investigated other transcription factors and showed that C/EBPbeta and C/EBPdelta expression was limited in MyD88- or IL-1R-associated kinase 4-deficient macrophages treated with LPS. Importantly, the absence of both C/EBPbeta and C/EBPdelta resulted in the impaired induction of proinflammatory cytokines stimulated by several TLR ligands. Our results identify c-Rel and C/EBPbeta/delta as important transcription factors in a MyD88-dependent pathway that regulate the induction of proinflammatory cytokines.

FIGURE 1

NF-κB subunit c-Rel fails to translocate into the nuclei of MyD88-deficient macrophages. A and B, To study NF-κB DNA binding activity, nuclear extract samples were obtained from macrophages after LPS stimulation and subjected to EMSA with a ³²P-labelled NF-κB probe. In C, a super-shift assay was performed with anti-p65 or anti-p50 antibody. The asterisk indicates super-shifted bands. D, RNA samples were obtained from macrophages after LPS treatment and probed with IκBα, IL-6 or TNFα cDNA. E and F, Total cell lysates or nuclear extract samples were immunoblotted with antibodies against each NF-κB subunit, in order to detect the total protein expression and nuclear translocation of NF-κB subunits.

FIGURE 2

NF-κB subunit c-Rel is essential for IL-12 p40 production. WT, MyD88- or c-Rel-deficient macrophages were stimulated with LTA, poly(I:C), Imiquimod, CpG (1 μg/mL each) or LPS (100 ng/mL). The secretion of IL-12 p40, IL-6 or TNFα was determined by ELISA.

FIGURE 3

Impaired induction of C/EBP^β and C/EBP^δ in MyD88-deficient macrophages. A, Nuclear extract samples were obtained from macrophages after LPS stimulation and subjected to EMSA with a C/EBP^β probe. In addition, a super-shift assay was performed with anti-C/EBP^β or anti-C/EBP^δ antibody. The asterisk indicates the super-shifted band. B and C, To detect C/EBP^β and C/EBP^δ expression, total cell lysates were obtained from MyD88+/− or MyD88−/− macrophages and analyzed by Western blot. C/EBP^δ or the three isoforms of C/EBP^β (LAP*, LAP, LIP) were detected by antibodies against C-terminal peptides. D, RNA samples were obtained from MyD88 +/+ or MyD88 −/− macrophages after LPS treatment and the transcription of C/EBPβ and C/EBPδ was determined by Northern blotting. E and F, In the subsequent experiments, macrophages were stimulated with LPS from different sources LPS from E. coli 0111:B4, LPS from Salmonella minnesota and synthetic Lipid A (100 ng/mL each) or various TLR ligands LTA, Imiquimod (R837), CpG (1 ^μg/mL each) for 8 hr. The expression of C/EBP^β and C/EBP^δ was detected as described above.

FIGURE 4

The induction of C/EBP^β and C/EBP^δ was also impaired in IRAK-4-deficient macrophages. A and B, Total cell lysates were obtained from IRAK-4 KO macrophages or IRAK-4 kinase-dead (KD) macrophages. The expression of C/EBP^β and C/EBP^δ was detected as described previously. C, RNA samples were obtained from WT macrophages after LPS treatment and the transcription of C/EBP^β, C/EBP^δ, IL-6 or TNF^α was determined by Northern blotting.

FIGURE 5

Characterization of C/EBP^β/δ protein expression and cytokine secretion in C/EBP^β or C/EBP^δ single knock-out macrophages. A and B, Total cell lysates were obtained from WT, C/EBP^β or C/EBP^δ single knock-out macrophages and subjected to Western blot analysis. C and D, WT or C/EB^δ-deficient macrophages were stimulated with LPS (100 ng/mL) or CpG (1 ^μg/mL) for 24 hr, and the secretion of IL-6 and TNF^α was determined by ELISA.

FIGURE 6

C/EBP^β and C/EBP^δ are critical for proinflammatory cytokine production in MEF. A and B, Total cell lysates were obtained from MyD88 +/+, MyD88 −/−, IRAK-4 +/+ and IRAK-4 −/− MEF cells after LPS treatment (10 ^μg/mL). These samples were subjected to Western blot analysis. C, To study the impact of C/EBP^β and C/EBP^δ double deficiency, WT or C/EBP^β/δ double knock-out (DKO) MEF cells were treated with IL-1b (10 ng/mL) or LPS (10 ^μg/mL) for 24 hr, and the concentration of IL-6 in the medium was determined by ELISA. D, RNA samples were obtained from WT or C/EBPβ/δ DKO MEF cells after treatment with IL-1β or LPS for 1 or 2 hr. IκBα induction was determined by Northern blot.

FIGURE 7

Impaired induction of proinflammatory cytokines upon TLR stimulation of C/EBP^β/δ-deficient macrophages. A and B, Fetal livers were obtained from WT or C/EBP^β/δ DKO embryos. Fetal liver-derived macrophages were stimulated with LTA, Imiquimod (R837), CpG (1 ^μg/mL each) or LPS (100 ng/mL) for 24 hr. The secretion of IL-6 and TNF^α was determined by ELISA. C, IκBα induction in LPS-stimulated macrophages was determined by Northern blot.