Littlewood-Evans et al. demonstrate that extracellular succinate leads to the propagation of inflammatory macrophage activation, providing translational evidence to support the development of GPR91 antagonists for the treatment of rheumatoid arthritis.

Runx1 is a developmentally regulated transcription factor that is essential for haemopoiesis. Runx1 can bind as a monomer to the core consensus sequence TGTGG, but binds more efficiently as a hetero-dimer together with the non-DNA binding protein CBFβ as a complex termed core binding factor (CBF). Here, we demonstrated that CBF can also assemble as a dimeric complex on two overlapping Runx1 sites within the palindromic sequence TGTGGCTGCCCACA in the human granulocyte macrophage colony-stimulating factor enhancer. Furthermore, we demonstrated that binding of Runx1 to the enhancer is rigidly controlled at the level of chromatin accessibility, and is dependent upon prior induction of NFAT and AP-1, which disrupt a positioned nucleosome in this region. We employed in vivo footprinting to demonstrate that, upon activation of the enhancer, both sites are efficiently occupied. In vitro binding assays confirmed that two CBF complexes can bind this site simultaneously, and transfection assays demonstrated that both sites contribute significantly to enhancer function. Computer modelling based on the Runx1/CBFβ/DNA crystal structure further revealed that two molecules of CBF could potentially bind to this class of palindromic sequence as a dimeric complex in a conformation whereby both Runx1 and CBFβ within the two CBF complexes are closely aligned.

The human interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating-factor (GM-CSF, or CSF2) gene cluster arose by duplication of an ancestral gene. Although just 10 kb apart and responsive to the same signals, the IL-3 and GM-CSF genes are nevertheless regulated independently by separate, tissue-specific enhancers. To understand the differential regulation of the IL-3 and GM-CSF genes we have investigated a cluster of three ubiquitous DNase I-hypersensitive sites (DHSs) located between the two genes. We found that each site contains a conserved CTCF consensus sequence, binds CTCF, and recruits the cohesin subunit Rad21 in vivo. The positioning of these sites relative to the IL-3 and GM-CSF genes and their respective enhancers is conserved between human and mouse, suggesting a functional role in the organization of the locus. We found that these sites effectively block functional interactions between the GM-CSF enhancer and either the IL-3 or the GM-CSF promoter in reporter gene assays. These data argue that the regulation of the IL-3 and the GM-CSF promoters depends on the positions of their enhancers relative to the conserved CTCF/cohesinbinding sites. We suggest that one important role of these sites is to enable the independent regulation of the IL-3 and GM-CSF genes.

Objective. Fcγ receptors (FcγR) play important roles in both protective and pathogenic immune responses. The assembly of the CBM signalosome encompassing caspase recruitment domain-containing protein 9, B cell CLL/lymphoma 10, and mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT-1) is required for optimal FcγR-induced canonical NF-κB activation and proinflammatory cytokine release. This study was undertaken to clarify the relevance of MALT-1 protease activity in FcγR-driven events and evaluate the therapeutic potential of selective MALT-1 protease inhibitors in FcγR-mediated diseases.Methods. Using genetic and pharmacologic disruption of MALT-1 scaffolding and enzymatic activity, we assessed the relevance of MALT-1 function in murine and human primary myeloid cells upon stimulation with immune complexes (ICs) and in murine models of autoantibody-driven arthritis and immune thrombocytopenic purpura (ITP).Results. MALT-1 protease function is essential for optimal FcγR-induced production of proinflammatory cytokines by various murine and human myeloid cells stimulated with ICs. In contrast, MALT-1 protease inhibition did not affect the Syk-dependent, FcγR-mediated production of reactive oxygen species or leukotriene B 4 . Notably, pharmacologic MALT-1 protease inhibition in vivo reduced joint inflammation in the murine K/BxN serum-induced arthritis model (mean area under the curve for paw swelling of 45.42% versus 100% in control mice; P = 0.0007) but did not affect platelet depletion in a passive model of ITP.Conclusion. Our findings indicate a specific contribution of MALT-1 protease activity to FcγR-mediated events and suggest that MALT-1 protease inhibitors have therapeutic potential in a subset of FcγR-driven inflammatory disorders.