Fig 2: IL-17Rb and CXCL5 up-regulation in injured white matter vasculatureSchematic representation of IL-17/CXCL5 signaling in chronically injured cerebral endothelia (A). TRAP-qPCR fold expression compared with average fpkm of top DEGs from white matter endothelia (*adjusted p < 0.05) (B). Weight-adjusted ELISA values (pg/mL) for murine CXCL5 in retro-orbital blood samples from CFD (black) and HFD (red) animals (n = 4/grp, p = 0.0355) (C). Immunofluorescence labeling for IL-17Rb (green, D) and CXCL5 (green, E) is absent in white matter vasculature of Tie2-Cre;tdTomato mice on CFD (left panels) and abundant in white matter vasculature of Tie2-Cre;tdTomato mice on HFD (right panels). Single-channel labeling for IL17Rb (bottom panels, D) and CXCL5 (bottom panels, E) show heterogeneous endothelial expression. Labeling for GLUT-1 (blue), CXCL5 (red), and PDGFRa (green) at 7 days post-stroke in animals on CFD (left) and HFD (right). Inset boxes from the peri-infarct tissue (top) masked for GLUT-1 (white) with only co-localized CXCL5 (purple) (bottom). Graph of percentage of co-localized CXCL5+/GLUT-1+ voxels (****p < 0.0001) (F). Error bars represent S.E.M.Scale bars: 50 µm (F), 20 µm (D), and 10 µm (E).

Fig 3: Reactive astrocytes. (A) Control astrocytes stained with IL6 (green), GFAP (red), vimentin (magenta), and DAPI (blue). (B) IL1ß reactive astrocytes stained with IL6, GFAP, vimentin and DAPI. (C) Control astrocytes evaluated for CXCL5 + 6 immunocytochemistry, as compared to reactive astrocyte (D). IL1ß reactive astrocytes immunocytochemistry staining for CXCL5 + 6 (green). (E) The fraction of GFAP+ astrocytes population was significantly increased as compared to control. (p = 0.00152, 2558 total cells counted in reactive astrocytes and 1962 total cells counted in control) (F) The overall population fraction positive for either IL6 (p = 3.4 × 10 -6, 1299 cells counted in reactive astrocytes, and 965 cells counted in control) or CXCL5 + 6 (p = 0.00571, 1059 cells counted in reactive astrocytes, 1297 cells counted in control) were significantly increased in reactive astrocytes as compared to control. (G) Within the GFAP+ cells, 94.9 ± 1% were also co-labelled with IL6, while 66.7 ± 4% were co-labelled with CXCL5 + 6 immunocytochemistry. (H) As a confirmation, a qRT-PCR was carried out for IL6, CXCL6 and NMES1, of which all were upregulated in comparison to control. IL6 (p = 7.01 × 10-7), CXCL6 (p = 2.34 × 10-6) and NMES1 (p = 6.4 × 10-7). The fold changes were respectively (IL6) 159.8 ± 37, (CXCL6) 53.0 ± 9 and (NMES1)133.1 ± 15 folds from control. Scale bar 20 µm. (n = 4 independent experiments).

Fig 4: CXCL5 as a biomarker of cerebral small vessel diseasePlasma levels of log10-CXCL5 in ASPIRE cohort subjects separated by detectable plasma IL-17B (n = 32; median 1043.0 pg/mL) compared with those with undetectable plasma IL-17B (n = 99; median 515.3 pg/mL; *p < 0.0001). Plasma log10-CXCL5 levels in subjects with MRI-confirmed acute microvascular ischemia (IL-17B + subjects; n = 9; 978.2 pg/mL versus IL-17B- subjects; n = 24; 539.7 pg/mL) (**p = 0.0157) (A). Ordinal shift analysis of modified Fazekas scale scores from plasma IL-17B+ and IL-17B- subjects (p < 0.0001) (B). Representative immunohistochemical detection of CXCL5 in human frontal white matter vasculature in subjects with cerebrovascular pathology (C). Percentage of CXCL5+ vessel segments in peri-ventricular white matter (n = 10) (p = 0.0005). Error bars represent S.E.M.Scale bar: 10 µm

Fig 5: IL-17B/IL-17Rb/CXCL5 signaling is a vessel-to-OPC signal in white matter vasculatureHuman brain microvascular endothelial cells were stimulated with IL-17 ligands A–E (250 ng/mL) and CXCL5 levels measured in conditioned media 48 h after stimulation (*p = 0.0372 by Kruskal-Wallis H test; **post-hoc comparison for IL-17B versus no ligand, adjusted p = 0.0178) (A). Phalloidin+ cellular area in O4+ OPCs grown in vitro exposed to vehicle (top panel) or recombinant CXCL5 (bottom panel) for 48 h (p < 0.0001, F = 9.82 by one-way ANOVA) (B). Approach for CXCL5 transgenic-viral gain of function in subcortical white matter of Tie2-Cre;tdTomato mice (top panel) (C). PDGFRa+ OPC (green) labeling in GFP-transduced Tie2-Cre;tdTomato mice (red, left panel) and CXCL5-GFP-transduced Tie2-Cre;tdTomato mice (right panel). Representative masked cellular profiles of PDGFRa+ cell area (bottom panels). Schematic of anti-IL-17B antibody treatment (top panel) (D). PDGFRa+ OPC (green) labeling in control IgG-treated Tie2-Cre:tdT mice (left panel) and anti-IL-17B IgG-treated Tie2-Cre:tdT mice (right panel). Representative masked cellular profiles of PDGFRa+ cell area (bottom panels). Proportion of OPCs per unit distance from vessel (0–35 µm) in each condition (total measured cell number per condition in parentheses) (E). Average distance of OPCs to vessel (***p = 0.0005, F = 6.06 by one-way ANOVA; **adjusted p = 0.0039; *adjusted p = 0.0168) (F). Average in vivo PDGFRa+ OPC cell area (**p = 0.0068, F = 7.38 by one-way ANOVA; **adjusted p = 0.002) (G). Graph of co-localized CXCL5+/GLUT-1+ voxels in anti-IL-17B IgG-treated animals (n = 4/grp; *p = 0.018) (H). Error bars represent S.E.M.Scale bars: 10 µm