It is becoming increasingly apparent that the tools of systems biology are invaluable in deciphering the complexity of the immune system and in predicting novel drug targets20-24
. TLR4 stimulation results in the induction of a complex gene regulatory network that programs macrophage activation resulting in an effective host response to pathogens12,13,15,16
. We have shown that the TLR4 agonist, LPS, regulates the transcription of approximately 2000 genes within 24 hrs in macrophages32
. It is well established that transcriptional programs are propagated by sequential cascades of transcription factors25,26
. We showed here that LPS-stimulation of macrophages induces the transcription of two clusters of transcription factors within 3 hrs; the first cluster contains 23 TFs and the second cluster contains 55 TFs. Next we used a combination of mathematical and biological experiments to predict and validate a transcriptional network involved in TLR4 activation. The power of the approach lies in its ability to rapidly uncover complex interactions between transcription factors, and to define the functional emergent properties of the system which, in turn, suggest the molecular underpinnings of the biological response. An analysis of the transcription factors in clusters 1 and 2 predicted a number of networks involved in the TLR4 response.
We focused on an NF-κB(Rel)/ATF3/C/EBPδ sub-network; each of these transcription factors had previously been shown to participate in host defense27,33,34
, but their interaction, and the consequences of this interaction in the innate immune response, was not previously described. High density temporal measurements of LPS-induced binding of these transcription factors to the Il6
promoter, combined with gene deletion studies, enabled us to construct a model of a regulatory circuit that participates in the transcription of this cytokine gene. In this model TLR4 stimulates the translocation of NF-κB to the nucleus where it activates weak transcription of Il6
. Concomitantly NF-κB induces C/EBPδ, which then binds to the Il6
promoter and cooperates with NF-κB to stimulate maximal transcription of the cytokine gene. At a later time point ATF3 attenuates Cebpd
transcription. We previously demonstrated that ATF3 recruits histone deacetylase 1 (HDAC1) to the Il6
promoter in an LPS-dependent manner. The ATF3-associated HDAC1 then deacetylates histones, resulting in the closure of chromatin and the inhibition of Il6
. It is known that C/EBPδ binds to and recruits the histone acetylase CBP to its target promoters, thus leading to the increased histone acetylation and to the opening of chromatin35
. It is therefore possible that the NF-κB (Rel)-ATF3-C/EBPδ regulatory network is regulated by epigenetic chromatin remodeling. The relationship between NFkB and C/EBPδ suggests coherent feed-forward type I regulation30
. This type of regulation has been suggested to protect biological systems from unwanted responses to fluctuating inputs30
. The inflammatory response is a two-edged sword and it is therefore critical that inflammatory cells be able to discriminate between real and perceived threats. The coherent feed-forward type I regulatory circuit described above could, in principle, enable immune cells to filter transient insults from more dangerous persistent attacks. Exploration of this concept necessitates computational simulation of the system; therefore we used time-delay differential equations to simulate pulses of NF-κB activation and to examine transcriptional responses in silico
. These simulations demonstrated a threshold effect in the transcriptional regulation of Il6
and a critical role for C/EBPδ in a regulatory circuit that discriminates transient and persistent TLR4-stimulation. The predictions were validated in LPS-stimulated macrophages and in an in vivo
model of bacterial infection.
We used a combination of motif scanning, microarray and ChIP-to-chip analysis and identified a large number of LPS-induced C/EBPδ targets. These genes demonstrated differential transcriptional responsiveness to persistent and transient LPS-dependent stimulation of macrophages in vitro, and many have ascribed roles in host defense to bacterial infection. Consistent with the in vitro studies, Cebpd-null mice were able to resist low dose, transient, infection with E. coli H9049, but were highly susceptible to higher dose, persistent infection. In summary, we have used the tools of systems biology to demonstrate that TLR4-induced inflammatory responses are regulated by the integration of transcriptional “on” and “off” switches with “amplifiers” and “attenuators”. In addition, we have demonstrated a mechanism by which the macrophages are able to discriminate between real and perceived threats. Collectively these regulatory elements may facilitate the maintenance of effective host defense and the prevention of inflammatory disease.