case_images: PMC11770959_01
case_images: Immunophenotyping analysis of PBMCs from the Case, SLE patients and healthy controls expanded granulocytic populations and dampened T effector populations. A Heatmap of the median marker intensities (Arcsinh, 0-1 transformed marker median expression calculated over cellular events from all the samples in the data set, Healthy Controls, HC, n = 4, SLE patients, n = 3 and the Case) of the 29 markers across the 31 cell populations obtained with FlowSOM (cluster id, colour annotated). The intensity of the green colour is indicative of low to high marker expression respectively. Hierarchical similarity between the expression of the 29 markers is noted with a dendrogram on the top (Euclidean distance; average linkage). B Dimensionality reduction analysis, t-SNE plot, visualizing cluster assignments of cell subtypes, representative of all samples (left panel) and each sample individually (right panel). C Boxplots showing relative frequency (proportion, of live singlet PBMCs) of the 31 identified FlowSOM metaclusters for the three conditions, HC, SLE and the Case
case_images: 12969_2024_1050_Fig4_HTML.jpg
case_images: PMC11770959_01_12969_2024_1050_Fig4_HTML.jpg
case_images: Fig4
case_images: T-cells, dendritic cells and distinct monocytic subsets did not show the same trend (Supplementary Fig. 4). Together, our data corroborate that increased apoptosis, driven by enhanced DNA-damage response, takes place in the peripheral blood of AGS patient and is mediated via the ATM pathway, likely fueling the inflammatory phenotype of the disease.
case_images: Increased apoptosis in AGS correlates with ATM-mediated DNA damage response A. Apoptosis levels as determined by assessment of cleaved caspase 3 (CC3) and DNA damage response via examination of the following phosphorylated proteins in PBMCs under immunofluorescence confocal microscopy: gammaEta2AlphaChi, p53 (S15), ATM (S1981) and ATR (T1989) in AGS and HC (n = 1/condition). Analyzed results for gammaEta2AlphaChi, p-p53, p-ATM and p-ATR are depicted as foci per cell. For CC3 staining, positive signal is depicted with green and cells are quantified as positive or negative utilizing dapi nuclear staining (blue). Statistical significance was obtained by unpaired Student's t test, p >= 0.05 (ns), *p < 0.05, **p < 0.01 and ***p < 0.001. Scale bar: 10 muM. (B) B Schematic representation of the results indicating that apoptosis occurs due to DNA damage response. C Granulocytes (derived of peripheral blood) from CdL/AGS patient although exhibit higher basal level of DNA damage compared to healthy individuals and SLE patients, p-gammaEta2AlphaChi levels are approximately equal to the controls, after induction of DNA breaks with 50 uM of Etoposide
case_images: 12969_2024_1050_Fig5_HTML.jpg
case_images: PMC11770959_01_12969_2024_1050_Fig5_HTML.jpg
case_images: Fig5
case_images: To further explore this, we performed an extended analysis of DDR-related apoptosis pathway on PBMCs of the Case and healthy control, monitoring major signal transducers such as phospho-ATR, phosphor-ATM, phospho-p-53, gamma-H2AX and cleaved caspase 3 through immunofluorescence confocal microscopy (Fig. 5A-B). In spite of this, after etoposide incubation only SLE granulocytes exhibit induced levels of gamma-H2AX, while DNA-damage levels fall to levels equal to healthy control in AGS patient (Fig. 5C).