Background Clinical evidence connects hyperinsulinemia with obesity, and development of type 2 diabetes (T2D). However, its role in autoimmune conditions was questioned. We investigated consequences of hyperinsulinemia for development of T2D and CD4 T cell function in rheumatoid arthritis (RA). Methods Incident T2D was prospectively studied in two independent RA cohorts and in gout patients matched to RA by age and gender, for 10 years. Effect of hyperinsulinemia and JAK-STAT signaling inhibition (JAKi) in CD4 T cells was studied by integrating transcriptional sequencing with direct effect of insulin, and JAKi on cell proliferation, DNA enrichment, and cytokine production. Results T2D was 3.2-2.5 times less prevalent in RA compared to gout, particularly in females. Hyperinsulinemia predicted the development of T2D, regardless of metabolic parameters and insulin resistance. Additionally, hyperinsulinemia correlated with the senescence-associated high serum levels of IL6, IL8, and VEGF. Hyperinsulinemia, along with ex-vivo exposure of CD4 cells to insulin, inhibited cell cycle progression and induced DNA enrichment through the suppression of the PI3K-Src kinases and cell cycle promoting genes. It also reduced IFNγ production. JAKi-treated CD4+ cells regained insulin sensitivity, which activated glucose metabolism and facilitated senescence. This insulin-dependent mechanism promoted the accumulation of naïve CD4 cells in JAKi-treated patients. Conclusions This study shows that insulin has important immunosuppressive ability controlling the adaptive immunity by suppressing IFNγ production and inducing senescence in the effector CD4 T cells. Inhibition of JAK-STAT signaling enhances insulin sensitivity and rejuvenates CD4 cell population in RA patients. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work has been funded by grants from the Swedish Research Council (MB, 2017-03025 and 2017-00359), the Swedish Association against Rheumatism (MB, R-566961, R-751351 and R-860371; MD, R-968867; RP, R-969562, R-862061), the King Gustaf V:s 80-year Foundation (MB, FAI-2018-0519, FAI-2020-0653, FAI-2022-0882), the Regional agreement on medical training and clinical research between the Western Gotaland county council and the University of Gothenburg (MB, ALFGBG-717681, ALFGBG-965623; RP, ALFGBG-965012, ALFGBG-926621; MD, ALFGBG-888321), the University of Gothenburg. The authors declare that the funding sources have no role in study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The included swedish RA study is approved by the Swedish Ethical Review Authority and was registered at the Clinical Trials.gov with ID [NCT03449589][1]. The data collection of gout patients and their comorbidities was approved by the Ethical Review Board of Gothenburg, Sweden. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes This study includes publically available transcriptional data which is deposited at the Gene Expression Omnibus at the National Centre for Biotechnology Information, accession numbers GSE201669 and GSE138747. Other data from the present study are available upon request to the authors [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT03449589&atom=%2Fmedrxiv%2Fearly%2F2024%2F03%2F10%2F2024.03.08.24303970.atom

Background: Clinical evidence connects hyperinsulinemia with obesity, poor lipid profile, and development of type 2 diabetes. However, its role in autoimmune conditions was questioned. Objectives: In this study, we investigated effect of hyperinsulinemia on CD4+ T cell function in rheumatoid arthritis (RA) and the ability of the Jak-STAT inhibiting treatment (JAKi) to counteract this effect. Methods: Plasma insulin levels were measured after an overnight lent in 56 female non-diabetic RA patients, by ELISA, and identified 12 patients with insulin levels above 157 pmol/L, indicating hyperinsulinemia (Table 1). Transcriptional sequencing profile CD4+ cells and cytokine (IFNg, IL10, TNF, IL8, IL6) protein profile of CD4+ cells of patients with hyperinsulinemia using JAKi or no-JAKi treatment were compared. To investigate a direct effect of insulin and JAKi, on CD4+ cells proliferation by Cell-TraceViolet dilution and cell cycle and DNA enrichment by 7AAD staining of CD4+ cells from 14 healthy donors was studied by flow cytometry. Gene transcription was analyzed by qPCR and cytokine production by ELISA. Results: CD4+ cells of patients with hyperinsulinemia had suppression of the PI3K-Src kinases and lower expression of Th subset specific transcription factors RORC, PRDM1 and STAT4, and cytokines IFNG, and IL10, suggesting a less aggressive CD4+ cell phenotype. Hyperinsulinemia patients had also lower serum levels of IL6 and lower erythrocyte sedimentation rate. Insulin exposure of CD4+ cells ex vivo decreased production of IFNγ, TNFα and IL6, along with decreased expression of IRS1 and PIK3CG, suggesting immunosuppressive abilities of insulin.CD4+ cells of JAKi treated patients were recognized by increased insulin signaling by upregulating IRS1, IRS2 and AKT1, and a high glycolytic index, which indicate higher insulin sensitivity and activated glucose metabolism, suggesting that CD4+ cells of JAKi-treated patients regained insulin sensitivity. CD4+ cells of JAKi-treated patients displayed a decreased expression of cyclin-dependent kinase (CDK)1 and increased transcription of CDK inhibitors CDKN1A/p21 and CDKN2A/p16. This pattern was replicated in CD4+ cells treated with JAKi ex vivo and resulted in G1/S phase transition arrest, characterized by accumulation of cells in G1 phase and retention of Cell-Trace Violet, measured by flow cytometry. This was consequently supported by an increased expression of CDKN1A and CDKN2A genes. Flow cytometry analysis showed that insulin slowed down cell cycle by prolonging G2 phase. JAKi acted more potently by enhancing cell cycle arrest in every step of cell phase transition. In patients with hyperinsulinemia, the insulin-induced effect on cell cycle was seen in upregulation of CDKN2D gene and suppression of genes in the chromosomal passenger complex needed to accomplish mitosis. Conclusion: This study shows that insulin has important immunosuppressive ability restricting activity of the effector Th1 cells. Hyperinsulinemia controls the adaptive immunity by suppressing IFNγ production and inducing senescence and in CD4+ T cells. Inhibition of JAK-STAT signaling enhances insulin sensitivity of CD4+ cells in RA patients. REFERENCES: NIL. Acknowledgements: NIL. Disclosure of Interests: None declared.Table 1Characteristics of RA patients.HyperinsulinemiaHyperinsulinemiaNormal insulinNormal insulinJAKi, n=7No-JAKi, n=5JAKi, n=17No-JAKi, n=27Age, yr52.0 ± 18.263.8 ± 9.052.0 ± 10.765.0 ± 7.6DD, yr10.6 ± 8.521.2 ± 13.413.9 ± 9.010.6 ± 8.5DAS282.01 ± 0.952.68 ± 1.142.47 ± 0.972.80 ± 1.10Insulin levels, pmol/L359 ± 192602 ± 65965 ± 3790 ± 36Treatment (n)MTX (6)bDMARD (1)MTX (3)bDMARD (2)MTX (6)cDMARD (4)bDMARD (2)MTX (15)bDMARD (9)no DMARD (7)Values are shown as mean ± SD

Background: Epigenetic processes promote development of pathogenic cell types in rheumatoid arthritis (RA), but precise molecular mechanisms behind these effects remain largely unexplored. Survivin has recently been identified as important regulator of histone deposition on chromatin preventing accumulation of the repressive H3K27me3 mark (1,2). Objectives: In this study, we investigate if survivin affects acetylation of histone H3K27 in CD4+ T cells, and how this is associated with effect of immunosuppressive RA treatment. Methods: Chromatin of CD4+ cells (n=12) treated with or without the survivin-inhibitor YM155 was immunoprecipitated with antibodies to histone H3K27ac and sequenced (ChIP-seq, Illumina). Peaks with change >30% in deposition of H3K27ac upon YM155-treatment were annotated to the genomic regulatory elements (RE) via GeneHancer database, which also gave information of genes connected to these RE. Transcriptomics of CD4+ cells isolated from RA patients treated with MTX (n=18), TNFi (n=10), JAKi (n=24) and having no DMARDs (n=7), by RNA sequencing, deposited GSE201669. The genes differentially expressed (DEG, nominal p<0.05) upon JAKi-treatment compared to other treatments were identified by DESeq2 (R-studio, Bioconductor). External transcriptome datasets of CD4+ cells isolated from patients before and after treatment with MTX (n = 28, GSE176440), abatacept (n = 14, GSE121827), and tocilizumab (n = 12, GSE113156). Treatment affected DEG were identified by DESeq2 (R-studio, Bioconductor). The enrichment analysis of DEG for biological processes within Gene ontology library were analyzed through String database. Over-representation analysis was done at http://cpdb.molgen.mpg.de. Results: Inhibiting survivin with YM155 in CD4+ cells induced a significant (>30%) change in 17% (1967 of 11529 peaks) of H3K27ac-peaks. These survivin-sensitive H3K27ac peaks were located within 339 cis-RE, connected to 1022 protein-coding genes expressed in CD4+ cells. Biological pathway analysis revealed that the top biological processes enriched among the these 1022 genes were mRNA metabolic process (GO:0016071 FDR = 0.0043), Cellular response to DNA damage stimulus (GO:0006974 FDR = 0.0029), DNA metabolic process (GO:0006259 FDR 0.033), RNA processing (GO:0006396 FDR 0.00058), RNA metabolic process (GO:0016070 FDR = 2.70e-05), and Cellular response to stress (GO:0033554 FDR = 0.00099). Together, these processes has a significant overlap and comprised 28% (286 of 1022) of the genes connected to the changed H3K27ac- genes. Immunosuppressive treatment had a significant imprint on H3K27ac connected genes, which were found over-represented among the DEG after treatment with MTX (p = 6e-49, OR = 1.77), abatacept (p = 1.6e-25, OR = 2.31), tocilizumab (p = 1.9e-7, OR = 1.51) and JAKi (p = 0, OR = 3.52) (all by Enrichr: Epigenomics roadmap). Furthermore, we found that JAKi and Mtx affected the same processes of transcription, RNA processing and translation; TGFb and SMAD signaling; NOTCH signaling; and DNA damage response as controlled by the genes connected to changed H3K27ac (JAKi p = 0.010 OR = ∞, Mtx p = 0.0001 OR = 3.2). In the top enriched GO:BP of the genes connected to changed H3K27ac, the DEG of the treatments were enriched to different extent (Table 1). In total, JAKi affected 219, abatacept 17, tocilizubam 17 and MTX 48 of the genes within the top GO:BP. Conclusion: Immunosuppressive treatment affects genes under the epigenetic control in transcription, RNA processing and DNA damage response exerted through the survivin-sensitive H3K27ac deposition. Molecular signature of CD4+T cells reflecting activation of these processes could assist individual treatment choice in RA patients. Table 1. REFERENCES: [1] Jensen, M.. et al. (2023) Survivin prevents the polycomb repressor complex 2 from methylating histone 3 lysine 27. iScience, 26, 106976. [2] Erlandsson, M.C.. et al. (2022) Survivin promotes a glycolytic switch in CD4(+) T cells by suppressing the transcription of PFKFB3 in rheumatoid arthritis. iScience, 25, 105526. Acknowledgements: NIL. Disclosure of Interests: None declared.

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.

Background: Rheumatoid arthritis today has no cure. Several clinical trials [1] have demonstrated ability of immunosuppressive drugs to prevent development of arthritis whereas others did not. Accurate selection of individuals at risk to develop arthritis from arthralgia is the key to increase efficacy and choice of prevention treatment. This enables an intensive search for molecular characteristics of processes preceding arthritis. Objectives: To identify inflammatory subtypes with highest and lowest probability of arthritis development. Methods: The study includes the cohort of 1012 patients assessed for joint pain at the Rheumatology Clinic during the period of two calendar years. The records of the 1st visit were carefully revised to exclude 303 patients with diagnosis of gout, polymyalgia rheumatica, spondylarthritis, primary Sjögren’s syndrome, SLE, trauma and paraneoplastic skeletal complaints. The remaining 709 patients with arthritis (ARTH) and clinically suspect arthralgia (ALG) were included in the 5-year prospective follow-up for new cases of arthritis. In addition to traditional inflammation parameters of CRP, ESR, hemoglobin, WBC, and platelet counts, blood samples of the 1st visit were investigated for autoantibodies ACPA, RF, cytokines IFNγ and IL8, growth factors Flt3-ligand, insulin, and oncoprotein survivin. These variables combined with gender and age, were put in the unsupervised learning algorithm to identify groups with similar profiles. Clinical characteristics of the formed clusters were analyzed to identify subtypes with high and low probability for arthritis development. Results: The unsupervised clustering resulted in 6 groups with diverse inflammatory profiles as summarized in Table 1. The largest group 5 had almost equal proportion of Arthritis and ALG diagnosis at the 1st visit, two of 6 groups were dominated by ARTH, and three of 6 groups were dominated by ALG. Individuals in the ALG groups 1,2,3 were significantly younger compared to the ARTH groups 4,6 (p<0.0001) and had higher frequency of females (p=0.0012). The ARTH groups had high IL8 and were remarkably different in autoantibody profile, and levels of IFNγ and insulin. The ALG groups were varying in WBC count, survivin and Flt3-ligand levels and, also, in presence of autoantibodies. In total, 83% of the new arthritis cases developed within 2 years and were most prevalent in the ARTH group 4, which dominated by autoantibody presence compared to all other groups (OR 4.25[2.33-7.69], p<0.0001) including the ALG group 2 with autoantibodies (OR 4.25[2.33-7.69], p=0.00027). The group with highest prevalence of new arthritis cases was also recognized by a combination of highest levels of IFNγ, IL8 and insulin compared to all other groups, while traditional inflammatory parameters CRP, ESR, platelet and WBC counts were not different from other groups. The highest WBC count coexisted with high survivin found in ALG group 1. The ALG group 3 had the longest time to new arthritis and was recognized by a combination of high Flt3-ligand and survivin. Conclusion: Here, we demonstrated that the presence of autoantibodies shared with high levels of Insulin, IFNγ and IL8 characterize Arthritis and identify the ALG patients at risk of progressing to arthritis. High levels of survivin and Flt3-ligand are valuable for long-term prognosis in arthritis development. REFERENCES: [1] Van der Helm-van Mil, A.H.M. Joint Bone Spine 2023 Jul;90(4):105543. doi: 10.1016/j.jbspin.2023.105543. Acknowledgements: NIL. Disclosure of Interests: None declared.Group 1Group 2Group 3Group 4Group 5Group 6Patients, n30999019927021Age, y47,2±16.847,8±13.942,7±16.154,7±15.450,1±14.757,4±16.8Gender, F90%81%78%70%70%71%Diagnosisat 1st visitALG, 93%Arth, 7%ALG, 100%Arth, 0ALG,83%Arth, 17%ALG, 35%Arth, 65%ALG, 59%Arth, 41%ALG, 33%Arth, 67%Presence of autoantibodies017%0100%00New ARTH14%11%12%34,8%10%14%Dominating variableWBCsurvivinaAB 17%Flt3-ligandsurvivinaAB 100%,InsulinIFNγ, IL8NoneESR+CRPPlateletsIL8

Hyperinsulinemia connects obesity, and a poor lipid profile, with type 2 diabetes (T2D). Here, we investigated consequences of insulin exposure for T cell function in the canonical autoimmunity of rheumatoid arthritis (RA). We observed that insulin levels correlated with the glycolytic index of CD4+ cells but suppressed transcription of insulin receptor substrates, which was inversely related to insulin sensitivity. This connection between insulin levels and the glycolytic index was not seen in CD4+ cells of healthy controls. Exposure of CD4+ cells to insulin induced a senescent state recognized by cell cycle arrest and DNA content enrichment measured by flow cytometry. It also resulted in accumulation of DNA damage marker γH2AX. Insulin suppressed IFNγ production and induced the senescence-associated secretome in CD4+ cell cultures and in patients with hyperinsulinemia. Inhibition of JAK-STAT signaling (JAKi) improved insulin signaling, which activated the glycolytic index and facilitated senescence in CD4+ cell cultures. Treatment with JAKi was associated with an abundance of naïve and recent thymic emigrant T cells in the circulation of RA patients. Thus, we concluded that insulin exerts immunosuppressive ability by inducing senescence and inhibiting IFNγ production in CD4+ cells. JAKi promotes insulin effects and supports elimination of the pathogenic CD4+ cell in RA patients.

This study explores a regulatory role of oncoprotein survivin on the bivalent regions of chromatin (BvCR) characterized by concomitant deposition of trimethylated lysine of histone H3 at position 4 (H3K4me3) and 27 (H3K27me3). Intersect between BvCR and chromatin sequences bound to survivin demonstrated their co-localization on cis -regulatory elements of genes which execute DNA damage control in primary human CD4+ cells. Survivin anchored BRG1-complex to BvCR to repress DNA damage repair genes in IFNγ-stimulated CD4+ cells. In contrast, survivin inhibition shifted the functional balance of BvCR in favor of H3K4me3, which activated DNA damage recognition and repair. Co-expression of BRG1, survivin and IFNγ in CD4+ cells of patients with rheumatoid arthritis identified arthritogenic BRG1hi cells abundant in autoimmune synovia. Immunomodulating drugs inhibited the subunits anchoring BRG1-complex to BvCR, which changed the arthritogenic profile. Together, this study demonstrates the function of BvCR in DNA damage control of CD4+ cells offering an epigenetic platform for survivin and BRG1-complex targeting interventions to combat autoimmunity. Summary This study shows that bivalent chromatin regions accommodate survivin which represses DNA repair enzymes in IFNγ-stimulated CD4+ T cells. Survivin anchors BAF/SWI complex to these regions and supports autoimmune profile of T cells, providing novel targets for therapeutic intervention. ### Competing Interest Statement G. Katona and M.I. Bokarewa submitted a patent application for the machine learning method described in the paper. The remaining authors have no competing interests.

Background: Immunomodulating treatment in rheumatoid arthritis (RA) prevent aberrant proliferation and activation of CD4+T cells by various mechanisms. Yet, drug choice remains empirical. In RA, unrepaired DNA damage promote the arthritogenic potential of effector T cells [1]. DNA damage response (DDR) combines cell cycle check points, DNA repair and DNA damage tolerance. It is controlled epigenetically by the Switch/Sucrose NonFermentable complex (SWI/SNF or BRG1) at chromatin marked by histone H3 tail modifications. DDR has a strong IFN adherence [2]. Objectives: This study investigates if the molecular signature of DNA damage is connected to high BRG1 expression in CD4+ cells of RA patients and how it is affected by immunomodulating drugs. Methods: Transcriptome of BRG1hi CD4+ cells of RA patients (n=24) were identified by RNAseq followed by DESeq2 analysis. RA treatment effect was identified in paired CD4+ T cells of patients pre- and post-treatment with abatacept (n=14, GSE121827, 24 weeks), tocilizumab (n=6, GSE113156, 24 weeks), and methotrexate (MTX, n=28, GSE176440, 12 weeks), by RNAseq. Treatment responsive differentially expressed genes (trDEG) were identified by DESeq2.DNA sequences binding H3K4me3, H3K27me3 and H3K27ac were obtained through immunoprecipitation and sequencing (ChIPseq). ChIPseq overlap indicating bivalent chromatin regions (BvCR) was identified by R package ChIPPeakAnno. Tag normalization enabled comparison between different ChIPseq to identify the dominant H3 modification. Genes connected to overlapped ChIPseq peaks (BvCR-dependent) within cis-regulatory elements were retrieved from GeneHancer database. Annotation to DDR pathway was done using Enrichr. Weighted gene correlation network analysis was performed using R package WGCNA. To identify IFN-sensitive genes, CD4+ cells sorted from blood (n=4) were cultured with IFNγ (50ng/mL) for 72h. Transcriptome was analysed by RNAseq followed by DESeq2. Results: Analysis of BRG1hiCD4+ cells revealed 8358 DEG compared to BRG1loCD4+ cells, which had accumulation of immune checkpoint proteins PD1, PD-L1 and CTLA4, and chemokine receptors CCR1, CCR5 and CD69, indicating a PD1+CD4+ cell phenotype promoting RA pathogenesis. The DDR pathway was significantly enriched in DEG of BRG1hiCD4+ cells (FDR=1.5e-23) and included the canonical SWI complex (ARID1A/B, SMARCA2), PBAF complex (PBRM1, ARID2) and the common subunits (BRG1, SMARCC1/C2, SMARCD1/D3). DNA damage marker γH2AX and classic DNA repair proteins FANCI, MSH2, MSH6, and MRE11 were significantly upregulated in BRG1hiCD4+ cells, while ATM was repressed.Immunomodulating treatment affected 40% of BRG1hi DEG. Among those, MTX reduced expression of PD1 (log2FC=-0.35, p=4.2e-4) and increased CD86 (log2FC=0.63, p=0.054), while abatacept and tocilizumab had no effect on PD1 expression.One-fourth of trDEG were BvCR-dependent and 40% (682 genes) were IFNγ−sensitive, with a majority regulated by H3K4me3-dominated BvCR. MTX affected 71% of these IFNγ-sensitive genes (484 genes) making a significant impact in epigenetic processes in RA CD4 cells. Among the DDR pathway trDEG were numerous SWI complex subunits, while BRG1 expression was unaffected. WGCNA of IFNγ-sensitive genes in BRG1hi cells (682 genes) showed downregulation of chromatin remodeling (GO:0006338, ARID1B, CHD7, SATB1) and upregulation of cell cycle phase transition (GO:0044772, CDKN1A, WEE1, MYC).In the trDEG under BvCR control in BRG1hi cells, H3K4me3-BvCR regulated 15 genes annotated to apoptotic DNA damage signaling (GO:0042771, DYRK2, PML). Genes controlled by H3K4me3-BvCR were mostly downregulated by immunomodulation, with MTX having the largest effect. Conclusion: BRG1hiCD4+ cells embody the activated DNA damage response and promote RA pathogenesis. MTX treatment affected the DNA damage response controlled by IFN signaling in CD4+T cells through the SWI/BRG1 complex. This makes a significant impact in epigenetic processes in RA CD4 cells and implies that high expression of BRG1 in RA recognize MTX-responsive RA patients. REFERENCES: [1] Li, Y. et al. Immunity 45, 903–916 (2016).[2] Begg, K. A. G., et al. DNA Repair 133, 103609 (2024). Acknowledgements: NIL. Disclosure of Interests: None declared.

Background: Neurological involvement in rheumatoid arthritis (RA) is understudied despite a high prevalence of depression, cognitive deficits, chronic pain, and fatigue reported in RA patients. Furthermore, there is increasing interest in the role of peripheral and brain-resident innate immune cells in aging and neurodegenerative disease. Systemic immune activation has been shown to activate microglia and affect brain region size in models of RA [1,2], but how chronic inflammation affects the aging human brain has not been previously studied. Objectives: We investigate regional brain volumes in RA patients compared to healthy age- and sex-matched controls and relate differences to neuropsychiatric symptoms and peripheral blood CD14+ monocyte phenotypes. Methods: We included 71 female patients (median age 64 years, range 23-76) with established RA (disease duration 10 years, range 0-45) and 268 healthy women (median age of 54 years (21-82)) who served as controls (C). T1-weighted cranial magnetic resonance images were used to measure 120 brain regions. For analysis of brain region differences in relation to age, RA patients and controls were binned into age intervals of 5 years: 0-45y (RA: n=7, C: n=98), 45-50y (RA: n=4, C: n=15), 50-55y (RA: n=11, C: n=26), 55-60y (RA: n=7, C: n=32), 60-65y (RA: n=13, C: n=41), 65-70y (RA: n=21, C: n=25), older than 70y (RA: n=8, C: n=31). Patient-reported neuropsychiatric symptoms were assessed with the Fibromyalgia Impact Questionnaire (FIQ). Peripheral blood CD14+ monocytes were isolated and analysed with RNA sequencing. Genes and pathways associated with lateral ventricle size was identified by comparing patients with lateral ventricle size smaller and larger than 30 cm3. Results: RA patients over 65 had significantly enlarged lateral ventricles of the brain compared to healthy controls of the same age range (Figure 1). While there were no differences in lateral ventricle size between RA patients and controls in participants under 65 years, the lateral ventricles were enlarged by 25% (p=0.020) in patients 65-70, and 42% (p=0.00020) in patients over 70. Next, we analysed regional brain volumes in the 71 RA patients compared to 71 age-matched controls. We found that 7 limbic and 5 cortical regions were significantly different in both the left and right hemisphere. Of these regions, the thalamus, the middle frontal gyrus and the superior frontal gyrus had strong inverse correlations to the lateral ventricle volume (thalamus: Spearman r=-0.46, p=0.0002; middle frontal gyrus: r=-0.56, p<0.0001; superior frontal gyrus: r=-0.36, p=0.0052). Interestingly, the reduced thalamus size was associated with depression reported in the FIQ questionnaire according to a linear model controlling for the patients age (Beta=-21, p=0.0080). Monocytes share characteristics with microglia and can migrate to the brain. We found that CD14+ monocytes from patients with larger lateral ventricles had several activated pathways related to neuroinflammation, including neuroinflammation signalling and multiple-sclerosis signalling. Upregulated genes included CYBB, MSR1, VEGFA, HLA-DRA, A2M and CD9, which implies neurodegenerative microglia phenotypes [3]. Conclusion: RA patients over the age of 65 had larger lateral cerebral ventricles, indicating brain atrophy possibly caused by accelerated aging or neurodegeneration. Enlarged ventricles were likely a consequence of shrinkage of the thalamus as well as frontal cortical regions and were associated with neuropsychiatric symptoms in RA patients. Monocytes of patients with enlarged lateral ventricles had a phenotype associated with neuroinflammation and disease-associated microglia. A limitation to our study is an insufficient sample size for rigorous statistical testing using a split-sample approach. We therefore present our generated hypotheses along with statistical test results as suggestions for future rigorous testing on independent data. REFERENCES: [1] Anderson & Wasén et al., PNAS 2019 [2] Süß et al., Cell Reports 2020 [3] Butovsky & Weiner, Nature Reviews Neuroscience 2018 Acknowledgements: NIL. Disclosure of Interests: None declared.

Background Rheumatoid arthritis (RA) is associated with aberrant epigenomic control which obstructs the proper function of CD4 T cells. Bivalent chromatin regions (BvCR), containing histone H3 tail trimethylation at lysine 4 (H3K4me3) and lysine 27 (H3K27me3), act as a major organizer of the 3D nuclear environment required for efficient T cell development. Survivin has gained importance in RA pathogenesis. Survivin is shown to interact with chromatin genome-wide and regulate gene transcription in the IFNg producing CD4 T cells [1]. Objectives This study investigates survivin cooperation with nucleosome in BvCR and functional effect of this cooperation on CD4 cell phenotype. Methods BvCR were identified in CD4 cells by annotation of H3K4me3, H3K27me3 and H3K27ac binding through chromatin immunoprecipitation and sequencing (ChIPseq). Protein binding was quantified through peak score. An overlap of the H3 peaks and Survivin ChIPseq (12 individual CD4 cultures) by at least one bp was identified through R package ChIPPeakAnno. Changes in H3 peaks in CD4 cells treated with a survivin inhibitor YM155 were assessed. Tag normalization was applied to enable comparison between different ChIPseq. Tag prevalence within the BvCR defined the dominant H3 group. Changeable BvCR were defined by alteration in the dominant-H3 group after YM155 treatment. TF ChIPseq colocalization analysis was performed through ReMap2022 database. To analyse effect of BvCR changeability on transcriptome, we performed RNAseq of YM155-treated CD4 cells (n=4). Overlap of BvCR with regulatory elements (RE) was done through GeneHancer database. TF enrichment analysis was performed through GSEA (Broad Institute). Results Integration of H3K4me3, H3K27me3 and H3K27ac ChIP-seq identified 6199 BvCR, 66% of those contained survivin peaks. Fluorescence imaging of effector T cells validated nuclear colocalization of survivin with histone proteins. Of those 6199 BvCR, 43.8% were dominated by H3K4me3, which had also the highest survivin peak scores. Complexes of histone modifiers COMPASS, responsible for H3K4 methylation, and SWI/SNF, responsible for counteracting PRC2-guided H3K27me3, colocalized with BvCR. YM155-treatment significantly increased deposition of both H3K4me3 and H3K27me3. Changeable BvCR frequently acquired a K4me3-dominant status after YM155 treatment. This indicates that survivin contributes to regulation of methylation of lysines in H3 tail within BvCR. TF enrichment analysis of genes connected to the BvCR revealed the role of histone demethylases KDM7A (FDR=1.03e-32), PHF2 (FDR=3.47e-19), KDM5D (FDR=1.51e-15) in their transcription. Strongest overall enrichment was identified in H3K4me3-dominant BvCR colocalized with survivin. Integration of BvCR with CD4 RNA-seq revealed 2469 protein-coding genes connected through RE to H3K4me3-dominant BvCR, which sustained transcriptional activity and 346 of those were differentially expressed (DEG, nom p<0.05) in YM155-treated cells. DEG transcriptionally dependent on the TF families of histone modifiers built a strong SWI/SNF-dependent interaction network acting in biological processes of immune system regulation (FDR=1.07e-6. FOXP1, KLF3, FYB1, BCL2), DNA repair (FDR=5.57e-6. MSH6, MGMT, MCM8, UPF1), and RNA polymerase transcription (FDR=1.22e-8. TCF3, ARID5A, RARA). CD4 cell transcriptomics of randomly selected RA patients demonstrated that a) DEG controlled by the H3K4me3-dominant BvCR were upregulated in survivin-high CD4 cells with proinflammatory Th1 phenotype b) DEG had altered expression in CD4 cells of patients treated with JAK-inhibitors. Conclusion This study demonstrates that survivin contributes to regulation of lysine methylation in H3 tail within BvCR, which coordinates activity of the SWI/SNF complex in chromatin remodelling and transcription. We demonstrate that survivin inhibition affects this coordinated activity, and thereby impinge on CD4 activation in RA. Reference [1]Erlandsson et al. iScience 2022;25:105526. Acknowledgements: NIL. Disclosure of Interests None Declared.

This study investigates the role of survivin in epigenetic control of gene transcription through interaction with the polycomb repressive complex 2 (PRC2). PRC2 is responsible for silencing gene expression by trimethylating lysine 27 on histone 3. We observed differential expression of PRC2 subunits in CD4+ T cells with varying levels of survivin expression, and ChIP-seq results indicated that survivin colocalizes with PRC2 along DNA. Inhibition of survivin resulted in a significant increase in H3K27 trimethylation, implying that survivin prevents PRC2 from functioning. Peptide microarray showed that survivin interacts with peptides from PRC2 subunits, and machine learning revealed that amino acid composition contains relevant information for predicting survivin interaction. NMR and BLI experiments supported the interaction of survivin with PRC2 subunit EZH2. Finally, protein-protein docking revealed that the survivin-EZH2 interaction interface overlaps with catalytic residues of EZH2, potentially inhibiting its H3K27 methylation activity. These findings suggest that survivin inhibits PRC2 function.

Background We have recently shown that Rho GTPase activation in macrophages results in arthritis development in mice. These macrophages show a tight interaction with T cells leading to suppression of cell homing and regulate migration of thymic T regulatory cells to periphery contributing arthritis development[1]. Objectives To translate findings in experimental arthritis to human RA, we explore the phenotype of CDC42hiCD14+ cells in blood and synovial tissue of RA patients and investigate how anti-rheumatic treatment intervene with the Rho GTPase-dependent molecular mechanisms of arthritis. Methods We used transcriptome (RNAseq) of CD14+ and CD4+ cells of 77 active RA patients naïve to TNF-inhibitors (TNFi) and of 59 inactive RA patients treated with methotrexate (MTX, n=18), TNFi (n=10) and JAK-inhibitors (JAKi, n=24), or having no DMARD treatment (n=7). In both cohorts, patients were stratified by mean expression of CDC42 in CD14+ cells to mimic activation of Rho GTPases. Differentially expressed genes (DEG) were identified by DESeq2 (nominal p<0.05). Transcriptomics of CD14+ and CD4+ cells were analyzed and translated into clinical correlates of inflammation and disease activity by DAS28. Additionally, we investigated a cluster of CDC42hi macrophages in synovial tissue (STM) using single cell RNAseq in 25 patients[2]. Results In two independent sets, CDC42hiCD14+ cells were characterized by enrichment of DEG operating in the oxidative phosphorylation, proteasome activity and RNA transcription. Many DEG were under transcriptional control of NFE2L1, known to supervise those processes. DEG with strong correlation to CDC42 defined the metabolic signature (MetSig) of CDC42hiCD14+ cells, which summarized the expression of ATP5BP, COX7A2, PSMB6, PSME3, GTF3C6, and GTF2E2. High MetSig recognized CD14+ cells enriched with MHC transcripts, components in peptide loading and expression of proteasome subunits. MetSighiCD14+ cells had high chemokine and cytokine production and migratory phenotype. Analyzing STM, we identified CDC42hiSTM with high MetSig expressing complete set of immunoproteasome genes, high levels of MHC receptors and peptide loading proteins. Additionally, this STM cluster was affected by IFN-γ. We experimentally confirmed that immunoproteasome expression in CD14+ cells was dependent on IFN-γ and not hypoxia, common at inflammation. Examining effect of antirheumatic treatment, we found CDC42hiCD14+ cells were infrequent (OR= 11.9, [3.4, 49.7], p<0.0001) and the MetSig reduced (p=0.0004) in patients treated with JAKi compared to other DMARDs. Concurrently, CD14+ cells of those patients had low production of TNF-α, IL-6, IL-1β, CXCL8 and IL-10 (all, p<0.05). mRNA of immunoproteasome subunits, peptide loading proteins and MHC-II (including HLA-DRB1) were downregulated. Analysis of CD4+ cells of JAKi treated patients showed upregulation of TCR components and high expression of Rho GTPases. This indicated a disruption of CD14+/CD4+ interaction in the immunological synapse. To investigate a direct effect of JAKi on the MetSig genes and immunoproteasome, we treated CD14+ cell cultures with tofacitinib. JAKi upregulated NFE2L1 and decreased expression of the immunoproteasome subunits, but expression of MetSig remained unchanged. Lastly, we investigated connection between the MetSig and RA disease activity (by DAS28) in a regression model. The model identified patients, which had a strong dependence between these parameters. At bottom of the model were patients with low DAS28 and MetSig, mostly treated with JAKi. Consequently, patients at the upper part may represent suitable candidates for JAKi-treatment. Conclusion This study demonstrates that CDC42-related metabolic signature identifies antigen-presenting CD14+ cells that migrate to joints to coordinate autoimmunity. JAKi suppress the antigen presenting capacity of CD14+ cells. The MetSig in CD14+ cells may identify candidate patients for JAKi treatment. References [1]Malmhäll-Bah, et al. J Autoimmun. 130, 102843 2022. [2]Alivernini, et al. Nat Med. 26(8), 1295-1306 2020. Acknowledgements We thank the research nurses Anneli Lund and Marie-Louise Andersson at Rheumatology Clinic, Sahlgrenska University Hospital, Gothenburg, for blood sampling. We thank all RA patients who participated. We appreciate support of Aridaman Pandit and Weiyang Tao at Center for Translational Immunology, University Medical Center Utrecht, NL, sharing data of the BiOCURA cohort. We are thankful to Prof. Marcela Pekna at Clinical Neuroscience, University of Gothenburg, assisting with hypoxia experiments. Disclosure of Interests None Declared.

Background Rheumatoid arthritis (RA) and diabetes mellitus (DM) have common genetic risk factors, while molecular mechanisms behind this commonality have not been studied. Objectives To study non-metabolic effects of insulin in CD4+ T cells of RA patients. Methods Incidental DM was studied in a consecutive cohort of 330 (256 female, 74 male) RA patients and 1011 (798 female, 213 male) gout patients matched to RA by age and gender (1 to 3) retrieved from the Health Care Register of Western Sweden during 10 years of follow-up. Insulin was measured at baseline and analyzed in relation to clinical and serological disease parameters. Transcriptomics by RNAseq was done on CD4+T cells of 57 RA patients. Differentially expressed genes (DEG, nominal p<0.05) in patients with hyperinsulinemia (n=11, median 344 pmol/L) were identified using DESEq2 (R studio, Bioconductor) and analyzed for enrichment of biological processes and transcription factor (TF) families using GSEA (Broad Institute). To study direct effect of insulin on CD4+T cell function, in vitro culture experiments were performed. Analysis of CD4+Tcell phenotype for senescence, exhaustion and apoptosis was done by flow cytometry, transcriptomics and cytokine measurement. Results In total, 102 new cases of DM were registered, significantly less often in RA patients compared to gout patients (p=3.2e-4, OR=0.34 [0.18-0.64]). The new DM were more prevalent in men (p=0.012) with no difference between RA and gout patients (9.8 vs 7.5%, OR=1.34 [0.46-3.52] p=0.55). In females, RA patients developed significantly fewer cases of DM than gout patients (2.1 vs 9.4%, OR=0.21 [0.07; 0.47], p=3.2e-5). In RA patients, the insulin levels correlated positively to inflammation measured by serum levels of IL6, VEGF and IL8 followed by IFNγ total, 102 new cases of DM were registered, significantly less often in RA patients compared to gout patients (p=3.2e-4, OR=0.34 [0.18-0.64]). The new DM were more prevalent in men (p=0.012) with no difference between RA and gout patients (9.ol/L). Transcription profile of CD4+T cells revealed 688 (131 upregulated, 557 downregulated) protein-coding DEG in patients with hyperinsulinemia. The down-regulated DEG showed enrichment for T cell activation (GO:0042110, FDR 2.6e-26) and inflammatory response (GO:0006954, FDR 2.9e-21). Analysis for TF families showed that the down-regulated DEG were controlled by TFs HMGB1, NFAT and NFκB (all FDR<e-6), which were significantly enriched for Th1 T cell markers including TBX21, EOMES, CXCR3 and IL2. The upregulated DEG were controlled by TFs MORC2, which regulates response to DNA damage, and ZBTB7B essential for differentiation and commitment of CD4+T cells. DEG included GART gene controlling the folate biosynthesis and the transcription regulating proteins MRPL3 and DAP3. Patients with hyperinsulinemia had significantly higher serum levels of IL6, IL8 and VEGF (all p<0.05), suggesting an inflammatory environment. However, they were not different in DAS28, CRP, WBC and platelet counts. Stimulation of CD4+T cell cultures with insulin (n=12) resulted in a significant suppression of IFNγ cell cultures with insulin (n=12) resulted in a significant suppression of IFNscription regulating proteins MRPL3 and DAP3. Patients with hyperinsulinemia had significantly higher serum levels of IL6, IL8 and VEGF (all p<0.05), suggesting an inflammatoryrichment for DNA (7AAD+ cells) in flow cytometry, which does not support induction of senescence by insulin in CD4+ cells. In contrast, CD4+ cells of hyperinsulinemic RA patients expressed lower levels of CCL chemokines, IL2, and TNF, which suggests that insulin induced exhaustion of CD4+T cells. Conclusion This study shows that hyperinsulinemia has non-metabolic functions under conditions of chronic inflammation. It could indicate residual RA activity. Insulin controls overactivation of adaptive immunity and causes exhaustion of pro-inflammatory Th1 cells in RA patients. REFERENCES: NIL. Acknowledgements: NIL. Disclosure of Interests None Declared.

Background Multiple genetic polymorphisms are associated with high risk for rheumatoid arthritis (RA) [1]. The importance of epigenetic processes has been postulated, but precise molecular mechanisms behind these associations remain largely unexplored. Survivin is an important player in RA, which has been recently shown essential for IFNg signaling in CD4 T lymphocytes acting in partnership with IRF1 [2]. Objectives We study how survivin-dependent epigenetic deposition of histone H3 tails in RA risk loci affects function of CD4+ T cells and response to JAK inhibition. Methods Chromatin of CD4+ cells (n=12) was immunoprecipitated with antibodies to histone H3K27ac, H3K4me3, and survivin, and sequenced (ChIP-seq, Illumina). Parallel ChIP-seq and RNA-seq was done in CD4+ cells treated with survivin inhibitor YM155. Peaks with change >30% in deposition of H3K27ac and/or H3K4me3 upon YM155-treatment were annotated to the genomic regulatory elements (RE) via GeneHancer database. Single nucleotide polymorphism (SNP) related to RA risk were identified within those RE. CD4+ T cell transcriptomics by RNA-seq was done in 24 random RA patients and in 59 RA patients treated with MTX (n=18), TNFi (n=10), JAKi (n=24) and having no DMARDs (n=7). The genes differentially expressed (DEG, nominal p<0.05) in BIRC5hi and JAKi-treated CD4+ cells were identified by DESeq2 (R-studio, Bioconductor). Results Deposition of 15% (1705/11152) of H3K4me3 and 17% (1943/11530) of H3K27ac peaks changed >30% upon survivin-inhibition. Approximately 50% of these peaks overlapped with survivin peaks. The change in H3K4me3 peaks was significantly larger if the peak colocalized with survivin peak. These peaks were located within 228 RE, 28 of these RE contained 52 RA risk SNPs. Among others, changeable peaks were accumulated within ICOS/CTLA4/CD28 locus and contained 7 SNPs. Majority of genes connected to the SNP-containing RE (110/153 genes) were transcriptionally different in BIRC5hiCD4+T cells and participated in the regulation of immune system (GO:0002682, FDR=4.3e-4), regulation of IL2 production (GO:0032663, FDR=0.019), TNF mediated signaling (GO:0033209, FDR=5.4e-3). These SNP-RE connected genes formed three independent clusters 1) TCR receptor co-stimulators (CD40, ICOS, CTLA4, CD28, CD244, TRAF6 and TBX21, SLAMF1, TNFRSF9), 2) connected to the LCK interacting transmembrane adaptor 1 (LIME1, SLC2A4RG, ZGPAT, GMEB2, TNFRSF6B, RTEL1) and 3) heat-shock proteins (HSPD1, HSP90AB1, HDAC7, KPNB1). The clusters were functionally connected to survivin partners IRF1 and SMAD3. Notably, transcription of CD28, ICOS, and SLAMF1 functionally connected to IRF1, and CHERP, HDAC7, PRPF6 and SRSF10 functionally connected to SMAD3 were significantly changed (all, nominal p<0.05) in CD4+ cells upon survivin inhibition by YM155. Since survivin mediates IFNg-dependent processes [2], we analyzed transcription of 110 SNP-RE connected DEG in CD4+ cells of JAKi-treated RA patients. We found that DEG in the IRF1 and SMAD3 clusters were inversely affected by JAKi, e.g., IRF1-related CD28, ICOS, and SLAMF1 upregulated after survivin inhibition were suppressed by JAKi, while SMAD3 related CHERP, PRPF6 down-regulated by survivin inhibition were upregulated in CD4+ cells of JAKi-treated patients. Effect of JAKi in the IRF1-related genes was executed only in BIRC5loCD4+ cells and remained unchanged in BIRC5hiCD4+ cells. Conclusion This study shows that survivin contributes to deposition of histone H3K27ac and H3K4me4 marks in proximity of RA-risk genes and control immune system regulators in CD4 T cells. JAK-inhibitors operate downstream of survivin and high levels of survivin may impede JAKi-treatment. References [1]Okada Y et al. Ann Rheum Dis 2019, 78(4):446-453 [2]Erlandsson MC et al. iScience 2022, 25(12):105526 Acknowledgements We thank the research nurses Anneli Lund and Marie-Louise Andersson at Rheumatology Clinic, Sahlgrenska University Hospital, Gothenburg, for blood sampling. We thank all RA patients who participated. Disclosure of Interests None Declared.

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.

OBJECTIVES:MicroRNAs (miRs) are non-translated RNA sequences that elicit negative control over protein expression. The adipose tissue (AT) is considered the major producer of miRs and inflammatory interleukin 6 (IL-6). This study aims to investigate the relationship between production of IL-6 and miRs in AT. METHODS:IL-6 gene expression was analysed in RNA extracts from subcutaneous AT of 75 patients with rheumatoid arthritis (RA), with qPCR. Genome-wide profile of human miRs (2565 miRs, 96.6%) was analysed in 35 AT samples on 3D microarray. The miR-processing proteins Dicer, Drosha and DGCR8 were analysed with qPCR. In silico prediction of protein targets for the differentially expressed (DE) miRs (p<0.05; log2FC >±0.5) was conducted by DIANA software. Seven AT samples were stimulated in vitro with IL-6 or IL-6+IL-6R antibody tocilizumab and analysed for the miR processing proteins. RESULTS:We identified 30 DE miRs between AT with high and low IL-6 mRNA, of which 26 miRs were inversely related with IL-6 levels. DE miRs were predicted to interfere in oestrogen (p=0.001), FoxO (p=0.006) and insulin (p=0.03) signalling pathways. High expression of IL-6 in AT was associated with significantly higher expression of Dicer (p=0.04) and Drosha (p=0.04), while inhibition of IL-6 signalling with tocilizumab decreased the levels of total miRs processing enzymes (p=0.003). CONCLUSIONS:IL-6 mRNA production in AT has a negative effect on the miRs expression profile and it increases miR-production capacity.