David Saliba

Extensive characterization of NF-κB binding uncovers non-canonical motifs and advances the interpretation of genetic functional traits

BackgroundGenetic studies have provided ample evidence of the influence of non-coding DNA polymorphisms on trait variance, particularly those occurring within transcription factor binding sites. Protein binding microarrays and other platforms that can map these sites with great precision have enhanced our understanding of how a single nucleotide polymorphism can alter binding potential within an in vitro setting, allowing for greater predictive capability of its effect on a transcription factor binding site.ResultsWe have used protein binding microarrays and electrophoretic mobility shift assay-sequencing (EMSA-Seq), a deep sequencing based method we developed to analyze nine distinct human NF-κB dimers. This family of transcription factors is one of the most extensively studied, but our understanding of its DNA binding preferences has been limited to the originally described consensus motif, GGRRNNYYCC. We highlight differences between NF-κB family members and also put under the spotlight non-canonical motifs that have so far received little attention. We utilize our data to interpret the binding of transcription factors between individuals across 1,405 genomic regions laden with single nucleotide polymorphisms. We also associated binding correlations made using our data with risk alleles of disease and demonstrate its utility as a tool for functional studies of single nucleotide polymorphisms in regulatory regions.ConclusionsNF-κB dimers bind specifically to non-canonical motifs and these can be found within genomic regions in which a canonical motif is not evident. Binding affinity data generated with these different motifs can be used in conjunction with data from chromatin immunoprecipitation-sequencing (ChIP-Seq) to enable allele-specific analyses of expression and transcription factor-DNA interactions on a genome-wide scale.

Disturbed blood flow induces RelA expression via c-Jun N-terminal kinase 1: a novel mode of NF-κB regulation that promotes arterial inflammation.

Rationale: The nuclear factor (NF)-κB pathway is involved in arterial inflammation. Although the signaling pathways that regulate transcriptional activation of NF-κB are defined, the mechanisms that regulate the expression levels of NF-κB transcription factors are uncertain. Objective: We studied the signaling mechanisms that regulate RelA NF-κB subunit expression in endothelial cells (ECs) and their role in arterial inflammation. Methods and Results: Gene silencing and chromatin immunoprecipitation revealed that RelA expression was positively regulated by c-Jun N-terminal kinase (JNK) and the downstream transcription factor ATF2 in ECs. We concluded that this pathway promotes focal arterial inflammation as genetic deletion of JNK1 reduced NF-κB expression and macrophage accumulation at an atherosusceptible site. We hypothesized that JNK signaling to NF-κB may be controlled by mechanical forces because atherosusceptibility is associated with exposure to disturbed blood flow. This was assessed by positron emission tomography imaging of carotid arteries modified with a constrictive cuff, a method that was developed to study the effects of disturbed flow on vascular physiology in vivo. This approach coupled to en face staining revealed that disturbed flow elevates NF-κB expression and inflammation in murine carotid arteries via JNK1. Conclusions: We demonstrate that disturbed blood flow promotes arterial inflammation by inducing NF-κB expression in endothelial cells via JNK-ATF2 signaling. Thus, our findings illuminate a novel form of JNK–NF-κB crosstalk that may determine the focal nature of arterial inflammation and atherosclerosis. # Novelty and Significance {#article-title-52}Rationale: The nuclear factor (NF)-&kgr;B pathway is involved in arterial inflammation. Although the signaling pathways that regulate transcriptional activation of NF-&kgr;B are defined, the mechanisms that regulate the expression levels of NF-&kgr;B transcription factors are uncertain. Objective: We studied the signaling mechanisms that regulate RelA NF-&kgr;B subunit expression in endothelial cells (ECs) and their role in arterial inflammation. Methods and Results: Gene silencing and chromatin immunoprecipitation revealed that RelA expression was positively regulated by c-Jun N-terminal kinase (JNK) and the downstream transcription factor ATF2 in ECs. We concluded that this pathway promotes focal arterial inflammation as genetic deletion of JNK1 reduced NF-&kgr;B expression and macrophage accumulation at an atherosusceptible site. We hypothesized that JNK signaling to NF-&kgr;B may be controlled by mechanical forces because atherosusceptibility is associated with exposure to disturbed blood flow. This was assessed by positron emission tomography imaging of carotid arteries modified with a constrictive cuff, a method that was developed to study the effects of disturbed flow on vascular physiology in vivo. This approach coupled to en face staining revealed that disturbed flow elevates NF-&kgr;B expression and inflammation in murine carotid arteries via JNK1. Conclusions: We demonstrate that disturbed blood flow promotes arterial inflammation by inducing NF-&kgr;B expression in endothelial cells via JNK-ATF2 signaling. Thus, our findings illuminate a novel form of JNK–NF-&kgr;B crosstalk that may determine the focal nature of arterial inflammation and atherosclerosis.

IRF5:RelA Interaction Targets Inflammatory Genes in Macrophages

Summary Interferon Regulatory Factor 5 (IRF5) plays a major role in setting up an inflammatory macrophage phenotype, but the molecular basis of its transcriptional activity is not fully understood. In this study, we conduct a comprehensive genome-wide analysis of IRF5 recruitment in macrophages stimulated with bacterial lipopolysaccharide and discover that IRF5 binds to regulatory elements of highly transcribed genes. Analysis of protein:DNA microarrays demonstrates that IRF5 recognizes the canonical IRF-binding (interferon-stimulated response element [ISRE]) motif in vitro. However, IRF5 binding in vivo appears to rely on its interactions with other proteins. IRF5 binds to a noncanonical composite PU.1:ISRE motif, and its recruitment is aided by RelA. Global gene expression analysis in macrophages deficient in IRF5 and RelA highlights the direct role of the RelA:IRF5 cistrome in regulation of a subset of key inflammatory genes. We map the RelA:IRF5 interaction domain and suggest that interfering with it would offer selective targeting of macrophage inflammatory activities.

IRF5 controls both acute and chronic inflammation

Significance Many of the world’s major chronic diseases are driven by inflammation. The most abundant inflammatory cells in these diseases are myeloid cells, such as macrophages and neutrophils. Both cell types show remarkable phenotypic diversity among tissues. Defining molecular factors that control this diversity provides abundant scope for the generation of more specific and effective therapeutics, as the lack of specificity of the current most widely used antiinflammatory approaches, such as glucocorticoids and nonsteroidal antiinflammatory molecules, leads to widespread problems if used long term, even at relatively low doses. In this study we demonstrate that a transcription factor called IFN regulatory factor 5 controls macrophage and neutrophil aspects of inflammation, and thus its blockade might be an effective therapeutic strategy for multiple indications. Whereas the importance of macrophages in chronic inflammatory diseases is well recognized, there is an increasing awareness that neutrophils may also play an important role. In addition to the well-documented heterogeneity of macrophage phenotypes and functions, neutrophils also show remarkable phenotypic diversity among tissues. Understanding the molecular pathways that control this heterogeneity should provide abundant scope for the generation of more specific and effective therapeutics. We have shown that the transcription factor IFN regulatory factor 5 (IRF5) polarizes macrophages toward an inflammatory phenotype. IRF5 is also expressed in other myeloid cells, including neutrophils, where it was linked to neutrophil function. In this study we explored the role of IRF5 in models of acute inflammation, including antigen-induced inflammatory arthritis and lung injury, both involving an extensive influx of neutrophils. Mice lacking IRF5 accumulate far fewer neutrophils at the site of inflammation due to the reduced levels of chemokines important for neutrophil recruitment, such as the chemokine (C-X-C motif) ligand 1. Furthermore we found that neutrophils express little IRF5 in the joints and that their migratory properties are not affected by the IRF5 deficiency. These studies extend prior ones suggesting that inhibiting IRF5 might be useful for chronic macrophage-induced inflammation and suggest that IRF5 blockade would ameliorate more acute forms of inflammation, including lung injury.

KAP1/TRIM28: An inhibitor of IRF5 function in inflammatory macrophages

IRF5 plays a key role in the induction of pro-inflammatory cytokines, contributing to the plasticity and polarisation of macrophages to an M1 phenotype and initiation of a potent T(H)1-T(H)17 response. To better understand the means of IRF5 transcriptional action, we conducted a screen for IRF5-interacting partners by affinity purification coupled to mass spectrometry and identified KAP1/TRIM28 as a novel protein-protein interaction partner of IRF5. KAP1 acts as a transcriptional co-repressor, chiefly via recruitment of complexes involved in chromatin silencing, such as histone deacetylases and methyltransferases. We mapped the N-terminus of IRF5, encompassing its DNA-binding domain together with a highly intrinsically disordered region, as crucial for the IRF5-KAP1 interaction interface, and demonstrated that IRF5 can also form complexes with the methyltransferase SETDB1. Knockdown of KAP1 (TRIM28) gene expression in human M1 macrophages potentiated IRF5-mediated expression of TNF and other M1 macrophage markers. This effect may be linked to methyltransferase activity of SETDB1, such as trimethylation of lysine 9 of histone 3 (H3K9me3), deposition of which was decreased at the human TNF locus upon KAP1 knockdown. Our study furthers an understanding of the complex molecular interactions between the TRIM and IRF protein families, and highlights a role of the inhibitory properties of KAP1 in association with IRF5-mediated gene expression.

FOXO3 as a new IKK-ε-controlled check-point of regulation of IFN-β expression

Cell survival transcription factor FOXO3 has been recently implicated in moderating pro‐inflammatory cytokine production by dendritic cells (DCs), but the molecular mechanisms are unclear. It was suggested that FOXO3 could antagonize NF‐κB activity, while IKK‐β was demonstrated to inactivate FOXO3, suggesting a cross‐talk between the two pathways. Therefore, FOXO3 activity must be tightly regulated to allow for an appropriate inflammatory response. Here, we show that in human monocyte‐derived DCs (MDDCs), FOXO3 is able to antagonize signaling intermediates downstream of the Toll‐like receptor (TLR) 4, such as NF‐κB and interferon regulatory factors (IRFs), resulting in inhibition of interferon (IFN)‐β expression. We also demonstrate that the activity of FOXO3 itself is regulated by IKK‐ε, a kinase involved in IFN‐β production, which phosphorylates and inactivates FOXO3 in response to TLR4 agonists. Thus, we identify FOXO3 as a new IKK‐ε‐controlled check‐point of IRF activation and regulation of IFN‐β expression, providing new insight into the role of FOXO3 in immune response control.

IRF5 governs liver macrophage activation that promotes hepatic fibrosis in mice and humans

Hepatic fibrosis arises from inflammation in the liver initiated by resident macrophage activation and massive leukocyte accumulation. Hepatic macrophages hold a central position in maintaining homeostasis in the liver and in the pathogenesis of acute and chronic liver injury linked to fibrogenesis. Interferon regulatory factor 5 (IRF5) has recently emerged as an important proinflammatory transcription factor involved in macrophage activation under acute and chronic inflammation. Here, we revealed that IRF5 is significantly induced in liver macrophages from human subjects developing liver fibrosis from nonalcoholic fatty liver disease or hepatitis C virus infection. Furthermore, IRF5 expression positively correlated with clinical markers of liver damage, such as plasma transaminase and bilirubin levels. Interestingly, mice lacking IRF5 in myeloid cells (MKO) were protected from hepatic fibrosis induced by metabolic or toxic stresses. Transcriptional reprogramming of macrophages lacking IRF5 was characterized by immunosuppressive and antiapoptotic properties. Consequently, IRF5 MKO mice respond to hepatocellular stress by promoting hepatocyte survival, leading to complete protection from hepatic fibrogenesis. Our findings reveal a regulatory network, governed by IRF5, that mediates hepatocyte death and liver fibrosis in mice and humans. Therefore, modulating IRF5 function may be an attractive approach to experimental therapeutics in fibroinflammatory liver disease.

Disturbed Blood Flow Induces RelA Expression via c-Jun N-Terminal Kinase 1 A Novel Mode of NF-kappa B Regulation That Promotes Arterial Inflammation

Rationale: The nuclear factor (NF)-κB pathway is involved in arterial inflammation. Although the signaling pathways that regulate transcriptional activation of NF-κB are defined, the mechanisms that regulate the expression levels of NF-κB transcription factors are uncertain. Objective: We studied the signaling mechanisms that regulate RelA NF-κB subunit expression in endothelial cells (ECs) and their role in arterial inflammation. Methods and Results: Gene silencing and chromatin immunoprecipitation revealed that RelA expression was positively regulated by c-Jun N-terminal kinase (JNK) and the downstream transcription factor ATF2 in ECs. We concluded that this pathway promotes focal arterial inflammation as genetic deletion of JNK1 reduced NF-κB expression and macrophage accumulation at an atherosusceptible site. We hypothesized that JNK signaling to NF-κB may be controlled by mechanical forces because atherosusceptibility is associated with exposure to disturbed blood flow. This was assessed by positron emission tomography imaging of carotid arteries modified with a constrictive cuff, a method that was developed to study the effects of disturbed flow on vascular physiology in vivo. This approach coupled to en face staining revealed that disturbed flow elevates NF-κB expression and inflammation in murine carotid arteries via JNK1. Conclusions: We demonstrate that disturbed blood flow promotes arterial inflammation by inducing NF-κB expression in endothelial cells via JNK-ATF2 signaling. Thus, our findings illuminate a novel form of JNK–NF-κB crosstalk that may determine the focal nature of arterial inflammation and atherosclerosis. # Novelty and Significance {#article-title-52}Rationale: The nuclear factor (NF)-&kgr;B pathway is involved in arterial inflammation. Although the signaling pathways that regulate transcriptional activation of NF-&kgr;B are defined, the mechanisms that regulate the expression levels of NF-&kgr;B transcription factors are uncertain. Objective: We studied the signaling mechanisms that regulate RelA NF-&kgr;B subunit expression in endothelial cells (ECs) and their role in arterial inflammation. Methods and Results: Gene silencing and chromatin immunoprecipitation revealed that RelA expression was positively regulated by c-Jun N-terminal kinase (JNK) and the downstream transcription factor ATF2 in ECs. We concluded that this pathway promotes focal arterial inflammation as genetic deletion of JNK1 reduced NF-&kgr;B expression and macrophage accumulation at an atherosusceptible site. We hypothesized that JNK signaling to NF-&kgr;B may be controlled by mechanical forces because atherosusceptibility is associated with exposure to disturbed blood flow. This was assessed by positron emission tomography imaging of carotid arteries modified with a constrictive cuff, a method that was developed to study the effects of disturbed flow on vascular physiology in vivo. This approach coupled to en face staining revealed that disturbed flow elevates NF-&kgr;B expression and inflammation in murine carotid arteries via JNK1. Conclusions: We demonstrate that disturbed blood flow promotes arterial inflammation by inducing NF-&kgr;B expression in endothelial cells via JNK-ATF2 signaling. Thus, our findings illuminate a novel form of JNK–NF-&kgr;B crosstalk that may determine the focal nature of arterial inflammation and atherosclerosis.

Interferon Regulatory Factor 5 Controls Necrotic Core Formation in Atherosclerotic Lesions by Impairing Efferocytosis.

Background: Myeloid cells are central to atherosclerotic lesion development and vulnerable plaque formation. Impaired ability of arterial phagocytes to uptake apoptotic cells (efferocytosis) promotes lesion growth and establishment of a necrotic core. The transcription factor interferon regulatory factor (IRF)-5 is an important modulator of myeloid function and programming. We sought to investigate whether IRF5 affects the formation and phenotype of atherosclerotic lesions. Methods: We investigated the role of IRF5 in atherosclerosis in 2 complementary models. First, atherosclerotic lesion development in hyperlipidemic apolipoprotein E-deficient (ApoE-/-) mice and ApoE-/- mice with a genetic deletion of IRF5 (ApoE-/-Irf5-/-) was compared and then lesion development was assessed in a model of shear stress-modulated vulnerable plaque formation. Results: Both lesion and necrotic core size were significantly reduced in ApoE-/-Irf5-/- mice compared with IRF5-competent ApoE-/- mice. Necrotic core size was also reduced in the model of shear stress-modulated vulnerable plaque formation. A significant loss of CD11c+ macrophages was evident in ApoE-/-Irf5-/- mice in the aorta, draining lymph nodes, and bone marrow cell cultures, indicating that IRF5 maintains CD11c+ macrophages in atherosclerosis. Moreover, we revealed that the CD11c gene is a direct target of IRF5 in macrophages. In the absence of IRF5, CD11c- macrophages displayed a significant increase in expression of the efferocytosis-regulating integrin-&bgr;3 and its ligand milk fat globule-epidermal growth factor 8 protein and enhanced efferocytosis in vitro and in situ. Conclusions: IRF5 is detrimental in atherosclerosis by promoting the maintenance of proinflammatory CD11c+ macrophages within lesions and controlling the expansion of the necrotic core by impairing efferocytosis.

Cross-species Analysis Reveals Evolving and Conserved Features of the Nuclear Factor κB (NF-κB) Proteins

Background: NF-κB regulates transcription via binding to DNA and interactions with cofactors. Results: NF-κB binding to DNA and cytosolic IκBs is conserved, whereas binding to nuclear IκBs has evolved. Conclusion: There is distinct evolutionary pressure on two NF-κB/IκB binding interfaces. Significance: The results provide inroads into IκB-specific modulation of NF-κB activity. NF-κB is a key regulator of immune gene expression in metazoans. It is currently unclear what changes occurred in NF-κB during animal evolution and what features remained conserved. To address this question, we compared the biochemical and functional properties of NF-κB proteins derived from human and the starlet sea anemone (Nematostella vectensis) in 1) a high-throughput assay of in vitro preferences for DNA sequences, 2) ChIP analysis of in vivo recruitment to the promoters of target genes, 3) a LUMIER-assisted examination of interactions with cofactors, and 4) a transactivation assay. We observed a remarkable evolutionary conservation of the DNA binding preferences of the animal NF-κB orthologs. We also show that NF-κB dimerization properties, nuclear localization signals, and binding to cytosolic IκBs are conserved. Surprisingly, the Bcl3-type nuclear IκB proteins functionally pair up only with NF-κB derived from their own species. The basis of the differential NF-κB recognition by IκB subfamilies is discussed.