| 1. |
- Andersson, KM, et al.
(författare)
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GGTASE DEFICIENT MACROPHAGES ALTER INTEGRIN EXPRESSION ON LYMPHOCYTES AND FACILITATE DEVELOPMENT OF ARTHRITIS
- 2020
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Ingår i: ANNALS OF THE RHEUMATIC DISEASES. - : BMJ. - 0003-4967 .- 1468-2060. ; 79, s. 205-206
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Geranylgeranyltransferase type I (GGTaseI) is the enzyme responsible for the prenylation/ lipidation of the RhoA family proteins, which keeps them attached to the cell membrane. We reported that GGTaseI-deficient (GLC) mice develop a spontaneous and age-dependent arthritis, reproducing the pathology of RA1. Targeting GGTaseI activates RhoA proteins.Objectives:To study which of the activated Rho proteins is responsible for development of arthritis, we deleted individual RhoA, Rac1 or Cdc42 genes in GLC mice. We study consequences of GGTaseI deficiency for lymphocyte function.Methods:Double deficient mice that lack Rac1 (GLC Rac1fl/fl), RhoA (GLC RhoAfl/fl) and Cdc42 (GLC Cdc42fl/fl) were developed by Cre-technology using the LysM-promotor, and were on a mixed genetic background (129Ola/Hsd-C57BL/6)2. Joints of the hind paws were assessed for signs of arthritis histologically and by micro CT at age of 16 weeks. Phenotype of spleen CD4 and CD8 T cells was analysis by flow cytometry. Proliferation and cytokine production was assessed in spleen cultures by ELISA. Gene expression profile was analyzed by RT-PCR.Results:Deletion of Rho proteins had divergent effect on development of arthritis in GLC mice. We observed a reduction of the arthritis index in GLC Rac1fl/fl (n=19, p=0.027) and GLC RhoAfl/fl (n=4, p=0.007) mice compared to GLC (n=16), while GLC Cdc42fl/fl (n=4) had no change in arthritis development. GLC RhoAfl/fl mice increased the bone mass compared to GLC (p=0.029).Flow cytometry analysis showed that RA-prone GLC and GLC Cdc42fl/fl mice had lower number of CD4 cells in spleen. CD4 cells of RA-prone GLC and GLC Cdc42 mice had significantly higher subsets of the regulatory FoxP3+ and FOXp3+CD25+ cells (p=0.016-0.029 and p=0.016-0.029 respectively) compared to control and GLC RhoAfl/fl mice. Additionally, RA-prone mice had higher expression of receptors to extracellular matrix proteins collagen (α2β2) and fibronectin (α5β1) compared to control mice (p=0.016 and p=0.011 resp) and to RA-protected mice (GLC Rac1fl/fl and GLC RhoAfl/fl, p=0.0004 and p=0.011, resp). In total, both the number of FoxP3+ CD4 cells and the expression of α5β1 receptors on CD4 cells correlated strongly with the synovitis score (r=0.72, p=0.0017 and r=0.59, p=0.012, respectively).GGTaseI gene lays under the control of HOX proteins essential for cell homing. Importantly, HOX regulate the expression of integrins. Studying the expression of HoxA genes in spleen, we found that RA prone GLC and GLC Cdc42 mice tended to have lower expression of HoxA2 and higher expression of HoxA9 compared to RA-protected GLC Rac1 and GLC RhoA and to control mice. The Hoxa9/Hoxa2 ratio was significantly higher in RA prone mice compared to RA-protected mice (p=0.0085) and control mice (p=0.019). This ratio correlated with α5β1 receptors (r=0.55, p=0.0084), FOXP3+ CD4 cells (r=0.50, p=0.017), and the arthritis index (r=0.50, p=0.033).Conclusion:Taken together this study shows that Rho proteins play divergent role in development of arthritis. Activation of Rac1 and RhoA by GGTaseI deletion changes the pattern of HOXA proteins and increases expression of integrin receptors, which facilitates leukocyte influx in the paw joints. Deletion of Rac1 and RhoA has RA-protective effect in GLC mice.References:[1]Khan, O.M., et al.J Clin Invest121, 628 (2011).[2]Akula, M.K., et al.Nat Commun10, 3975 (2019).Disclosure of Interests:None declared
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| 2. |
- Andersson, KM, et al.
(författare)
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SURVIVIN INHIBITS TRANSCRIPTION OF PBX1 AND SUPPORTS THE EFFECTOR PHENOTYPE OF THE MEMORY CD4 T CELLS IN RHEUMATOID ARTHRITIS
- 2020
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Ingår i: ANNALS OF THE RHEUMATIC DISEASES. - : BMJ. - 0003-4967 .- 1468-2060. ; 79, s. 227-228
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The oncogenic protein survivin is a marker of severe rheumatoid arthritis (RA). High serum levels of Survivin predict progressive joint damage1and poor treatment response2.Objectives:To study the role of survivin in the transcriptional regulation of phenotype in CD4+T cells.Methods:CD4+T cells of RA female patients were isolated from the perpheral blood. Activated CD4+cells were treated with survivin inhibitor YM155. Transcriptional analysis was done by RNAseq (Illumina) and conventional qPCR. Chromatin of CD4 cells was immunoprecipitated using polyclonal antibodies to survivin and subjected to deep sequencing (survivin ChIPseq, Hiseq2000, Illumina) and aligned to GRCh38. Statistical analysis of differentially expressed genes (DEG) was done in R-studio using Benjamini-Hochberg adjustment for multiple testing (Bioconductor, DESeq2 package).Results:Survivin ChIPseq of the activated CD4+T cells was enriched with the genes engaged in regulatory transcription factor specific DNA binding (GO:0000987, adj p=0.0005) and RNA polymerase II regulatory transcription (GO:0000978, adj p = 0.0004). Among survivin targets were the genes of HOX-B cluster and TALE family proteins MEIS, PKNOX and PBX1 controlling early leukopoesis and T cell maturation. Inhibition of survivin in PBMC resulted in significant upregulation of PBX1 (p=0.023), MEIS3 (p=0.0036), similar tendency was observed for HOXB6 and HOXC4 genes. RNAseq analysis CD4 cells of RA patients with different transcription of PBX1, identified 1636 genes (adj p<10-5). BIRC5, coding for survivin, was 8.3 folds higher in CD4+cells with low PBX1 (p=0.0005). Among the core transcription factors of T helper cell differentiation, we identifed NF-kB1 and NF-kB2, TBX21, IRF4, IRF8 and STAT3, BATF and BATF3. This followed by significantly higher TNF, IFNg and IL17A and IL17F in PBX1lo CD4 T cells. The pathway enrichment analysis of DEG identified strong over-representation of cytokine-specific genes (GO:005125, GO:0005126, GO:0048018, GO:0030545, FDR q-values 10-12-10-9). The genes of IL4, IL5, IL13, IL9, IL3 and CSF2 located within the chromosome 5 were common for all GO-lists, and were higher in PBX1lo, but none of those genes was identified by survivin-ChIPseq or PBX1-ChIPseq. Analysis of ChIPseq data identified the genes of STAT3, IRF4, IRF8 and BATF as common targets for PBX1 and survivin.Conclusion:This genome-wide analysis indicates that survivin regulates transcription of the TALE family protein PBX1 in CD4+ T cells, which has essential effect for differentiation and phenotype of Th subsets. Low PBX1 in RA patients is associated with terminally differentiated effector CD4+ T cells.References:[1]Svensson, B.et.al.Smoking in combination with antibodies to cyclic citrullinated peptides is associated with persistently high levels of survivin in early rheumatoid arthritis: a prospective cohort study.Arthritis Res Ther16, R12 (2014).[2]Levitsky, A.et.al.Serum survivin predicts responses to treatment in active rheumatoid arthritis: a post hoc analysis from the SWEFOT trial.BMC Med13, 247 (2015).Disclosure of Interests:None declared
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| 12. |
- Bokarewa, MI, et al.
(författare)
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Reactivity against phospholipids during pregnancy
- 1998
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Ingår i: Human reproduction (Oxford, England). - : Oxford University Press (OUP). - 0268-1161 .- 1460-2350. ; 13:9, s. 2633-2635
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Tidskriftsartikel (refereegranskat)
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| 19. |
- Chandrasekaran, V, et al.
(författare)
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AGGREGATED SURVIVIN BINDING AROUND HISTONE H3 EPIGENETIC MODIFICATIONS IN RISK LOCI ASSOCIATED WITH RHEUMATOID ARTHRITIS
- 2021
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Ingår i: ANNALS OF THE RHEUMATIC DISEASES. - : BMJ. - 0003-4967 .- 1468-2060. ; 80, s. 428-428
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Survivin is an integral part of the Chromosomal Passenger Complex (CPC) which plays a vital role in mitosis. Experiments have demonstrated that survivin can physically bind to DNA. Crystallographic studies show that survivin binds to Threonine-3 of histone H3. In patients with autoimmune diseases, increased survivin expression contributes to an aggravated disease phenotype. Thus, functional, and mechanistic data point to a potential chromatin regulatory role for survivin, possibly in combination with the established gene regulatory function carried out by histone epigenetic modifications (EM).Objectives:The objective of the study was to analyse the co-localization of chromatin bound survivin with three histone H3 epigenetic modifications – acetylated lysine 27 (K27ac) and trimethylated lysine 4 (K4me3) and lysine-27 (K27me3). The second objective was to analyse if survivin-bound DNA sequences overlapped with sequences in the vicinity of 106 GWAS SNPs that are associated with a risk of developing rheumatoid arthritis (RA).Methods:Chromatin from CD4 T cells of 14 female subjects was immunoprecipitated with survivin antibodies and each of the histone H3 antibodies, and coupled with sequencing (ChIPseq, Hiseq2000, Illumina). After mapping the annotations of sequenced regions to the human reference genome hg38, enriched peaks were identified through Homer software. The identified survivin ChIP peaks were analysed for colocalization with peaks of the three histone H3 EMs and with RA risk loci, using the Bioconductor package ‘ChIPPeakAnno’ through RStudio.Results:Among the total of ~13,000 individual survivin ChIP-peaks, 33% colocalized with histone H3 EM peaks. The overlapping peaks show a linear increase in average peak size compared with the peaks showing no colocalization with any H3 EM peak. A maximum of 5.5-fold increase in average peak size was observed when survivin bound peaks overlap with peaks of all three H3 EMs. A major proportion (86%) of top RA risk SNPs was associated with either binding of survivin or H3 EMs. In this subset, 63% of RA risk SNPs were found within an area of 100 kilobases from survivin ChIP-peaks, with preferential enrichment of high-scoring peaks when survivin colocalizes with all 3 H3 EMs. Survivin was bound to risk SNPs annotated to, among others, the major immunological genes CD83, IRF4, CD28, ICOS and IL2RAConclusion:This study presents experimental evidence that survivin binding to DNA preferentially occurred in regions with high density of histone EMs. The increased aggregation of survivin around histone H3 EMs point to its potential regulatory function in gene transcription. Since regions around RA risk SNPs overlap with survivin peaks, survivin’s nuclear function could have immunologically important effects in mechanisms of autoimmune diseases.Disclosure of Interests:None declared
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| 20. |
- Chandrasekaran, V, et al.
(författare)
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FUNCTIONAL ROLE OF SURVIVIN IN ORGANIZATION OF BIVALENT CHROMATIN REGIONS AND CONSEQUENCE FOR ARTHRITIS-RELEVANT GENE EXPRESSION
- 2022
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Ingår i: ANNALS OF THE RHEUMATIC DISEASES. - : BMJ. - 0003-4967 .- 1468-2060. ; 81, s. 231-231
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Bivalent chromatin (BvCR) is characterized by the presence of simultaneous active and repressive modifications on histone H3 proteins. Influencing expression of the genes, BvCR determine cell fate and direct differentiation and lineage commitment in primary T cells and contribute to autoimmunity. Survivin is highly expressed during cell division and in effector Th1 cells contributing to aggravation of autoimmune inflammation. Survivin can physically bind to DNA, specifically to Threonine-3 of histone H3 (1). Thus, functional, and mechanistic data point to a potential chromatin regulatory role for survivin, potentially acting in combination with histone epigenetic modifications (EMs).ObjectivesThe goal of our study is to establish the colocalization of survivin with BvCRs and to deduce functional effects of this collaboration on chromatin organization and gene expression.MethodsChromatin from CD4+ T cells of 14 female subjects was immunoprecipitated with survivin antibodies and histone H3K27ac, H3K27me3, H3K4me3 antibodies, and coupled with DNA sequencing (ChIPseq, Hiseq2000, Illumina). BvCR were identified as exact overlaps of the three histone EM peaks and the overlapping regions were searched for co-localization with survivin using the ‘ChIPPeakAnno’ Bioconductor package. Tag counts K27me3>K27ac were defined as inactive/poised BvCR, while tag count K27me3<K27ac were identified asactive BvCR. Motif search was done through the MEME tool, and high/moderate complexity motifs with E-value >10e-5 were selected and scanned through the HOCOMOCO database to identify consensus transcription factor (TF) motifs. TFs co-localized with the BvCD were identified through ReMap database. To identify survivin sensitive genes, CD4+ T cells were treated with survivin inhibitor YM155 and a list of reproducible DEG (log2FC>[0.4], >1 experiment) was mapped and analysed for clustering with BvCR.ResultsCo-localization of survivin ChIP peaks with individual H3-peaks was significantly less frequent compared to overlap with all three (a3)-H3 BvCR (7.1 vs 29.8%, p=8.9e-13). Overlap of a3-H3 peaks not containing survivin was less frequent (34%) compared to those which contained survivin (66%). Notably, survivin peak size was 5.5-fold higher when colocalized with a3-H3 peaks, compared to no, or any single H3 (p<2.2e-16). In contrast, no size difference for any of the H3 EM peaks was found.Further analysis of two non-redundant groups of BvCR that contain (survivin-a3H3, n=4085), and not containing survivin (a3H3noSurv, n = 2131) demonstrated that survivin was mostly associated with inactive BvCR (OR1.29, p=6.6e-6), while no such specificity was found for BvCR with no survivin. Additionally, survivin containing BvCR contained abundant binding sites matching known consensus TF motifs. No sequence-specific motifs were identified in BvCR with no survivin. Comparison of results obtained through HOCOMOCO and ReMap databases resulted in a list of 68 unique TFs. Many of those are key regulators of adaptive immune responses, cellular metabolism, and pluripotency. Differentially expressed genes mapped to BvCR demonstrated enrichment for cellular hormone metabolic processes, regeneration and DNA biosynthesis.ConclusionThis study provides experimental evidence that survivin defines binding specificity in bivalent chromatin regions being associated with regulation of cellular metabolism and renewal of CD4+ T cells that are functionally important to resist autoimmunity.References[1]Kelly AE, Ghenoiu C, Xue JZ, Zierhut C, Kimura H, Funabiki H. Survivin reads phosphorylated histone H3 threonine 3 to activate the mitotic kinase Aurora B Science. 2010 Oct 8; 330(6001): 235–239.Disclosure of InterestsNone declared
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| 21. |
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| 22. |
- Erlandsson, M, et al.
(författare)
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TRANSCRIPTIONAL ACTIVITY OF SURVIVIN CONTRIBUTES TO MATURATION AND FUNCTION OF THE INTERFERON-GAMMA PRODUCING T CELLS IN RHEUMATOID ARTHRITIS
- 2020
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Ingår i: ANNALS OF THE RHEUMATIC DISEASES. - : BMJ. - 0003-4967 .- 1468-2060. ; 79, s. 83-84
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Interferon gamma (IFNg) signalling and downstream effects make important contribution in pathogenesis of rheumatoid arthritis (RA). Here, we propose a mechanism by which oncoprotein survivin participates in development of IFN-dependent repertoire of T cells in RA patients.Objectives:We study the role of survivin in the phenotype of CD4 T cells of RA patients.Methods:CD4 cells of RA patients and healthy controls were purified from blood, activated and subjected to RNAseq, ChIPseq with antibodies to survivin (BIRC5) was performed on CD4+ cells. Histone H3 ChIPseq was performed using antibodies to H3K27ac, H3K27me3 and H3K4me3. Statistical analysis was performed In R-studio using the Bioconductor package DESeq2, clustering using Spearman and Ward.D2.Results:Unsupervised clustering of CD4 samples by expression of 48 core Th cell markers identified subsets of CD28hiCD27hiIFNnegcentral memory cells (Tcm), CD28loCD27loIFNloeffector memory cells (Tem) and CD28nullCD27nullIFNhiterminal effector cells (Tte). Tte cells showed classical features of Th1 cells including high levels of TBX21, TNFa and IL32 and signs of activation in IFN signalling machinery. Interestingly, they combined the features of peripheral Tregs CD25hiFoxp3hiIKZF2negand IL10 producing cells together with type 1 regulatory cells, which rely on transcription factors BATF and IRF1 for the differentiation and produced high amounts of perforin and granzyme B. Importantly, Tte CD4 cells had also high transcription of BIRC5 (p=1.15e-18).To study if BIRC5 is a part of IFN signalling, CD4 cells were cultured with survivin inhibitor YM155 and activated with IFNg. RNAseq analysis revealed 2033 (FC<2.0, n=336) differentially expressed genes in the IFN stimulated cultures. Interestingly, a substantial number of these IFN-dependent genes was significantly reduced in the survivin-deficient cultures and included among others CD28, FoxP3, IKZF2, ICOS, BATF, PRDM1, CXCR3, IRF4 and IRF8. Analysis of the peak sequences identified enrichment for composite motifs for IRFs (ETS:IRF, p1.0e-124; bZIP:IRF, p=1.0e-640), indicating that survivin is important for IFNg signalling. Numerically, the peaks containing ETS:IRF motifs were most prevalent and identified in total within 49.7% of all survivin-ChIP peaks. Frequent was co-localisation of the IRF:bZIP and IRF:ETS motifs within the survivin peaks. Among the IRF motifs dominated those suitable for IRF1 (p=1,0e-127) and IRF8 (p=1,0e-84). However, the DNA binding motifs of these two are alike.Encouraged by the survivin ChIPseq results, we wanted to know its relation to histone marks. We observed that 50% of survivin peaks containing both IRF:bZIP and IRF:ETS motifs are co-localized with the H3K27ac marks. In total, 16 of 48 core Th cell markers used for patients clustering were identified by survivin ChIPseq, co-localized with IRF composite motifs and histone marks. They were also dependent of survivin for expression.Conclusion:his study showed that survivin binds to DNA and regulates the core gene expression contributing to maturation and function of the IFNg producing Th1 cells.References:-Disclosure of Interests:Malin Erlandsson: None declared, Karin ME Andersson: None declared, Nisha Nair: None declared, Anastasius Damdimopoulos: None declared, Sofia Töyrä Silfverswärd: None declared, Rille Pullerits: None declared, Anne Barton Consultant of: AbbVie, Maria I Bokarewa: None declared
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| 23. |
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| 24. |
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| 25. |
- Magnusson, Mattias, et al.
(författare)
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Epstein–Barr virus in bone marrow of rheumatoidarthritis patients predicts response to rituximabtreatment
- 2010
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Ingår i: British Journal of Rheumatology. - : Oxford University Press (OUP). - 0263-7103 .- 1460-2172. ; 49, s. 1911-1919
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Tidskriftsartikel (refereegranskat)abstract
- Objectives. Viruses may contribute to RA. This prompted us to monitor viral load and response to anti-CD20 therapy in RA patients.Methods. Blood and bone marrow from 35 RA patients were analysed for CMV, EBV, HSV-1, HSV-2, parvovirus B19 and polyomavirus using real-time PCR before and 3 months after rituximab (RTX) treatment and related to the levels of autoantibodies and B-cell depletion. Clinical response to RTX was defined as decrease in the 28-joint disease activity score (DAS-28) >1.3 at 6 months.Results. Before RTX treatment, EBV was identified in 15 out of 35 patients (EBV-positive group), of which 4 expressed parvovirus. Parvovirus was further detected in eight patients (parvo-positive group). Twelve patients were negative for the analysed viruses. Following RTX, EBV was cleared, whereas parvovirus was unaffected. Eighteen patients were responders, of which 12 were EBV positive. The decrease in the DAS-28 was significantly higher in EBV-positive group compared with parvo-positive group (P = 0.002) and virus-negative patients (P = 0.04). Most of EBV-negative patients that responded to RTX (75%) required retreatment within the following 11 months compared with only 8% of responding EBV-positive patients. A decrease of RF, Ig-producing cells and CD19+ B cells was observed following RTX but did not distinguish between viral infections. However, EBV-infected patients had significantly higher levels of Fas-expressing B cells at baseline as compared with EBV-negative groups.Conclusions. EBV and parvovirus genomes are frequently found in bone marrow of RA patients. The presence of EBV genome was associated with a better clinical response to RTX. Thus, presence of EBV genome may predict clinical response to RTX.
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| 26. |
- Malmhall-Bah, E, et al.
(författare)
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RHO EXPRESSION FACILITATES T CELL MIGRATION TO LYMPH NODES IN RESPONSE INFLAMMATION
- 2021
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Ingår i: ANNALS OF THE RHEUMATIC DISEASES. - : BMJ. - 0003-4967 .- 1468-2060. ; 80, s. 12-13
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Deficiency in geranylgeranyltransferase type I (GGTase-I) results in accumulation of active Rho family proteins RhoA, Rac1 and Cdc42, responsible for cell communication and migration. We reported that mice with GGTase-I deficient macrophages (GLC mice) develop a spontaneous and age-dependent arthritis, reproducing pathology of RA [1].Objectives:We study how GGTase-I deficiency in Mø changes T cell phenotype to facilitate their translocation to joints and the development of arthritis.Methods:GLC mice were developed on a mixed genetic background (129Ola/Hsd-C57BL/6) by Cre-technology using LysM-promotor to knockout the Pggt1b gene in Mø[2]. CD4+ cells were isolated from spleen and lymph node (LN) of 16 weeks-old mice (GLC n=7, wt n=5) expected to have high prevalence of arthritis. RNA was extracted to measure expression of the Rho proteins and signature genes to characterize differences in Th-subtypes and migration abilities of CD4+ cells between GLC and wt mice. Furthermore, Illumina RNAseq analyzed the transcriptome of LN CD4+ cells. In a separate experiment we treated GLC mice with CTLA4-FP (n=12) or PBS (n=11) for 20 weeks from the age of 5 weeks. Rationale was to disrupt Mø/T cell contact to prevent arthritis. To study Rho-protein dependent phenotype in human RA, we performed RNAseq of sorted CD4+ cells of RA patients.Results:RNAseq showed that CD4+ cells in LN of GLC mice had IFN-γ dependent cytotoxic profile and upregulated numerous pro-inflammatory genes including Eomes, Cxcr3, Tigit, Tnfsf10, Il-1rl1, Stat1, Jak3, Irf7, Irf5, Ptpn13. Furthermore, the over-represented genes often depended on the IRF family in their transcription.GLC mice overexpressed Cdc42 and Rac1 in spleen CD4+ compared to wt (p=0.005 and p=0.048 resp.). Spleen GLC CD4+ cells had higher levels of α5β1 and α2β2 integrins, strongly correlating to Cdc42 (r= 0.61 p=0.0027 and r=0.50, p=0.018) and arthritis (r=0.64, p=0.0015 and r=0.69, p=0.0004). Importantly, Cdc42, Rac1, and RhoA were higher expressed in LN CD4+ compared to spleen (p=0.016, p=0.031 and p=0.016). In addition, Itgb1 coding for β1 integrin, was upregulated in GLC CD4+ cells of both spleen and LN (p=0.003 p=0.03, resp.), suggesting Rho proteins are important for migration of CD4+ cells to the joint draining LN and for arthritis development. CD4+ cells that migrated to the LN had high proportion of Foxp3+ cells. This also correlated to the expression of Itgb1 (r=0.84, p=0.0012) presenting a plausible mechanism for increased influx of Tregs into joints. Several observations are in favor of this notion. First, GLC mice expressed more Foxp3 in LN compared to spleen CD4+ cells (p=0.016). Second, transcription of Foxp3 in LN CD4+ cells was higher in GLC mice compared to wt (p=0.015). Third, this high Foxp3 coexisted with low transcription of Lef1 (p=0.03), required for Treg immunosuppression. Last, Foxp3 correlated negatively to both Lef1 (r=-0.72, p=0.017), and its cofactor Tcf1 (r=-0.75, p=0.01).CTLA4-FP reduced inflammation in GLC mice evident as lower IFN-γ, IL-6 and TNF-α production (p=0.0002, p<0.0001 and p<0.0001 resp.) and the number of CD25+CD4+cells in spleen (p=0.027). In contrast, we observed increased IL-17A production (p=0.056). However, CTLA4-FP treatment did not affect migration of CD4+ cells enriched with Rho-protein into draining LN nor alleviate arthritis.Similar to the GLC mice, CD4+ cells of RA patients with high expression of RhoA, Rac1 and Cdc42 demonstrated enrichment for Th1 signature genes including IFNG, TBX21, Eomes, IL2RA, IL2RB, IL12RB2, TNF, IL18RAP (all, adj. p<0.05).Conclusion:This study shows that accumulation of Rho-proteins in CD4+ cells results in pro-inflammatory IFN-γ dependent phenotype in mice and human RA. Accumulation of RhoA, Rac1 and Cdc42 proteins trigger the migration of CD4+ cells into joint draining LN and facilitates arthritis. Inhibiting Mø/T cell contact in GLC mice did not suffice to prevent migration of Rho-protein expressing cells and arthritisReferences:[1]Khan, O.M., et al. J Clin Invest, 2011. 121(2): p. 628-39.[2]Akula, M.K., et al. Nat Commun, 2019. 10(1): p. 3975.Disclosure of Interests:None declared
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| 27. |
- Oparina, N, et al.
(författare)
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COMPLEX LANDSCAPE OF BIRC5/SURVIVIN GENOME BINDING IN HUMAN CD4+T CELLS
- 2021
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Ingår i: ANNALS OF THE RHEUMATIC DISEASES. - : BMJ. - 0003-4967 .- 1468-2060. ; 80, s. 410-410
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Survivin, coded by BIRC5 gene, is a multitasking protein essential for cell renewal and homeostasis. In autoimmune conditions as rheumatoid and psoriasis arthritis, survivin was associated with inflammation severity and joint damage. Importantly, inhibition of survivin alleviated experimental arthritis in mice. We have recently shown survivin to be essential for T cell differentiation and micro-RNA processing. The known anti-apoptotic and proliferation facilitating functions of survivin does not explain the nuclear localization of survivin in interphase.Objectives:We aimed to uncover nuclear functions of BIRC5/survivin in CD4 cell of RA patients and healthy.Methods:CD4 T cells were isolated from the peripheral blood using positive selection on magnetic beads (EasySep) and activated for 48h with ConA+LPS. Chromatin immunoprecipitation (ChIP) with polyclonal anti-survivin antibodies was done in four independent samples of healthy donors (n=5), healthy smokers (n=3), rheumatoid arthritis (n=3) and breast cancer (n=2). Pooled libraries were constructed for each group and ChIPseq was carried out (Illumina). For comparative RNAseq analysis, activated CD4 T cells were incubated with or without survivin inhibitor (YM155) for 24h. State-of-the-art bioinformatics pipelines were applied for NGS data and the survivin-binding peaks were used for comparison with genes, chromatin state annotation and functional gene- and regulatory regions-based functional analysis. Co-localization of peaks in the whole genome and in vicinity of the differentially expressed genes (DEG) was done using ReMap integrated ChIPseq datasets for all human cells and tissues.Results:We identified 13 thousands non-overlapping survivin ChIP-peaks (>3000 peaks were present in at least 3 samples). Survivin-bound regions were enriched near the genes and promoters (p=e-30 and p=e-8), which implied that survivin role in transcription could be mediated by known transcription factors. Thus, we analyzed survivin peaks vs binding regions of 1135 transcription regulators (TR) available in ReMap.Potential partner proteins of survivin were selected based on the enrichment of the overlapping peaks in the whole genome and in CD4-active regulatory areas. Both, strict overlaps and location within 10 and 100kb survivin peak vicinity were analyzed. This approach allowed us to select >150 TRs enriched in all tests. The enriched TRs were involved in immunity and RA-relevant pathways including cytokine response and production, JAK-STAT signaling, etc. Among the TRs co-localized with survivin were CHD8, MAX, EP300, BRD2, CTCF and RAD21, all responsible for chromatin architecture. Several TRs were massively enriched in the vicinity of DEGs after survivin depletion including MAX, AR, CTCF, MYC and IRF1. Search for TR binding motifs in survivin peaks supported over-representation of binding sites for IRFs (p=e-5) and several proteins of the bZIP-family (p=e-5).Conclusion:Analysis of the survivin bound DNA in CD4 cells demonstrated the nonrandom distribution with specific enrichment within the regulatory elements of the genes and co-localizeation with protein partners to regulate their transcription.Disclosure of Interests:None declared
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