Irina A. Udalova

IRF5 promotes inflammatory macrophage polarization and TH1-TH17 responses.

Polymorphisms in the gene encoding the transcription factor IRF5 that lead to higher mRNA expression are associated with many autoimmune diseases. Here we show that IRF5 expression in macrophages was reversibly induced by inflammatory stimuli and contributed to the plasticity of macrophage polarization. High expression of IRF5 was characteristic of M1 macrophages, in which it directly activated transcription of the genes encoding interleukin 12 subunit p40 (IL-12p40), IL-12p35 and IL-23p19 and repressed the gene encoding IL-10. Consequently, those macrophages set up the environment for a potent T helper type 1 (TH1)-TH17 response. Global gene expression analysis demonstrated that exogenous IRF5 upregulated or downregulated expression of established phenotypic markers of M1 or M2 macrophages, respectively. Our data suggest a critical role for IRF5 in M1 macrophage polarization and define a previously unknown function for IRF5 as a transcriptional repressor.

A polymorphism that affects OCT-1 binding to the TNF promoter region is associated with severe malaria

Genetic variation in cytokine promoter regions is postulated to influence susceptibility to infection, but the molecular mechanisms by which such polymorphisms might affect gene regulation are unknown. Through systematic DNA footprinting of the TNF (encoding tumour necrosis factor, TNF) promoter region, we have identified a single nucleotide polymorphism (SNP) that causes the helix-turn-helix transcription factor OCT-1 to bind to a novel region of complex protein-DNA interactions and alters gene expression in human monocytes. The OCT-1-binding genotype, found in approximately 5% of Africans, is associated with fourfold increased susceptibility to cerebral malaria in large case-control studies of West African and East African populations, after correction for other known TNF polymorphisms and linked HLA alleles.

Functional consequences of a polymorphism affecting NF-kappaB p50-p50 binding to the TNF promoter region.

ABSTRACT Stimulation of the NF-κB pathway often causes p65-p50 and p50-p50 dimers to be simultaneously present in the cell nucleus. A natural polymorphism at nucleotide −863 in the human TNF promoter (encoding tumor necrosis factor [TNF]) region provides an opportunity to dissect the functional interaction of p65-p50 and p50-p50 at a single NF-κB binding site. We found that this site normally binds both p65-p50 and p50-p50, but a single base change specifically inhibits p50-p50 binding. Reporter gene analysis in COS-7 cells expressing both p65-p50 and p50-p50 shows that the ability to bind p50-p50 reduces the enhancer effect of this NF-κB site. Using an adenoviral reporter assay, we found that the variant which binds p50-p50 results in a reduction of lipopolysaccharide-inducible gene expression in primary human monocytes. This finding adds to a growing body of experimental evidence that p50-p50 can inhibit the transactivating effects of p65-p50 and illustrates the potential for genetic modulation of inflammatory gene regulation in humans by subtle nucleotide changes that alter the relative binding affinities of different forms of the NF-κB complex.

Complex NF-kappaB interactions at the distal tumor necrosis factor promoter region in human monocytes

We describe a dense cluster of DNA-protein interactions located 600 nucleotides upstream of the transcriptional start site of the human tumor necrosis factor (TNF) gene. This area was identified as being of potential importance for lipopolysaccharide-inducible TNF expression in the human monocyte cell line Mono Mac 6, based on reporter gene analysis of point mutations at a number of nuclear factor κB (NF-κB)-like motifs within the human TNF promoter region. The area contains two NF-κB sites, which are here shown by DNase I and methylation interference footprinting to flank a novel binding site. UV cross-linking studies reveal that the novel site can also bind NF-κB as well as an unknown protein(s) of approximately 40 kDa. We show that these three adjacent κB-binding sites differ markedly in their relative affinities for p50/p50, p65/p65, and p65/p50, yet this 39-nucleotide segment of DNA appears capable of binding up to three NF-κB heterodimers simultaneously. Reporter gene studies indicate that each element of the cluster contributes to lipopolysaccharide-induced transcriptional activation in Mono Mac 6 cells. These findings suggest that NF-κB acts in a complex manner to activate TNF transcription in human monocytes.

Evidence for a dual mechanism for IL-10 suppression of TNF-alpha production that does not involve inhibition of p38 mitogen-activated protein kinase or NF-kappa B in primary human macrophages.

IL-10 is a potent anti-inflammatory cytokine and inhibitor of TNF-α production. The molecular pathways by which IL-10 inhibits TNF-α production are obscure, with diverse mechanisms having been published. In this study, a new approach has been taken for the study of human cells. Adenovirus was used to deliver TNF-α promoter-based luciferase reporter genes to primary human monocytic cells. The reporter genes were highly responsive to macrophage activation and appeared to mirror the behavior of the endogenous TNF-α gene. When added, either with or after the stimulus, IL-10 required the 3′ untranslated region of the TNF-α gene to inhibit luciferase mRNA and protein expression, indicating a posttranscriptional mechanism. However, if macrophages were incubated with IL-10 before activation, inhibition of gene expression was also mediated by the 5′ promoter, suggesting a transcriptional mechanism. To our knowledge, this is the first time that a dual mechanism for IL-10 function has been demonstrated. Studies to elucidate the mechanisms underlying the inhibition of TNF-α production addressed the effect of IL-10 on the activation of p38 mitogen-activated protein kinase and NF-κB. However, these studies could demonstrate no requirement for the inhibition of p38 mitogen-activated protein kinase or NF-κB activation as potential mechanisms. Overall, these results may explain the diversity previously ascribed to the complex mechanisms of IL-10 anti-inflammatory activity.

Principles of dimer-specific gene regulation revealed by a comprehensive characterization of NF-κB family DNA binding

The unique DNA-binding properties of distinct NF-κB dimers influence the selective regulation of NF-κB target genes. To more thoroughly investigate these dimer-specific differences, we combined protein-binding microarrays and surface plasmon resonance to evaluate DNA sites recognized by eight different NF-κB dimers. We observed three distinct binding-specificity classes and clarified mechanisms by which dimers might regulate distinct sets of genes. We identified many new nontraditional NF-κB binding site (κB site) sequences and highlight the plasticity of NF-κB dimers in recognizing κB sites with a single consensus half-site. This study provides a database that can be used in efforts to identify NF-κB target sites and uncover gene regulatory circuitry.

Transcriptional Regulation of the Endogenous Danger Signal Tenascin-C: A Novel Autocrine Loop in Inflammation

Inappropriate expression of proinflammatory mediators underpins the pathogenesis of autoimmune disease and tumor metastasis. The extracellular matrix glycoprotein tenascin-C is an endogenous activator of innate immunity that promotes the synthesis of inflammatory cytokines via activation of TLR4. Little tenascin-C is observed in most healthy adult tissues, but expression is specifically upregulated at sites of inflammation. Moreover, high levels of tenascin-C are associated with chronic inflammation and found in the tumor stroma. In this study, we show that the expression of tenascin-C is induced in immune myeloid cells activated by a variety of inflammatory stimuli, including specific TLR ligands. Its synthesis is transcriptionally regulated and requires the specific activation of AKT/PI3K and NF-κB signaling pathways. Using a bioinformatic approach, we identified a large number of conserved noncoding regions throughout the tenascin-C genomic locus that may contribute to its transcriptional regulation during inflammation. We also demonstrate that tenascin-C expression is transient during acute inflammation. In contrast, persistently high levels of expression occur in the inflamed synovium of joints from rheumatoid arthritis patients. Thus, misregulated expression of this endogenous danger signal may promote an autocrine loop of inflammation and contribute to the persistence of inflammation in autoimmune diseases or to tumor egress and invasion during metastasis.

The role of transposable elements in the regulation of IFN-lambda1 gene expression.

IFNs λ1, λ2, and λ3, or type III IFNs, are recently identified cytokines distantly related to type I IFNs. Despite an early evolutionary divergence, the 2 types of IFNs display similar antiviral activities, and both are produced primarily in dendritic cells. Although virus induction of the type I IFN-β gene had served as a paradigm of gene regulation, relatively little is known about the regulation of IFN-λ gene expression. Studies of virus induction of IFN-λ1 identified an essential role of IFN regulatory factors (IRF) 3 and 7, which bind to a regulatory DNA sequence near the start site of transcription. Here, we report that the proximal promoter region of the IFN-λ1 regulatory region is not sufficient for maximal gene induction in response to bacterial LPS, and we identify an essential cluster of homotypic NF-κB binding sites. Remarkably, these sites, which bind efficiently to NF-κB and function independently of the IRF3/7 binding sites, originate as transposable elements of the Alu and LTR families. We also show that depletion of the NF-κB RelA protein significantly reduces the level of the IFN-λ1 gene expression. We conclude that IFN-λ1 gene expression requires NF-κB, and we propose a model for IFN-λ1 gene regulation, in which IRF and NF-κB activate gene expression independently via spatially separated promoter elements. These observations provide insights into the independent evolution of the IFN-λ1 and IFN-β promoters and directly implicate transposable elements in the regulation of the IFN-λ1 gene by NF-κB.

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.

Macrophage heterogeneity in the context of rheumatoid arthritis

Macrophages are very important in the pathogenesis of rheumatoid arthritis (RA). The increase in the number of sublining macrophages in the synovium is an early hallmark of active rheumatic disease, and high numbers of macrophages are a prominent feature of inflammatory lesions. The degree of synovial macrophage infiltration correlates with the degree of joint erosion, and depletion of these macrophages from inflamed tissue has a profound therapeutic benefit. Research has now uncovered an unexpectedly high level of heterogeneity in macrophage origin and function, and has emphasized the role of environmental factors in their functional specialization. Although the heterogeneous populations of macrophages in RA have not been fully characterized, preliminary results in mouse models of arthritis have contributed to our understanding of the phenotype and ontogeny of synovial macrophages, and to deciphering the properties of monocyte-derived infiltrating and tissue-resident macrophages. Elucidating the molecular mechanisms that drive polarization of macrophages towards proinflammatory or anti-inflammatory phenotypes could lead to identification of signalling pathways that inform future therapeutic strategies.