Fig 1: Induction of acute hypoxia of healthy donor (HD) natural killer (NK) cells or high affinity NK (haNK) cells exposed to 0% oxygen. (A) HD NK cells or haNK cells were incubated in ambient (20%) oxygen or 0% oxygen in the presence of the hypoxia indicator pimonidazole for 5 hours at 37°C. Cells were stained for Hypoxyprobe (green, 63×), DAPI nuclear stain (blue) and visualized by microscopy. Insets: average pimonidazole positive staining cells positive per high power field; (right) secondary antibody control (40×); (B) haNK cells continue to produce endogenous interleukin (IL)-2 under hypoxic conditions. Cells were incubated in 20% oxygen or 0% oxygen for 5 hours at 37°C. IL-2 expression was assessed by western blot analysis and quantified using band densitometry analysis normalizing to GAPDH (inset panels), and (C) quantified by ELISA. This experiment was repeated three times with similar results.
Fig 2: Comparative analysis of cytokine levels in patients with arboviral infections and controls. Boxplots illustrate serum concentrations of IL-2, IL-6, IL-10, TNF-α, and IFN-γ across patients infected with Dengue virus (DENV), Chikungunya virus (CHIKV), Zika virus (ZIKV), Mayaro virus (MAYV), and healthy controls (HC). Cytokine levels were measured in pg/mL using high-sensitivity ELISA assays. The boxes represent the interquartile range (IQR), the horizontal line indicates the median, and whiskers denote the range within 1.5 times the IQR. Individual data points are shown as black dots. Statistical comparisons between groups were performed using the Kruskal-Wallis test followed by Dunn’s post hoc test. Significant p-values (< 0.05) are indicated above the corresponding comparisons, highlighting differential cytokine expression patterns among arboviral infections and controls
Fig 3: Temporal dynamics of serum cytokine levels in patients with arboviral infections according to days post symptom onset. Median concentrations and interquartile ranges (IQR) of IL-2, IL-6, IL-10, TNF-α, and IFN-γ in patients infected with DENV, CHIKV, ZIKV, and MAYV, stratified by days post symptom onset (1–2, 3–4, and 5–9 days) compared to healthy controls (HC). Statistical comparisons were performed using the Kruskal-Wallis test followed by Dunn’s post hoc test (adjusted Bonferroni method). Only statistically significant p-values (p < 0.05) are shown for days 3–4 and 5–9. All comparisons for day 1–2 versus controls were significant across cytokines and viruses, except for MAYV, where IL-2 and IFN-γ levels did not differ significantly from controls
Fig 4: Hierarchical clustering heatmap of standardized cytokine expression across patients with arboviral infections and controls. Z-score standardized values of IL-2, IL-6, IL-10, TNF-α, and IFN-γ were compared among patients infected with Dengue virus (DENV), Chikungunya virus (CHIKV), Zika virus (ZIKV), Mayaro virus (MAYV), and healthy controls. Hierarchical clustering was applied to both cytokines and patient groups using Euclidean distance and the average linkage method. The color scale represents standardized cytokine expression levels, where purple indicates higher expression and yellow indicates lower expression relative to the mean of each cytokine
Fig 5: Correlation analysis between cytokine levels and patient variables: gender, age, and days post symptom onset. Spearman’s correlation coefficients (r) between serum cytokine concentrations (IL-2, IL-6, IL-10, TNF-α, IFN-γ) and patient characteristics. Significant positive correlations (p-value < 0.05) are shown in blue, while non-significant correlations are displayed in black. No significant negative correlations were observed
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