Function of C/EBPdelta in a regulatory circuit that discriminates between transient and persistent TLR4-induced signals

Abstract

The innate immune system is like a double-edged sword: it is absolutely required for host defense against infection, but when uncontrolled, it can trigger a plethora of inflammatory diseases. Here we use systems-biology approaches to predict and confirm the existence of a gene-regulatory network involving dynamic interaction among the transcription factors NF-kappaB, C/EBPdelta and ATF3 that controls inflammatory responses. We mathematically modeled transcriptional regulation of the genes encoding interleukin 6 and C/EBPdelta and experimentally confirmed the prediction that the combination of an initiator (NF-kappaB), an amplifier (C/EBPdelta) and an attenuator (ATF3) forms a regulatory circuit that discriminates between transient and persistent Toll-like receptor 4-induced signals. Our results suggest a mechanism that enables the innate immune system to detect the duration of infection and to respond appropriately.

Figure 1. Prediction and validation of LPS-induced transcription factor network involving NF-κB, C/EBPδ and ATF3

a, Macrophages from wild-type mice were stimulated with 10 ng/ml LPS for the indicated times. mRNA was isolated and subjected to microarray analysis. A total of 78 TFs were detected in cluster 1 (red line) and cluster 2 (blue line) kinetic clusters. Shown are normalized log fold-change gene expression data over time. Each profile shows the cluster-average expression for a single cluster over 6 h after LPS-stimulation. Data represent the average of three independent experiments. b, ATF3 and NF-κB binding sites were identified in the cis-regulatory regions of transcription factor genes (cluster 2). Predicted targets were filtered using the additional constraint of a 150bp proximity limit (gray bars) between putative ATF3 and NF-κB binding sites. c, Macrophages from wild-type mice were stimulated with 10ng/ml LPS, for the indicated time periods. Nuclear Rel and ATF3 were immunoprecipitated and the indicated genes were amplified by via PCR from transcription factor-bound DNA. IgG immunoprecipitates, negative control. Data are representative of two independent experiments. d, WT and Atf3^-/- macrophages were stimulated with 10 ng/ml LPS in the presence or absence of the NF-κB inhibitor sc-514 (25μM) for the indicated times. Data represent the average of three independent experimental values ± standard error. e, Macrophages from wild-type and Atf3^-/- mice were stimulated with 10ng/ml LPS. Cells were harvested 4 h after LPS stimulation and the lysates were subjected to immunoblotting with the indicated antibodies. Actin, loading control. Data are representative of three independent experiments. f, Macrophages from wild-type mice were stimulated with 10 ng/ml LPS for the indicated times. Kinetics of Rel, C/EBPδ, and ATF3 recruitment to the Cebpd promoter were assayed by ChIP followed by PCR amplification, as in c. Data are representative of three independent experiments. g, Transcriptional factor transcriptional network model depicted as a BioTapestry diagram.

Figure 2. Mathematical model characterizing Il6 transcriptional regulation in TLR4-stimulated macrophages

a, Shown are NF-κB, ATF3 and C/EBPδ binding sites locations in the Il6 gene promoter relative to transcription start site, as predicted by motif scanning. b, Macrophages from wild-type mice were stimulated with 10 ng/ml LPS for 6 h and processed for ChIP assays as in Fig. 1c. The binding of immunoprecipitated NF-κB (Rel), ATF3 and C/EBPδ to the Il6 promoter was measured by PCR. InG, negative control immunoprecipitation. Data are representative of three independent experiments. c, Macrophages from wild-type mice were stimulated with 10 ng/ml LPS for the indicated times and processed for ChIP assays as in Fig. 1c. The binding of immunoprecipitated NFκB (Rel), C/EBPδ and ATF3 to the Il6 promoter was measured by quantitative real-time RT-PCR. Transcription factor binding was normalized to the amount of PCR product loaded. Data represent the average of three independent experiments. d, Predicted Il6 expression amounts in wild-type, Cebpd^-/- and Atf3^-/- cases of the kinetic model are shown as lines and measured Il6 mRNA quantities in wild-type, Cebpd^-/- and Atf3^-/- macrophages are shown as points. Data represent the average of three independent experimental values ± standard error. e, Extended transcriptional network model depicted as a BioTapestry diagram: Il6 gene expression is controlled by superposition of three network motifs: first, positive auto-regulation which is mediated by C/EBPδ binding its own promoter; second, feed-forward transcriptional activation of Il6, mediated by NF-κB and C/EBPδ; third, feed-forward transcriptional inhibition mediated by ATF3 binding to Cebpd and Il6 promoters.

Figure 3. Computational simulations of the transcriptional response of Il6 to TLR4 signals of varying duration reveal a threshold effect

a, Computational simulation of transient and persistent NF-κB signals. To simulate LPS pulsing, the NFκB signal was computationally varied over time. NF-κB signals of the same amplitude but different duration are shown. b,c Outputs of computationally simulated Il6 transcriptional response to transient LPS signals in WT (b) and Cebpd^-/- (c) macrophages. d,e Macrophages from wild-type (d) and Cebpd^-/- (e) mice were stimulated for 1 or 2 h or persistently with 10ng/ml LPS. Cells were harvested at the indicated time points and Il6 mRNA was measured by quantitative real-time RT-PCR. Data points represent the average of three independent experimental values and error bars indicate ± standard error. f, Macrophages from wild-type mice were stimulated for 1 h or 2 h or persistently with 10 ng/ml LPS. Cells were harvested at the indicated times and processed for ChIP assays as in Fig. 1c. Presence of immunoprecipitated Rel, on the Il6 promoter was measured by quantitative real-time RT-PCR. Transcription factor binding was normalized to the amount of PCR product loaded. Data points represent the average of three independent experimental values and error bars indicate ± standard error.

Figure 4. Identification of C/EBPδ-direct targets

a, Macrophages from wild-type mice were stimulated for 6 h with 10 ng/ml LPS. Cells were harvested and processed for immunoprecipitation with polyclonal antibodies against C/EBPδ. The binding of immunoprecipitated C/EBPδ to the promoters of target genes was detected using the Affymetrix GeneChip Mouse Promoter 1.0R Array. Averaged and normalized C/EBPδ binding across indicated promoters are shown for LPS-stimulated (red line) and unstimulated (blue line) cells. Arrow, transcription start site. Data represent the average of two independent experimental values. b, Macrophages from wild-type and Cebpd^-/- mice were stimulated with 1ng/ml LPS for the indicated time periods. Four hours after LPS stimulation cells were harvested and indicated mRNA transcripts were measured by quantitative real-time RT-PCR. Data points represent the average of three independent experimental values and error bars indicate ± the standard error.

Figure 5. The role of C/EBPδ in the restriction of transient and persistent bacterial infections

a, Wild-type mice were challenged intraperitoneally with low (1×10⁶ colony-forming units (cfu)) or high (1×10⁸ cfu) doses of Escherichia coli, H9049. Shown are averaged peritoneal bacterial counts (± standard error) at the indicated time points after infection (n=6 mice for each data point). b, Bacterial burden in the blood was measured 18 h after intraperitoneal (i.p.) infection of wild-type and Cebpd^-/- mice with 1×10⁶ c.f.u. E. coli H9049. Individual c.f.u. values and the average (horizontal line) from one representative experiment out of three are shown (n=6 mice for each group) c, Survival curve comparing wild type and Cebpd^-/- mice infected i.p. with 1×10⁶ c.f.u. E. coli H9049 (n =10 for each group). Data are representative of three independent experiments. d, Bacterial burden in the blood was measured 18 h after i.p. infection of wild-type and Cebpd^-/- mice with 1×10⁸ c.f.u. E. coli H9049. Individual c.f.u. values and the average (horizontal line) from one representative experiment out of three are shown (n=6 mice for each group) e, Survival curve comparing wild type and Cebpd^-/- after i.p. challenge with 5×10⁸ c.f.u. E. coli H9049 per mouse (n =10 for each group). Data are representative of three independent experiments.