Insulin signaling regulates cellular metabolism in an epigenetic manner, but its role in the immune cell homeostasis remains unknown. High plasma insulin obstructs efficient insulin signaling and rewires metabolic activity in autoimmunity. In this study, we explored the functional consequences of insulin signaling for the metabolism and phenotype of effector CD4+ T cells in blood and synovial tissue of patients with rheumatoid arthritis (RA). Transcriptome profiling of CD4+ cells in RA blood and synovia revealed high metabolic activity and effector function of the survivin/BIRC5hiPD1hi T peripheral helper cell population. Low insulin signaling and deficient histone acetylation in RA T cells amplified proinflammatory IFNγ and TNF expression. Co-deposition of survivin with acetylated histone H3K27 on regulatory chromatin controlled the transcription of histone acetylation complex subunits and insulin-dependent genes. Insulin stimulation and histone deacetylase inhibition induced an increase in histone acetylation. In CD4+ cell cultures and in aggressive PD1hiTph cells in RA synovial tissue, exposure to insulin synergized with inhibition of histone deacetylation to upregulate IL7 production suppressing IFNγ and PD1. This activated IL7R-signaling mediators STAT5A/B, BCL2, and promoted acquisition of CD27+CD45RO+ central memory phenotype in the PD1hiTph cells. Likewise, the CD4+ cells in hyperinsulinemic T2D patients showed enrichment of IL7R+T cell cluster. In RA patients, antagonizing folate transport and JAK/STAT signaling activated insulin signaling and histone acetylation-dependent metabolism of CD4+ cells. Concomitant with CTLA4-dependent signaling, this enabled the adoption of an incipient IL7R+ T cell phenotype. This study demonstrates that insulin binds together metabolic activity and histone acetylation in CD4+ cells. Sufficient insulin signaling promotes IL7R+ memory phenotype accrual in aggressive PD1hiTph cells. Hence, achieving insulin sensitivity via histone acetylation disarms effector CD4+ T cell function and presents an attractive interventional goal to restore immune cell homeostasis in RA.

Abstract Background Handgrip strength (HGS) in rheumatoid arthritis (RA) is commonly attributed to joint pathology, but may also reflect extra-articular manifestations, including atrophy of motor-related brain regions. We investigated HGS as a marker of peripheral joint status, systemic immune regulation, and central motor integrity. Methods Maximal HGS was assessed using a dynamometer. Joint pathology was evaluated using radiographic and clinical measures, and upper limb disability using questionnaire. Brain volumes were quantified using MRI and MAPER software. Transcriptome sequencing was performed on circulating CD4⁺ and CD14⁺ cells. In a six-month, single-arm pilot trial, a subgroup of patients performed daily hand exercises. Associations and longitudinal changes were analysed using linear mixed-effects models accounting for repeated measurements across hands and timepoints, with variable selection performed using LASSO regression. Results A total of 59 women with established RA were included in the cross-sectional analysis (median age 64 years [range 23–76], DAS28 2.46 [1.1–5.8], disease duration 11 years [0–45]). Lower HGS was associated with greater disability. HGS was also independently associated with premotor and supplementary motor cortex (PMA/SMA) volume after adjustment for age, hand dominance, and joint pathology. Among joint pathology measures, tender joint count showed a significant negative association with HGS. Transcriptome analyses of CD4⁺ and CD14⁺ cells indicated that lower HGS was associated with reduced immune responsiveness and altered cytokine signalling pathways. In a six-month pilot hand exercise trial (n = 12; median age 54 years [28–68], DAS28 2.87 [1.2–3.6], disease duration 14 years [1–40]), HGS increased at 3 months, with a non-significant trend at 6 months. Baseline PMA/SMA volume showed a non-significant trend towards predicting HGS improvement. Longitudinal analyses revealed region-specific brain changes, with a decrease in PMA/SMA volume and an increase in insular volume over time. Conclusions Handgrip weakness in RA may reflect both joint pathology and motor cortex atrophy in the PMA/SMA. Hand exercise improved HGS and induced certain structural changes in the brain, though effects on motor regions remain uncertain and warrant further study. Trial registration Clinical trial registration: ClinicalTrials.gov, NCT04378621. Registration date: May 5, 2020.

CD5L is an immunoregulatory protein induced during inflammation and makes a life-saving contribution in infection and sepsis. Here, we explored the overall impact of CD5L in rheumatoid arthritis (RA). Using experimental RA models, we demonstrated an early surge of CD5L expression in wild-type mice, as well as a higher incidence and increased severity of arthritis in CD5L-deficient mice. In the blood, CD5L-deficient mice exhibited enhanced inflammation, a higher proportion of CD11b⁺ mononuclear cells, and elevated levels of IL-1β, IL-17, and IFN-ψ, particularly at the pre-clinical stage of arthritis. In human RA, mononuclear cells conditioned with CD5L, as well as endogenous CD5L production, were associated with reduced CD11b expression, increased IL-10 and PD-L1 production, and an enrichment of non-classical CD16 + monocytes. Analysis of the CD5L-dependent transcriptome in CD14⁺ cells from RA patients revealed the acquisition of an efferocytosis profile, with upregulation of C1Q subunits, GAS6, AXL, and ALOX15B. It also showed signs of insufficient cargo processing, which dampens the resolution of inflammation, permits the expansion of IFN-primed non-classical monocytes, and correlates strongly with joint damage accrual. Despite markedly low CD5L levels in the synovium, RA synovial tissue was enriched with non-classical monocytes carrying the GAS6-AXL signature, which fosters osteoclast progenitors. In conclusion, this study demonstrates that the anti-inflammatory and inflammation-resolving properties of CD5L in experimental and human RA are mediated through the induction of efferocytosis-related and IFN-primed monocyte programs. This harbors a potential risk of uncontrolled invasion of non-classical monocytes into the synovial tissue, leading to joint structural damage. The complex role of CD5L in inflammation and disease outcomes requires careful consideration of the disease phase and experimental context.

Abstract Chronic systemic inflammation has been implicated in age-related neurodegeneration, but whether rheumatoid arthritis (RA) is associated with accelerated brain aging remains unclear. We combined structural magnetic resonance imaging (MRI), circulating neurodegeneration biomarkers, and peripheral monocyte transcriptomics to investigate brain aging in RA across two independent cohorts. A brain-age prediction model trained in healthy controls from the IXI imaging dataset was applied to RA patient cohorts from Gothenburg (n = 71) and Glasgow (n = 50). RA was associated with significantly elevated corrected brain-age gap relative to healthy controls (+6.5 years, 95% CI 4.2–8.8 years, p = 1.2 × 10 −7 ), with substantially stronger effects in patients ≥60 years. Older RA patients demonstrated a significant ventricular enlargement together with reduced frontal and parietal lobe volumes. Serum brain-derived tau and glial fibrillary acidic protein levels were elevated in RA. The increased brain-age gap was associated with altered myeloid transcriptional signatures. These findings demonstrate that RA is associated with age-related neurostructural alterations consistent with accelerated brain aging.

Radiographic axial spondyloarthritis (r-axSpA) is a chronic inflammatory disease that primarily affects the axial skeleton and entheses, leading to pathological spinal bone formation and systemic bone loss. Treatments with tumor necrosis factor inhibitors (TNFi) and interleukin-17 inhibitors (IL-17i) have shown efficacy in reducing inflammation and potentially impacting bone remodeling in r-axSpA. Osteoclasts, crucial for bone resorption, are derived from the monocytic cell lineage and regulated by proinflammatory cytokines. This study aimed to evaluate the osteoclast development capacity from peripheral blood monocytes in patients with r-axSpA with different treatment strategies and compare it to controls. This study included 28 patients with long-standing r-axSpA receiving various treatments, including disease-modifying anti-rheumatic drugs (DMARDs) and NSAIDs, as well as 16 blood-donor controls. Disease activity was assessed using the Ankylosing Spondylitis Disease Activity Score (ASDAS). CD14 + monocytes were isolated from blood samples and differentiated into osteoclasts in vitro by stimulation with three different conditions: (I) macrophage colony-stimulating factor (M-CSF), (II) M-CSF and receptor activator of nuclear factor-κβ (RANKL), and (III) M-CSF, RANKL, and tumor necrosis factor-alpha (TNF). Osteoclast and osteoclast precursor formation were assessed using tartrate-resistant acid phosphatase (TRAP) staining, and TRAP5b concentration in supernatants was measured by ELISA. The frequency of CD14 + monocytes was similar in patients with r-axSpA and controls, but the capacity to develop osteoclasts and osteoclast precursors was significantly decreased in the r-axSpA patients. Stratification of the patients based on treatment with or without biological DMARDs (bDMARDs) revealed no significant differences in ASDAS or frequency of CD14 + monocytes. Notably, only r-axSpA patients receiving bDMARDs exhibited a reduced ability to develop osteoclasts and osteoclast precursors compared to those not on bDMARDs and controls. Lower Trap5b concentrations in supernatants corroborated these findings. Our study demonstrates that patients with r-axSpA exhibit a reduced capacity for osteoclast formation from CD14 + monocytes isolated from peripheral blood. The process was modulated by treatment with bDMARDs, which might explain the previously shown sparing effect of bDMARDs on bone mineral density in r-axSpA.

Methotrexate (MTX), the most common first-line treatment in rheumatoid arthritis, is often insufficient, with no model capable of predicting response. The RA classification criteria, including autoantibodies and inflammation, were applied to 257 patients with newly diagnosed inflammatory arthritis in the cohort study, estimating MTX response. A total of 172 patients received MTX as the first anti-rheumatic drug and response was recorded at 1 year follow-up. A multivariable logistic regression used variables distinct between MTX-responders and non-responders to build the predictive model of response. Overall, 53.5% of MTX treated patients responded. Non-responders were frequently autoantibody positive, and responders were older, had lower RA classification scores, frequent corticosteroid use, and high insulin levels at baseline. Inflammation parameters were comparable between the groups. In the multiple regression analysis, the RA classification score and age at the first visit were strong predictors of MTX response (AUC 0.697, p < 0.0001). Including blood levels of insulin and IFNg improved AUC to 0.782 (p < 0.0001), offering early discrimination between responders and non-responders with high accuracy. Cellular experiments showed that insulin could be used to estimate MTX response by demonstrating that insulin changed the transcription of MTX target genes in the folate metabolism after exposing CD4+ cells ex vivo, which could facilitate MTX response in immune cells.

Background Insulin has epigenetic effect influencing gene expression. High peripheral insulin concentrations promote insulin resistance in autoimmunity. Oncoprotein survivin/ BIRC5 modulates glucose metabolism through chromatin binding and propagates IFNg effects in CD4+ cells. In this study, we explored how insulin influences chromatin binding and metabolic activity in autoimmune CD4+ cells of patients with rheumatoid arthritis (RA). Methods We profiled the metabolic activity of CD4+ cell clusters using single-cell transcriptome analysis in blood, synovial fluid and synovial tissue of RA patients. Through chromatin immunoprecipitation and sequencing, we identified the genes controlled by deposition of survivin and acetylated lysine 27 on histone H3 (H3K27ac) in CD4+ cells. Treating CD4+ cells with insulin and histone deacetylase inhibitors (HDACi), we identified changes in H3K27ac, linked those to transcription of the H3K27-survivin-controlled genes and the pathogenic phenotype of CD4+ cells using flow cytometry. Finally, we explored if anti-diabetic and anti-rheumatic drugs affect the metabolic profile and memory phenotype of the metabolic active CD4+ cells. Results Transcription of survivin/ BIRC5 and histone acetylation enzymes strongly correlate with active metabolism in blood CD4+ cells of RA patients. In RA synovial tissue, these BIRC5hi active T cell clusters are inflammatory, exhausted, and memory-like. Genome co-deposition of H3K27ac-survivin pinpointed the insulin-dependent genes in metabolic active CD4+ cells. These genes favored histone acetylation by suppressing methylating enzymes EZH2 and KMT2A , and T cell development by activating CD27, CD3G, and SCIMP . Inhibition of histone deacetylation reverted these transcriptional effects and supported cellular sensitivity to insulin. Insulin stimulation increased H3K27ac and together with HDACi, suppressed PDCD1 and IFNg transcription and production in CD4+CD27+CD45RO+ memory T cells. Immune modulation impacted metabolic activity and synergized with the effect of histone acetylation on insulin responsiveness in RA patients. Conclusions RA synovia is enriched with the metabolic active BIRC5 hiCD4+ T cell clusters. The metabolic activity of these cells is histone acetylation-dependent and mediates insulin effects through the H3K27ac-survivin epigenetic mechanism. Increasing plasma insulin levels when combined with insulin sensitivity, can be protective in RA dearmoring effector T cell function. Hence, increasing the insulin sensitivity by enabling histone acetylation presents a reasonable interventional goal to restore immune cell homeostasis in RA. ### Competing Interest Statement The authors have declared no competing interest.

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

This work describes a comprehensive study of the vascular tree and perfusion characteristics of normal kidney and renal cell carcinoma. Methods: Nephrectomy specimens were perfused ex-vivo, and the regional blood flow was determined by infusion of radioactive microspheres. The vascular architecture was characterized by micronized barium sulphate infusion. Kidneys were subsequently sagitally sectioned, and autoradiograms were obtained to show the perfusate flow in relation to adjacent contact X-ray angiograms. Vascular resistance in defined tissue compartments was quantified, and finally, the tumor vasculature was 3D reconstructed via the micro-CT technique. Results show that the vascular tree of the kidney could be distinctly defined, and autoradiograms disclosed a high cortical flow. The peripheral resistance unit of the whole perfused specimen was 0.78 ± 0.40 (n = 26), while that of the renal cortex was 0.17 ± 0.07 (n = 15 with 114 samples). Micro-CT images from both cortex and medulla defined the vascular architecture. Angiograms from the renal tumors demonstrated a significant vascular heterogeneity within and between different tumors. A dense and irregular capillary network characterized peripheral tumor areas, whereas central parts of the tumors were less vascularized. Despite the dense capillarity, low perfusion through vessels with a diameter below 15 µm was seen on the autoradiograms. We conclude that micronized barium sulphate infusion may be used to demonstrate the vascular architecture in a complex organ. The vascular resistance was low, with little variation in the cortex of the normal kidney. Tumor tissue showed a considerable vascular structural heterogeneity with low perfusion through the peripheral nutritive capillaries and very poor perfusion of the central tumor, indicating intratumoral pressure exceeding the perfusion pressure. The merits and shortcomings of the various techniques used are discussed.

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 an inflammatory joint disease that leads to significant impairment in hand function. Chronic systemic inflammation in RA has been shown to change the activity of specific brain structures [1]. The relationship between brain structure and hand dysfunction in RA is underexplored. Objectives: We investigated structural changes in the brain of RA patients in relation to grip strength (GS), aiming to identify peripheral and brain immune responses associated with these changes. Methods: Based on GS measurement using a dynamometer, we analysed 60 RA patients with an mean age 60 years (range 23-73), disease duration 14 years (range 0-45), and mean GS 197 N (range 29-400). Patients were dichotomized by the median GS to compare the groups with weak GS versus strong GS. A subset of 12 patients underwent hand training consisting of 5 simple exercises performed separately for each hand for 10 minutes daily during 6 months. Instructions were given on an individual basis. Each patient wrote a training diary. Patients reported their functional disability using the Disabilities of the Arm, Shoulder, and Hand (DASH) questionnaire, the Health Assessment Questionnaire (HAQ) and the Fibromyalgia Impact Questionnaire (FIQ). Brain tissue was assessed by T1-weighted magnetic resonance imaging (MRI) at the baseline and after 6 months of hand training for the subset of 12 patients. Volumes of 121 brain regions were analysed using MAPER software with respect to grey matter (GM), white matter (WM), and cerebrospinal fluid (CSF) compartments. Levels of IFNγ in serum and supernatants of CD4+ T cells were determined by ELISA. RNA sequencing was used to analyse the transcriptional profile of peripheral blood CD4+ T cells. Results: RA patients with weak GS had significantly higher functional disability according to DASH and HAQ, and experienced more pain according to FIQ scores (all, p<0.001). There was no significant difference in age, disease duration or disease activity by DAS28 between the weak GS and strong GS patients. We found that the weak-GS patients had significantly increased WM volume in the putamen (L, p=0.002; R, p=0.0046) and caudate nucleus (L, p=0.0082; R, p=0.067) and decreased GM volume in the thalamus (L, p=0.038; R, p=0.028), regions which play a role in planning the execution of movement. Weak-GS patients also had significantly smaller WM volumes of the sensorimotor network, including the precentral gyrus (PG) (L, p=0.025; R, p=0.083), middle frontal gyrus (MFG) (L, p=0.0023; R, p=0.0099) and superior temporal gyrus anterior part (STGAP) (R, p=0.0051). Interestingly, after 6 months of hand training, some of the sensorimotor regions were increased in 12 patients who completed the training (MFG: 0.75% increase, p=0.0024; STGAP: 2.7% increase, p=0.033). Furthermore, weak-GS patients had lower levels of IFNγ in both serum (p=0.0006) and CD4+ T cell supernatants (p=0.0018). In the CD4+ T cells of the weak-GS patients, the downregulated genes were involved in T-cell activation and migration, and IFNγ production and response, while the upregulated genes were related to ribosome biogenesis. Further correlation analysis showed that the IFNγ sensitive genes were significantly correlated with the WM volume of the PG and putamen, and genes involved in ribosome biogenesis were correlated with WM volume of the PG and MFG. Conclusion: Hand GS loss in RA patients is associated with measurable changes in the brain volume of motor cortex and the dorsal striatum. These changes can be mitigated by daily hand training. Regional brain changes were associated with dysregulated immune system and impaired IFNγ production and signalling in CD4+ T cells. This study provides new clues for understanding the interaction between the immune and nervous systems in RA, which should be taken into account during treatment of RA patients. REFERENCES: [1] Schrepf et al. Nature Communications. 2018. Acknowledgements: NIL. Disclosure of Interests: None declared.