Function of C/EBPd in a regulatory circuit that discriminates between transient and persistent TLR4-induced signals Vladimir Litvak, Stephen A Ramsey, Alistair G Rust, Daniel E Zak, Kathleen A Kennedy, Aaron E Lampano, Matti Nykter, Ilya Shmulevich & Alan Aderem 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-jB, C/EBPd and ATF3 that controls inflammatory responses. We mathematically modeled transcriptional regulation of the genes encoding interleukin 6 and C/EBPd and experimentally confirmed the prediction that the combination of an initiator (NF-jB), an amplifier (C/EBPd) 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. The innate immune system must provide stable, specific and protec- tive responses in a diverse pathogenic environment while at the same time attenuating the collateral damage inflicted by the inflam- mation associated with such responses 1–8 . Much has been learned about the recognition mechanisms that facilitate the specificity of innate immune responses. In general, pattern-recognition receptors such as the Toll-like receptors (TLRs) recognize microbial compo- nents 9–11 and activate intracellular signaling pathways that lead to the transcriptional induction of genes critical for protective inflam- matory responses 12,13 . Bacterial lipopolysaccharide (LPS) is a principal surface compo- nent of Gram-negative bacteria and is detected by TLR4 (A002296) 14 . LPS stimulation leads to macrophage activation characterized by changes in extracellular and intracellular microbe-killing systems, the production and secretion of proin- flammatory cytokines and chemokines, enhanced expression of costimulatory receptors essential for efficient T cell activation and enhanced production of arachidonic acid metabolites 15,16 . These and other inflammatory responses in macrophages are driven mainly at the level of transcription 17,18 . However, the gene-regula- tory program of TLR-induced activation of macrophages is not well understood. It is known that macrophages express more than 500 transcription factors 19 , of which approximately 100 are induced by LPS; this suggests a high degree of complexity in the regulation of TLR4-induced responses. In this report we use the tools of systems biology 20–24 to identify a transcriptional circuit leading to the TLR4-activated state in macrophages. We analyzed temporal activation of macrophages by LPS by microarray and then clustered these data to show regulated ‘waves’ of transcription. It is well established that genes that are regulated together often share cis-regulatory elements and that transcriptional programs are propagated by sequential cascades of transcription factors 25,26 . We therefore identified transcription factors in the first cluster of expressed genes (cluster 1) and used computational motif scanning to predict which genes in cluster 2 contained in their promoters binding sites for cluster 1 transcrip- tion factors. We then confirmed those predictions by chromatin immunoprecipitation (ChIP), a method that also allowed us to establish the kinetics of promoter occupancy. These kinetic data allowed mathematical modeling of the transcriptional circuitry, which in turn allowed the prediction of previously unknown functions not easily identified by conventional approaches. We then tested the functional predictions in cell culture systems and in mice. We used this strategy to identify a previously unknown regulatory circuit involving the transcription factors NF-kB (A002052), ATF3 (A003217) and C/EBPd . We predicted and confirmed that C/EBPd acts as an amplifier of NF-kB responses and that it discriminates between transient and persistent TLR4 signals. By combining ChIP with microarray technology (‘ChIP-on-chip’ analysis), we identified 63 LPS-induced C/EBPd targets, many of which are linked to host defenses against bacterial infection. Integration of the kinetic and functional data suggested a mechanism by which C/EBPd participates in the control of persistent bacterial infection. Received 16 October 2008; accepted 19 February 2009; published online 8 March 2009; doi:10.1038/ni.1721 Institute for Systems Biology, Seattle, Washington, USA. Correspondence should be addressed to A.A. (aaderem@systemsbiology.org). NATURE IMMUNOLOGY VOLUME 10 NUMBER 4 APRIL 2009 437 ARTICLES © 2009 Nature America, Inc. All rights reserved.