Fig 1: Cytokine expression in the spinal cord of wild-type mice and IL-20RB–/– mice at peak of EAE. Cytokines and VEGF expression within spinal cord tissues was detected by multianalyte bead-based immunoassay (A–M) and ELISA (N–P), n = 4, for wild-type mice at peak of EAE (peak, ∼14 dpi) (black circle) and sham-immunized, IL-20RB–/– mice (red circle), and sham-immunized (sham-immunized not shown). One-way ANOVA analysis followed by Sidak’s post hoc test was perform comparing primarily wild-type mice and IL-20RB–/– mice at peak of EAE and showed that mean differences to be significant for many cytokines: IL-1β (mean ± SEM: 1.10 ± 0.21 vs. 0.39 ± 0.08, p < 0.01, A), IL-2 (mean ± SEM: 0.67 ± 0.05 vs. 0.44 ± 0.06, p < 0.01, B), IL-4 (mean ± SEM: 0.96 ± 0.17 vs. 0.24 ± 0.03, p < 0.001, C), IL-6 (mean ± SEM: 1.36 ± 0.10 vs. 0.36 ± 0.03, p < 0.0001, D), IL-10 (mean ± SEM: 0.80 ± 0.17 vs. 0.31 ± 0.03, p < 0.01, E), IL-12 (mean ± SEM: 0.24 ± 0.001 vs. 0.26 ± 0.01, p < 0.001, F), IFN-γ (mean ± SEM: 0.50 ± 0.07 vs. 0.33 ± 0.03, p < 0.05, I), GM-CSF (mean ± SEM: 0.30 ± 0.01 vs. 0.26 ± 0.003, p < 0.001, J), TNF-α (mean ± SEM: 0.54 ± 0.06 vs. 0.29 ± 0.02, p < 0.001, L), IL-20 (mean ± SEM: 14.83 ± 3.54 vs. 7.85 ± 1.09, p < 0.05, O), and IL-24 (mean ± SEM: 25.43 ± 5.59 vs. 11.04 ± 0.91, p < 0.05, P). Results are shown as mean ± SEM, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ****p < 0.0001.
Fig 2: IL-20 receptor subunit β expression on rhesus macaque CNS microvessels. CNS region-specific microvessels were isolated from macaque tissue, n = 3, to elucidate IL-20RB protein expression by immunolabeling for VEGFR-2 (white, endothelial marker), IL-20RB (red), AQP4 (green, astrocytic end-feet marker), and DAPI (blue), scale bar = 10 μm (A). VEGFR-2 colocalized with IL-20RB confirming endothelial cells expression of IL-20RB whereas AQP4 appears to envelop both. Histogram analysis was performed to 50 microvessels per specimen and CNS region to determine microvessels diameter and AUI for IL-20RB expression (representative AUI vs. axis plot, B). One-way ANOVA analysis followed by Sidak’s post hoc test showed no statistical significance between AUC means for the different CNS regions (mean ± SD cortex: 6.4 × 10–4 ± 2.9 × 10–5, cerebellum: 6.0 × 10–4 ± 7.3 × 10–5, brainstem: 6.4 × 10–4 ± 2.5 × 10–5 and spinal cord: 7.1 × 10–4 ± 6.7 × 10–5, C). Results are shown as mean ± SD.
Fig 3: Human CNS microvasculature expresses IL-20 receptor subunits. Human microvessels were isolated from PVWM post-mortem biopsies, n = 3, to elucidate IL-20 receptor subunits protein expression by immunolabeling for IL-22RA1 (white), IL-20RB (red), IL-20RA (green), and DAPI (blue), scale bar = 10 μm (A). Histogram analysis was performed to 50 microvessels per specimen to determine AUI and AUC of each receptor subunit (representative AUI vs. axis plot histogram, B). Although AUC means exhibit a trend IL-22RA1 > IL-20RB > IL-20RA, one-way ANOVA analysis followed by Sidak’s post hoc test showed that mean AUC differences between subunits are not statistically significant (mean ± SD IL-22RA1: 1.2 × 10–3 ± 2.1 × 10–4, IL-20RB: 9.9 × 10–4 ± 2.0 × 10–4, and IL-20RA: 8.1 × 10–4 ± 1.7 × 10–4, C). Results are shown as mean ± SD.
Fig 4: Expression of IL-20 receptors and IL-20-mediated distortion in CXCL12 polarity in HCMEC/D3 cells. HCMEC/D3 seeded on eight-well slides were incubated for 24 h in the presence or absence of 1 ng/ml rhIL-1β, n = 3. After 24 h, cells were immunolabeled for IL-22RA1 (white), IL-20RB (red), and IL-20RA (green) using DAPI (blue) for nuclear staining (A). A total of 50 cells per group were analyzed to determine AUC as estimate for receptor subunits expression levels (dashed bracket shown a representative single cell, scale bar = 10 μm) (A). Graphic summary of AUI for control or rhIL-1β-treated HCMEC/D3 cells, n = 50, used to calculate AUC (B). Heatmap of change in AUC distribution of IL-20RA, IL-20RB, and IL-22RA1 between control and rhIL-1β (C). Two-way ANOVA followed by Sidak’s post hoc test showed that mean differences are statistically significant for all receptor subunits. Results are shown as mean ± SD, p < 0.0001. A similar approach was used to elucidate if IL-20 exerted changes in CXCL12 apicobasal polarity (D). HCMEC/D3 were treated with 10 ng/ml rhIL-20, n = 3, for 24 h, and immunolabeled for basal marker CXCL12 (red), apical marker GGT1 (green), and DAPI (blue) for nuclear staining (E). White dashed bracket shows representative single cell used for histogram analysis, scale bar = 10 μm (E). Graphic summary of AUI used to determine AUC, n = 25, for control or rhIL-20-treated HCMEC/D3 cells (F). Heatmap of mean change in AUC distribution of GGT1 and CXCL12 between control and IL-20-treated cells (G). Two-way ANOVA followed by Sidak’s post hoc test, showed a statistically significant change for CXCL12 and not for GGT1 (H). Results are shown as mean ± SD, **p < 0.01 and ****p < 0.0001.
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