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. 2022 Dec 1;41(23):e111192.
doi: 10.15252/embj.2022111192. Epub 2022 Oct 31.

Maternal immune activation leads to defective brain-blood vessels and intracerebral hemorrhages in male offspring

Marco Rasile  1   2 , Eliana Lauranzano  2 , Elisa Faggiani  2 , Margherita M Ravanelli  1   2 , Federico S Colombo  2 , Filippo Mirabella  1   2 , Irene Corradini  2   3 , Maria L Malosio  2   3 , Antonella Borreca  2   3 , Elisa Focchi  3 , Davide Pozzi  1   2 , Toni Giorgino  4 , Isabella Barajon  1   2 , Michela Matteoli  1   3
Affiliations

Maternal immune activation leads to defective brain-blood vessels and intracerebral hemorrhages in male offspring

Marco Rasile et al. EMBO J. .

Erratum in

Abstract

Intracerebral hemorrhages are recognized risk factors for neurodevelopmental disorders and represent early biomarkers for cognitive dysfunction and mental disability, but the pathways leading to their occurrence are not well defined. We report that a single intrauterine exposure of the immunostimulant Poly I:C to pregnant mice at gestational day 9, which models a prenatal viral infection and the consequent maternal immune activation, induces the defective formation of brain vessels and causes intracerebral hemorrhagic events, specifically in male offspring. We demonstrate that maternal immune activation promotes the production of the TGF-β1 active form and the consequent enhancement of pSMAD1-5 in males' brain endothelial cells. TGF-β1, in combination with IL-1β, reduces the endothelial expression of CD146 and claudin-5, alters the endothelium-pericyte interplay resulting in low pericyte coverage, and increases hemorrhagic events in the adult offspring. By showing that exposure to Poly I:C at the beginning of fetal cerebral angiogenesis results in sex-specific alterations of brain vessels, we provide a mechanistic framework for the association between intragravidic infections and anomalies of the neural vasculature, which may contribute to neuropsychiatric disorders.

Keywords: CD146; MIA; NVU; intracerebral hemorrhages; sex dimorphism.

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Figures

Figure 1
Figure 1. GD9 Poly I:C‐induced MIA causes sex‐specific remodeling of the brain vessels and BBB breakdown

  1. 3D rendering of the cerebrovascular tree in brain slices from male P90 Ctrl and Poly I:C MIA offspring stained with intravascular DiI (red). Scale bar = 50 μm.

  2. DiI‐positive blood vessel geometry analysis—quantification of blood vessel number, diameter (mean and cumulative frequency distribution), length, number of branch segments, and average branch length—of P90 male Ctrl and Poly I:C MIA offspring. Vessel number for Ctrl (20.93 ± 1.73) and Poly I:C (27.41 ± 1.99) MIA offspring, Mann–Whitney U‐test, **P < 0.01. Mean diameter for Ctrl (5.58 ± 0.01) and Poly I:C (4.96 ± 0.01) MIA offspring, Mann–Whitney U‐test, ****P < 0.0001. The total length of vessel per image for Ctrl (6.12 ± 0.76) and Poly I:C (9.83 ± 1.48) MIA offspring, Mann–Whitney U‐test, **P < 0.01. A number of branches for Ctrl (9.60 ± 0.54) and Poly I:C (15.80 ± 1.21) MIA offspring, Mann–Whitney U‐test, *P < 0.05. Average branch length for Ctrl (34.07 ± 1.35) and Poly I:C (33.47 ± 1.67) MIA offspring.

  3. 3D rendering of the cerebrovascular tree in brain slices from female P90 Ctrl and Poly I:C MIA offspring stained with intravascular DiI (red). Scale bar = 50 μm.

  4. DiI‐positive blood vessel geometry analysis—quantification of blood vessel number, diameter (mean and cumulative frequency distribution), length, number of branch segments, and average branch length—of P90 female Ctrl and Poly I:C MIA offspring. Vessel number for Ctrl (9.97 ± 1.18) and Poly I:C (8.61 ± 0.78) MIA offspring. Mean diameter for Ctrl (5.77 ± 0.03) and Poly I:C (6.16 ± 0.02) MIA offspring, Mann–Whitney U‐test, ****P < 0.0001. The total length of vessel per image for Ctrl (3.23 ± 0.50) and Poly I:C (2.97 ± 0.30) MIA offspring. Number of branches for Ctrl (10.50 ± 0.87) and Poly I:C (7.24 ± 0.44) MIA offspring. Average branch length for Ctrl (38.06 ± 2.17) and Poly I:C (46.93 ± 1.49) MIA offspring, Mann–Whitney U‐test, ***P < 0.001.

  5. Quantification of in vivo brain permeability at P30 in male Ctrl (0.00 ± 0.00) and Poly I:C (3.68 ± 1.98)‐treated offspring, Mann–Whitney U‐test, P = 0.060.

  6. Quantification of Evans blue (EB) analysis of in vivo brain permeability at P90 (top) and representative pictures (bottom) of perfused brains from male Ctrl (left; 0.00 ± 0.00) and Poly I:C (right; 20.32 ± 10.95) offspring. Mann–Whitney U‐test, *P < 0.05. In accompanying pseudocolor images, blue pixels are represented as a gradient of light‐to‐dark blue depending on the intensity of the color in the original photo while the absence of blue tones is represented as green.

  7. Quantitative analysis of the optical density of claudin‐5 (CLDN5) immunoreactive bands normalized to calnexin (CANX) in cortices from P90 male Ctrl (0.99 ± 0.09) and Poly I:C (0.58 ± 0.05) offspring. Mann–Whitney U‐test, **P < 0.01.

  8. Quantitative analysis of the optical density of ZO‐1 immunoreactive bands normalized by α‐Tubulin (αTUB) in cortices from P90 male Ctrl (1.00 ± 0.04) and Poly I:C (0.75 ± 0.08) offspring. Student's t‐test, *P < 0.05.

  9. Quantification of in vivo brain permeability at P30 in female Ctrl (0.58 ± 0.17) and Poly I:C (0.31 ± 0.14)‐treated offspring.

  10. Quantification of Evans blue (EB) analysis of in vivo brain permeability at P90 (top) and representative pictures (bottom) of perfused brains from female Ctrl (left; 0.70 ± 0.47) and Poly I:C (right; 0.10 ± 0.10) offspring. In accompanying pseudocolor images, blue pixels are represented as a gradient of light‐to‐dark blue depending on the intensity of the color in the original photo while the absence of blue tones is represented as green.

  11. Quantitative analysis of the optical density of claudin‐5 (CLDN5) immunoreactive bands normalized by CANX in cortices from P90 female Ctrl (1.00 ± 0.16) and Poly I:C (1.39 ± 0.13) offspring.

  12. Quantitative analysis of the optical density of ZO‐1 immunoreactive bands normalized by αTUB in cortices from P90 female Ctrl (1.00 ± 0.11) and Poly I:C (1.19 ± 0.19) offspring.

  13. Representative images of P90 Ctrl and Poly I:C male offspring cortices stained for TJs proteins. Claudin‐5 (top green) and ZO‐1 (bottom red)—scale bar = 10 μm.

  14. Quantitative analysis of the mean intensity of CLDN5 in cortices from P90 male Ctrl (75.14 ± 1.08) and Poly I:C (67.39 ± 2.00) offspring. Student's t‐test, *P < 0.05.

  15. Quantitative analysis of the CLDN5 signal volume in cortices from P90 male Ctrl (5.54 ± 0.59) and Poly I:C (3.70 ± 0.48) offspring. Student's t‐test, *P < 0.05.

  16. Quantitative analysis of the mean intensity of ZO‐1 in cortices from P90 male Ctrl (73.16 ± 0.59) and Poly I:C (73.86 ± 1.94) offspring.

  17. Quantitative analysis of the ZO‐1 signal volume in cortices from P90 male Ctrl (23.29 ± 1.42) and Poly I:C (16.78 ± 2.18) offspring. Student's t‐test, *P < 0.05.

  18. Graphical representation of MIA experimental design. Pregnant mice at GD9 were exposed to vehicle (Ctrl) or Poly I:C and the progeny, separated by sex, were analyzed at different time points (i.e., E17, P30, and P90). The figures were adapted and modified from Servier Medical Art (http://smart.servier.com/).

  19. Graphical representation of adult immune‐challenge experimental design. P20 male mice were exposed to vehicle (Ctrl) or Poly I:C to be analyzed at P90. The figures were adapted and modified from Servier Medical Art (http://smart.servier.com/).

  20. [Left] Quantification of in vivo cerebrovascular permeability to EB of P90 Ctrl (0.16 ± 0.16) and Poly I:C‐treated (0.89 ± 0.89) males injected at P20. [Right] Representative pictures of saline‐perfused brains 24 h after the EB injection accompanied by pseudocolor images—blue pixels are represented as a gradient of light‐to‐dark blue depending on the intensity of the color in the original photo while the absence of blue tones is represented as green—from Ctrl (top) and Poly I:C (bottom) male mice.

  21. Quantitative analysis of the optical density of CLDN5 immunoreactive bands normalized to CANX in cortices from P90 Ctrl (0.99 ± 0.12) and Poly I:C (0.98 ± 0.10) males treated at P20.

  22. Quantitative analysis of the optical density of ZO‐1 immunoreactive bands normalized to CANX in cortices from P90 Ctrl (1.00 ± 0.11) and Poly I:C (0.73 ± 0.05) males treated at P20.

Data information: Ctrl males = white bars with blue border. Poly I:C males = blue bars with blue border. Ctrl females = white bars with a purple border. Poly I:C females = purple bars with a purple border. In Fig 1B and D, black dotted lines represent the Ctrl while the blue and purple solid lines represent the male Poly I:C and female Poly I:C offspring, respectively. Numbers in bars indicate the number of animals (N) and images (n). Bars represent mean ± SEM. Source data are available online for this figure.
Figure 2
Figure 2. Maternal immune activation causes sex‐specific alterations in the expression of vascular and inflammatory controller molecules
  1. A

    qPCR analyses of GD9 + 6 h male embryos for: Pecam1 (Ctrl = 1.00 ± 0.17 N = 8, Poly I:C = 0.89 ± 0.23 N = 13), CD248 (Ctrl = 1.00 ± 0.20 N = 6, Poly I:C = 1.35 ± 0.31 N = 11), Vegfa (Ctrl = 1.00 ± 0.19 N = 8, Poly I:C = 0.15 ± 0.04 N = 8, Student's t‐test, ***P < 0.001), Pdfgb (Ctrl = 1.00 ± 0.23 N = 5, Poly I:C = 1.10 ± 0.18 N = 13), Tgfb1 (Ctrl = 1.00 ± 0.21 N = 7, Poly I:C = 1.76 ± 0.22 N = 10, Mann–Whitney U‐test, *P < 0.05), Tgfb2 (Ctrl = 1.00 ± 0.28 N = 6, Poly I:C = 1.37 ± 0.28 N = 13), Tgfb3 (Ctrl = 1.00 ± 0.51 N = 6, Poly I:C = 0.73 ± 0.19 N = 10), and Foxf2 (Ctrl = 1.00 ± 0.45 N = 8, Poly I:C = 0.46 ± 0.18 N = 13) mRNAs.

  2. B

    qPCR analyses of E17 male cortices for: Pecam1 (Ctrl = 1.00 ± 0.63 N = 10, Poly I:C = 3.07 ± 1.58 N = 8; Mann–Whitney U‐test, *P < 0.05), CD248 (Ctrl = 1.00 ± 0.23 N = 10, Poly I:C = 3.33 ± 0.79 N = 9; Mann–Whitney U‐test, **P < 0.01), Vegfa (Ctrl = 1.00 ± 0.10 N = 8, Poly I:C = 1.70 ± 0.29 N = 5; Mann–Whitney U‐test, *P < 0.05), Pdfgb (Ctrl = 1.00 ± 0.75 N = 9, Poly I:C = 2.05 ± 0.96 N = 9; Mann–Whitney U‐test, *P < 0.05), Tgfb1 (Ctrl = 1.00 ± 0.16 N = 7, Poly I:C = 15.27 ± 5.75 N = 7; Mann–Whitney U‐test, ***P < 0.001), Tgfb2 (Ctrl = 1.00 ± 0.16 N = 8, Poly I:C = 3.82 ± 1.05 N = 6; Mann–Whitney U‐test, *P < 0.05), Tgfb3 (Ctrl = 1.00 ± 0.10 N = 9, Poly I:C = 10.58 ± 6.73 N = 8; Mann–Whitney U‐test, ***P < 0.001); and Foxf2 (Ctrl = 1.00 ± 0.06 N = 5, Poly I:C = 2.60 ± 0.57 N = 8; Mann–Whitney U‐test, *P < 0.05) mRNAs.

  3. C

    qPCR analyses of P90 male cortices for: Pecam1 (Ctrl = 1.00 ± 0.18 N = 8, Poly I:C = 0.85 ± 0.08 N = 8), CD248 (Ctrl = 1.00 ± 0.20 N = 9, Poly I:C = 0.93 ± 0.11 N = 8), Vegfa (Ctrl = 1.00 ± 0.07 N = 8, Poly I:C = 0.75 ± 0.06 N = 8; Mann–Whitney U‐test, *P < 0.05); Pdfgb (Ctrl = 1.00 ± 0.20 N = 8, Poly I:C = 1.11 ± 0.17 N = 4), Tgfb1 (Ctrl = 1.00 ± 0.29 N = 5, Poly I:C = 2.37 ± 0.47 N = 7; Mann–Whitney U‐test, *P < 0.05); Tgfb2 (Ctrl = 1.00 ± 0.29 N = 4, Poly I:C = 1.19 ± 0.26 N = 7), and Tgfb3 (Ctrl = 1.00 ± 0.18 N = 10, Poly I:C = 1.78 ± 0.50 N = 11) mRNAs.

  4. D

    qPCR analyses of GD9 + 6 h female embryos for: Pecam1 (Ctrl = 1.00 ± 0.27 N = 10, Poly I:C = 2.22 ± 0.50 N = 6), CD248 (Ctrl = 1.00 ± 0.27 N = 10, Poly I:C = 2.32 ± 0.57 N = 6; Mann–Whitney U‐test, *P < 0.05), Vegfa (Ctrl = 1.00 ± 0.38 N = 8, Poly I:C = 0.29 ± 0.03 N = 5; Mann–Whitney U‐test, *P < 0.05), Pdfgb (Ctrl = 1.00 ± 0.36 N = 10, Poly I:C = 1.31 ± 0.47 N = 6), Tgfb1 (Ctrl = 1.00 ± 0.16 N = 8, Poly I:C = 1.63 ± 0.42 N = 7), Tgfb2 (Ctrl = 1.00 ± 0.33 N = 9, Poly I:C = 1.75 ± 0.29 N = 6; Mann–Whitney U‐test, *P < 0.05), Tgfb3 (Ctrl = 1.00 ± 0.29 N = 9, Poly I:C = 1.33 ± 0.51 N = 6), and Foxf2 (Ctrl = 1.00 ± 0.28 N = 9, Poly I:C = 1.71 ± 0.52 N = 6) mRNAs.

  5. E

    qPCR analyses of E17 female cortices for: Pecam1 (Ctrl = 1.00 ± 0.34 N = 6, Poly I:C = 0.27 ± 0.08 N = 8), CD248 (Ctrl = 1.00 ± 0.27 N = 8, Poly I:C = 1.04 ± 0.28 N = 9), Vegfa (Ctrl = 1.00 ± 0.70 N = 5, Poly I:C = 0.32 ± 0.13 N = 5), Pdfgb (Ctrl = 1.00 ± 0.38 N = 6, Poly I:C = 0.19 ± 0.07 N = 9), Tgfb1 (Ctrl = 1.00 ± 0.33 N = 5, Poly I:C = 0.09 ± 0.03 N = 6), Tgfb2 (Ctrl = 1.00 ± 0.32 N = 7, Poly I:C = 0.45 ± 0.11 N = 8), Tgfb3 (Ctrl = 1.00 ± 0.38 N = 7, Poly I:C = 0.40 ± 0.20 N = 9), and Foxf2 (Ctrl = 1.00 ± 0.18 N = 5, Poly I:C = 1.04 ± 0.28 N = 8) mRNAs.

  6. F

    qPCR analyses of P90 female cortices for: Pecam1 (Ctrl = 1.00 ± 0.15 N = 5, Poly I:C = 0.69 ± 0.15 N = 5), CD248 (Ctrl = 1.00 ± 0.13 N = 5, Poly I:C = 0.58 ± 0.09 N = 5), Vegfa (Ctrl = 1.00 ± 0.38 N = 6, Poly I:C = 1.53 ± 0.38 N = 8), Pdfgb (Ctrl = 1.00 ± 0.16 N = 9, Poly I:C = 1.77 ± 0.35 N = 17), Tgfb1 (Ctrl = 1.00 ± 0.26 N = 13, Poly I:C = 1.30 ± 0.23 N = 14), Tgfb2 (Ctrl = 1.00 ± 0.27 N = 11, Poly I:C = 0.59 ± 0.06 N = 4), and Tgfb3 (Ctrl = 1.00 ± 0.45 N = 11, Poly I:C = 0.51 ± 0.06 N = 4) mRNAs.

  7. G

    qPCR analyses of GD9 + 6 h male embryos for Il1α (Ctrl = 1.00 ± 0.29 N = 7, Poly I:C = 2.10 ± 0.57 N = 17), Il1β (Ctrl = 1.00 ± 0.39 N = 6, Poly I:C = 10.84 ± 4.70 N = 6; Mann–Whitney U‐test, **P < 0.01), Il1rn (Ctrl = 1.00 ± 0.27 N = 7, Poly I:C = 3.60 ± 1.58 N = 11), and Il6 (Ctrl = 1.00 ± 0.17 N = 6, Poly I:C = 0.82 ± 0.12 N = 7) mRNAs.

  8. H

    qPCR analyses of E17 male cortices for: Il1α (Ctrl = 1.00 ± 0.27 N = 9, Poly I:C = 1.77 ± 0.53 N = 11), Il1β (Ctrl = 1.00 ± 0.21 N = 7, Poly I:C = 1.33 ± 0.33 N = 8), Il1rn (Ctrl = 1.00 ± 0.25 N = 10, Poly I:C = 0.82 ± 0.40 N = 18), and Il6 (Ctrl = 1.00 ± 0.24 N = 7, Poly I:C = 2.17 ± 0.43 N = 8) mRNAs.

  9. I

    qPCR analyses of P90 male cortices for: Il1α (Ctrl = 1.00 ± 0.24 N = 8, Poly I:C = 1.51 ± 0.26 N = 10), Il1β (Ctrl = 1.00 ± 0.27 N = 8, Poly I:C = 1.19 ± 0.34 N = 8), Il1rn (Ctrl = 1.00 ± 0.31 N = 18, Poly I:C = 1.30 ± 0.50 N = 14), and Il6 (Ctrl = 1.00 ± 0.27 N = 7, Poly I:C = 2.40 ± 0.70 N = 8) mRNAs.

  10. J

    qPCR analyses of GD9 + 6 h female embryos for: Il1α (Ctrl = 1.00 ± 0.93 N = 6, Poly I:C = 0.03 ± 0.00 N = 4), Il1β (Ctrl = 1.00 ± 0.61 N = 5, Poly I:C = 0.17 ± 0.04 N = 7), Il1rn (Ctrl = 1.00 ± 0.25 N = 9, Poly I:C = 0.33 ± 0.11 N = 7), and Il6 (Ctrl = 1.00 ± 0.48 N = 7, Poly I:C = 0.59 ± 0.24 N = 5) mRNAs.

  11. K

    qPCR analyses of E17 female cortices for: Il1α (Ctrl = 1.00 ± 0.31 N = 5, Poly I:C = 1.38 ± 0.20 N = 12), Il1β (Ctrl = 1.00 ± 0.30 N = 5, Poly I:C = 1.05 ± 0.35 N = 7), Il1rn (Ctrl = 1.00 ± 0.55 N = 6, Poly I:C = 0.45 ± 0.22 N = 6), and Il6 (Ctrl = 1.00 ± 0.26 N = 7, Poly I:C = 0.94 ± 0.14 N = 8) mRNAs.

  12. L

    qPCR analyses of P90 female cortices for Il1α (Ctrl = 1.00 ± 0.08 N = 5, Poly I:C = 1.32 ± 0.11 N = 9), Il1β (Ctrl = 1.00 ± 0.13 N = 5, Poly I:C = 0.65 ± 0.09 N = 8), Il1rn (Ctrl = 1.00 ± 0.61 N = 4, Poly I:C = 0.85 ± 0.29 N = 7), and Il6 (Ctrl = 1.00 ± 0.11 N = 9, Poly I:C = 1.28 ± 0.20 N = 20) mRNAs. Values are normalized over control (dashed line).

  13. M

    ELISA quantification of total TGF‐β1 in E17 male embryos brains as pg/ml (Ctrl = 132.6 ± 18.14 and Poly I:C = 129.6 ± 8.62).

  14. N

    ELISA quantification of active free TGF‐β1 in E17 male embryos brains as pg/ml (Ctrl = undetectable and Poly I:C = 0.92 ± 0.25).

  15. O

    ELISA quantification of total TGF‐β1 in E17 female embryos brains as pg/ml (Ctrl = 177.6 ± 10.88 and Poly I:C = 134.4 ± 13.59).

  16. P

    ELISA quantification of active free TGF‐β1 in E17 female embryos brains as pg/ml (undetectable).

  17. Q–S

    Western Blotting quantification of SMAD proteins in E17 male cortices for (Q) pSMAD1‐5 (Ctrl = 0.99 ± 0.21, Poly I:C = 2.67 ± 0.46); Mann–Whitney U‐test, *P < 0.05, (R) pSMAD2 (Ctrl = 1.00 ± 0.04, Poly I:C = 0.88 ± 0.24), and (S) SMAD4 (Ctrl = 1.00 ± 0.12, Poly I:C = 0.96 ± 0.21). Protein levels were normalized to GAPDH.

  18. T–V

    Western blotting quantification of SMAD proteins in E17 female cortices for (T) pSMAD1‐5 (Ctrl = 1.00 ± 0.02, Poly I:C = 0.70 ± 0.18), (U) pSMAD2 (Ctrl = 1.00 ± 0.01, Poly I:C = 1.09 ± 0.12), and (V) SMAD4 (Ctrl = 1.00 ± 0.16, Poly I:C = 1.46 ± 0.57). Protein levels were normalized to GAPDH.

Data information: Ctrl males = white bars with blue border. Poly I:C males = blue bars with blue border. Ctrl females = white bars with purple border. Poly I:C females = purple bars with purple border. Numbers in bars indicate the number of animals (N). Bars represent mean ± SEM. Source data are available online for this figure.
Figure 3
Figure 3. TGF‐β1 dampens the expression of CD146 and claudin‐5 and reduces the tightness of in vitro barrier models—independently of the sex

  1. 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), and female Poly I:C (1.00 ± 0.07) offspring. Data are normalized to male Ctrl.

  2. 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), and female Poly I:C (0.95 ± 0.01) offspring. Mann–Whitney U‐test, *P < 0.05. Data are normalized to male Ctrl.

  3. 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), and female Poly I:C (1.15 ± 0.08) offspring. Mann–Whitney U‐test, *P < 0.05. Data are normalized to bEnd.3.

  4. 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), and female Poly I:C (0.65 ± 0.05) offspring. One sample t‐test, **P < 0.01. Data are normalized to bEnd.3.

  5. TEER (Ω.cm2) was monitored over 72 h treatment with TGF‐β1 and normalized to baseline in BBB models composed by a monolayer of sorted ECs from male (72 h TGF‐β1 = 82.85 ± 1.56) or female (72 h TGF‐β1 = 85.92 ± 2.03) embryos. The insert shows the values at 72 h (bars) normalized to the 0 h values (dashed line). One sample t‐test, ***P < 0.001 and ****P < 0.0001.

  6. TEER (Ω.cm2) was monitored over 72 h treatment with TGF‐β1 and normalized to baseline in barrier models composed by a monolayer of bEND.3 alone (72 h TGF‐β1 = 89.85 ± 2.05) or co‐cultured with sorted PCs from male (72 h TGF‐β1 = 88.83 ± 1.93) or female (72 h TGF‐β1 = 89.02 ± 2.22) embryos. The insert shows the values at 72 h (bars) normalized to the 0 h values (dashed line). Wilcoxon signed‐rank test, *P < 0.05.

  7. 10 kDa dextran permeability after 72 h 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), and female TGF‐β1 (0.47 ± 0.01).

  8. 10 kDa dextran permeability 72 h 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 < 0.05.

  9. 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 < 0.05) and representative histogram of untreated (solid gray) and TGF‐β1‐treated cells (hollow black bold line).

  10. 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 < 0.01) and representative histogram of untreated (solid gray) and TGF‐β1‐treated cells (hollow black bold line).

  11. 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.

  12. Quantitative analysis of CD146 immunofluorescence mean intensity measured in primary male brain EC monolayers under control conditions or after 72 h treatments with different cytokines: NT = 100.00 ± 6.55, TGF‐β1 = 94.03 ± 2.01, IL‐1β + TGF‐β1 = 84.87 ± 4.59, and IL‐1β = 92.34 ± 2.65; Kruskal–Wallis, *P < 0.05.

  13. Quantitative analysis of claudin‐5 immunofluorescence mean intensity in primary male brain EC monolayers under control conditions or after 72 h treatments with different cytokines: NT = 99.76 ± 5.19, TGF‐β1 = 86.74 ± 6.76, IL‐1β + TGF‐β1 = 51.64 ± 3.31, and IL‐1β = 107.3 ± 7.37; ordinary one‐way ANOVA, **P < 0.01, ****P < 0.0001.

  14. Quantitative analysis of claudin‐5 immunofluorescence area (μm2) in primary male brain EC monolayers under control conditions or after 72 h treatments with different cytokines: NT = 39,781 ± 2,342, TGF‐β1 = 31,707 ± 5,939, IL‐1β + TGF‐β1 = 5,415 ± 1,037, and IL‐1β = 46,303 ± 2,986; ordinary one‐way ANOVA, *P < 0.05, **P < 0.01, ****P < 0.0001.

  15. Graph showing TEER measured by volt ohmmeter at 72 h 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 < 0.01. Data are normalized to t0.

  16. Permeability to 3 kDa 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 < 0.05.

  17. Permeability to 10 kDa 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 < 0.05.

Data information: A‐H. 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. K‐Q. Cortex‐derived ECs isolated from male mice at 2 months were used to build in vitro barrier models and analyzed. bEnd.3 cells = bars with black border. Cells derived from Ctrl males = white bars with blue border. Cells derived from Poly I:C males = blue bars with blue border. Cells derived from Ctrl females = white bars with purple border. Cells derived from Poly I:C females = purple bars with purple border. In Fig 3E and F, blue and purple dotted lines represent cells derived from male and female brains, respectively. The black dotted line represents bEnd.3 cultures. Numbers in bars indicate the number of animals (N) or independent cultures/pictures. Bars represent mean ± SEM. Source data are available online for this figure.
Figure 4
Figure 4. Maternal immune activation induces sex‐specific alterations of the neurovascular unit changing the ratio between ECs and PCs

  1. [left] Representative dot plots showing the gating strategy for E17 samples to distinguish NVU cells. The cells were pre‐gated on single cells and live cells (Zombie Aqua negative). Live cells were pre‐gated on CD45 (CD45−). [right] Brain pericytes (PC) were defined as CD146+CD31− cells and brain endothelial cells (EC) as CD31+cells.

  2. [left] Quantification of the ratio between PC number and EC numbers for male (blue) and female (purple) E17 offspring cortex samples. Male Ctrl = 1.10 ± 0.08, male Poly I:C = 1.34 ± 0.09, female Ctrl = 1.02 ± 0.08, and female Poly I:C = 1.12 ± 0.09. Mann–Whitney U‐test, *P < 0.05. Bars represent average ratio of individual embryos ± SEM, and numbers in bars indicate the number of embryos. [right up] Percentages of ECs in male Ctrl (0.79 ± 0.05), male Poly I:C (0.90 ± 0.09), female Ctrl (0.65 ± 0.07), and female Poly I:C (0.80 ± 0.07) samples, and [right down] PCs in male Ctrl (0.60 ± 0.05), male Poly I:C (0.57 ± 0.06), female Ctrl (0.53 ± 0.07), and female Poly I:C (0.62 ± 0.06) samples.

  3. [left] Gating strategy used to analyze P30 samples: single cells, live (Zombie Aqua negative), and CD45− cells. [right] PCs are identified as CD146+CD13+CD31−; and ECs are CD13−CD31+.

  4. [left] Quantification of EC/PC number ratio for male and female P30 offspring cortex samples, obtained by manual gating. Male Ctrl = 1.78 ± 0.29, male Poly I:C = 1.74 ± 0.32, female Ctrl = 1.49 ± 0.11, and female Poly I:C = 1.60 ± 0.19. [right up] Percentages of ECs in male Ctrl (17.07 ± 1.88), male Poly I:C (17.52 ± 1.90), female Ctrl (14.58 ± 0.66), and female Poly I:C (15.73 ± 1.07) samples, and [right down] percentage of PCs in male Ctrl (10.55 ± 1.89), male Poly I:C (10.67 ± 1.04), female Ctrl (10.07 ± 0.85), and female Poly I:C (10.20 ± 0.79).

  5. Normalized percentage of live (CD45−) EdU‐positive cells at E17 in male Ctrl (1.00 ± 0.05), male Poly I:C (0.62 ± 0.11), female Ctrl (0.61 ± 0.04), and female Poly I:C (0.59 ± 0.11) samples. Mann–Whitney U‐test and Kruskal–Wallis test, *P < 0.05, **P < 0.001.

  6. Percentage of PCs EdU‐positive cells at E17 in male Ctrl (7.88 ± 1.57), male Poly I:C (4.55 ± 0.70), female Ctrl (7.03 ± 1.04), and female Poly I:C (4.93 ± 0.83) samples. Student's t‐test, *P < 0.05.

  7. Percentage of ECs EdU‐positive cells at E17 in male Ctrl (4.30 ± 0.71), male Poly I:C (2.80 ± 0.53), female Ctrl (2.84 ± 0.43), and female Poly I:C (2.29 ± 0.77) samples.

  8. Normalized percentage of live (CD45−) EdU‐positive cells at P30 in male Ctrl (1.16 ± 0.14), male Poly I:C (0.66 ± 0.16), female Ctrl (1.00 ± 0.08), and female Poly I:C (0.91 ± 0.13) samples. Mann–Whitney U‐test, *P < 0.05.

  9. Percentage of PCs EdU‐positive cells at P30 in male Ctrl (0.80 ± 0.24), male Poly I:C (0.24 ± 0.06), female Ctrl (0.26 ± 0.08), and female Poly I:C (0.38 ± 0.13) samples. Mann–Whitney U‐test, *P < 0.05.

  10. Percentage of ECs EdU‐positive cells at P30 in male Ctrl (0.58 ± 0.10), male Poly I:C (0.44 ± 0.19), female Ctrl (0.38 ± 0.22), and female Poly I:C (0.25 ± 0.06) samples.

  11. [left] CD146‐normalized MFI of the EC cells at P30 in male Ctrl (100.00 ± 8.27), male Poly I:C (65.73 ± 3.63), female Ctrl (57.08 ± 14.10), and female Poly I:C (43.07 ± 8.34). Mann–Whitney U‐test and ordinary one‐way ANOVA, **P < 0.01. [right] Representative histograms CD146 expression on ECs in male Ctrl (blue hollow), male Poly I:C (solid light blue), female Ctrl (purple hollow), and female Poly I:C (solid purple) samples.

  12. Percentages of PC CD146++, CD146+, and CD146− cells, respectively, at P30 in male Ctrl (26.32/36.75/23.48 ± 2.84/3.31/4.62), male Poly I:C (27.53/42.40/20.60 ± 2.25/0.58/1.94), female Ctrl (29.05/40.98/2.19 ± 1.99/1.32/2.19), and female Poly I:C (27.17/46.60/17.10 ± 3.09/3.86/1.96) samples.

  13. [left] CD105‐normalized MFI of the EC cells at P30 in male Ctrl (100.00 ± 7.39), male Poly I:C (99.58 ± 6.23), female Ctrl (100.30 ± 1.61), and female Poly I:C (96.67 ± 3.72). [right] Representative histograms indicating mean fluorescence intensity (MFI) of CD105 on EC in male Ctrl (blue hollow), male Poly I:C (solid light blue), female Ctrl (purple hollow), and female Poly I:C (solid purple) samples.

  14. [left] Quantitative data of the normalized MFI of CD105 in PCs at P30 in male Ctrl (100.00 ± 3.43), male Poly I:C (98.01 ± 5.14), female Ctrl (98.57 ± 4.16), and female Poly I:C (87.99 ± 4.21). Mann–Whitney U‐test, *P < 0.05. [right] Representative histograms for CD105 expression on PCs in male Ctrl (blue hollow), male Poly I:C (solid light blue), female Ctrl (purple hollow), and female Poly I:C (solid purple) samples.

  15. [top] Representative images of CD13 (red) positive pericytes covering lectin‐positive vessels (green) in sections of P90 untreated male brains. [bottom] 3D rendering, scale bar = 20 μm, and enlargement scale bar = 10 μm.

  16. Percentage of CD13‐positive pericyte coverage at P90 in male Ctrl (53.42 ± 9.22) and Poly I:C (27.73 ± 5.29). Student's t‐test, *P < 0.05.

  17. Percentage of PDGFrB‐positive pericyte coverage at P90 in male Ctrl (66.15 ± 3.09) and Poly I:C (55.08 ± 3.10). Student's t‐test, *P < 0.05.

  18. Representative images of PDGFrB (red) and NG2 (white)‐positive pericytes covering lectin‐positive vessels (blue) in sections of P90 untreated male brains. Scale bar = 10 μm and enlargement scale bar = 5 μm.

  19. Percentage of CD13‐positive pericyte coverage at P90 in female Ctrl (71.57 ± 8.37) and Poly I:C (69.13 ± 6.63).

  20. Percentage of PDGFrB‐positive pericyte coverage at P90 in female Ctrl (66.77 ± 11.76) and Poly I:C (70.56 ± 8.32).

Data information: Embryos prenatally exposed to vehicle (Ctrl) or Poly I:C on GD9 were dissected, and cortex single‐cell suspension was analyzed by multi‐color flow cytometry. Ctrl males = white bars with blue border. Poly I:C males = blue bars with blue border. Ctrl females = white bars with purple border. Poly I:C females = purple bars with purple border. CD13‐positive pericytes were represented ad CD146‐ (dark green bars), CD146+ (green bars), and CD46++ (light green bars). Bars represent mean ± SEM, and numbers in bars indicate the number of animals. Source data are available online for this figure.
Figure 5
Figure 5. MIA‐Poly I:C causes acute and chronic intracerebral bleedings

  1. Representative images of brain acute bleedings detected through hemoglobin IHC in P30 Ctrl and Poly I:C MIA male offspring. Scale bar = 500 μm and enlargement scale bar = 50 μm.

  2. Representative images of brain acute bleedings detected through hemoglobin IHC in P90 Ctrl and Poly I:C MIA male offspring. Scale bar = 500 μm and enlargement scale bar = 50 μm.

  3. Quantification of hemoglobin (Hb)‐positive signal in the brain of P30 and P90 male MIA offspring, expressed as a percentage of the area. P30 Ctrl = 0.00 ± 0.00, P30 Poly I:C = 0.01 ± 0.00, P90 Ctrl = 0.00 ± 0.00, and P90 Poly I:C = 0.01 ± 0.00. Mann–Whitney U‐test, ***P < 0.001.

  4. Representative 3D rendering of iron deposits detected by DAB‐enhanced Perls staining in P30 Ctrl and Poly I:C MIA male offspring brains. Brain volume is rendered gold transparent with a positive signal for the staining color coded by surface dimension. Scale bar = 100 μm.

  5. Quantification of DAB‐enhanced histochemical iron stains in the brain of P30 Ctrl (0.00 ± 0.00) and Poly I:C (0.01 ± 0.00) male MIA offspring, expressed as Perls‐positive percentage of the volume. Mann–Whitney U‐test, *P < 0.05.

  6. Poisson distribution R‐value of Perls/DAB‐positive staining of P30 male MIA offspring, respectively, in the coronal (Ctrl = 1.19 ± 0.04, Poly I:C = 0.94 ± 0.01; Mann–Whitney U‐test, **P < 0.01), axial (Ctrl = 1.16 ± 0.08, Poly I:C = 1.03 ± 0.03), and sagittal (Ctrl = 1.09 ± 0.09, Poly I:C = 1.01 ± 0.05) planes.

  7. Representative images of iron deposits detected through Perls staining in P90 Ctrl and Poly I:C MIA male offspring brains. Scale bar = 500 μm and enlargement scale bar = 100 μm.

  8. Quantification of Perls‐positive staining in the brain of P90 Ctrl (0.25 ± 0.05) and Poly I:C (0.59 ± 0.10) male MIA offspring, expressed as a percentage of the area. Mann–Whitney U‐test, **P < 0.01.

  9. Representative image of marble‐burying test at 0 and 30 min. Fifteen‐glass marbles were evenly spaced on a 5 × 3 grid. Marbles buried by two‐thirds at least are indicated by a red circle or by a cross within a red circle when fully covered by the sawdust.

  10. Quantification of buried marble (n°) by P90 Ctrl male (3.33 ± 1.67), Poly I:C male (5.72 ± 2.10), Ctrl female (3.83 ± 1.72), and Poly I:C female (3.00 ± 2.00) mice. Student's t‐test, **P < 0.01.

  11. Quantification of self‐grooming time (sec) by P90 Ctrl males (19.34 ± 13.00), Poly I:C males (43.38 ± 35.21), Ctrl females (19.86 ± 18.06), and Poly I:C females (16.93 ± 11.19) mice. Student's t‐test, *P < 0.05.

Data information: Ctrl males = white bars with blue border. Poly I:C males = blue bars with blue border. Ctrl females = white bars with purple border. Poly I:C females = purple bars with purple border. Numbers in bars indicate the number of animals (N) and slices (n). Bars represent mean ± SEM. Source data are available online for this figure.
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