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Macrophages are key drivers of inflammation and tissue damage in autoimmune diseases including rheumatoid arthritis. The rate-limiting step for transcription of more than 70% of inducible genes in macrophages is RNA polymerase II (Pol II) promoter-proximal pause release; however, the specific role of Pol II early elongation control in inflammation, and whether it can be modulated therapeutically, is unknown. Genetic ablation of a pause-stabilizing negative elongation factor (NELF) in macrophages did not affect baseline Pol II occupancy but enhanced the transcriptional response of paused anti-inflammatory genes to lipopolysaccharide followed by secondary attenuation of inflammatory signaling in vitro and in the K/BxN serum transfer mouse model of arthritis. To pharmacologically disrupt the Pol II transcription cycle, we used two covalent inhibitors of the transcription factor II H-associated cyclin-dependent kinase 7 (CDK7), THZ1 and YKL-5-124. Both reduced Pol II pausing in murine and human macrophages, broadly suppressed induction of pro- but not anti-inflammatory genes, and rapidly reversed preestablished inflammatory macrophage polarization. In mice, CDK7 inhibition ameliorated both acute and chronic progressive inflammatory arthritis. Lastly, CDK7 inhibition down-regulated a pathogenic gene expression signature in synovial explants from patients with rheumatoid arthritis. We propose that interfering with Pol II early elongation by targeting CDK7 represents a therapeutic opportunity for rheumatoid arthritis and other inflammatory diseases.

Macrophages adopt distinct phenotypes in response to environmental cues, with type-2 cytokine interleukin-4 promoting a tissue-repair homeostatic state (M2 IL4 ). Glucocorticoids (GC), widely used anti-inflammatory therapeutics, reportedly impart a similar phenotype (M2 GC ), but how such disparate pathways may functionally converge is unknown. We show using integrative functional genomics that M2 IL4 and M2 GC transcriptomes share a striking overlap mirrored by a shift in chromatin landscape in both common and signal-specific gene subsets. This core homeostatic program is enacted by transcriptional effectors KLF4 and the glucocorticoid receptor, whose genome-wide occupancy and actions are integrated in a stimulus-specific manner by the nuclear receptor cofactor GRIP1. Indeed, many of the M2 IL4 :M2 GC -shared transcriptomic changes were GRIP1-dependent. Consistently, GRIP1 loss attenuated phagocytic activity of both populations in vitro and macrophage tissue-repair properties in the murine colitis model in vivo. These findings provide a mechanistic framework for homeostatic macrophage programming by distinct signals, to better inform anti-inflammatory drug design., (© 2024. The Author(s).)

Macrophages (MФ) and microglia (MG) are critical in the pathogenesis of multiple sclerosis (MS) and its mouse model, experimental autoimmune encephalomyelitis (EAE). Glucocorticoids (GCs) and interferon β (IFN-β) are frontline treatments for MS, and disrupting each pathway in mice aggravates EAE. Glucocorticoid receptor-interacting protein 1 (GRIP1) facilitates both GR and type I IFN transcriptional actions; hence, we evaluated the role of GRIP1 in neuroinflammation. Surprisingly, myeloid cell-specific loss of GRIP1 dramatically reduced EAE severity, immune cell infiltration of the CNS, and MG activation and demyelination specifically during the neuroinflammatory phase of the disease, yet also blunted therapeutic properties of IFN-β. MФ/MG transcriptome analyses at the bulk and single-cell levels revealed that GRIP1 deletion attenuated nuclear receptor, inflammatory and, interestingly, type I IFN pathways and promoted the persistence of a homeostatic MG signature. Together, these results uncover the multifaceted function of type I IFN in MS/EAE pathogenesis and therapy, and an unexpectedly permissive role of myeloid cell GRIP1 in neuroinflammation., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2020 Mimouna et al.)

Studies on macrophage gene expression have historically focused on events leading to RNA polymerase II recruitment and transcription initiation, whereas the contribution of post-initiation steps to macrophage activation remains poorly understood. Here, we report that widespread promoter-proximal RNA polymerase II pausing in resting macrophages is marked by co-localization of the negative elongation factor (NELF) complex and facilitated by PU.1. Upon inflammatory stimulation, over 60% of activated transcriptome is regulated by polymerase pause-release and a transient genome-wide NELF dissociation from chromatin, unexpectedly, independent of CDK9, a presumed NELF kinase. Genetic disruption of NELF in macrophages enhanced transcription of AP-1-encoding Fos and Jun and, consequently, AP-1 targets including Il10. Augmented expression of IL-10, a critical anti-inflammatory cytokine, in turn, attenuated production of pro-inflammatory mediators and, ultimately, macrophage-mediated inflammation in vivo. Together, these findings establish a previously unappreciated role of NELF in constraining transcription of inflammation inhibitors thereby enabling inflammatory macrophage activation.

The glucocorticoid receptor (GR) potently represses macrophage-elicited inflammation, however, the underlying mechanisms remain obscure. Our genome-wide analysis in mouse macrophages reveals that pro-inflammatory paused genes, activated via global negative elongation factor (NELF) dissociation and RNA Polymerase (Pol)2 release from early elongation arrest, and non-paused genes, induced by de novo Pol2 recruitment, are equally susceptible to acute glucocorticoid repression. Moreover, in both cases the dominant mechanism involves rapid GR tethering to p65 at NF-kB-binding sites. Yet, specifically at paused genes, GR activation triggers widespread promoter accumulation of NELF, with myeloid cell-specific NELF deletion conferring glucocorticoid resistance. Conversely, at non-paused genes, GR attenuates the recruitment of p300 and histone acetylation, leading to a failure to assemble BRD4 and Mediator at promoters and enhancers, ultimately blocking Pol2 initiation. Thus, GR displays no preference for a specific pro-inflammatory gene class; however, it effects repression by targeting distinct temporal events and components of transcriptional machinery., Competing Interests: MS, BT, MC, DR, DD, BB, LY, BZ, XH, RL, YC, IR No competing interests declared, (© 2018, Sacta et al.)

The glucocorticoid (GC) receptor (GR) suppresses inflammation by activating anti-inflammatory and repressing pro-inflammatory genes. GR-interacting protein-1 (GRIP1) is a GR corepressor in macrophages, however, whether GRIP1 mediates GR-activated transcription, and what dictates its coactivator versus corepressor properties is unknown. Here we report that GRIP1 loss in macrophages attenuates glucocorticoid induction of several anti-inflammatory targets, and that GC treatment of quiescent macrophages globally directs GRIP1 toward GR binding sites dominated by palindromic GC response elements (GRE), suggesting a non-redundant GRIP1 function as a GR coactivator. Interestingly, GRIP1 is phosphorylated at an N-terminal serine cluster by cyclin-dependent kinase-9 (CDK9), which is recruited into GC-induced GR:GRIP1:CDK9 hetero-complexes, producing distinct GRE-specific GRIP1 phospho-isoforms. Phosphorylation potentiates GRIP1 coactivator but, remarkably, not its corepressor properties. Consistently, phospho-GRIP1 and CDK9 are not detected at GR transrepression sites near pro-inflammatory genes. Thus, GR restricts actions of its own coregulator via CDK9-mediated phosphorylation to a subset of anti-inflammatory genes.

Diet-induced obesity causes chronic macrophage-driven inflammation in white adipose tissue (WAT) leading to insulin resistance. WAT macrophages, however, differ in their origin, gene expression and activities: unlike infiltrating monocyte-derived inflammatory macrophages, WAT-resident macrophages counteract inflammation and insulin resistance, yet, the mechanisms underlying their transcriptional programming remain poorly understood. We recently reported that a nuclear receptor cofactor-glucocorticoid receptor (GR)-interacting protein (GRIP)1-cooperates with GR to repress inflammatory genes. Here, we show that GRIP1 facilitates macrophage programming in response to IL4 via a GR-independent pathway by serving as a coactivator for Kruppel-like factor (KLF)4-a driver of tissue-resident macrophage differentiation. Moreover, obese mice conditionally lacking GRIP1 in macrophages develop massive macrophage infiltration and inflammation in metabolic tissues, fatty livers, hyperglycaemia and insulin resistance recapitulating metabolic disease. Thus, GRIP1 is a critical regulator of immunometabolism, which engages distinct transcriptional mechanisms to coordinate the balance between macrophage populations and ultimately promote metabolic homeostasis.

Background: Inflammation triggered by infection or injury is tightly controlled by glucocorticoid hormones which signal via a dedicated transcription factor, the Glucocorticoid Receptor (GR), to regulate hundreds of genes. However, the hierarchy of transcriptional responses to GR activation and the molecular basis of their oftentimes non-linear dynamics are not understood., Results: We investigated early glucocorticoid-driven transcriptional events in macrophages, a cell type highly responsive to both pro- and anti-inflammatory stimuli. Using whole transcriptome analyses in resting and acutely lipopolysaccharide (LPS)-stimulated macrophages, we show that early GR target genes form dense networks with the majority of control nodes represented by transcription factors. The expression dynamics of several glucocorticoid-responsive genes are consistent with feed forward loops (FFL) and coincide with rapid GR recruitment. Notably, GR binding sites in genes encoding members of the KLF transcription factor family colocalize with KLF binding sites. Moreover, our gene expression, transcription factor binding and computational data are consistent with the existence of the GR-KLF9-KLF2 incoherent FFL. Analysis of LPS-downregulated genes revealed striking enrichment in multimerized Zn-fingers- and KRAB domain-containing proteins known to bind nucleic acids and repress transcription by propagating heterochromatin. This raises an intriguing possibility that an increase in chromatin accessibility in inflammatory macrophages results from broad downregulation of negative chromatin remodelers., Conclusions: Pro- and anti-inflammatory stimuli alter the expression of a vast array of transcription factors and chromatin remodelers. By regulating multiple transcription factors, which propagate the initial hormonal signal, GR acts as a coordinating hub in anti-inflammatory responses. As several KLFs promote the anti-inflammatory program in macrophages, we propose that GR and KLFs functionally cooperate to curb inflammation.

Type I interferon (IFN) is essential for host defenses against viruses; however, dysregulated IFN signaling is causally linked to autoimmunity, particularly systemic lupus erythematosus. Autoimmune disease treatments rely on glucocorticoids (GCs), which act via the GC receptor (GR) to repress proinflammatory cytokine gene transcription. Conversely, cytokine signaling through cognate Jak/STAT pathways is reportedly unaffected or even stimulated by GR. Unexpectedly, we found that GR dramatically inhibited IFN-stimulated gene (ISG) expression in macrophages. The target of inhibition, the heterotrimeric STAT1-STAT2-IRF9 (ISGF3) transcription complex, utilized the GR cofactor GRIP1/TIF2 as a coactivator. Consequently, GRIP1 knockdown, genetic ablation, or depletion by GC-activated GR attenuated ISGF3 promoter occupancy, preinitiation complex assembly, and ISG expression. Furthermore, this regulatory loop was restricted to cell types such as macrophages expressing the GRIP1 protein at extremely low levels, and pharmacological disruption of the GR-GRIP1 interaction or transient introduction of GRIP1 restored RNA polymerase recruitment to target ISGs and the subsequent IFN response. Thus, type I IFN is a cytokine uniquely controlled by GR at the levels of not only production but also signaling through antagonism with the ISGF3 effector function, revealing a novel facet of the immunosuppressive properties of GCs.

The kinetics and magnitude of cytokine gene expression are tightly regulated to elicit a balanced response to pathogens and result from integrated changes in transcription and mRNA stability. Yet, how a single microbial stimulus induces peak transcription of some genes (TNFalpha) within minutes whereas others (IP-10) require hours remains unclear. Here, we dissect activation of several lipopolysaccharide (LPS)-inducible genes in macrophages, an essential cell type mediating inflammatory response in mammals. We show that a key difference between the genes is the step of the transcription cycle at which they are regulated. Specifically, at TNFalpha, RNA Polymerase II initiates transcription in resting macrophages, but stalls near the promoter until LPS triggers rapid and transient release of the negative elongation factor (NELF) complex and productive elongation. In contrast, no NELF or polymerase is detectible near the IP-10 promoter before induction, and LPS-dependent polymerase recruitment is rate limiting for transcription. We further demonstrate that this strategy is shared by other immune mediators and is independent of the inducer and signaling pathway responsible for gene activation. Finally, as a striking example of evolutionary conservation, the Drosophila homolog of the TNFalpha gene, eiger, displayed all of the hallmarks of NELF-dependent polymerase stalling. We propose that polymerase stalling ensures the coordinated, timely activation the inflammatory gene expression program from Drosophila to mammals.

Glucocorticoids dramatically inhibit cytokine and chemokine production. They act through the glucocorticoid receptor (GR), a ligand-dependent transcription factor that binds to and represses activities of other DNA-bound regulators, activator protein 1 and nuclear factor kappaB, utilizing a p160 GRIP1 as a corepressor. A yeast two-hybrid screen with the GRIP1 corepression domain (RD) yielded interferon (IFN) regulatory factor (IRF)3-a downstream effector of Toll-like receptors (TLR) 3/4 and an essential activator of several IFN and chemokine genes. We defined the GRIP1:IRF3 interface and showed that endogenous GRIP1 and IRF3 interact in mammalian cells. Interestingly, GR and IRF3 competed for GRIP1 binding; GR activation or GRIP1 knockdown in macrophages blocked whereas GRIP1 overexpression rescued IRF3-dependent gene expression. GR interference persisted in MyD88- and IFNA receptor-deficient mice, suggesting a specific disruption of TLR3-IRF3 pathway, not of autocrine IFN signaling. Finally, IRF3-stimulated response elements were necessary and sufficient for TLR3-dependent induction and glucocorticoid inhibition. Thus, GRIP1 plays a cofactor role in innate immunity. Competition with GR for GRIP1 antagonizes IRF3-mediated transcription, identifying the GRIP1:IRF3 interaction as a novel target for glucocorticoid immunosuppression.