Journal: Nature Aging

Article Title: DNA damage in macrophages drives immune autoreactivity via nuclear antigen presentation

doi: 10.1038/s43587-025-01053-3

Figure Lengend Snippet: (A.) Immunofluorescence staining of γ-Η2A.X and 53BP1 in wt untreated, wt LPS-treated (wt LPS ) and Er1 Lyz2/− BMDMs. Cells with over 2 colocalized foci of the two proteins were labeled positive and indicated using white arrows. The percentage of positive cells is plotted. (At least 4 independent optical fields were counted from n = 3 biological replicates.) Single-channel images of Extended Data Fig. 7A are shown in Supplementary file ( B.) Flow cytometry analysis of the MHC-II expression levels in wt untreated, wt LPS and Er1 Lyz2/− BMDMs. Representative histograms and MFIs are plotted (n = 3-5). (C.) Immunoprecipitation and western blot detection of the MHC-II protein in BMDMs using an anti-MHC-II (IP) or isotype control antibody (IgG). (D.) Volcano plot of differentially presented peptides in wt (downregulated, blue) and wt LPS cells (upregulated, red). Log 2 (Fold Change) of -6 or 6 represents peptides uniquely identified in the wt or wt LPS MHC-peptidome. Statistical significance (ANOVA analysis) was set at p-value ≤ 0.05 (horizontal black dashed line) and peptide enrichment at Log 2 (Fold Change)≥0.3 (upregulated in wt LPS ) or Log 2 (Fold Change)≤-0.3 (downregulated in wt LPS ) (vertical black dashed line). Extracellular matrix proteins are labeled with their corresponding gene symbol. (E.) Bubble plot of the Gene Ontology (GO) term enrichment analysis (cellular component, Mann-Whitney U test) of significantly over-represented wt LPS peptides, when compared to wt controls (p-value ≤ 0.05 and Log 2 Fold change≥0.3). The dot size shows the total count of genes per annotated pathway and the blue color scale indicates the statistical significance as per the p-value of the enriched pathways. The x axis indicates the fold enrichment derived from pathway analysis. (F.) IFN-γ ELISpot analysis of splenocytes isolated from either 8-month-old wt or Er1 Lyz2/− mice and pulsed with the indicated peptides. Representative images are shown. Error bars indicate S.E.M. among replicates. Asterisk indicates the significance set at p-value: *≤0.05, **≤0.01 (two-tailed Student’s t-test). Scale bars: 10μm.

Article Snippet: Antibodies and isotype controls were purchased from BioLegend and Proteintech: anti-CD19 (BioLegend, 152410; clone 1D3/CD19), anti-CD138 (BioLegend, 142503; clone 281-2), anti-CD11b (BioLegend, 101212; clone M1I70), anti-CD4 (BioLegend, 100406, 100412 and 100432; clone GK1.5), anti-CD25 (BioLegend, 102012; clone PC61), anti-FOXP3 (Proteintech, PE-65089; clone 3G3), anti-MHC-II (BioLegend, 107606, 107636 and 107631; clone M5/114.15.2), anti-CD86 (BioLegend, 105026; clone GL-1), anti-Ly6G (BioLegend, 127654 and 127607; clone 1A8), anti-CD62L (BioLegend, 104412; clone MEL-14), anti-CD44 (BioLegend, 103036; clone IM7), anti-T-bet (BioLegend, 644812; clone 4B10), anti-PD-1 (BioLegend, 135214; clone 29F.1A12), anti-CD69 (BioLegend, 104507; clone H1.2F3), anti-F4/80 (BioLegend, 123110; clone BM8), anti-CD115 (BioLegend, 135512; clone AFS98), anti-mouse Lineage Cocktail with Isotype Ctrl (BioLegend, 133305; clones 17A2, RB6-8C5, RA3-6B2, Ter119 and M1/70), anti-mouse CD34 antibody (BioLegend, 119307; clone MEC14.7), anti-mouse Ly-6A/E (Sca-1) (BioLegend, 108123; clone D7), anti-mouse CD16/32 (BioLegend, 101325; clone 93), anti-mouse c-Kit (BioLegend, 105815; clone 2B8), anti-mouse CD170 (Siglec-F) antibody (BioLegend, 155523; clone S17007L), anti-mouse NK-1.1 (BioLegend, 108705; clone PK316), anti-mouse CD3a (Proteintech, PE-65060), anti-mouse FoxP3 (BioLegend, 126409; clone MF-14), i-mouse CD8a (BioLegend, 100712; clone 53-6.7), PerCP rat IgG2a (BioLegend, 400529; clone RTK2758) and FITC rat IgG2a (Proteintech, FITC-65209; clone 2A3) were used as isotype control antibodies.

Techniques: Immunofluorescence, Staining, Labeling, Flow Cytometry, Expressing, Immunoprecipitation, Western Blot, Control, MANN-WHITNEY, Derivative Assay, Enzyme-linked Immunospot, Isolation, Two Tailed Test