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.

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.macrophages | neutrophils | IRF5 | acute inflammation | arthritis

Protective high-affinity antibody responses depend on competitive selection of B cells carrying somatically mutated B-cell receptors by follicular helper T (TFH) cells in germinal centres. The rapid T-B-cell interactions that occur during this process are reminiscent of neural synaptic transmission pathways. Here we show that a proportion of human TFH cells contained dense-core granules marked by chromogranin B, which are normally found in neuronal presynaptic terminals storing catecholamines such as dopamine. TFH cells produce high amounts of dopamine and released it upon cognate interaction with B cells. Dopamine causes rapid translocation of intracellular ICOSL (inducible T-cell co-stimulator ligand, also known as ICOSLG) to the B-cell surface, which enhances accumulation of CD40L and chromogranin B granules at the human TFH cell synapse and increases the synapse area. Mathematical modelling suggests that faster dopamine-induced T-B-cell interactions increase total germinal centre output and accelerate it by days. Delivery of neurotransmitters across the T-B-cell synapse may be advantageous in the face of infection.

SummaryInterferon 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.

Spatially and temporally controlled expression of inflammatory mediators is critical for an appropriate immune response. In this study, we define the role for interferon regulatory factor 5 (IRF5) in secretion of tumor necrosis factor (TNF) by human dendritic cells (DCs). We demonstrate that DCs but not macrophages have high levels of IRF5 protein, and that IRF5 is responsible for the late-phase expression of TNF, which is absent in macrophages. Sustained TNF secretion is essential for robust T IntroductionTumor necrosis factor (TNF) is one of the major cytokines responsible for effector immune functions. As well as playing a central role in host defense against infection, TNF is a major factor in the pathogenesis of chronic inflammatory disease such as rheumatoid arthritis (RA). Consequently, tightly controlled regulation of its expression is critical for an appropriate immune response. This occurs at the transcriptional and posttranscriptional levels, with transcriptional regulation showing specificity for both stimulus and cell type. 1 The NF-B family of transcription factors (TFs) plays a major role in transcriptional up-regulation of the TNF gene by lipopolysaccharide (LPS) in both mouse and human myeloid cells. [2][3][4][5] Regulation of transcription for many immune genes in response to Toll-like receptor (TLR) signaling involves a combination of NF-B and interferon regulatory factor (IRF) factors. 6 IRFs appear to provide a mechanism for conferring signal specificity to a variety of target gene subsets, with IRF3 being essential for type I interferon (IFN) response, 7 and IRF5 playing a key role in induction of proinflammatory cytokines, including TNF, Consequently, IRF5 Ϫ/Ϫ mice show resistance to lethal shock induced by CpG-B or LPS. 8 Unlike other IRF family members, IRF5 contains 2 nuclear localization signals (NLSs), 1 in the N-terminus and the other in the C-terminus of the protein. This results in low levels of nuclear translocation and therefore weak trans-activation activity of IRF5, even in unstimulated cells. 9 The molecular pathways leading to IRF5 activation are not well understood, but it was shown that TLR signaling induces the formation of MyD88-IRF5-TRAF6 complexes, 8 and is probably followed by phosphorylation of specific sites within the IRF5 C-terminal autoinhibitory domain. 10 Human IRF5 is expressed as multiple spliced variants with distinct cell type-specific expression, cellular localization, differential regulation, and dissimilar functions. 11 Moreover, genetic polymorphisms in the IRF5 gene leading to expression of several unique isoforms have been implicated in autoimmune diseases, including systemic lupus erythematosis (SLE), RA and Sjogren syndrome. 12-15 IRF5 mRNA expression has been detected in B cells, dendritic cells (DCs), monocytes, and natural killer (NK) cells but not in T cells, 11 yet little is known about the IRF5 protein expression in these cells.Here, we demonstrate that human monocytes acquire high levels of IRF5 protein during differentiation into monocyte-d...