Figure 3A. TEER of the in vitro BBB models composed by a monolayer of ECs sorted from E17 Male Ctrl (1.00 ± 0.02), Male Poly I:C (0.92 ± 0.04), Female Ctrl (1.15 ± 0.04), Female Poly I:C (1.00 ± 0.07) offspring. Data are normalized to male Ctrl. Data information: Embryos prenatally exposed to vehicle (Ctrl) or Poly I:C on GD9 were dissected, and cortex derived vascular cells were used to build in-vitro barrier models. Cells derived from Ctrl males = white bars with blu border. Cells derived from Poly I:C males = blu bars with blu border. Cells derived from Ctrl females = white bars with purple border. Cells derived from Poly I:C females = purple bars with purple border. Numbers in bars indicate the number of animals (N) or independent cultures/pictures. Bars represent mean ± SEM.B. Permeability to 10 kDa Dextran of the in vitro BBB models composed by a monolayer of sorted ECs from Male Ctrl (1.00 ± 0.06), Male Poly I:C (1.13 ± 0.02), Female Ctrl (0.97 ± 0.05), Female Poly I:C (0.95 ± 0.01) offspring. Mann-Whitney U test, *p < .05. Data are normalized to male Ctrl. Data information: Embryos prenatally exposed to vehicle (Ctrl) or Poly I:C on GD9 were dissected, and cortex derived vascular cells were used to build in-vitro barrier models. Cells derived from Ctrl males = white bars with blu border. Cells derived from Poly I:C males = blu bars with blu border. Cells derived from Ctrl females = white bars with purple border. Cells derived from Poly I:C females = purple bars with purple border. Numbers in bars indicate the number of animals (N) or independent cultures/pictures. Bars represent mean ± SEM.C. TEER of barriers composed of bEND.3 alone (1.00 ± 0.00) or co-cultured with sorted PCs from Male Ctrl (1.17 ± 0.07), Male Poly I:C (0.95 ± 0.05), Female Ctrl (1.15 ± 0.08), Female Poly I:C (1.15 ± 0.08) offspring. Mann-Whitney U test, *p < .05. Data are normalized to bEnd.3. Data information: Embryos prenatally exposed to vehicle (Ctrl) or Poly I:C on GD9 were dissected, and cortex derived vascular cells were used to build in-vitro barrier models. Cells derived from Ctrl males = white bars with blu border. Cells derived from Poly I:C males = blu bars with blu border. Cells derived from Ctrl females = white bars with purple border. Cells derived from Poly I:C females = purple bars with purple border. Numbers in bars indicate the number of animals (N) or independent cultures/pictures. Bars represent mean ± SEM.D. Permeability to 10 kDa Dextran of barriers composed by a monolayer of bEND.3 alone (1.00 ± 0.06) or co-cultured with sorted PCs from Male Ctrl (0.47 ± 0.09), Male Poly I:C (0.46 ± 0.07), Female Ctrl (0.46 ± 0.11), Female Poly I:C (0.65 ± 0.05) offspring. One sample t test, **p < .01. Data are normalized to bEnd.3. Data information: Embryos prenatally exposed to vehicle (Ctrl) or Poly I:C on GD9 were dissected, and cortex derived vascular cells were used to build in-vitro barrier models. Cells derived from Ctrl males = white bars with blu border. Cells derived from Poly I:C males = blu bars with blu border. Cells derived from Ctrl females = white bars with purple border. Cells derived from Poly I:C females = purple bars with purple border. Numbers in bars indicate the number of animals (N) or independent cultures/pictures. Bars represent mean ± SEM.E. TEER (Ω.cm2) was monitored over 72h treatment with TGF-β1 and normalized to baseline in BBB models composed by a monolayer of sorted ECs from Male (72h TGF-β1 = 82.85 ± 1.56) or Female (72h TGF-β1 = 85.92 ± 2.03) embryos. The insert shows the values at 72h (bars) normalized to the 0h values (dashed line). One sample t test, ***p < .001, ****p < .0001. Data information: Embryos prenatally exposed to vehicle (Ctrl) or Poly I:C on GD9 were dissected, and cortex derived vascular cells were used to build in-vitro barrier models. In Fig.3 E blue and purple dotted lines represent cells derived from male and female brain respectively.F. TEER (Ω.cm2) was monitored over 72h treatment with TGF-β1 and normalized to baseline in barrier models composed by a monolayer of bEND.3 alone (72h TGF-β1 = 89.85 ± 2.05) or co-cultured with sorted PCs from Male (72h TGF-β1 = 88.83 ± 1.93) or Female (72h TGF-β1 = 89.02 ± 2.22) embryos. The insert shows the values at 72h (bars) normalized to the 0h values (dashed line). Wilcoxon signed-rank test, *p < .05. Data information: Embryos prenatally exposed to vehicle (Ctrl) or Poly I:C on GD9 were dissected, and cortex derived vascular cells were used to build in-vitro barrier models. In Fig.3 F blue and purple dotted lines represent cells derived from male and female brain respectively.G. 10 kDa Dextran permeability after 72h TGF-β1 treatment of the in vitro BBBs prototypes composed by a monolayer made of sorted ECs from Male NT (0.43 ± 0.02), Male TGF-β1 (0.43 ± 0.03); Female NT (0.45 ± 0.00), Female TGF-β1 (0.47 ± 0.01). Data information: Embryos prenatally exposed to vehicle (Ctrl) or Poly I:C on GD9 were dissected, and cortex derived vascular cells were used to build in-vitro barrier models. Cells derived from Ctrl males = white bars with blu border. Cells derived from Ctrl females = white bars with purple border. Numbers in bars indicate the number of animals (N) or independent cultures/pictures. Bars represent mean ± SEM.H. 10 kDa Dextran permeability 72h after TGF-β1 treatment of NVU prototypes composed by a monolayer made of bEND.3 alone (NT, solid black bar = 0.45 ± 0.01, TGF-β1, dotted black bar = 0.49 ± 0.00) and co-cultured with sorted PCs from Male (NT = 0.45 ± 0.04, TGF-β1 = 0.48 ± 0.00), or Female (NT = 0.49 ± 0.03, TGF-β1 = 0.53 ± 0.02) embryos. Student's t test, *p < .05. Data information: Embryos prenatally exposed to vehicle (Ctrl) or Poly I:C on GD9 were dissected, and cortex derived vascular cells were used to build in-vitro barrier models. bEend.3 cells = bars with black border. Cells derived from Ctrl males = white bars with blu border. Cells derived from Ctrl females = white bars with purple border. Numbers in bars indicate the number of animals (N) or independent cultures/pictures. Bars represent mean ± SEM.I. FACS quantitative data of the normalized MFI of CD146 (NT = 100.00 ± 1.14, TGF-β1 = 54.08 ± 9.42; Student's t test, *p < .05) and representative histogram of untreated (solid gray) and TGF-β1 treated cells (hollow black bold line). J. FACS quantitative data of the normalized MFI of Claudin-5 (CLDN5; NT = 100.00 ± 8.94, TGF-β1 = 65.22 ± 5.29; Student's t test, **p < .01) and representative histogram of untreated (solid gray) and TGF-β1 treated cells (hollow black bold line). Data information: bEend.3 cells = bars with black border. The black dotted line represent bEnd.3 cultures. Numbers in bars indicate the number of animals (N) or independent cultures/pictures. Bars represent mean ± SEM.K. Representative images of monolayers of primary endothelial cells isolated from male brains and stained for CD146 (red) and CLDN5 (green) under control conditions (NT) or upon treatment with different cytokines. Scale bar = 50 µm. L. Quantitative analysis of CD146 immunofluorescence mean intensity measured in primary male brain EC monolayers under control conditions or after 72h treatments with different cytokines: NT = 100.00 ± 6.55, TGF-β1 = 94.03 ± 2.01, IL-1β + TGF-β1 = 84.87 ± 4.59, IL-1β = 92.34 ± 2.65; Kruskal-Wallis, *p < .05. M. Quantitative analysis of Claudin-5 immunofluorescence mean intensity in primary male brain EC monolayers under control conditions or after 72h treatments with different cytokines: NT = 99.76 ± 5.19, TGF-β1 = 86.74 ± 6.76, IL-1β + TGF-β1 = 51.64 ± 3.31, IL-1β = 107.3 ± 7.37; Ordinary one-way ANOVA, **p < .01, ****p < .0001. N. Quantitative analysis of Claudin-5 immunofluorescence area (µm2) in primary male brain EC monolayers under control conditions or after 72h treatments with different cytokines: NT = 39781 ± 2342, TGF-β1 = 31707 ± 5939, IL-1β + TGF-β1 = 5415 ± 1037, IL-1β = 46303 ± 2986; Ordinary one-way ANOVA, *p < .05, **p < .01, ****p < .0001. Data information: Cortex-derived-EC isolated from male mice at 2 mouths were used to build in-vitro barrier models and analyzed. Cells derived from Ctrl males = white bars with blu border. Numbers in bars indicate the number of animals (N) or independent cultures/pictures. Bars represent mean ± SEM.O. Graph showing TEER measured by volt-ohmmeter at 72h normalized in NT (100.00 ± 7.46) and IL-1β + TGF-β1 (49.93 ± 10.96) treated primary male brain EC cultures. Student's t test, **p < .01. Data are normalized to t0. Data information: Cortex-derived-EC isolated from male mice at 2 mouths were used to build in-vitro barrier models and analyzed. Cells derived from Ctrl males = white bars with blu border. Numbers in bars indicate the number of animals (N) or independent cultures/pictures. Bars represent mean ± SEM.P. Permeability to 3kDa dextran quantified by fluorescence spectrophotometry in NT (1.00 ± 0.10) and IL-1β + TGF-β1 (1.92 ± 0.33) treated primary male brain EC cultures. Student's t test, *p < .05. Q. Permeability to 10kDa dextran quantified by fluorescence spectrophotometry in NT (1.00 ± 0.11) and IL-1β + TGF-β1 (1.80 ± 0.26) treated primary male brain EC cultures. Student's t test, *p < .05. Data information: Cortex-derived-EC isolated from male mice at 2 mouths were used to build in-vitro barrier models and analyzed. Cells derived from Ctrl males = white bars with blu border. Numbers in bars indicate the number of animals (N) or independent cultures/pictures. Bars represent mean ± SEM.