Bivalent chromatin accommodates survivin and BRG1/SWI complex to activate DNA damage response in CD4+ cells.

BACKGROUND: Bivalent regions of chromatin (BvCR) are characterized by trimethylated lysine 4 (H3K4me3) and lysine 27 on histone H3 (H3K27me3) deposition which aid gene expression control during cell differentiation. The role of BvCR in post-transcriptional DNA damage response remains unidentified. Oncoprotein survivin binds chromatin and mediates IFNγ effects in CD4+ cells. In this study, we explored the role of BvCR in DNA damage response of autoimmune CD4+ cells in rheumatoid arthritis (RA). METHODS: We performed deep sequencing of the chromatin bound to survivin, H3K4me3, H3K27me3, and H3K27ac, in human CD4+ cells and identified BvCR, which possessed all three histone H3 modifications. Protein partners of survivin on chromatin were predicted by integration of motif enrichment analysis, computational machine-learning, and structural modeling, and validated experimentally by mass spectrometry and peptide binding array. Survivin-dependent change in BvCR and transcription of genes controlled by the BvCR was studied in CD4+ cells treated with survivin inhibitor, which revealed survivin-dependent biological processes. Finally, the survivin-dependent processes were mapped to the transcriptome of CD4+ cells in blood and in synovial tissue of RA patients and the effect of modern immunomodulating drugs on these processes was explored. RESULTS: We identified that BvCR dominated by H3K4me3 (H3K4me3-BvCR) accommodated survivin within cis-regulatory elements of the genes controlling DNA damage. Inhibition of survivin or JAK-STAT signaling enhanced H3K4me3-BvCR dominance, which improved DNA damage recognition and arrested cell cycle progression in cultured CD4+ cells. Specifically, BvCR accommodating survivin aided sequence-specific anchoring of the BRG1/SWI chromatin-remodeling complex coordinating DNA damage response. Mapping survivin interactome to BRG1/SWI complex demonstrated interaction of survivin with the subunits anchoring the complex to chromatin. Co-expression of BRG1, survivin and IFNγ in CD4+ cells rendered complete deregulation of DNA damage response in RA. Such cells possessed strong ability of homing to RA joints. Immunomodulating drugs inhibited the anchoring subunits of BRG1/SWI complex, which affected arthritogenic profile of CD4+ cells. CONCLUSIONS: BvCR execute DNA damage control to maintain genome fidelity in IFN-activated CD4+ cells. Survivin anchors the BRG1/SWI complex to BvCR to repress DNA damage response. These results offer a platform for therapeutic interventions targeting survivin and BRG1/SWI complex in autoimmunity.

Rho-GTPase dependent leukocyte interaction generates pro-inflammatory thymic Tregs and causes arthritis.

Conditional mutation of protein geranylgeranyltransferase type I (GGTase-I) in macrophages (GLC) activates Rho-GTPases and causes arthritis in mice. Knocking out Rag1 in GLC mice alleviates arthritis which indicates that lymphocytes are required for arthritis development in those mice. To study GLC dependent changes in the adaptive immunity, we isolated CD4+ T cells from GLC mice (CD4+GLCs). Spleen and joint draining lymph nodes (dLN) CD4+GLCs exhibited high expression of Cdc42 and Rac1, which repressed the caudal HOXA proteins and activated the mechanosensory complex to facilitate migration. These CDC42/RAC1 rich CD4+GLCs presented a complete signature of GARP+NRP1+IKZF2+FOXP3+ regulatory T cells (Tregs) of thymic origin. Activation of the ß-catenin/Lef1 axis promoted a pro-inflammatory Th1 phenotype of Tregs, which was strongly associated with arthritis severity. Knockout of Cdc42 in macrophages of GLC mice affected CD4+ cell biology and triggered development of non-thymic Tregs. Knockout of Rac1 and RhoA had no such effects on CD4+ cells although it alleviated arthritis in GLC mice. Disrupting macrophage and T cell interaction with CTLA4 fusion protein reduced the Th1-driven inflammation and enrichment of thymic Tregs into dLNs. Antigen challenge reinforced the CD4+GLC phenotype in non-arthritic heterozygote GLC mice and increased accumulation of Rho-GTPase expressing thymic Tregs in dLNs. Our study demonstrates an unexpected role of macrophages in stimulating the development of pro-inflammatory thymic Tregs and reveal activation of Rho-GTPases behind their arthritogenic phenotype.

Metabolic signature and proteasome activity controls synovial migration of CDC42hiCD14+ cells in rheumatoid arthritis.

Objective: Activation of Rho-GTPases in macrophages causes inflammation and severe arthritis in mice. In this study, we explore if Rho-GTPases define the joint destination of pathogenic leukocytes, the mechanism by which they perpetuate rheumatoid arthritis (RA), and how JAK inhibition mitigates these effects. Methods: CD14+ cells of 136 RA patients were characterized by RNA sequencing and cytokine measurement to identify biological processes and transcriptional regulators specific for CDC42 hiCD14+ cells, which were summarized in a metabolic signature (MetSig). The effect of hypoxia and IFN-γ signaling on the metabolic signature of CD14+ cells was assessed experimentally. To investigate its connection with joint inflammation, the signature was translated into the single-cell characteristics of CDC42 hi synovial tissue macrophages. The sensitivity of MetSig to the RA disease activity and the treatment effect were assessed experimentally and clinically. Results: CDC42 hiCD14+ cells carried MetSig of genes functional in the oxidative phosphorylation and proteasome-dependent cell remodeling, which correlated with the cytokine-rich migratory phenotype and antigen-presenting capacity of these cells. Integration of CDC42 hiCD14+ and synovial macrophages marked with MetSig revealed the important role of the interferon-rich environment and immunoproteasome expression in the homeostasis of these pathogenic macrophages. The CDC42 hiCD14+ cells were targeted by JAK inhibitors and responded with the downregulation of immunoproteasome and MHC-II molecules, which disintegrated the immunological synapse, reduced cytokine production, and alleviated arthritis. Conclusion: This study shows that the CDC42-related MetSig identifies the antigen-presenting CD14+ cells that migrate to joints to coordinate autoimmunity. The accumulation of CDC42 hiCD14+ cells discloses patients perceptive to the JAKi treatment.

Cohesin-Mediated Chromatin Interactions and Autoimmunity.

Proper physiological functioning of any cell type requires ordered chromatin organization. In this context, cohesin complex performs important functions preventing premature separation of sister chromatids after DNA replication. In partnership with CCCTC-binding factor, it ensures insulator activity to organize enhancers and promoters within regulatory chromatin. Homozygous mutations and dysfunction of individual cohesin proteins are embryonically lethal in humans and mice, which limits in vivo research work to embryonic stem cells and progenitors. Conditional alleles of cohesin complex proteins have been generated to investigate their functional roles in greater detail at later developmental stages. Thus, genome regulation enabled by action of cohesin proteins is potentially crucial in lineage cell development, including immune homeostasis. In this review, we provide current knowledge on the role of cohesin complex in leukocyte maturation and adaptive immunity. Conditional knockout and shRNA-mediated inhibition of individual cohesin proteins in mice demonstrated their importance in haematopoiesis, adipogenesis and inflammation. Notably, these effects occur rather through changes in transcriptional gene regulation than through expected cell cycle defects. This positions cohesin at the crossroad of immune pathways including NF-kB, IL-6, and IFNγ signaling. Cohesin proteins emerged as vital regulators at early developmental stages of thymocytes and B cells and after antigen challenge. Human genome-wide association studies are remarkably concordant with these findings and present associations between cohesin and rheumatoid arthritis, multiple sclerosis and HLA-B27 related chronic inflammatory conditions. Furthermore, bioinformatic prediction based on protein-protein interactions reveal a tight connection between the cohesin complex and immune relevant processes supporting the notion that cohesin will unearth new clues in regulation of autoimmunity.

Composition and structure of cell wall ulvans recovered from Ulva spp. along the Swedish west coast.

The cell wall polysaccharide ulvan was isolated from two species of the seaweed Ulva collected along the Swedish west coast. Acidic extraction was benchmarked against hot water extraction with enzymatic purification and against commercial ulvan. Extracted ulvan contained 11-18 % g/g of ash, some protein (up to 1.3 % g N/g) but minimal colored impurities. The ulvans had high molecular weights (660,000-760,000 g/mol) and were composed of 77-79 % g/g carbohydrates, mainly rhamnose, xylose, glucose, glucuronic acid, and iduronic acid. The extraction protocol and the ulvan source strongly impact the molecular weight and the chemical composition. Acidic extraction caused almost complete desulfation of the isolated ulvan while the other method preserved a significant degree of SO3 substituents. Elemental analysis of ash remaining after thermal degradation showed presence of common mineral elements such as Na, Ca, Mg, Al, and K, but none of the heavy metals Pb, Hg, or As.