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Transcriptional control of the inflammatory response

Ruslan Medzhitov* and Tiffany Horng

Abstract | Inflammation is a multicomponent response to tissue stress, injury and infection, and a crucial point of its control is at the level of gene transcription. The inducible inflammatory gene expression programme such as that triggered by Toll-like receptor signalling in macrophages is comprised of several coordinately regulated sets of genes that encode key functional programmes; these are controlled by three classes of transcription factors, as well as various transcriptional co-regulators and chromatin modifications. Here, we discuss the mechanisms of and the emerging principles in the transcriptional regulation of inflammatory responses in diverse physiological settings.
Inflammation is a fundamental adaptation to the loss of cellular and tissue homeostasis with many important physiological roles, including host defence, tissue remodelling and repair, and the regulation of metabolism13. The complexity of the inflammatory response requires that its many functional programmes are controlled coordinately in some situations but independently in others. This is achieved through multiple mechanisms that operate at different levels, including alterations in the composition of immune cells in tissues, changes in cell responsiveness to inflammatory stimuli, regulation of signalling pathways and control at the level of gene expression. So, the mechanisms that regulate inflammatory responses can be divided into cell-specific, signal-specific and gene-specific mechanisms. Cell-specific mechanisms operate at the level of different cell types, and include regulation of their recruitment and activation. Signal-specific mechanisms operate at the level of signalling pathways: for example, by terminating activation of the key transcription factor nuclear factor-B (NF-B) as a part of a negative feedback mechanism. Finally, gene-specific mechanisms operate at the level of individual genes and gene subsets. For example, interleukin-10 (IL-10) and many nuclear receptors negatively regulate the transcription of specific subsets of inflammatory genes. As such, gene-specific mechanisms are particularly well suited to provide functional specificity in an inflammatory response. Here, we review the recent progress in understanding the transcriptional regulation of the inflammatory response. In addition, we discuss the key factors and molecular mechanisms that regulate inflammatory gene expression, with the objective of defining some general principles that govern this important physiological process. Macrophages are crucial mediators of the inflammatory response, and Toll-like receptors (TLRs) are the best-characterized inducers of acute inflammation. Therefore, much of this Review focuses on the transcriptional regulation of inflammation by TLR ligands and in particular by lipopolysaccharide (LPS) in macrophages. After only a few hours of LPS stimulation, the expression of several hundred genes is induced (and repressed) in macrophages4,5. This is a complex transcriptional response, consisting of multiple gene sets that encode functional programmes controlling cell migration, tissue repair and remodelling, antimicrobial defence, phagocytosis, metabolic reprogramming and the regulation of adaptive immune responses. These gene sets, or transcriptional modules, are often coordinately regulated by dedicated transcription factors. This feature of the transcriptional response enables autonomous control of individual transcriptional modules, because the transcriptional regulators that control their expression can be differentially regulated by positive and negative signals (BOX 1). Several examples of transcriptional regulators that control distinct transcriptional modules are currently known, both within and outside of the LPS-induced transcriptional response. These include class II transactivator (CIITA), which is a master regulator of genes involved in the MHC class II-restricted pathway of antigen processing and presentation6; interferon (IFN)stimulated gene factor 3 (ISGF3), which controls the expression of type I IFN-induced antiviral genes; sterol regulatory element binding protein 2 (SREBP2), which controls the expression of genes involved in cholesterol biosynthesis7; and several stress-induced transcriptional
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*Howard Hughes Medical Institute and Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut 06510, USA. Department of Genetics and Complex Diseases, Harvard School of Public Health, Boston, Massachusetts 02115, USA. e-mails: ruslan.medzhitov@yale.edu; thorng@hsph.harvard.edu doi:10.1038/nri2634

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Box 1 | Module-specific transcriptional regulation of inflammatory gene expression
Many of the mechanisms that control gene expression operate in a gene-specific manner, which indicates that they might control specific modules of the inflammatory response. Although module-specific regulatory mechanisms have not yet been explored in detail, we describe a couple of examples to illustrate the biological contexts in which module-specific control has an obvious advantage. Lipopolysaccharide (LPS) tolerance is a state of hyporesponsiveness to LPS (and other inflammatory stimuli) that is induced during conditions of excessive inflammation (such as sepsis) to limit inflammation-associated pathology. LPS-tolerant cells are refractory to the induction of expression of inflammatory cytokines such as tumour necrosis factor and interleukin-6 (IL-6), and this is due, at least in part, to the downregulation of expression of many inflammatory signalling proteins80. Importantly, however, LPS-induced signalling in LPS-tolerant cells can still induce the expression of genes that encode anti-inflammatory cytokines and antimicrobial peptides. The differential inducibility of these classes of genes (or modules) is associated with distinct patterns of chromatin remodelling8183. This indicates that the transcriptional regulation of LPS tolerance enables the inhibition of some functional programmes (for example, those encoding inflammatory cytokines) while inducing other programmes (for example, antimicrobial effector functions), which could be advantageous when a host has to deal with a persistent infection82. As another example of module-specific transcriptional regulation, we consider the multiple functions of different LPS Inflammatory cytokines tissue-resident macrophage populations. All macrophages are key orchestrators of the inflammatory response, Chemotactic factors but they also have tissue-specific functions that are and receptors TLR4 programmed by local factors (see figure). For example, Coagulation factors IL-10 induces colonic epithelium macrophages to carry out immunomodulatory functions that are appropriate at this Antimicrobial effector functions hostcommensal interface, whereas white adipose tissue macrophages seem to have a role in metabolic regulation Pathogen recognition that is also tightly linked to the control of inflammation. and phagocytosis These tissue-specific functional programmes are Antigen processing transcriptionally regulated and are conferred by the and presentation expression of transcription factors that are unique to these macrophage populations inhibitor of nuclear factor-B Tissue repair factors NS (IBNS) and peroxisome proliferator-activated Metabolic regulators receptor- (PPAR) in macrophages of the colonic epithelium and white adipose tissue, respectively57,84.
Nature Reviews | Immunology

Adipose tissue
A type of connective tissue that is specialized for the storage of neutral lipids.

Transcription factor
A specialized nuclear protein that can bind to DNA and regulate gene expression. Most transcription factors have transactivation or repressor domains but, in addition, they can function as architectural proteins and promote chromatin remodelling by recruiting additional activator or repressor complexes.

Chromatin
Chromation is composed of DNA together with histones and other associated proteins.

Transcriptional co-regulator
Transcriptional co-regulators lack DNA-binding specificity and must be recruited to their target genes through interactions with transcription factors or by binding to particular chromatin modifications. Co-regulators have an important role in modulating gene expression and in many cases couple transcription factors to downstream effector mechanisms for gene regulation.

regulators (such as N-ethylmaleimide-sensitive factor 2 for cellular stress induced by reactive oxygen species8, hypoxia-inducible factor 1 for the hypoxic response9, X-box-binding protein 1 for the unfolded protein response 10 and aryl hydrocarbon receptor for the xenobiotic-induced response11). In each case, the genes that comprise a given transcriptional module are functionally related, which explains the requirement for their coordinated control by dedicated transcriptional regulators. Importantly, the full repertoire of TLR-induced transcriptional modules is currently unknown, as are the transcriptional master regulators of these modules. Nevertheless, the concept of transcriptional modules is useful when considering the heterogeneity of a complex transcriptional response, such as that induced by LPS in macrophages. Here, we first review what is known regarding the regulation of the LPS-induced transcriptional response by transcription factors, chromatin modifications and transcriptional co-regulators in macrophages, and the relative roles of each of these components in the inflammatory response. Then, we describe the mechanisms by which various signalling pathways modulate this transcriptional programme in distinct biological contexts. Finally, we emphasize the modular nature of the transcriptional control of inflammation as being central to its physiological regulation and therapeutic manipulation.

Transcription factors Induction of the LPS-dependent transcriptional response in macrophages is orchestrated by many transcription factors, consistent with the complexity of the response. These transcription factors can be divided into three categories on the basis of their mode of activation and function. This classification is not intended to demarcate mutually exclusive groups of transcription factors, but to illustrate general principles regarding their mechanisms of action and their role in the control of various inducible transcriptional modules in macrophages. The first category (class I) consists of transcription factors that are constitutively expressed by many cell types and that are activated by signal-dependent posttranslational modifications. In most cases, these transcription factors are retained in the cytoplasm in the basal state and their signal-dependent activation involves their nuclear translocation. This class is the best characterized of the three categories of transcription factors and it includes proteins that are known to have important roles in inflammation, such as NF-B, IFN-regulatory factors (IRFs) and cAMP-responsive-element-binding protein 1 (CREB1). The genes that are induced most rapidly by LPS stimulation (the so called primary response genes) are regulated by these transcription factors (FIG. 1). There are multiple mechanisms that quickly terminate the activation of NF-B and IRFs; for example, inhibitor of NF-B- (IB) exports NF-B from the nucleus
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LPS TLR4

ATF3 NF-B Class I transcription factors IRF C/EBP

Cytoplasm

PU.1 RUNX1 Primary response genes 0.52 hours Class II transcription factors 28 hours Secondary response genes

C/EBP IRF8 Nucleus Macrophagespecific gene expression Chromatin remodelling

Class III transcription factors

Figure 1 | lipopolysaccharide (lPS)-induced primary and secondary response genes are regulated by three categories of transcription factors. The first category (class I) consists of transcription factors that are activated post-translationally by Toll-like receptor (TLR) signalling, often at the step of nuclear translocation. Examples include Nature Reviews | Immunology nuclear factor-B (NF-B) and interferon-regulatory factor (IRF) proteins. These transcription factors control the induction of the primary response genes. The second category (class II) is comprised of transcription factors that are induced during the primary response, such as CCAAT/enhancer-binding protein- (C/EBP), which control induction of the secondary response genes. A third category of transcription factors (class III), which includes PU.1, C/EBP, runt-related transcription factor 1 (RUNX1) and IRF8, is not directly targeted by pro-inflammatory signals but is induced during macrophage differentiation, and has a key role in specifying macrophage-specific patterns of inducible gene expression. ATF3, activating transcription factor 3.

and/or facilitates its removal from the promoters of target genes12,13. However, positive feed-forward mechanisms might ensure the sustained activation of these transcription factors and their participation in subsequent waves of gene induction; for example, the production of tumour necrosis factor (TNF) triggered by LPS stimulation seems to be crucial for autocrine signalling and induction of a second wave of NF-B activation14,15. The second category of transcription factors (class II) are synthesized de novo after LPS stimulation. It is estimated that approximately 50 proteins make up this group, many of which have poorly defined functions in the context of the LPS-induced transcriptional response. These transcription factors regulate subsequent waves of gene expression after the primary response genes, and they can do so over a prolonged period of time5. The activity of these transcription factors is often subject to positive feedback control, and because these proteins are transcriptionally upregulated, a general principle here seems to be transcriptional autoregulation. For example, the amplification of the LPS-induced transcriptional response by CCAAT/enhancer-binding protein- (C/EBP) requires its autoinduction16. The stable upregulation of expression of these transcription factors could also enable the reprogramming of macrophage functions. For this reason, we speculate that some transcription factors in this category might function as master regulators of distinct functional modules. The third category of transcription factors (class III) consists of lineage-specific transcriptional regulators, the expression of which is turned on during macrophage differentiation. Notable members of this group
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include Pu.1 (also known as SPI1) and C/EBP, as well as runt-related transcription factor 1 (RuNX1) and IRF8 (REFS 17,18). Although none of these transcription factors is exclusive to macrophages (for example, Pu.1 is also expressed by B cells), they are induced during macrophage differentiation and their combinatorial expression specifies the macrophage phenotype. These proteins turn on constitutively expressed genes in macrophages, remodel chromatin at inducible genes and silence genes that are associated with alternative cell fates. In mature macrophages, these transcription factors mediate cell type-specific responses to inflammatory signals and other stimuli, presumably by conferring a permissive chromatin state on macrophage-specific inducible genes. A unique mode of action of at least some of the transcription factors in this category is the organization of cell type-specific, higher order chromatin structure or chromosomal domains. In particular, RuNX1 has been shown to anchor specific genomic loci to the nuclear matrix (the architectural scaffold of the nucleus) to assemble domains of active or inactive chromatin19 that could determine the macrophagespecific patterns of active, silenced and inducible genes on a global scale. The transcription factors of the three categories mentioned above do not act independently, but function coordinately to control the LPS-induced transcriptional response. using a systems biology approach, Aderem and colleagues have identified some of the regulatory circuits that are shedding light on the logic of this combinatorial control4,16,20. They have found, using global kinetic profiling, that LPS-induced gene expression in macrophages
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Table 1 | Three main classes of transcription factors regulate the lipopolysaccharide-induced transcriptional programme
Class of transcription factor
Class I: constitutively expressed but latent Class II: synthesized de novo

Transcription factor activation

Signal-dependent post-translational modifications

Intracellular localization Function of transcription factors of transcription factors

Often regulated at the point of nuclear translocation Responsible for the primary phase of gene induction; there are multiple mechanisms for rapidly terminating signal-dependent transcription factor activation Regulate subsequent waves of gene expression; might stably reprogramme gene expression

mode of action

Examples

Integrate signals from diverse signalling pathways

NF-B, IRF3

Signal-dependent transcriptional induction

Most are constitutively nuclear

Some might specify transcriptional modules that encode functional programmes in macrophages Might organize higher-order chromatin structure or chromosomal domains

C/EBP

Class III: induced during cell differentiation

Constitutively expressed and active

Constitutively nuclear

Regulate constitutive, as well as inducible, gene expression; establish macrophage-specific patterns of chromatin remodelling during cell differentiation

RUNX1, PU.1, C/EBP

C/EBP, CCAAT/enhancer-binding protein; IRF3, interferon-regulatory factor 3; NF-B, nuclear factor-B; RUNX1, runt-related transcription factor 1.

can be divided into a limited number of distinct patterns of gene induction (and repression). using this approach, combined with motif scanning of the promoters of these genes in silico, they can define sets of genes that seem to be coordinately regulated and the transcription factors that are likely to control their expression. In this way, they showed that the sustained expression of several inflammatory genes is mediated by a transcriptional network that consists of three transcription factors: NF-B, which functions as the activator; activating transcription factor 3 (ATF3), which functions as the inducible repressor; and C/EBP, which functions as the inducible activator and amplifier16. These observations also illustrate how combinatorial control by multiple transcription factors enables NF-B, which is required for the induction of most of the LPS-induced genes, to engage in module-specific regulation of inflammatory gene expression. In addition, the temporal characteristics of transcription factor activation (and attenuation) might be essential for determining the repertoire and specificity of a complex transcriptional response. A series of elegant studies by Hoffmann and colleagues have shown that perturbations in the dynamics of NF-B activation can result in profound, qualitative changes in gene expression15,21. This is associated not only with stimulusintrinsic alterations in inducible gene expression, but also results in the modification of transcriptional responses triggered by other signalling pathways that activate NF-B22,23. So, the complex transcriptional programme induced in macrophages after LPS stimulation is a product of the coordinated action of the three categories of transcription factors described above (TABLE 1).

interplay between these processes in the control of inflammation. Here, we discuss the role of chromatin modifications in regulating inflammatory gene expression, with a focus on covalent histone modifications (BOX 2). we describe what is known about how differential histone modifications can mediate gene-specific transcriptional regulation and discuss how chromatin architecture delimits cell type and signal specificity in the transcriptional control of inflammation. Histone modifications. Several histone modifications have been shown recently to differentially regulate subsets of LPS-induced genes and they are of particular interest because of their role in regulating specific transcriptional modules within the multicomponent transcriptional response mediated by LPS-induced signalling (BOX 2) . one of the first studies in this area indicated that phosphorylation of histone 3 at serine 10 (H3S10) might have a gene-specific role in NF-B recruitment 25. After LPS stimulation, the genes encoding IL-6, IL-12p40 and CC-chemokine ligand 2 (CCL2), but not TNF and CCL3, undergo H3S10 phosphorylation at their promoters. This phosphorylation event depends on the mitogen-activated protein kinase p38, and specific blockade of p38 activation inhibits H3S10 phosphorylation, NF-B recruitment and gene induction25. It is unclear how the phosphorylation of H3S10 is coupled to NF-B recruitment, but this model is consistent with other studies showing that H3S10 phosphorylation is associated with transcriptional activity 26. Moreover, the inhibition of inflammatory gene induction by some pathogens is associated with disruption of H3S10 phosphorylation (see below). It is not known whether H3S10 is a direct substrate of p38, but the data indicate that p38 or a p38-regulated kinase, such as mitogen- and stress-activated kinase 1 (MSK1) or MSK2, might be recruited to a subset of genes to confer H3S10-dependent transcriptional regulation. IB kinase- (IKK) can also be recruited to gene
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Dynamic chromatin remodelling Recent studies have highlighted an important role for chromatin in the control of inflammatory gene expression24. DNA methylation and RNA interference are other epigenetic mechanisms for regulating gene expression, and future studies will shed light on the functional
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Box 2 | Covalent histone modifications and the control of gene expression
Histones, the proteins that package the genetic material, are subject to a large number of covalent modifications, including lysine and arginine methylation, lysine acetylation, serine phosphorylation and lysine ubiquitylation85. The counteracting activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs) establish the levels of histone acetylation, whereas lysine methylation is regulated by SET (suppressor of variegationenhancer of zestetrithorax) domain family proteins (for methylation) and lysine-specific demethylase 1 (LSD1) and Jumonji C (JMJC) domain-containing proteins (for demethylation)85. Strahl and Allis first proposed that histone modifications are found in non-random patterns in the genome to form a histone code, with distinct combinations of modifications specifying unique states of gene expression86. Reader proteins bind to and specifically discriminate between different histone modifications, and have a crucial role in coupling the recognition of histone modifications to the downstream effector mechanisms that regulate gene expression; they can recruit other chromatin-modifying factors or the transcriptional machineries that control the initiation and elongation phases of transcription and mRNA processing86. In the past few years, genome-wide maps of histone modifications coupled with transcriptional profiling have identified many histone modifications as being either active or inactive. Histone 3 lysine 4 (H3K4) trimethylation, for example, is associated with transcriptionally active or poised loci, whereas H3K27 and/or H3K9 trimethylation correlate with gene silencing85. Moreover, whereas some histone modifications seem to associate strongly with a particular state of gene expression (for example, active, poised or silent loci), others seem to mark much smaller subsets of active or inactive genes. Histone modifications that associate strongly with a particular state of gene expression probably regulate general features of gene induction or repression, often because they couple directly to various transcriptional machineries. For example, transcription factor IID (TFIID), which is essential for promoter recognition by RNA polymerase II, is recruited to inducible genes in part through promoter-localized H3K4 trimethylation87. This provides the rationale for the nearly invariant distribution of H3K4 trimethylation at the promoters of transcriptionally active and poised genes. Conversely, modifications such as H3K27 trimethylation and H2AK119 ubiquitylation might function more exclusively to control only subsets of inducible or repressible genes, and therefore might regulate transcriptional modules within a more complex transcriptional response.

Small interfering RNA (siRNA)-mediated knockdown

Double-stranded RNAs (dsRNAs) with sequences that precisely match a given gene can knockdown the expression of that gene by directing RNA-degrading enzymes to destroy the encoded mRNA transcript. The two most common forms of dsRNA used for gene silencing are short usually 21-base pair long siRNAs or the plasmid-delivered short hairpin RNAs (shRNAs).

Polycomb proteins
A group of proteins that are required to maintain the silencing of genes encoding key developmental regulators, such as Hox.

promoters in a signal-dependent manner to phosphorylate H3S10 (REFS 27,28). These examples illustrate that chromatin can be a direct, albeit distal, target of signal transduction cascades, and they underscore the fundamental role of chromatin in regulating geneenvironment interactions; however, it is not known what directs the promoter-specific targeting of these kinases. Perhaps even more interesting are the inhibitory histone modifications that mark subsets of inducible genes. ubiquitylation of H2A at lysine 119 (H2AK119) is one such modification that inhibits the basal expression level of some LPS-inducible genes for example, CCL5, CXC-chemokine ligand 10 (CXCL10) and CXCL2, but not CXCL1 in a macrophage cell line29. This study proposed that ubiquitylation of H2AK119 at these gene promoters is necessary to prevent recruitment of the elongation factor FACT (facilitates chromatin transcription) complex. LPS-induced signalling triggers the gene-specific recruitment of the deubiquitylating enzyme 2A-HuB (also known as DZIP3), the activity of which is necessary for H2AK119 deubiquitylation and hence gene induction29. Consistent with this model, small interfering RNA (siRNA)-mediated knockdown of 2A-HuB is sufficient to decrease the level of basal H2AK119 ubiquitylation and leads to the recruitment of FACT to gene promoters and small but significant increases in the level of basal gene expression29. This indicates that at these promoters, H2AK119 ubiquitylation might be necessary to prevent low-level, leaky gene expression. It is interesting that some, but not all, inflammatory genes seem to require this additional regulatory mechanism for maintaining basal repression; perhaps these genes encode proteins with biological functions that must be particularly tightly regulated.

Another subset of LPS-inducible genes undergoes signal-dependent demethylation of trimethylated H3K27 as a prerequisite for induction. The enzyme responsible for this activity, lysine-specific demethylase 6B (KDM6B; also known as JMJD3), is itself transcriptionally upregulated after LPS stimulation, and siRNA-mediated knockdown of KDM6B inhibits demethylation and the induction of Bmp2 (bone morphogenetic protein 2) expression30. This is consistent with previous studies that have established a role for H3K27 trimethylation in maintaining gene silencing through the recruitment of Polycomb proteins. As removal of the H3K27 inhibitory modification enables gene expression during cellular differentiation31, KDM6B-dependent regulation of an LPS-inducible gene30 indicates that macrophage activation has some features that are similar to differentiation. It is important to point out that repressive histone modifications, such as H2AK119 ubiquitylation and H3K27 trimethylation, are additional regulatory checkpoints of inducible gene expression. Stimulusdependent induction of genes bearing these modifications requires recruitment of the appropriate chromatin-modifying enzymes; hence, these histone modifications restrict both the types of biological signal and the classes of genes that are induced. Moreover, these repressive histone modifications could be targeted by anti-inflammatory signalling pathways as a means to enforce gene-specific inhibition of the inflammatory response (see below). It should be interesting to characterize further the LPSinduced transcriptional response with respect to other inhibitory epigenetic modifications, including H3 arginine 2 dimethylation, H3K9 dimethylation and DNA methylation.
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a CpG island-associated primary response gene
Basal transcription Methylation Acetylation Nucleosome H3K4 H3K9 H4 HAT SP1 Pol II Unspliced transcript Stimulus-dependent transcription Acetylation H4 HAT P-TEFb NF-B Ser5 Spliced transcript

P Ser5

P Ser2
Pol II

b Secondary response gene

Inaccessible chromatin Remodelling Methylation Acetylation HAT NF-B BRG1 IRF3 H3K4 H3K9 Ser5 Acetylation H4 HAT P-TEFb NF-B

P Ser2
Pol II

BRG1 IRF3

Figure 2 | Two distinct modes for regulating inducible genes. a | At primary response genes that have promoters containing CpG islands, Toll-like receptor (TLR) signalling induces a switch from basal gene transcription (mediated by serine 5 (Ser5)-phosphorylated RNA polymerase II (Pol II)) to the stimulus-dependent production of Nature Reviews | Immunology mature, processed transcripts, which depends on the recruitment of positive transcription elongation factor b (P-TEFb). b | By contrast, secondary response genes require chromatin remodelling as a prerequisite for transcription factor binding and the recruitment of histone-modifying enzymes and the general transcription initiation machinery. BRG1, BRM/SWI2-related gene 1 (also known as SMARCA4); HAT, histone acetyltransferase; IRF3, interferon-regulatory factor 3; NF-B, nuclear factor-B; SP1, specificity protein 1.

Nucleosome
This is the basic repeating unit of eukaryotic genomes. Nucleosomes consist of 146 base pairs of DNA wound around an octamer of histone proteins.

SWISNF
(switching-defective sucrose non-fermenting). An ATP-dependent chromatin remodelling protein complex that was first identified in yeast. Related complexes exist in mammals (where they are known as BAF) and are involved in the chromatin remodelling of various genes.

CpG island
A sequence of 0.52 kilobases that is rich in CpG dinucleotides. They are mostly located upstream of housekeeping genes and also of some tissue-specific genes, and they coincide with gene promoters in these contexts. In mammalian cells, most CpG islands are hypomethylated with respect to the rest of the genome.

Chromatin architecture. Chromatin remodelling 32 can also regulate the dynamic induction of inflammatory gene expression. This activity is mediated by chromatin remodelling complexes, which use ATP to slide nucleosomes relative to DNA or to alter nucleosome DNA contacts, thereby modulating the accessibility of chromatin-associated DNA to transcriptional regulators33. Chromatin remodelling complexes are generally thought to be regulated mainly at the level of recruitment to target gene promoters. However, a recent study showed that the chromatin remodelling complex SWISNF (switching-defectivesucrose non-fermenting; also known as BAF) can also be regulated after recruitment to a target gene by a Ca2+calmodulin-dependent signal in LPS-stimulated macrophages34. Smale and colleagues35 have recently shown that the requirements for chromatin remodelling differ between LPS-inducible primary and secondary response genes (FIG. 2) . The induction of secondary response genes requires de novo protein synthesis, whereas the upregulation of expression of primary response genes does not (although this does not preclude the regulation of primary response genes in the secondary phase of the transcriptional response)35. Chromatin remodelling by SwISNF is necessary for the induction of secondary response genes (and the delayed primary response genes), but not for the early primary response genes35. Indeed, subsequent studies showed that whereas the promoters of secondary response genes undergo LPSdependent H3K4 trimethylation and H3 acetylation, the promoters of SwISNF-independent primary response genes have high basal levels of these active histone modifications, even before LPS stimulation32,36,37. Importantly, the presence of active chromatin correlates closely with

promoter GC content, which indicates that the underlying DNA sequence at promoters with CpG islands, which are found in many SwISNF-independent primary response genes, could direct chromatin remodelling to the active state37. This is supported by the observations that these CpG islands are associated with low nucleosome density and do not assemble stable nucleosomes in vitro 37, and that these promoters also bear active chromatin even in embryonic stem cells32,37, which indicates that chromatin remodelling is not a consequence of cellular differentiation. Previous studies 38,39 have indicated a role for chromatin in restricting the accessibility of transcription factors to their target genes, and these studies expand on this concept, showing that active chromatin might enable more promiscuous induction of GC-rich promoters of primary response genes, whereas inactive chromatin at the promoters of secondary response genes restricts gene induction to a limited range of stimuli in specialized cell types32,37. Along these lines, it is noteworthy that GC-rich primary response genes, but not most secondary response genes, are regulated by the transcriptional co-repressors nuclear receptor co-repressor (NCoR) and REST co-repressor (RCoR; also known as CoREST)32 (see below), perhaps to counter the active chromatin that is associated with these genes in the basal state. It will be interesting to further characterize primary and secondary response genes with respect to other features of chromatin, and to understand the consequences of these chromatin modifications for gene induction. Further analyses of the GC-rich primary response genes have uncovered additional surprises (FIG. 2) . In the basal state, these genes (but not secondary response genes) are transcribed at low levels by RNA
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Transcriptional co-repressor
Transcriptional co-repressors are protein complexes, including NCOR and SMRT, that associate with nuclear hormone receptors and other transcriptional regulators to inhibit gene transcription. A common mechanism of repression is by recruiting histone-deacetylase complexes to reverse the actions of histone acetyltransferases.

CBPp300
CREB-binding protein (CBP) and p300 are transcriptional co-activators that interact with many transcription factors to promote recruitment of the RNA polymerase holoenzyme and other transcriptional regulators, thereby allowing transcriptional induction. In addition, p300 and CBP have histone acetyltransferase activity, such that these proteins can influence chromatin activity by modulating nucleosomal histones.

polymerase II (Pol II) that is phosphorylated on serine 5 (Ser5P) but not on Ser2 of its carboxy terminal domain. Consistent with a crucial role of Ser2P in recruiting the RNA processing and splicing machinery, these basal transcripts of the primary response genes are extensively elongated but are not spliced32. LPS-induced signalling recruits positive transcription elongation factor b (P-TEFb), which phosphorylates Pol II on Ser2 and results in the generation of high levels of mature, spliced transcripts of the primary response genes. So, an important checkpoint for inducible gene expression seems to involve signal-dependent engagement of P-TEFb, conversion of Ser5P Pol II to the Ser2P form, and the production of mature, spliced transcripts of primary response genes32 (BOX 3). The organization of inducible transcriptional responses into a primary and a secondary component was first described for the mitogen-induced response40, and it is probably generally applicable to all other inducible stimuli. It will be interesting to see whether the different mechanisms described here for the regulation of CpG-associated primary response genes and secondary response genes will also apply to other inducible gene expression programmes.

Histone acetyltransferase
A protein that acetylates core histones on lysine residues, which has important regulatory effects on chromatin structure and assembly, and on gene transcription. In general, increased levels of histone acetylation are associated with activation of gene expression.

Co-regulators of the inflammatory response Co-regulators are transcriptional regulators that, unlike transcription factors, lack DNA-binding specificity and must be recruited to their target genes through other mechanisms. Because many co-activators and corepressors (co-regulators that activate and inhibit gene expression, respectively) function in the LPS-induced transcriptional response, here we focus on only a few of the most important or interesting examples.
Co-activators. Inflammatory gene induction by transcription factors such as NF-B depends on co-activator proteins. These transcriptional regulators can promote inflammatory gene expression in multiple ways. Many
Box 3 | Transcription initiation and elongation

co-activators have histone-modifying activities and can remodel chromatin at target genes to promote gene induction. For example, CBPp300-mediated histone acetylation can be coupled to the recruitment of SwISNF and other factors41, whereas PCAF (p300 CBP-associated factor) and GCN5 (general control of amino-acid synthesis 5) histone acetyltransferases can direct transcription elongation factors to target genes32. other co-activators lack intrinsic enzymatic activity and might promote the assembly of a transactivating complex. Yet another example of a co-activator function is provided by the IB family member IB. This protein is transcriptionally induced by TLR signalling and promotes the exchange of inhibitory p50 (also known as NF-B1) homodimers for transcriptionally active p50p65 (also known as RELA) heterodimers on some target gene promoters42. Intriguingly, recent studies indicate that p65 and IRF3 have additional roles in the LPS-induced transcriptional programme as co-activators as well as DNAbinding transcription factors. Leung et al. showed that IRF3-dependent regulation of Ccl2 expression requires IRF3 functioning as a co-activator for p65 (REF. 43) . Consistent with this finding, Glass and colleagues showed that the glucocorticoid receptor inhibits a subset of LPS-inducible genes by blocking the coactivator function of IRF3. In addition, p65 and IRF3 were shown to interact, and the glucocorticoid receptor could disrupt this binding 44. Conversely, p65 was suggested to be a co-activator for IRF3 and to facilitate IRF3 binding to DNA45. why do some but not all p65-regulated genes engage IRF3 as a co-activator? It is possible that subtle differences in the NF-B-binding site in a gene can induce conformational changes in p65 in an allosteric manner, thereby conferring specificity of co-activator binding 43,46. p65 and IRF3 might have evolved mutually co-activating functions because of their parallel roles in many aspects of the inflammatory response, and the

In the classical model of transcription initiation, the key regulated step is the signal-dependent engagement of RNA polymerase II (Pol II) at inducible promoters. Transcription factors that are activated by a triggering signal are recruited to target promoters, where they have an essential role in directing the recruitment of the transcription initiation machinery. However, it has also been appreciated for some time that at heat shock-inducible promoters in Drosophila melanogaster, Pol II is constitutively bound but stalled88. The essential elongation factor positive transcription elongation factor b (P-TEFb) is recruited following heat shock, and its kinase activity is necessary to inactivate inhibitors of elongation and to activate positive regulators, thereby triggering productive transcription elongation. In addition, the carboxy terminal domain of Pol II contains heptapeptide repeats that are also important targets of P-TEFb; when phosphorylated at serine 2 (Ser2) in the heptapeptide repeats, the carboxy terminal domain of Pol II can function as a platform for the recruitment of various mRNA-processing machineries. So, at heat shock-inducible promoters of D. melanogaster, signal-dependent gene induction seems to be regulated at the level of P-TEFb-mediated transcription elongation88. Interestingly, recent studies have indicated that this phenomenon might be much more prevalent than originally thought. In several studies, Pol II was shown to bind many promoters across the genome in the absence of active gene induction. This led to the proposal that many genes are actually associated with promoter-bound, transcriptionally stalled Pol II in the basal state, and that signal-dependent recruitment of P-TEFb is the key regulatory checkpoint8991. Interestingly, however, a recent study showed that signal-independent binding of Pol II to promoters in the basal state is associated with basal transcription that produces full-length but unspliced transcripts32. This indicates that Pol II binding in some other contexts could also be associated with basal transcription. It will be important to determine whether signal-independent Pol II recruitment is associated with a block of elongation versus basal transcription in a cell type-specific or gene-specific manner.

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Histone deacetylase
A protein that removes the acetyl groups from lysine residues that are located at the amino termini of histones. In general, decreased levels of histone acetylation are associated with the repression of gene expression. The balance of histone acetylation is maintained by the interplay between histone deacetylases and histone acetyltransferases.

combined transactivation potential of the two factors could result in increased inducible transcription of target genes. It would be interesting to determine if p65 and IRF3 can co-activate other transcription factors in the regulation of additional functional programmes. Co-repressors. NCoR and the closely related protein SMRT (silencing mediator of retinoid and thyroid receptors) have emerged as important regulators of inflammatory gene expression. The NCoR and SMRT multiprotein co-repressor complexes contain histone deacetylases (HDACs) and potentially other activities for inhibiting gene expression, and their stimulusdependent dismissal from the promoters of inflammatory genes (a phenomenon known as derepression) is a prerequisite for the inducible expression of these genes. NCoR is directed to many inflammatory genes, in part by the transcription factor JuN (also known as AP1), and its clearance is triggered by signal-induced exchange of JuN homodimers for transcriptionally active JuNFoS heterodimers47,48. By contrast, SMRT is recruited to gene promoters by the transcriptional repressor TEL (translocationETSleukaemia) 47 . Interestingly, some inflammatory genes are regulated by both NCoR and SMRT co-repressor complexes,

indicating that this group of genes can be regulated by a greater diversity of signals47,49. In this regard, it is interesting that another co-repressor complex, RCoR, is also associated with inflammatory genes and is cleared from target promoters after LPS-induced signalling 32, which indicates that it might also provide a stimulus-specific regulatory checkpoint. Importantly, whereas LPS stimulation dismisses NCoR from target genes before their induction, a counteracting activity is provided by nuclear receptor-mediated stabilization of NCoR (FIG. 3 and see below). with respect to the control of inflammatory gene expression by these co-repressors, several interesting questions remain. what is the repertoire of genes that are regulated by each co-repressor? why are some genes controlled by more than one co-repressor? How do these corepressors mediate repression, and do they have distinct modes of repression? How are they recruited in the basal state and dismissed in a signal-dependent manner?

Basal state Methylation H3K4 H3K9 H4

HDAC Co-repressor

Acetylation Pol II

Repression of LPS-inducible inflammatory genes

Inactive transcription factor complex PPAR LXRs Others TLR ligands or other pro-inflammatory signals

Ser5 HAT Co-activator Pol II Active transcription factor complex

Transcript

P Ser2
Activation of LPS-inducible inflammatory genes

Figure 3 | Control of inflammatory gene expression by co-activators and co-repressors. In the basal state, co-repressors such as nuclear receptor co-repressor (NCOR), silencing mediator of retinoid and thyroid receptorsNature Reviews | Immunology (SMRT) and REST co-repressor (RCOR) are recruited to target promoters by various transcription factors, where they counter inflammatory gene expression by inhibiting histone acetylation (and possibly also other activating histone modifications such as H3K4 trimethylation). Toll-like receptor (TLR) signalling and other pro-inflammatory signals induce the exchange of co-repressors for co-activators on target promoters, resulting in the activation of gene expression. Nuclear receptors such as peroxisome proliferatoractivated receptor- (PPAR), glucocorticoid receptor and liver X receptors (LXRs) constitute an important class of anti-inflammatory regulators, which block inflammation in part by inhibiting this exchange. In the best studied case of NCOR, inflammatory signals trigger its proteasomal degradation, and this can in turn be inhibited by PPAR and LXRs. HAT, histone acetyltransferase; HDAC, histone deacetylase; Pol II, RNA polymerase II.
NATuRE REVIEwS | Immunology

Negative regulation of inflammatory genes The induction of an inflammatory response is essential for host defence during infection, but timely resolution is also important to limit the detrimental effects of inflammation, particularly when it is inappropriately sustained or increased. For this reason, acute inflammation leads to the upregulation of expression of many negative regulators of inflammation. These negative regulators fall into two main categories: signal-specific regulators and gene-specific regulators. The first category consists of regulators that inhibit signal transduction by TLRs and other inflammatory pathways, and includes A20, ST2, IL-1R-associated kinase M (IRAKM) and suppressor of cytokine signalling (SoCS) proteins50. Although these proteins inhibit inflammatory signalling through various mechanisms, they all function proximal to the receptor, and so are expected to block gene induction by that receptor in a global manner. The second category includes transcriptional repressors or other negative regulators that function to modulate gene expression. There are two types of transcriptional negative regulator: basal repressors and inducible repressors. Basal repressors are constitutively expressed and are important for the basal repression of many inflammatory genes. For example, homodimers of the NF-B family member p50 can function as transcriptional repressors that are exchanged for transcriptionally active p65p50 heterodimers as a result of signalling induced by LPS51. JuN49, SIRT6 (silent mating type information regulation 2 homologue 6)52 and co-repressors such as NCoR47,49, SMRT47 and RCoR32 have also been shown to mediate basal repression of a subset of LPS-inducible inflammatory genes (FIG. 3) . By contrast, inducible repressors are normally expressed only at low levels or not at all, but are transcriptionally induced by LPSinduced signalling, indicating that they are part of a negative feedback mechanism that limits the inflammatory response. Importantly, inducible repressors block the expression of secondary response genes, whereas basal repressors inhibit the expression of primary
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response genes associated with CpG islands. These primary response genes might require repression in the basal state to prevent low-level constitutive expression, whereas the inaccessible nature of the chromatin at secondary response genes is generally refractory to basal gene expression due, in part, to the occlusion of binding sites for transcription factors. Therefore, it is probable that different modes of transcriptional repression can operate to inhibit primary and secondary response genes. LPS-inducible negative feedback loops. Members of the IB family are noteworthy examples of inducible negative regulators. IB, which was the first identified member of this family, mainly inhibits the expression of NF-Bdependent genes on a global level, but IBNS and B cell lymphoma 3 (BCL-3) limit inflammation in a genespecific manner 5355. IBNS and BCL-3 are transcriptionally induced by LPS stimulation, and their genetic deletion results in the enhanced induction of inflammatory genes and increased susceptibility to endotoxic shock54,56. Both proteins mediate the negative regulation of inflammation by modulating the exchange of active NF-B dimers for their inactive counterparts at target gene promoters. Interestingly, IBNS and BCL-3 seem to control distinct sets of genes IL-6, IL-12p40 and IL-18 for IBNS, and TNF, IL-10 and IL-1 for BCL-3 although the basis of this specificity remains to be determined54,55. ATF3 is another transcriptional negative regulator that can be induced by LPS stimulation. Similarly to IBNS and BCL-3, loss of ATF3 leads to hyperinduction of a subset of LPS-inducible genes and increased susceptibility to endotoxic shock20. ATF3-mediated transcriptional inhibition can be reversed by HDAC inhibitors, which indicates that at least one key aspect of the function of ATF3 is to recruit HDACs to target genes20. Moreover, ATF3 is a member of the CREB family of basic leucine zipper transcription factors and has been shown to form a regulatory circuit with NF-B and C/EBP at some LPS-inducible genes16. The transcriptional programme induced by LPS is undoubtedly controlled by many other transcriptional repressors, the identity and mechanisms of function of which remain poorly characterized and warrant further study. Finally, it is worth noting that many of the same proteins that function in the LPS-induced negative feedback loops are also upregulated by other pathways that inhibit inflammatory gene expression. For example, IL-10, which is an important anti-inflammatory cytokine for many cells of the immune system including macrophages, can also induce the expression of both IBNS and BCL-3 (REFS 57,58). Interestingly, in colonic macrophages, IL-10-mediated induction of IBNS expression seems to be of particular importance in inhibiting the production of IL-6 and other inflammatory responses57. This is shown by the development of colitis in IBNS-deficient mice, similarly to IL-10deficient mice54. In other settings, however, BCL-3 or other IL-10-inducible negative regulators might be more important for mediating IL-10-dependent suppression of inflammation. Negative regulation by anti-inflammatory pathways. Inflammatory gene induction is subject to negative regulation by a large number of pathways and mechanisms that are important to limit the pathophysiological consequences of excessive inflammation. These include antiinflammatory cytokines (such as IL-10 and transforming growth factor- (TGF)), nuclear hormone receptors (including, but not limited to, glucocorticoid receptors, liver X receptors (LXRs), peroxisome proliferator-activated receptors (PPARs) and vitamin D receptor) and cAMP. Consistent with the crucial anti-inflammatory role of IL-10, mice lacking this cytokine mount exaggerated inflammatory responses to infection and in septic shock59 and develop spontaneous colitis due to perturbed host commensal homeostasis in the intestines59. An important mechanism by which IL-10 inhibits inflammation is at the level of transcription, as indicated by the ability of cycloheximide treatment to block IL-10-mediated inhibition of primary response genes60. As mentioned above, IL-10 induces the expression of several negative regulators that mediate gene-specific repression of the LPS-induced inflammatory response. IBNS and BCL-3 disrupt NF-B-mediated transcription, but the function of other IL-10-induced proteins and whether they modify chromatin in a gene-specific manner are not clear. cAMP is another negative regulator of inflammation61. It is activated by G-protein-coupled receptors for glucagon, acetylcholine, adenosine and many other biological signals. cAMP works mainly by activating protein kinase A (PKA), but the exact mechanism of cAMP-mediated inhibition of inflammatory gene expression is not known. Nuclear receptors are another major class of transcriptional negative regulators of inflammation. These include glucocorticoid receptors, PPARs and LXRs. These proteins therefore integrate the control of inflammation with various important physiological functions, such as metabolism62. Nuclear receptors are thought to inhibit inflammation by at least two distinct mechanisms. Activated nuclear receptors can directly induce gene expression programmes that are anti-inflammatory. For example, the activation of glucocorticoid receptor increases the uptake of apoptotic cells by macrophages concurrent with the inhibition of inflammatory signalling63. Nuclear receptors can also inhibit inflammation directly, in a gene-specific manner. For example, PPAR activation results in disassociation of the BCL-6 co-repressor from the Ccl2 promoter, thereby enabling recruitment of BCL-6 to inflammatory target genes to mediate transcriptional inhibition 64. Alternatively, activated nuclear receptors can be recruited to some inflammatory genes where they inhibit the clearance of the co-repressors NCoR and SMRT, a process known as transrepression62. Interestingly, transrepression by LXRs and PPAR results in the impaired induction of distinct subsets of inflammatory genes, but it is not known what dictates the differential gene-specific recruitment of LXRs and PPAR62. The multitude of mechanisms by which nuclear receptors can inhibit inflammatory gene expression underscores the importance of transcriptional control of inflammation by metabolism and other physiological processes.
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Endotoxic shock
A clinical condition that is induced by hyper-reaction of the innate immune system to bacterial LPS. It is mediated by the inflammatory cytokines IL-1 and TNF, which are produced in large amounts due to sustained stimulation of TLR4 by LPS.

Septic shock
A systemic response to severe bacterial infections, which is generally caused by Gram-negative bacterial endotoxins, that leads to a hyperactive and out-of-balance network of inflammatory cytokines, affecting vascular permeability, cardiac function and metabolic balance. This can lead to tissue necrosis, multiple-organ failure and death.

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SIRT proteins, which are nicotinamide adenine dinucleotide (NAD)-dependent deacetylases of the class III HDAC family, have also been implicated recently in the transcriptional control of inflammatory genes. unlike its yeast homologue Sir2 and the class I and class II HDACs, SIRT1 targets transcription factors and co-activators for deacetylation; SIRT1 counters p300-mediated acetylation of NF-B p65 in its transactivation domain, which leads to a block of p65-dependent gene induction that is independent of DNA binding 65. SIRT6 also inhibits NF-B activity, but in this context NF-B itself is not the target; instead, SIRT6 deacetylates H3K9 at the promoters of some NF-B-regulated genes66. Acetylated H3K9 is closely associated with transcriptional activation across the genome, and SIRT6-mediated deacetylation of H3K9 represses both basal and stimulus-dependent gene induction66. These studies raise the possibility that SIRT1 and SIRT6 (and perhaps other members of the family) might couple energy status (in the form of NAD sensing) to the control of inflammation. Finally, HES and HEY are two other LPS-inducible transcriptional repressors that function to attenuate the induction of a subset of inflammatory responses67. In an intriguing example of signalling pathway crosstalk, LPS-dependent upregulation of expression of HES and HEY by macrophages requires concomitant activation of the Notch pathway and the downstream transcriptional activator recombination-signal-binding protein-J (RBP-J). Moreover, IFN signalling blocks HES and HEY upregulation, illustrating a novel mechanism for the well-established priming effect of IFN on LPS-stimulated macrophages67. Global transcriptional profiling to define the repertoire of HES- and HEYrepressed genes should help to define the physiological contexts in which Notch signalling inhibits inflammatory gene expression. Pathogen-mediated chromatin remodelling. Pathogens can modulate inflammation at multiple levels. General inflammatory signalling pathways are a common target of pathogen virulence factors, but in addition, pathogens have evolved mechanisms to inhibit inflammation in a module-specific manner. For example, Mycobacterium tuberculosis subverts the IFN response in macrophages without disrupting the JAKSTAT (Janus kinasesignal transducer and activator of transcription) pathway, and it does so in a gene-specific manner that targets a subset of IFNregulated genes68. In addition, several recent studies have highlighted how pathogens can modify chromatin at inflammatory genes69. Inhibition of TNF (but not IL-10) production by infection with Toxoplasma gondii is associated with decreased H3 acetylation and H3S10 phosphorylation, and decreased recruitment of p65 to the Tnf promoter 70. Interestingly, several other pathogens also downregulate H3S10 phosphorylation, presumably for the purpose of blocking expression of a subset of inflammatory genes71. It is not known why or how different pathogens have converged on this particular chromatin modification to subvert the host inflammatory response. p38 is a common target of pathogen virulence factors, so it is possible that the downregulation of H3S10 phosphorylation is a consequence of p38 inhibition. It will be interesting to see if pathogens use other chromatin remodelling activities to subvert specific functional programmes of the host immune response.

Deacetylation
Acetylation is a post-translational modification of chromatin components, particularly histones, and other proteins. Histone deacetylases have been identified as components of nuclear co-repressor complexes.

Notch pathway
A pathway comprising highly conserved transmembrane receptors that regulate cell-fate choice in the development of many cell lineages, and so are crucial for the regulation of embryonic differentiation and development.

MicroRNAs
Single-stranded RNA molecules of approximately 2123 nucleotides in length that are thought to regulate the expression of other genes.

Additional control mechanisms Recent studies have provided evidence of a potential role for long non-coding RNAs (lncRNAs) in regulating inflammatory gene expression 72,73. The role of microRNAs in this process has been recently reviewed74 and will not be addressed here. Apart from their role in X chromosome inactivation and imprinting, the function of lncRNAs is not well understood. Broadly speaking, lncRNAs have been described that direct the expression of specific genomic loci (regulation in trans); alternatively, the process rather than the product of lncRNA transcription has been shown to inhibit or activate gene expression (regulation in cis)75. An example of regulation in cis is the requirement for the signaldependent transcription of a lncRNA for the induction of the closely juxtaposed lysozyme gene73. Lysozyme is an antimicrobial enzyme that hydrolyses bacterial cell wall peptidoglycan, the expression of which is induced during macrophage differentiation. In the basal state, the insulator protein CCCTC-binding factor (CTCF) is bound to regulatory elements of the lysozyme gene, but after LPS-induced signalling, transcription of a lncRNA through this regulatory region of the lysozyme gene displaces CTCF and leads to local chromatin remodelling and induction of the lysozyme gene. Insulator proteins can block the interactions between regulatory regions of a gene76, so the displacement of CTCF might enable the distal enhancer of the lysozyme gene to interact, in a signal-dependent manner, with the promoter 73. In light of this, a recent study identified ~1,600 highly conserved lncRNAs in mammalian cells, of which more than 20 can be induced by LPS stimulation of bone marrow-derived dendritic cells72. Some of these inducible lncRNAs might regulate other genes in cis, and it will be interesting to understand what categories of LPS-inducible genes are subject to this type of control. Previous studies have shown that many proinflammatory cytokine and chemokine genes are also regulated post-transcriptionally. These genes encode mRNAs with Au-rich elements (AREs) in the 3 untranslated regions, which are recognized by a network of ARE-binding proteins that control mRNA metabolism by distinct mechanisms, including regulation of translation and mRNA decay. So, the maximal induction of TNF, IL-1 and IL-6 by LPS requires inactivation of ARE-mediated mRNA destabilization in a manner that depends on the p38 mitogen-activated protein kinase pathway 77,78. Finally, the endonuclease ZC3H12A was recently shown to regulate the stability of inducible gene transcripts79. In its absence, macrophages produce increased levels of certain pro-inflammatory cytokines such as IL-6 and IL-12, and mice that lack ZC3H12A develop inflammation and severe immune diseases that
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lead to early death. ZC3H12A is thought to target a distinct repertoire of mRNAs, independently of ARE recognition, further underscoring the role of mRNA transcript stability in regulating the inflammatory response. not discussed here, although some of them (for example, IFN in host defence and other cellular stressors) must control inflammation in a module-specific manner, through transcriptional mechanisms that would be of great interest to understand. In addition, we have discussed how transcriptional modules encode functional programmes of the inflammatory response, and it will be important to further define these modules and the mechanisms that enable their autonomous regulation. The identification of module-specific transcriptional regulators and chromatin modifications is important not only for understanding the organization of this transcriptional programme, but would also enable the specific manipulation of various components of the inflammatory response. Finally, there is a growing awareness that many of the most prevalent human diseases are associated with pathophysiological chronic inflammation. This type of inflammation is persistent and long-lasting, and is associated with self-amplifying loops that maintain its expression. Given the role of chromatin in regulating both dynamic and stable patterns of gene expression, chronic inflammation is probably associated with a reprogramming of inflammatory gene expression that is mediated by alterations to chromatin. Therefore, it will be important to determine whether chromatin dysregulation underlies chronic inflammation in many disease settings.

Conclusions and future perspectives The LPS-inducible transcriptional programme has served as an excellent model for understanding the transcriptional control of inflammation. Many questions remain that would be of interest to address. First, we have focused on several pathways that negatively regulate inflammation to illustrate some general principles. But these are only a small fraction of the total repertoire of physiological signals that inhibit inflammation, underscoring the necessity of being able to fine-tune inflammation in a module-specific manner. Future studies that address the mechanisms of inhibition by these different signals will be important. For example, what sets of genes and functional programmes are inhibited by IL-10 compared with TGF? Are there transcriptional repressors that are shared by multiple negative regulatory pathways? of the several repressors that are activated or induced by a particular negative regulator of inflammation, what are the relative contributions of individual repressors and how do they differ in distinct cell types and physiological settings? Pathways that positively modulate inflammation undoubtedly exist but are

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Acknowledgements

We would like to thank D. Hargreaves, S. Foster and S. Smale for discussions. R.M. is an Investigator at the Howard Hughes Medical Institute.

DATABASES
UniProtKB: http://www.uniprot.org 2A-HUB | A20 | ATF3 | BCL-3 | Bmp2 | CBP | CCL2 | CCL3 | CCL5 | C/EBP | C/EBP | CREB1 | CXCL2 | CXCL10 | GCN5 | HES | HEY | IB | IB | IBNS | IKK | IL-6 | IL-10 | IL-12p40 | IRAKM | IRF3 | IRF8 | JUN | KDM6B | MSK1 | MSK2 | NCOR | p50 | p65 | p300 | PCAF | PPAR | PPAR | PU.1 | RBP-J | RCOR | RUNX1 | SIRT1 | SIRT6 | SMRT | ST2 | TEL | TGF | TNF | ZC3H12A

FURTHER INFORMATION
Ruslan Medzhitovs homepage: http://www.med.yale.edu/immuno/fac_medzhitov.html Tiffany Horngs homepage: http://www.hsph.harvard.edu/faculty/tiffany-horng
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