The present work aimed at modelling in vitro the interplay between immune complexes (ICs) containing scleroderma-specific antibodies, vasculopathy and skin fibrosis to reproduce the multifaceted etiopathogenesis of systemic sclerosis (SSc). Endothelial cells (ECs) were grown in a bioreactor comprising apical and basal chambers separated by a porose membrane reproducing the endothelial barrier. ECs were seeded in the apical chamber in hydrogel. Skin fibroblasts were isolated from normal healthy subjects (NHS) and cultivated in acellularized 3D scaffold in a single-chamber bioreactor. The two bioreactors were interconnected via a fluidic system supplied by a peristaltic pump. ICs were purified by PEG precipitation from sera of SSc patients with anti-centromere or anti-DNA topoisomerase antibodies, systemic lupus erythematosus (SLE) or NHS. ECs were treated with SSc-ICs, SLE-ICs, NHS-ICs, TNFα or culture medium. Sections of ECs/hydrogel and fibroblasts/scaffold were evaluated by histological/immunohistochemical and immunocytochemical analysis. ICs and soluble mediators (IL-6, IL-8, CCL2/MCP-1) were measured in supernatants collected at different sites along the fluidic circuit. Upon stimulation with SSc-ICs, ECs in hydrogel developed unorganized endothelial tubular structures. The porous membrane prevented the passage of ICs but not of soluble mediators. Treating ECs with SSc-ICs induced the formation of fibrous septa, increased expression of collagen IV and αSMA, and reduced expression of CD34 in fibroblast scaffold. This innovative dynamic bicompartmental in vitro system incorporating 5 degrees of complexity (5D) allows reproducing the SSc environment, since stimulation of ECs with SSc-ICs is sufficient not only to disrupt vascular architecture but also to induce skin fibrosis.
Rheumatoid arthritis (RA) is a chronic autoimmune disease primarily mediated by the abnormal proliferation and activation of fibroblast-like synoviocytes (SFs); this process is closely associated with ferroptosis. Previous studies have confirmed the therapeutic potential of artesunate (ART), a derivative of artemisinin, in alleviating RA. In this study, we further clarified that ART suppresses the abnormal activation of SFs and ameliorates RA progression through the JAK1/STAT3-GPX4-ferroptosis signaling pathway. First, we conducted a series of in vitro assays to evaluate the biological effects of ART on RASFs and osteoarthritis (OA) SFs, including cell proliferation, invasion and migration. Subsequently, we investigated the impact of ART on cell death, especially on ferroptosis-related indicators, including transmission electron microscopy (TEM), GPX4 expression, intracellular lipid peroxides, glutathione (GSH) and reactive oxygen species (ROS). To elucidate the underlying mechanism, we performed RNA sequencing to analyze differentially enriched signaling pathways and employed molecular docking to predict potential protein targets. Key findings were validated by Western blotting and ChIP-qPCR. Next, we established a collagen-induced arthritis (CIA) mouse model to compare arthritis symptoms, pathological changes, and immunological responses between ART-treated and control groups. Finally, we isolated primary synovial fibroblasts (CIA SFs) from synovial tissue to confirm ART’s effects and its molecular mechanism of abnormal activation of SFs. ART was shown to inhibit cytokine secretion, cell proliferation, and migration while promoting ferroptosis (increased ROS, MDA and lipid hydroperoxides, decreased GSH and GPX4). Notably, the suppressive effects of ART on cellular activation—including cytokine release, proliferation, and migration—were significantly reversed upon inhibition of ferroptosis. Mechanistically, ART downregulated GPX4 expression through suppression of the JAK1/STAT3 signaling pathway. In vivo experiments further demonstrated that ART ameliorated arthritis severity in CIA mice, as evidenced by reduced arthritis scores, joint swelling, as well as improved histopathological parameters and decreased cytokine levels in joint supernatants and serum. Part of this therapeutic effect may be attributed to the induction of ferroptosis in SFs within CIA joints. In conclusion, our study provided the evidence that ART inhibits the activation of SFs by inducing ferroptosis through the JAK1/STAT3-GPX4 signaling pathway, thereby attenuating joint damage. In RASFs, ART inhibits cell activation (cytokine secretion, cell proliferation and migration). In RASFs, ART inhibits cell activation by inducing ferroptosis through the JAK1/STAT3 signaling pathway. In animal models, ART attenuates collagen-induced arthritis by inducing the ferroptosis of CIA SFs.
RA and PsA share common pathogenic mechanisms but differ at clinical, cellular, and molecular levels. This study compares the inflammatory, macrophage, and osteogenic potential of monocytes in RA and PsA. CD14+ monocytes were isolated from RA, PsA and HC PBMCs and stimulated with LPS. Mo-MACs were generated using M-CSF and polarised to M1/M2. Inflammatory gene expression, endocytosis, monocyte/macrophage markers, metabolic, DNA-methylation and histone modifications were assessed by RT-PCR and flow-cytometry. Real-time metabolic profiles were analysed by XF-Seahorse. Mesenchymal stem cells (MSCs) were cultured with monocyte-conditioned media (CM) to assess osteogenesis, migration, and activation. LPS induced IL-6, IL-1β, CXCL10-11, SLAMF1 and SLAMF2 expression in RA > PsA> HC CD14+ monocytes, with SLAMF3 significantly decreased. This was paralleled by impaired endocytic capacity, a shift towards a glycolytic profile and a dysfunctional mitochondrion. RA monocytes displayed more pronounced metabolic reprogramming compared with PsA. Frequency/expression of activation and macrophage markers (CD40, CD64, CD206, CD163, SLAMF7) were higher on RA PsA classical and intermediate monocytes vs. HC. RA Mo-MACs retained the hyper-inflammatory and impaired endocytic phenotype of their precursor monocyte. PsA monocyte-CM preferentially enhanced MSC osteogenic function, whereas RA monocyte-CM predominantly induced MSC migration. H3K27Ac and H3K4M3e were increased on RA and PsA classical and intermediate monocytes consistent with baseline epigenetic priming of inflammatory gene programmes. TET2 and TET3 were further induced upon LPS stimulation in RA and PsA monocytes and were suppressed by the glucocorticoid budesonide, an effect paralleled by inhibition of pro-inflammatory cytokines. Finally, synovial monocytes displayed an activated, macrophage-like phenotype compared to their circulatory counterparts. RA and PsA CD14⁺ monocytes exhibit metabolic and functional reprogramming associated with distinct disease-biased effector profiles, alongside evidence of epigenetic priming at baseline and inducible responses following LPS stimulation. Collectively, these findings highlight circulating monocytes as key contributors to divergent pathology in inflammatory arthritis.
Patients with rheumatoid arthritis (RA) have increased oxidative stress, which may contribute to inflammation and elevated blood pressure. 2-hydroxybenzylamine acetate (2-HOBA) scavenges reactive downstream oxidative stress products, including isolevuglandins (IsoLGs), which covalently bind proteins and alter their structure. This pilot study evaluated the safety and tolerability of 2-HOBA, its effect on IsoLG-protein adducts, and potential effects on disease activity and blood pressure in RA. We conducted a single-center, randomized, double-blind, parallel-group, phase 2 clinical trial. Patients with RA (n = 32) were randomized to receive 2-HOBA 750 mg or matching placebo orally three times daily for 4 weeks. Primary outcomes were safety, tolerability, and change in cells with IsoLG-protein adducts. Exploratory outcomes included changes in disease activity and blood pressure. Linear mixed-effects models assessed treatment effects of 2-HOBA versus placebo. The modified intention-to-treat population included 30 participants (2-HOBA n = 16, placebo n = 14). 2-HOBA was safe and well tolerated, with similar adverse event rates between groups. Compared with placebo, 2-HOBA did not significantly alter cells with IsoLG-protein adducts (treatment effect 3.4
Systemic autoinflammatory diseases (SAIDs) have an intermittent and often unpredictable clinical course that can significantly impair patients’ quality of life (QoL). While QoL is commonly assessed in chronic diseases, the specific impact of SAIDs and the aspects of health that matter most to patients and families remain insufficiently described. We aimed to identify key elements required to assess health-related quality of life (HRQoL) in adults and children affected by FMF, MKD, TRAPS, CAPS, PFAPA, and Still’s disease. We conducted a qualitative multicenter prospective study using a phenomenological approach. Individual semi-structured interviews were conducted at three sites of the French National Reference Centre for SAIDs (CEREMAIA). Interviews were carried out with parents for children < 6 years, with patients and parents separately for those aged 6–17 years, and with patients alone for adults. We analyzed 58 interviews involving 39 patients and their parents. Three main dimensions emerged: (1) Self: Patients described a substantial emotional burden, including anxiety before diagnosis, fear of innovative treatments initiated at very young ages, and frustration related to pain, physical limitations, and body changes. Pain was often invasive and overwhelming, and the unpredictable nature of flares required patients to learn to anticipate triggers and adjust daily activities. Ambivalence toward medical care was frequent, with gratitude for effective treatments but concerns about long-term effects. (2) Others: SAIDs deeply affected family dynamics, with sibling jealousy or overprotection, unequal distribution of parental attention, and episodes of strain within conjugal relationships. Patients and parents also faced a lack of understanding from relatives and peers, due to the often-invisible nature of SAIDs, sometimes leading patients to hide symptoms or avoid discussing the disease; (3) Society: Unpredictable flares disrupted school attendance, shaped educational choices, limited career opportunities, and occasionally resulted in professional exclusion. Patients frequently adapted their aspirations to accommodate disease-related constraints. Our in-depth analysis shows that the QoL of patients with SAIDs was affected in multiple dimensions. These findings provide the conceptual foundation for developing SAID-specific, patient-reported outcome measures to better capture the lived experience of patients.
