Fig 2: SARS-CoV-2 and other RNA viruses activate IRFs differentially(A) Localization of endogenous transcription factors in resting and stimulated A549-ACE2 cells. Cells were infected with SeV, influenza A virus (H1N1 WSN) (IAV) at MOI 1, or SARS-CoV-2 at the indicated MOIs, for 6 h or left uninfected (NI). Alternatively, cells were infected in the presence of the PLP inhibitor GRL-0617 (50 μM). Cells fixed and labeled with anti-IRF1, IRF3, IRF5, or IRF7 were acquired with Airyscan mode. Fluorescence intensities in the nuclear and cytoplasmic regions of interest (ROIs) were measured using FiJi. Each point represents the nucleus/cytoplasm ratios of mean gray values for a single cell. The graph shows the median and interquartile range for ∼30 cells per condition from 3 independent experiments. ∗∗∗∗p < 0.0001, as determined by one-way ANOVA with Bonferroni post hoc test.(B) Representative images from (A). Scale bar: 5 μm.

Fig 3: Sex differences in the percentage of IRF5 positive B cells ex vivo following TLR7 and TLR9 stimulation. Percentages of IRF5 positive B cells were defined by Flow Cytometry with the aid of an isotype control. Upper row: percentages of IRF5 + CD20 + B cells are shown in an unstimulated condition and after 20 h of stimulation with a TLR7 agonist (CL097) and a TLR9 agonist (CpG (ODN 2216)). Percentages of IRF5 positive B cells increased significantly after TLR7 stimulation (p < 0.0001) and after TLR9 stimulation (p = 0.0002; two-tailed t test; women n = 11, men n = 11). Lower row left: women (pink dots) showed significantly higher percentages of IRF5 positive B cells than men (blue dots) after 20 h of TLR7 stimulation (p = 0.03; two-tailed t test; women n = 11, men n = 11). Lower row right: women (pink dots) showed significantly higher percentages of IRF5 positive B cells than men (blue dots) after 20 h of TLR9 stimulation (p = 0.02; two-tailed t test; women n = 11, men n = 11)

Fig 4: Applying different IRF-α/CBP AlphaCen assays to measure the sensing of viral infections(A) HEK-ACE2 cells were transfected with an empty (NS) or a 2CARD-expressing plasmid, together with Cen-CBP and either IRF1-α, IRF3-α, IRF5-α, or IRF7-α. AlphaCen signal was measured at 39 hpt. Data correspond to individual values and means ± SD from four independent experiments performed in triplicate.(B) Expression and localization of IRF-⍺ (green) and Cen-CBP (magenta) in HEK293T cells stimulated by SeV for 6 h or unstimulated (NI) and acquired with Airyscan processing. Representative images are shown with artificial coloring. Scale bar: 10 μm.(C) HEK293T cells were transfected with Cen-CBP and either IRF1-α, IRF3-α, IRF5-α, or IRF7-α. At 24 hpt, cells were infected with SeV, West Nile virus lineage 1 (WNV L1), or IAV (H1N1 WSN) at MOI 1 or left uninfected (NI). AlphaCen signal was measured at 24 hpi. Data correspond to means ± SD of three experiments performed in triplicate. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001, as determined by one-way ANOVA with Bonferroni post hoc test. ns, non-significant.(D) HEK-ACE2 cells were transfected with Cen-CBP and either IRF1-α, IRF3-α, IRF5-α, or IRF7-α and then treated with IFN-α2 overnight. At 24 hpt, cells were infected with SARS-CoV-2 at the indicated MOIs or left uninfected. Alternatively, cells were infected in the presence of GRL-0617 at 50 μM. AlphaCen signal was measured at 24 hpi. Data correspond to means ± SD of three experiments performed in triplicate. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001, as determined by one-way ANOVA with Bonferroni post hoc test. ns, non-significant.

Fig 5: Sex differences in the percentages of IRF5 positive B cells. A Flow cytometric plots showing the applied gating strategy for isolated B cells (CD3–CD20 + cells). FACS plots of a representative donor are shown (out of 22 experiments). B cells were purified with Human B Cell Enrichment Cocktail. The percentages of IRF5 positive B cells were analyzed ex vivo by flow cytometry. The first gate was set on physical parameters, then on SSC-W versus SSC-A and FSC-W versus FSC-A to eliminate doublets, then on CD3 negative events to exclude remaining CD3 positive cells. IRF5 levels (in blue) were determined by the use of an isotype control (in red) in B cells from women or age-matched men. B To confirm antibody specificity, we performed surface staining to detect IRF5 on the cell surface of CD20 + B cells. IRF5 staining was performed by surface staining (orange histogram) and after permeabilized in 0.01% Triton-X-100 for intracellular staining (blue histogram) or isotype control (red histogram). Surface staining of IRF5 served as a negative control as IRF5 is located in the cytoplasm and intranuclearly [25]. No IRF5 positive B cells were detected after surface staining alone. C Percentages of IRF5 positive B cells. Women (pink dots) showed significantly higher percentage of IRF5 positive B cells than men (blue dots) in unstimulated conditions (p = 0.01; two-tailed t test; women n = 11, men n = 11)