Document resource
Introduction Antinuclear autoantibodies (ANA) are significant hallmarks of systemic sclerosis (SSc), but their role in pathogenesis is uncertain. Purified IgG from SSc patients alter molecular signatures of effectors cells (fibroblasts [FB] and endothelial cells [EC]), and that ANA might interact with intracellular antigen. This study aims to evaluate ANA’s ability to penetrate cells, understand the internalization mechanisms and determine intracellular interactions.Material and Methods We employed a panel of antibodies (Ab) comprising total purified IgG from anti-topoisomerase I positive (IgG-ATA+) and anti-centromere positive (IgG-ACA+) patients, alongside IgG from healthy controls (IgG-HC). In addition, we utilized polyclonal SSc ATA (poly-ATA) and fluorochrome-conjugated poly-ATA antibodies (fluo-poly-ATA). Following a 72-hour culture period and 24 hours of serum deprivation, FB and EC were incubated with the aforementioned Ab for durations ranging from 5 minutes to 6 hours. For fixed-cell imaging, cells were washed after primary Ab incubation, fixed, permeabilized, and the primary Ab were revealed with anti-human IgG Ab. For live-cell imaging, fluo-poly-ATA were added to cultures, and time-lapse videos were captured via confocal videomicroscopy. Confocal images were acquired using a Zeiss LSM980 microscope.Results Following 1 hour of Ab incubation, intracellular fluorescent spots were detected in cytoplasmic and nuclear compartments of FB and EC exposed to total IgG from SSc patients and poly-ATA. These spots were more numerous in cells treated with IgG-ATA+ and poly-ATA compared to IgG-ACA+. In contrast, while some fluorescence was detected in cells incubated with igg-hc, nuclear localization was minimal to absent under this condition ( figure 1A and B). Using poly-ata, fluorescent signal appeared as early as 5 minutes post-incubation and increased progressively up to 6h (figure 1C). Live-cell videomicroscopy confirmed the rapid cellular uptake and nuclear penetration of fluo-poly-ATA. To verify nuclear localization of ANA, Z-stack confocal imaging generated three-dimensional cell reconstructions. These analyses confirmed nuclear localization of poly-ATA and fluo-poly-ATA both in FB and EC (figure 2A). Co-immunostaining with topoisomerase-1 protein revealed 28% and 31% colocalization with igg-ata+ and poly-ata, respectively, while cenp-b protein exhibited 22% colocalization with igg-aca+ (figure 2B). To investigate the mechanism of internalization, endocytic pathway inhibition assays were conducted. Pretreatment of FB with a clathrin-mediated endocytosis inhibitor significantly reduced intracellular fluorescence, whereas caveolin pathway inhibitor had no observable effect (figure 2C).Conclusions SSc ANA can penetrate cells and reach their target antigen in vitro. An Aab clathrin-mediated endocytosis mechanism is suggested. Further mechanistic studies are needed to assess ANA pathogenicity.Abstract P.062 Figure 1A. Representative image of FB and ECfollowing 1-hour incubation with IgG-HC, IgG ACA+, IgG ATA+ and poly-ATA (number of patients for each total igG condition=5) B. Quantification of fluorescently lebeled antibodies in FB and EC following 1-hour incubation with distinct IgG serolypes. Ab spot counts were quantified using Imaris in both cytoplasmic and nuclear compartments. A total of 30 cels per condition were analyzed across two to three independent experiments, depending on the serotype Data are presented as median * interquartile range (IQR). Statistical significance was assessed using the Kruskal-Wallis test followed by Dunn’s post hoctest; “*P< 0.001. C. Kinetic of fluorescence pattems in FB incubated with poly-ATA for durations ranging from 5 minutes to 6 hours.Abstract P.062 Figure 2A. Three-dimensional FB and EC incubated 1 hour with poly-ATA generated from Z-stack contocal imaging (0.2jm optical sechons). B. FB topo-isomerase I protein co-staining after 1-hour incubation of IgG-ATA+ and FB CENP-B protein co-staining after 1-hour incubation of IgG-ACA+. C. FB and EC after 1 hour with poly-ATA alone, poly- ATA+ cathrin endocytosis pathway blockage pretreatment [chlorpromazine) and poly-ATA + caveoin endocytosis pathway blockage pretreatment (genistein)