Fig 2: IL-6, sIL-6R, and IL-18 hypercytokinemia are associated with dysregulation of innate and adaptive immunity(A) Correlation matrix of cytokines in all acute samples. Statistical significance was defined as false discovery rate (FDR)-corrected p value q < 0.1.(B) Heatmap summarizing the correlations between cytokine levels and immune populations in acute COVID-19 based on manual gating strategy. Statistical significance was defined as FDR-corrected p value q < 0.1.(C and D) Representative plots of significant correlations between cytokine levels and immune cell populations from acute samples (n = 57) based on manual gating strategy and RBD-antibody titers (C) and FlowSOM analysis (D).

Fig 3: Broad immune activation in longitudinal COVID-19 samples(A and B) Overviews of (A) cohort and samples collected and (B) analyses performed.(C) UMAP plot from FlowSOM analysis for 3 flow-cytometric panels, with representative clusters and expression profiles.(D) Volcano plot of 184 immune features in acute and convalescent samples, with key representative features labeled.(E) TrackSOM analysis of samples stained with the immunophenotype panel, with plots showing time bins of lineage-defining markers and the activation markers CD38 and HLA-DR. Time bins were days 1–4, 5–8, 9–12, 13–17, 18–30, 31–35, 36–39, 41–45, 46–53, and 71–102 (Table S3).(F and G) Levels of cytokines (F) IL-6 and (G) IL-18, MCP-1, IFN-γ, and sIL-6R in COVID-19 plasma across time. Locally estimated scatterplot smoothing (LOESS) regression line and 95% confidence interval (CI) are shown, n = 119.

Fig 4: α-toxin contributes to the induction of IL-6 by S. aureus secretomes. (A) Keratinocyte secreted IL-6 levels after exposure to 19 different S. aureus secretomes with or without addition of anti-α-toxin polyclonal antibody (mean ± 1 SD: n = 3 per secretome). IL-6 absorbance values were normalized to baseline i.e., IL-6 value for untreated cells. Unpaired t-test; two-tailed ***p = 0.0003. (B) Validation of polyclonal antibody data, by testing different concentrations of anti-α-toxin monoclonal antibody in combination with secretome from a SCC-derived S. aureus isolate SSA105 known to contain high levels of α-toxin. Percentage of viable keratinocytes (left axis) and keratinocyte IL-6 response (IL-6 absorbance values baseline-corrected to untreated cells, right axis) after exposure to secretome from SSA105 ± anti-α-toxin monoclonal antibody. Neutralizing α-toxin results in loss of cytotoxic action of the tested S. aureus secretome and diminishes its ability to stimulate IL-6 secretion from HaCaT cells. Error bars indicate mean and standard deviation (n = 2). (C) Keratinocyte secreted IL-6 levels after exposure to S. aureus secretomes with or without addition of 100 or 400 ng/ml recombinant α-toxin (mean ± 1 SD: n = 3 per secretome), compared to untreated cells (gray) and control cells treated with uninoculated bacterial media DSM (green). Secretome from S. aureus isolates SSA55, SSA55, and SSA55 naturally contain very low levels of α-toxin (blue), whereas SSA110 naturally produces high levels of α-toxin (red).

Fig 5: The pro-inflammatory potential of S. aureus is determined by differences in gene regulation. Genetic and proteomic characteristics of statistically significant candidates (Table 1) that positively (n = 37, ordered by significance top to bottom) or negatively (n = 3, separated to bottom of heat map) correlated to S. aureus secretomes’ ability to induce IL-6 in keratinocytes (ordered by level of IL-6 induction left to right). Protein abundance, i.e., MS signal intensity of candidates within the different S. aureus secretome samples, is indicated by color scale, where white is no recorded signal. The cross signifies the absence of a functional gene.