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Cell. Author manuscript; available in PMC Apr 5, 2006.
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PMCID: PMC1430687
NIHMSID: NIHMS8229

Molecular Determinants of Crosstalk between Nuclear Receptors and Toll-like Receptors Mediating Counter-regulation of Inflammatory Responses

Summary

The ability of nuclear receptors (NRs) to repress transcriptional responses to diverse signaling pathways is an essential aspect of their biological activities, but mechanisms determining the specificity and functional consequences of transrepression remain poorly understood. Here, we report the identification of nuclear receptor-specific mechanisms that mediate signal- and gene-specific repression of transcriptional responses initiated by engagement of toll-like receptors (TLR) 3, 4 and 9. The glucocorticoid receptor (GR) was found to repress a large set of functionally related inflammatory response genes by disrupting p65/interferon regulatory factor (IRF) complexes required for TLR4- or TLR9-dependent, but not TLR3-dependent, transcriptional activation. This mechanism enables the GR to differentially regulate pathogen-specific programs of gene expression. In contrast, PPARγ and LXR repressed overlapping but functionally distinct sets of genes by p65/IRF3-independent mechanisms. In concert with their utilization of distinct transrepression mechanisms, simultaneous activation of GR and PPARγ or GR and LXRs resulted in additive/synergistic inhibition of subsets of TLR4 responses in vivo and in vitro. These findings reveal a molecular basis for combinatorial control of homeostasis and immune responses by NRs and suggest new approaches for treatment of inflammatory diseases.

Introduction

Members of the nuclear receptor superfamily play diverse roles in the regulation of development, homeostasis and immune responses by both positively and negatively regulating gene expression (Chawla et al., 2001; Evans, 1988; Kastner et al., 1995; Mangelsdorf and Evans, 1995). The glucocorticoid receptor (GR) is prototypic of a subset of ligand-dependent nuclear receptors that integrate host immune responses with physiological circuits that are required for maintenance of necessary organ functions. The ability of GR to repress transcriptional responses to inflammatory signals is an essential component of its homeostatic functions and a primary mechanism by which natural and synthetic GR agonists exert anti-inflammatory effects in a variety of disease settings (Reichardt et al., 1998; Reichardt et al., 2001). Negative regulation of inflammatory responses is thought to result, at least in part, from the ability of GR to interfere by transrepression, with the activities of other signal-dependent transcription factors that include NF-κB and activator protein-1 (AP-1) family members (Caldenhoven et al., 1995; Jonat et al., 1990; Scheinman et al., 1995b; Schüle et al., 1990; Yang-Yen et al., 1990) and reviewed in (De Bosscher et al., 2003). Numerous models have been proposed for GR-mediated transrepression, including direct interactions with NF-κB components (Caldenhoven et al., 1995; Liden et al., 1997; Scheinman et al., 1995b), regulation of components of signal transduction pathways involved in NF-κB and AP-1 activation (Auphan et al., 1995; Caelles et al., 1997; Scheinman et al., 1995a), competition for essential coactivators (Kamei et al., 1996; Sheppard et al., 1998), alternative utilization of coactivators (Kassel et al., 2004; Rogatsky et al., 2001; Scheinman et al., 1995b), recruitment of corepressors (Nissen and Yamamoto, 2000), and modifications of core transcription factors (De Bosscher et al., 2000; Nissen and Yamamoto, 2000). However, most of these models have been developed based on analysis of limited sets of specific target genes, often in transient transfection assays, and general applicability to broad programs of gene expression activated during inflammatory responses have not been established.

Anti-inflammatory activities have also been documented in vivo and/or in vitro for several other members of the nuclear receptor family, including estrogen receptors (ERs) (McKay and Cidlowski, 1999), vitamin D receptors (VDRs) (Nagpal et al., 2001), peroxisome proliferator-activated receptors (PPARs) (Devchand et al., 1996; Jiang et al., 1998; Marx et al., 2000; Ricote et al., 1998; Staels et al., 1998) and LXRs (Castrillo et al., 2003; Joseph et al., 2004; Joseph et al., 2003). PPARs and LXRs are regulated by fatty acid and cholesterol metabolites, respectively, and were initially characterized as nuclear receptors that play critical roles in lipid homeostasis (Forman et al., 1995; Issemann and Green, 1990; Janowski et al., 1996; Kliewer et al., 1995). Emerging evidence suggests that their ability to counter-regulate inflammatory responses plays important roles in both immunity and metabolic control (Joseph et al., 2003; Ricote et al., 1998). For example, PPARγ is thought to improve insulin resistance by both positively and negatively regulating gene expression, with PPARγ agonists suppressing the expression of inflammatory genes in adipocytes and adipose tissue-associated macrophages that are correlated with impaired insulin signaling (Pittas et al., 2004; Weisberg et al., 2003; Xu et al., 2003). While PPARs and LXRs have also been documented to antagonize the actions of NF-κB and AP-1 transcription factors (Ricote et al., 1998), the underlying mechanisms remain poorly understood.

Here, we have used toll-like receptor (TLR) signaling as a model system to explore mechanisms by which different members of the nuclear receptor superfamily repress pro-inflammatory programs of gene expression. TLR3, TLR4 and TLR9 are members of the TLR family of cell surface receptors that regulate transcriptional programs involved in innate immunity by recognizing pathogen-associated molecular patterns (Janeway and Medzhitov, 2002). TLR3 is activated by double stranded RNA and plays an important role in innate immunity directed against dsRNA viral pathogens by coupling to TRIF-dependent signal transduction pathways (Alexopoulou et al., 2001; Yamamoto et al., 2002). TLR9 is activated by immunostimulatory DNA sequences and is essential for innate immunity directed against certain double-stranded DNA viruses by coupling to the MyD88 signaling adapter (Hacker et al., 2000; Hemmi et al., 2000; Krug et al., 2004; Lund et al., 2003). TLR4 is activated by lipopolysaccharide (LPS) and mediates innate immunity against gram-negative bacterial infections by coupling to both MyD88 and TRIF (Alexopoulou et al., 2001; Poltorak et al., 1998). MyD88 and TRIF use overlapping but distinct signal transduction pathways to regulate the expression and function of numerous transcription factors that drive inflammatory responses, including NF-κB, AP-1, and interferon regulatory factors (IRFs) (Akira and Takeda, 2004; Pitha, 2004). In the present studies, GR, PPARγ and LXR agonists were found to repress both common and nuclear receptor-specific subsets of TLR target genes through the use of nuclear receptor- and TLR-specific transrepression mechanisms. Combinations of agonists for GR, PPARγ and LXRs resulted in additive or synergistic inhibition of a subset of TLR4-target genes both in cultured macrophages and in vivo, consistent with the simultaneous targeting of these genes by distinct mechanisms. These findings suggest that nuclear receptors function in a combinatorial manner to coordinately regulate the evolution of host immune responses.

Results

Nuclear Receptors Inhibit Overlapping but Distinct Subsets of LPS-Inducible Genes

Gene expression profiling experiments were initially performed to identify LPS-inducible genes in primary macrophages that were sensitive to transrepression by the GR agonist dexamethasone (Dex) (Figure 1A). The observation that about half of the LPS-inducible genes were Dex-sensitive raised the questions of how GR discriminated between sensitive and resistant genes and whether these two classes of genes exert distinct biological functions. Recent findings indicate that NCoR corepressor complexes occupy a subset of NF-κB and AP-1 target genes under basal conditions and are cleared in a signal-dependent manner as a prerequisite to transcriptional activation (Ogawa et al., 2004; Perissi et al., 2004). Because some NCoR target genes were also subject to Dex-mediated repression, microarray experiments were performed using NCoR−/− macrophages to determine whether NCoR was required for Dex-sensitivity (Ogawa et al., 2004). Lack of NCoR had no impact on GR-mediated repression (Figure 1B), indicating the utilization of NCoR-independent mechanisms. To investigate whether different nuclear receptors repress a common set of LPS target genes, gene expression profiling experiments were performed using nuclear receptor-specific agonists for PPARγ (rosiglitazone (Ro) and GW7845), LXRα/β (GW3965 and T1317), VDR (1,25-(OH)2vitamin D3), ER (17β-estradiol; E2), and RARs (all-trans retinoic acid; atRA). These experiments demonstrated that each agonist exerted an overlapping but distinct impact on LPS-dependent gene expression (Figures (Figures1C1C and and1D).1D). GR, LXR and PPARγ agonists were the most potent inhibitors of the LPS response, with VDR-, ER- and RAR-specific agonists exerting relatively modest inhibitory effects under these conditions (Figure 1C). Dex, GW3965 and Ro inhibited expression of a larger number of genes than they activated (Figures 1C and data not shown). Sensitivity to repressive effects of GR, PPARγ or LXR agonists did not correlate with degree of responsiveness to LPS (Figure 1C), absolute expression levels, or a requirement for the p65 compoment of NF-κB (Figure S1 and data not shown). In concert, these experiments suggest that negative regulation of gene expression is a dominant biological function of GR, LXR and PPARγ in activated macrophages and indicate that these nuclear receptors utilize distinct molecular mechanisms to repress TLR4-dependent gene expression.

Figure 1
Inhibition of LPS-Dependent Gene Expression by Nuclear Receptor Agonists (A) Relative expression of LPS-inducible genes in peritoneal macrophages under control conditions and after 6 h of LPS treatment in the absence or presence of 1 μM Dex. Left ...

p65/IRF3 Complexes Mediate Signal-specific Inhibition of Transcriptional Responses

Computational motif discovery methods were used to search for potential transcriptional regulatory elements mediating LPS-dependent activation and nuclear receptor-mediated transrepression. The sequence cAAActGAAAg was identified as the most highly significant motif enriched in the promoter sequences of LPS target genes (Figure 2A). This motif is nearly identical to consensus IRF3 and interferon (IFN)-sensitive response element (ISRE) sequences recognized by IRF3 and the type I IFN-inducible ISGF3 complex (Juang et al., 1998; Wathelet et al., 1998; Yoneyama et al., 1998). Recent findings indicate that binding of poly I:C to TLR3 and LPS to TLR4, respectively, activates IRF3 and induces ISRE-mediated gene activation (Fitzgerald et al., 2003; Juang et al., 1998; Navarro and David, 1999; Pitha, 2004; Wathelet et al., 1998; Yoneyama et al., 1998). We therefore performed gene expression profiling experiments to identify IRF3-target genes by comparing LPS responses of wild-type and IRF3−/− macrophages (Dang et al., 2004; Sato et al., 2000). In parallel, expression profiling experiments were performed to evaluate the impact of GR, PPARγ and LXR agonists on transcriptional responses of macrophages to LPS and poly I:C (Figure 2C). Although TLR3 and TLR4 differ with respect to use of MyD88 as an adapter protein for signal transduction (Akira and Takeda, 2004; Pitha, 2004), the qualitative and quantitative pattern of genes induced more than 3-fold by LPS and poly I:C at the 6 h time point were very similar (Figure 2B). Of the 543 genes induced more than 3-fold by LPS in wild-type macrophages, 159 genes exhibited a 90% or more loss of activation in IRF3−/− macrophages and an additional 160 genes exhibited at least a 40% reduction in LPS activation (Figure 2C, column 1). A complete listing of expression data and response element sequences is provided as Table S1.

Figure 2
Differential Repression of TLR Responses by GR, PPARγ, and LXRs (A) Identification of IRF3-binding sites as highly enriched sequence motifs in the promoters of LPS-inducible genes. The top sequence logo is representative of the most significant ...

As in the case of LPS-dependent gene expression, GR, PPARγ and LXR agonists repressed both common and nuclear receptor-specific targets of poly I:C-inducible genes (Figure 2C). However, despite the overall similarity in the sets of genes that were transcriptionally activated by LPS and poly I:C, the patterns of GR, PPARγ and LXR-mediated transrepression were significantly different, indicating that nuclear receptor transrepression is regulated in a signal-specific manner. For example, a substantial number of genes that were Dex-sensitive when activated by LPS became Dex-resistant when activated by poly I:C, illustrated for IP10 and Ifit1 in Figure 2D.

Unexpectedly, nearly all of the highly inducible LPS- and poly I:C-target genes that were Dex-sensitive when activated by LPS but Dex-resistant when activated by poly I:C were also highly dependent on IRF3 for LPS induction (Figure 3A and data not shown). The promoters for many of the genes exhibiting this pattern of expression contained proximal IRF3/ISRE sequences, exemplified by Ifit1 (Figure 3B). In contrast, while PPARγ and LXR agonists also inhibited a significant number of IRF3-dependent genes, the signal-specific pattern of sensitivity and resistance differed (Figures (Figures2C2C and and3A).3A). This pattern therefore suggested a mechanistic link between IRF3 and signal-specific transrepression by GR. To test this hypothesis, we evaluated the ability of GR to repress transcriptional activation of an artificial promoter constructed to exclusively contain ISRE elements, which was activated by LPS and poly I:C (Figure 3C). Significantly, the induction of the ISRE-dependent reporter was strongly inhibited by Dex when LPS was used as a stimulus, but not when poly I:C was used as a stimulus (Figure 3C).

Figure 3
Signal-specific Repression by GR Correlates with a Requirement for IRF3 for Transcriptional Activation (A) Transcriptional responses of 54 genes highly induced by LPS, poly I:C and CpG1668, exhibiting sensitivity to Dex when activated by LPS (column 2) ...

While TLR3 and TLR4 signaling both lead to activation of IRF3, recent studies suggest that p65/RelA functions as an essential coactivator of IRF3 in the case of TLR4 signaling, but not in case of TLR3 signaling (Wietek et al., 2003). The p65 requirement for LPS induction of an ISRE-dependent gene was confirmed by experiments demonstrating markedly impaired activation of the Ifit1 gene in NF-κB-deficient macrophages (Figure 3D). These observations raised the possibility that GR inhibited IRF3-target genes in response to TLR4 signaling, but not TLR3 signaling, by targeting the p65 requirement. We therefore characterized the composition of activation complexes bound to the proximal promoter region of Ifit1 in primary macrophages by chromatin immunoprecipitation (ChIP) assay. These experiments demonstrated that IRF3 and CBP were prominently recruited to the ISRE-containing promoter in response to both LPS and poly I:C (Figure 3E). In contrast, p65 was specifically recruited to the ISRE in response to LPS, but not in response to poly I:C. Interestingly, the recruitment of p65 to the ISRE in response to LPS was largely inhibited by Dex but not by PPARγ or LXR agonists (Figure 3E). Similar findings were obtained using ChIP to evaluate the composition of activation complexes bound to the artificial promoter construct exclusively containing ISRE elements (Figure S2).

Previous studies have suggested that nuclear receptor interactions with p65 are involved in transrepression (Liden et al., 1997; Scheinman et al., 1995b), but how this interaction could account for receptor-, signal- and gene-specific repression has not been established. We therefore evaluated the possibility that GR inhibited LPS induction of the ISRE promoter through direct interactions with p65. In vitro interaction assays confirmed that the GR-DBD strongly interacted with both full-length p65 and further narrowed this interaction to the N-terminal Rel-homology domain (RHD) (Figure 4A). In contrast, the PPARγ-DBD and the LXRα-DBD exhibited minimal interaction with p65 in vitro (Figure 4A). These results were confirmed by mammalian two-hybrid assays using a Gal4DBD-p65 fusion protein as bait and VP16 fusions with full-length GR, PPARγ or LXRα as preys (Figure 4B).

Figure 4
GR Specifically Inhibits the Interaction of p65 with IRF3 (A) GR-DBD preferentially interacts with the p65 RHD. GST pull-down assays were performed using the indicated GST-NR-DBD fusion proteins and in vitro translated full-length p65 or p65 RHD, respectively. ...

We next evaluated whether IRF3 interacted directly with p65 and whether this interaction was influenced by GR. In vitro binding assays demonstrated that a glutathione-S-transferase (GST)-p65 fusion protein interacted with IRF3 (Figure 4C). Addition of increasing amounts of GR-DBD to the binding reaction led to decreased IRF3 interaction with p65 and a concomitant increase in the binding of the GR-DBD (Figure 4C). Furthermore, IRF3 interacted with the RHD of p65 (Figure 4D). These findings suggest that GR and IRF3 compete for the same binding site and that GR preferentially interacts with p65.

To determine whether the interaction of GR with p65 was relevant to its repression function, we evaluated a GR mutant in which lysine 471 in the second zinc finger of the DBD was changed to alanine (GRK471A) based on a previous report that a corresponding mutant is defective for inhibition of p65 activity (Liden et al., 1997). In contrast to wild-type GR, GRK471A exhibited little interaction with p65 in vitro or in vivo in mammalian two-hybrid assays (Figures (Figures4B4B and and4E),4E), and GRK471A lacked inhibitory activity against Gal-p65 mediated transactivation (Figure 4F). Consistent with these in vitro findings, wild-type GR was able to inhibit p65-dependent transactivation of a Gal-IRF3 fusion gene, while GRK471A was not (Figure 4G), suggesting that direct interaction of GR and p65 is required for GR-mediated transrepression of IRF3. Taken together, these findings support a model in which the requirement of ISRE-containing genes for p65 as a coactivator following LPS activation, but not poly I:C stimulation, accounts for the LPS-specific sensitivity of these genes to transrepression by GR.

IRF3/p65 Complexes Mediate Gene-specific Inhibition of Transcriptional Responses

In addition to the IRF3-binding motif, κB elements were also highly enriched in promoter regions of LPS-inducible genes (Table S1). However, the presence of these sequences did not correlate with signal or gene-specific patterns of regulation. NF-κB binding sites were identified in promoters of genes that were sensitive to nuclear receptor agonists following activation by LPS or poly I:C (e.g., iNOS, Figure 2D), in promoters of genes that were nuclear receptor-sensitive following LPS but not poly I:C activation (e.g., Clic4, Figure 5A and data not shown), and in promoters of genes that were nuclear receptor-resistant regardless of the signal (e.g., Nfkbia and Gro1, Figure 5A and data not shown). In each case, transcriptional responses to LPS required p65 (Figure S1). The recent finding that a subset of NF-κB sites appear to determine the utilization of IRF3 as a coactivator of p65 (Leung et al., 2004) suggested the possibility that this might be a basis for gene-specific sensitivity to repression by GR. To examine this, we chose the Scyb9 and Clic4 genes because they were highly IRF3-dependent, Dex-sensitive NF-κB target genes that did not contain ISRE motifs within their proximal promoter or distal upstream regions (Figures (Figures5A5A and and5B).5B). The Nfkbia and Gro1 genes were chosen for comparison because they were highly induced by LPS in an IRF3-independent manner, were Dex-resistant, contained well-characterized κB elements and lacked proximal or distal ISRE elements (Figures (Figures5A5A and and5B).5B). ChIP experiments revealed that p65 was recruited to each of these genes in response to LPS, as expected (Figure 5C). IRF3 was recruited to the proximal promoter regions of Scyb9 and Clic4 in response to LPS, but not to the Nfkbia or Gro1 promoters, consistent with the requirement of Scyb9 and Clic4 for IRF3 for activation and confirming a gene-specific recruitment of IRF3 to a subset of p65-target genes. Significantly, treatment with Dex had no effect on the recruitment of p65 to any of these four target genes but inhibited the recruitment of IRF3 to the Scyb9 and Clic4 promoters, coincident with ligand-dependent recruitment of GR to these promoters (Figure 5C).

Figure 5
Utilization of IRF3 as a Coactivator of p65 Determines Gene-specific Sensitivity (A) Location of NF-κB sites in proximal promoter regions of Scyb9, Clic4, Nfkbia and Gro1, and their expression profiles in response to LPS in wild-type (WT) and ...

MyD88 Dictates GR-sensitivity of IRF3/7-dependent Gene Expression

Because TLR3 and TLR4 activate IRF3 and NF-κB through the TRIF-dependent pathway, while TLR4, in addition, activates NF-κB and MAP kinases via the MyD88-dependent pathway, these observations raised the possibility that glucocorticoid sensitivity was dictated by the utilization of the MyD88-dependent pathway. To initially test this hypothesis, we determined the profile of dexamethasone-sensitive genes in macrophages treated with immunostimulatory DNA (CpG1668) to activate TLR9, which exclusively couples to the MyD88-dependent pathway (Hacker et al., 2000; Krug et al., 2004). As in the case of polyI:C-stimulated cells, the overall profile of transcriptional activation induced by CpG1668 was very similar to that induced by LPS. Remarkably, of the genes that were highly induced by all three TLR agonists and were Dex-resistant when activated by polyI:C but Dex-sensitive when activated by LPS, 100% were also Dex-sensitive when activated by CpG1668 (Figure 3A). Furthermore, the quantitative extent of Dex-mediated repression was more pronounced following TLR9 stimulation than TLR4 stimulation in nearly every case (Figure 3A). TLR9 primarily induces ISRE gene expression through IRF7 (Kawai et al., 2004), suggesting that the mechanism of GR-mediated repression established for IRF3 extends to IRF7-dependent gene expression. To determine whether Dex-sensitivity of the response to LPS requires signaling through the MyD88 pathway, experiments were performed in MyD88−/− macrophages (Adachi et al., 1998). In these cells, LPS activation is entirely TRIF-dependent (Akira and Takeda, 2004; Kawai et al., 2001). Remarkably, IRF3-dependent genes that were Dex-sensitive in wild-type cells became Dex-resistant in MyD88−/− cells, regardless of whether they contained ISRE or κB elements (Figure 5D). These findings suggest that signaling through the MyD88-dependent pathway specifies Dex-sensitivity of this set of genes.

GR, PPARγ and LXR Function in a Combinatorial Manner to Inhibit LPS Responses

The observation that GR, PPARγ and LXR agonists repressed overlapping but distinct sets of LPS-target genes by p65-dependent and p65-independent mechanisms raised the possibility that they might exert combinatorial effects on inflammatory responses. To test this hypothesis, gene expression profiling experiments were performed to characterize LPS responses in the presence or absence of combinations of saturating concentrations of GR, PPARγ and LXR agonists. The results of this analysis for the combination of Dex and the PPARγ agonist GW7845 are illustrated in Figure 6A, restricted to the subset of genes transrepressed by at least one agonist. While several examples were observed in which nuclear receptor-specific inhibitory effects of one agonist were reversed by addition of the second agonist (red arrows), the major impact of the combination of agonists was to increase the strength of inhibition of a subset of LPS target genes (blue arrows). These results were confirmed by additional experiments that examined the concentration dependence of combinatorial interactions by Northern blot analysis and quantitative PCR analysis of representative target genes (Figures (Figures6B6B and and6D).6D). Low concentrations of Dex and GW7845 (10 nM) that exerted relatively little repressive effects when used individually could act synergistically in combination (Figure 6B). Parallel studies of combinations of Dex and GW3965 also demonstrated additive or synergistic effects on LPS-target genes (Figure 6C and data not shown).

Figure 6
GR and PPARγ Function in a Combinatorial Manner to Inhibit LPS Responses (A) Combinatorial interactions between GR and PPARγ agonists at a genome-wide level. Peritoneal macrophages were stimulated with LPS in the absence or presence of ...

To determine whether combinatorial effects of GR, PPARγ and LXR agonists acted at the promoter level, iNOS promoter activity was evaluated in RAW264.7 cells. The iNOS promoter was chosen for this analysis because the endogenous iNOS gene was subject to combinatorial inhibition by GR, PPARγ, and LXR agonists, its transcriptional activation requires binding sites for NF-κB (Lowenstein et al., 1993), and maximum responses to LPS required IRF3 (data not shown). As shown in Figure 6E, GR and PPARγ agonists inhibited iNOS promoter activity in a dose-dependent manner. When cells were treated with the combination of Dex and GW7845, at least additive effects with respect to inhibition of LPS response were observed at both saturating and non-saturating concentrations of ligands (Figure 6E). Similar results were observed for the combination of GR and LXR agonists (data not shown).

Although GR-mediated repression did not require NCoR (Figure 1B), we recently found that the ability of PPARγ to repress LPS activation of the iNOS promoter required NCoR (G.P., C.K.G., unpublished). Consistent with this, knockdown of NCoR expression with specific siRNAs had no effect on transrepression by Dex, but significantly impaired the ability of PPARγ and LXR agonists to repress the iNOS promoter (Figure S3). These findings indicate that at least two distinct, receptor-specific mechanisms are utilized by GR and PPARγ/LXR to repress LPS activation of the iNOS promoter, providing a potential explanation for synergistic repression when GR and PPARγ or LXR agonists are used in combination.

To investigate whether combinatorial interactions between PPARγ agonists and Dex observed in primary macrophages would also occur in an in vivo model system, we evaluated the IL-12 p40 subunit, as this was synergistically repressed in primary macrophages by GW7845 and Dex, but not by the combination of Dex and T1317. Consistent with these findings, treatment of mice with the combination of GW7845 (1 mg/kg/day) and Dex (1 mg/kg/day) prior to injection with LPS resulted in significantly greater inhibition of circulating IL-12 p40 than either agonist alone (Figure 6F). For the combination of GR and LXR agonists, we evaluated TNFα, based on synergistic inhibition by Dex and T1317 in primary macrophages. Treatment of mice with the combination of Dex (1 mg/kg/day) and T1317 (10 mg/kg/day) resulted in significantly greater inhibition of circulating tumor necrosis factor α (TNFα) levels following injection with LPS than observed following either agonist alone (Figure 6G).

Biological Functions Associated with Nuclear Receptor-sensitive and Nuclear Receptor-resistant TLR-target Genes

The identification of distinct subsets of TLR3- and TLR4-target genes exhibiting nuclear receptor-sensitive or nuclear receptor-resistant profiles raises the question of whether these genes participate in distinct biological processes. Venn diagrams illustrating the overlapping and distinct subsets of genes subject to transrepression by GR, PPAR and LXR following macrophage activation by LPS and poly I:C are illustrated in Figure 7A. To investigate the potential meaning of these findings at a biological level, statistical analysis of the functional annotations associated with specific sets of differentially regulated genes was performed using annotations provided by the Gene Ontology (GO) database (Gene Ontology Consortium, 2001). A subset of this analysis is presented in Figure 7B, illustrating major categories including immune cell homeostasis, response to virus, cytokine, chemokine signaling, etc. Although the effort to assign functional annotations to all mammalian gene products is at an early stage of development, the results of this analysis suggests significant functional differences in the sets of nuclear receptor-sensitive and nuclear receptor-resistant LPS-target genes. For example, the set of genes that was activated by LPS and resistant to GR, PPARγ and LXR agonists was enriched for functional annotations linked to metabolism (Figure 7B). In addition, transcriptional activation of the core components of the NF-κB pathway by LPS or poly I:C was almost completely resistant to repression by all three nuclear receptor agonists (Figure S4B). We also placed data for GR-mediated repression of LPS- and polyI:C-inducible genes on KEGG pathway maps (Kanehisa, 1996) and provide an example for the TLR signaling pathway in Figure S5. This figure indicates that components of the TLR signaling pathway in addition to NF-κB factors are Dex resistant, while TLR-activated chemokines and cytokines exhibit differential patterns of sensitivity that relate to proinflammatory effects and chemotaxis.

Figure 7
Receptor-, Signal- and Gene-specific Counter-regulation of Inflammatory Responses by GR, PPARγ and LXRs (A) Venn diagram indicating sensitivity of LPS-responsive genes to GR, PPARγ and LXR-specific agonists. Nuclear receptor sensitivity ...

GR, PPARγ and LXR regulated functionally overlapping sets of genes, but also targeted genes in functionally related groups in a nuclear receptor-specific manner. For example, the list of repressed genes with functional annotations linked to hemopoiesis by the Gene Ontology consortium reached statistical significance for Dex, but not LXR or PPARγ agonists (Figure 7B). Repressive actions of each nuclear receptor ligand on specific genes involved in immune cell migration, differentiation and activation are illustrated in Figure S4B. Overall, Dex inhibited a larger number of genes involved in immune cell activation to a greater extent than LXR or PPARγ agonists, which may explain in part why LXR and PPARγ agonists are not as effective as Dex in acute models of inflammation. Significant differences in effects of the three receptor-specific agonists on chemokine gene expression were observed, suggesting that each receptor may play a context-specific role in regulating recruitment of specific immune cells to sites of inflammation.

Discussion

Signal-specific, Gene-specific and Nuclear Receptor-specific Transrepression

The present studies have used a combination of gene expression profiling and molecular analysis to investigate nuclear receptor-specific and combinatorial mechanisms of transrepression by nuclear receptors. These observations extend the spectrum of nuclear receptor- and promoter-specific inhibition of signal-dependent gene expression, demonstrating that GR, PPARγ and LXR repress overlapping but distinct subsets of inflammatory response genes, consistent with the large number of mechanisms that have been proposed for negative regulation by nuclear receptors (De Bosscher et al., 2003). Although evidence supporting global inactivation of NF-κB activity by GR and ER has been reported in other cell types (Auphan et al., 1995; Ghisletti et al., 2005; Scheinman et al., 1995a), the present studies indicate that GR, LXRs and PPARγ target only a subset of the TLR-inducible NF-κB-dependent genes in macrophages. These findings indicate that the mechanisms of transrepression in this cell type must operate in a promoter-specific manner.

The observation that a significant set of genes that were sensitive to nuclear receptor-dependent repression when activated through TLR4 became resistant to repression when activated through TLR3 also indicates that transrepression programs mediated by GR, PPARγ and LXRs are regulated in a signal-specific manner. The finding of a highly significant correlation between IFR3-dependent transcriptional activation and signal-dependent transrepression by glucocorticoid receptor (GR) led to the discovery that GR effectively disrupts the formation of an IRF3/p65 activator/coactivator complex that is required for activation of ISRE-containing promoters by TLR4-but not TLR3-dependent signaling (Figures (Figures3E3E and and4C).4C). Of the 262 genes scored as being LPS-inducible and Dex-sensitive in these studies, at least 85 genes fit with the hypothesis that disruption of IRF3/p65 complexes is a quantitatively important component of the transrepression mechanism. Taken together with the results of studies in MyD88−/− macrophages (Figure 5D) and the patterns of gene expression following TLR9 activation (Figure 3A), these findings support a unifying model in which TLR signaling through MyD88 specifies glucocorticoid sensitivity of IRF-dependent genes through the utilization of IRF/p65 complexes (Figure 7C). As IRF7 is involved in TLR9-MyD88-dependent gene activation (Kawai et al., 2004), these results imply that the mechanism of GR-mediated repression operates through both IRF3 and IRF7. These findings thus reveal the molecular basis for an example of signal-specific transrepression that is utilized by a large group of functionally inter-related genes. Distinct regions of GR appear to be involved in mediating repression of AP-1 target genes (Bladh et al., 2005) and it will be of interest to explore signal-specific modulation of transrepression in response to other proinflammatory cytokines that induce AP-1 and STAT transcription factors, such as TNFα and IFNβ.

Conversely, the ability of IRF3 to function as an essential coactivator of p65 on a subset of NF-κB target genes provides an explanation for how transrepression by GR can be achieved in a gene-specific manner (Figure 7D). NF-κB target genes that are resistant to GR-mediated transrepression are predicted to utilize other classes of coactivators, such as Bcl3 (Leung et al., 2004), that may prevent the interaction of GR with DNA-bound NF-κB. Consistent with this, ChIP experiments demonstrated recruitment of GR to the Dex-sensitive Scyb9 and Clic4 promoters. A significant number of Dex-sensitive NF-κB target genes are not IRF3-dependent, indicating a requirement for additional mechanisms. Among the 100 most LPS and polyI:C-inducible genes, all of the genes that were Dex-sensitive following polyI:C stimulation were also Dex-sensitive following activation by LPS (Figure S4A). Similarly, among the 100 most polyI:C and CpG1668-inducible genes, all of the genes that were Dex-sensitive following polyI:C stimulation were also Dex-sensitive following activation by CpG1668 (Figure S4A). These results suggest that a common set of mechanisms is used for Dex repression of this subset of TLR-inducible genes. We anticipate that these mechanisms will include gene-specific recruitment of coactivators other than IRF3 that are also sensitive to competition with GR.

In contrast to GR, transrepression mediated by PPARγ and LXRs did not seem to involve p65. This observation is consistent with the overlapping but distinct sets of genes that were found to be sensitive to repression by PPARγ and LXRs and implies that distinct targeting mechanisms are employed by these nuclear receptors. We and others recently reported that a subset of genes induced by pro-inflammatory signals are occupied in the basal state by NCoR/SMRT corepressor complexes that act as transcriptional checkpoints to maintain these genes in a transcriptionally silent state (Hoberg et al., 2004; Ogawa et al., 2004; Perissi et al., 2004). Signal-dependent transcriptional activation of these genes requires clearance of NCoR/SMRT complexes by an ubiquitin/proteosome-dependent step as a prerequisite to transcriptional activation. Recent studies suggest that PPARγ represses LPS induction of the iNOS gene by preventing the clearance of the NCoR corepressor complex (G.P., C.K.G., unpublished). The convergence of NCoR-dependent and NCoR-independent transrepression mechanisms on the iNOS promoter support the hypothesis that synergistic repression of LPS target genes by GR and PPARγ is achieved by the combinatorial utilization of distinct, nuclear receptor-specific mechanisms.

Physiological Implications for Cellular Responses to Bacterial and Viral Pathogens

The observation that TLR-responsive genes exhibit different sensitivities to repression by nuclear receptors suggests that they play distinct biological roles in determining cellular responses to infection and other inflammatory processes. By specifically targeting p65/IRF3 complexes, GR is able to discriminate signals initiated by TLRs that either do or do not couple to the MyD88 signaling pathway, providing a biological rationale for the context-specific utilization of these complexes. For example, we predict that the antiviral program of TLR3–should be resistant to GR-mediated repression. In contrast, the antiviral program elicited by activation of TLR9, which is involved in innate responses to herpes simplex virus (HSV) (Krug et al., 2004; Lund et al., 2003), should be GR-sensitive. This prediction is in fact consistent with clinical experience and provides a potential molecular explanation for why steroid use is contraindicated in the presence of HSV-infected retinitis, encephalitis, and uveitis.

In addition to defining combinatorial control of inflammatory responses at the promoter level, these studies also suggest a higher level of regulation that serves to integrate both local and systemic signaling pathways. In the case of GR, the endogenous corticosteroid ligands are classic endocrine hormones, produced in the adrenal cortex in response to systemic physiological circuits (i.e., the hypothalamic-pituitary-adrenal axis). Circulating corticosteroids diffuse into cells and bind to GR in virtually all tissues with sub-nanomolar affinities, providing coordinate regulation of gene expression at the whole body level. In contrast, the adopted orphan receptors such as PPARγ and LXRs are activated by metabolites of fatty acids and cholesterol, respectively, that are produced locally within the cell and bind with relatively low affinity (Forman et al., 1997; Forman et al., 1995; Kliewer et al., 1995; Kliewer et al., 1997; Nagy et al., 1998). In addition, the expression of PPARγ and LXRα and the production of regulatory ligands are determined by local cytokines and other regulatory systems (Ricote et al., 1998). These results also suggest that different combinations of NR ligands will exert different biases with respect to evolution of inflammatory responses. Taken together, these observations suggest a model in which GR and PPARγ/LXR integrate systemic and local regulatory signals so as to coordinate transcriptional responses to infection throughout the body. In support of this, LXRs have recently been shown to play important roles in protection against bacterial infection (Joseph et al., 2004; Valledor et al., 2004).

Clinical Implications

Nuclear receptors are important targets of drugs used in a variety of human disease settings. In many cases, the ability to achieve desirable therapeutic effects with a natural or synthetic nuclear receptor agonist is limited by undesirable or unacceptable side effects. For example, glucocorticoids are potent anti-inflammatory drugs, but can cause or exacerbate hypertension, diabetes, obesity, and dyslipidemia. Emerging information on the ability of selective modulators of nuclear receptors to alter the specificity of coactivator and corepressor recruitment raises new possibilities for the development of novel pharmaceutical agents (Smith and O'Malley, 2004; Wagner et al., 2003). The present studies suggest an alternative, and potentially complementary, strategy to leverage desirable therapeutic effects while minimizing side effects. Using chronic, steroid-dependent inflammatory diseases as an example, it is possible that anti-inflammatory actions of synthetic glucocorticoids could be achieved at lower doses with fewer side effects by simultaneous administration of PPARγ or LXR agonists. The present findings may also influence decisions to use glucocorticoids or other classes of nuclear receptor agonists in the setting of viral or bacterial infection. Lack of efficacy of glucocorticoids in preventing some inflammatory complications of viral infections such as viral myocarditis (Vallejo and Mann, 2003), may potentially be explained by the viral activation of GR-resistant signaling pathways. Because PPARγ and LXR agonists exerted repressive effects on some of the GR-resistant inflammatory mediators induced by TLR3, it is possible that they might have therapeutic value in these settings.

Taken together, we propose that the GR, PPARs and LXRs function in a combinatorial manner to integrate systemic and local signals that control inflammatory gene expression and the evolution of innate and acquired immune responses. Further elucidation of these combinatorial mechanisms may provide further insights into how nuclear receptors control signal-activated transcription and lead to new strategies for treatment of inflammatory diseases.

Experimental Procedures

Cell Culture

Thioglycollate-elicited macrophages were isolated by peritoneal lavage 3 days following peritoneal injection of 2.5 ml 3% thioglycollate (DIFCO). Cells were plated in RPMI medium 1640 and 10% fetal bovine serum, washed after 5 h the medium was removed and cells were fed with fresh medium containing 0.5% fetal bovine serum. LPS (Sigma) was used at a concentration of 100 ng/ml. Fetal liver-derived macrophages generated from E14.5 embryo liver were plated and cultured in RPMI with 10% fetal bovine serum plus L-cell media for 7 days as described (Ogawa et al., 2004). Fetal liver-derived macrophages of TNF−/−cRel−/− (control) and TNF−/−cRel−/−RelA−/− (NF-κB−/−) were obtained by mating TNF−/−cRel−/−RelA+/− mice and by genotyping the embryos.

Expression Array Profiling

Cells were lysed with Trizol (Invitrogen) and total RNA was purified using RNeasy columns (Qiagen). cRNA was generated from 10 μg total RNA using Superscript (Invitrogen) and the High Yield RNA transcription labeling kit (Enzo). Fragmented cRNA was hybridized to Affymetrix Mu11 or Codelink mouse Uniset 1 microarrays according to manufacture's instructions. Data was analyzed with Microarray Suite (Affymetrix), GeneSpring (Silicongenetics) and in-house software developed as described (Ogawa et al., 2004; Sasik et al., 2002).

Computational Analysis

Proximal promoter regions were extracted for each gene represented on the microarray using the May 2004 mouse genome assembly with the method as described (Halees et al., 2003). Analysis was restricted to the region 1 kb upstream of the transcription start site. In cases were several possible alternative promoters may be present, analysis was focused on the most 5' transcription start site. Motif discovery was performed using a comparative algorithm previously described (Segal et al., 2002). Promoters were initially divided into two sets: those that were up regulated by LPS and those that were present on the array but did not change in response to LPS. An exhaustive search for all n-mers (6 < n < 12) was performed and each n-mer was scored for its enrichment in the promoters up regulated by LPS using the hypergeometric distribution. The top 500 n-mers with a p-value less than 0.01 were then clustered together and used to create position specific probability matrices. The matrices were then further optimized to discriminate between the LPS responsive and non-responsive genes by the methods as described (Segal et al., 2002).

Transient Transfection and Reporter Studies

Transient transfections were performed as described using Superfect reagent (Qiagen) (Ogawa et al., 2004; Ricote et al., 1998). RAW 264.7 cells were transfected with iNOS promoter-luciferase, pCMX-PPARγ and pCMX-RXRα plasmids. pRL-TK, a renilla luciferase reporter plasmid was also co-transfected as an internal control (Promega). GR, PPAR and LXR agonists were used at the indicated concentrations in 0.5% fetal bovine serum and cells were harvested 24 h later for analysis of luciferase activity. Double-stranded, short interfering RNAs (siRNA) were synthesized by Dharmacon Research (Lafayette) and were transfected for 48 h prior to activation with ligands and LPS induction as previously described (Perissi et al., 2004). Data are represented as mean ± SD.

GST Pull-down Assays

GST pull-down assays were carried out as described previously (Li et al., 2000). GST fusion proteins were produced as crude bacterial lysates and immobilized on glutathione agarose beads. p65, IRF3, full-length GR, GR(K471A), and GR-DBD proteins were translated in vitro using 35S-labeled methionine and TnT-coupled reticulocyte lysate system (Promega).

ChIP Assay

ChIP assay was conducted as previously (Baek et al., 2002; Perissi et al., 2004). Briefly, macrophages were fixed with 1% formaldehyde and cross-linked adducts were resuspended and sonicated resulting in DNA fragments of 300–900 bp. Protein-bound, immunoprecipitated DNA was reverse cross-linked at 65°C overnight and then purified using PCR purification Kit (Qiagen). 4 ul from a 50 ul DNA extraction were used for PCR amplification. Anti-IRF3 (Zymed), anti-CBP (Santa Cruz), anti-p65 (Santa Cruz), and anti-GR (Santa Cruz) antibodies were used in immunoprecipitation experiments.

RNA Analysis

RNA analysis by Northern blotting was performed as described (Ogawa et al., 2000). Five μg total RNA were separated by gel electrophoresis and transferred to nylon (Supercharge, Scheicher & Schuell). Prior to hybridization, membranes were UV cross-linked (Stratagene) and stained with Methylene Blue (Molecular Research Center, Cincinnati). Probes were generated by RT-PCR followed by random priming labeling (Invitrogen) and hybridization with QuickHyb (Stratagene). Real-time quantitative PCR (SYBRgreen) analysis was performed on an Applied Biosystems 7300 Real-Time PCR system.

LPS-Induced Endotoxin Shock and In Vivo Studies

Combinatorial effects of GR, PPARγ and LXR agonists in antagonism of LPS responses in vivo were evaluated by measuring TNFα and IL-12 p40 levels 6 and 8 hs after intraperitoneal injection of LPS at 1 mg/mouse, respectively. We initially performed a series of pilot studies to establish dosing schedules of Dex that would result in less than 50% inhibition of the LPS-induced TNFα and IL-12 p40 responses. Mice were orally dosed daily with Dex for 7 days and were intraperitoneally injected with LPS (1 mg/mouse). Blood was collected after LPS-stimulation, and analyzed for cytokine levels by ELISA. Based on the results of these pilot studies, two independent studies evaluating effects of combinations of PPARγ agonists and LXR agonists with Dex at the dosing levels indicated in Figure 6. At a minimum, six mice were used for each experimental condition.

Acknowledgements

We are grateful to U.C. San Diego BIOGEM Core Facility for assistance with microarray experiments, to S. Akira for MyD88−/− mice, to T. Taniguchi for IRF3−/− mice, to B. Beutler for CpG1668, and to T. Willson (GlaxoSmithKline) for GW7845. We thank A. Z. Howarth for figure preparation. M.G.R. is an investigator with the Howard Hughes Medical Institute. These studies were supported in part by NIH grants CA52599 and GM069338 and a grant from the Stanford Reynolds Center to C.K.G. These studies were supported in part by NIH grant and the Sandler Program for Asthma Research to M.G.R.

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