Research ArticleCEREBRAL CAVERNOUS MALFORMATION

Distinct cellular roles for PDCD10 define a gut-brain axis in cerebral cavernous malformation

See allHide authors and affiliations

Science Translational Medicine  27 Nov 2019:
Vol. 11, Issue 520, eaaw3521
DOI: 10.1126/scitranslmed.aaw3521
  • Fig. 1 Comparison of PDCD10, KRIT1, and CCM2 deficiency states in mice and humans.

    (A) CCM lesion burden in iBECre;Pdcd10fl/fl and iBECre;Pdcd10fl/fl Map3k3fl/+ mice at P10, assessed visually (left) and using microCT imaging (right). Scale bars, 1 mm. (B) Blinded microCT quantitation of CCM lesion burden in P10 iBECre;Pdcd10fl/fl and iBECre;Pdcd10fl/fl;Map3k3fl/+ littermates. n > 10 animals per genotype and 4 distinct litters. (C) Principle coordinates analysis (PCoA) of weighted UniFrac bacterial composition distances between the feces of individuals with familial CCM disease associated with mutations in KRIT1, CCM2, and PDCD10, individuals with sporadic CCM disease, and age/sex-matched healthy controls. P value compares bacterial compositions between healthy and CCM disease groups using PERMANOVA. (D) Relative abundance boxplots of Gram-negative Bacteroides in individuals with CCM disease and healthy controls. Significance (false discovery rate, q) determined by linear models of logit-transformed relative abundances with Benjamini-Hochberg correction for multiple comparisons. (E) PCoA of weighted UniFrac bacterial composition distances between the feces of only individuals with genotyped mutations in KRIT1, CCM2, and PDCD10. P values compare bacterial compositions in all groups using PERMANOVA. (F) Relative abundance boxplots of Gram-negative Bacteroides comparing genotyped familial KRIT1, CCM2, or PDCD10 patients with sporadic cases or healthy volunteers. Significance was determined by pairwise testing of estimated marginal means from a linear model comparing the genetic categories of CCM disease, controlling for age and sex. All associated P > 0.5 and not significant (n.s.). Error bars shown as SEM and significance (B) determined by unpaired, two-tailed Student’s t test. ***P < 0.001; n.s., P > 0.05.

  • Fig. 2 Effect of dextran sulfate sodium–induced colitis on CCM formation.

    (A) Schematic of the experimental design in which littermates receive an injection of tamoxifen 1 day after birth (P1), daily gavage of dextran sulfate sodium (DSS) or vehicle from P10 to P15, and tissue harvest at P21. 4OHT, 4-hydroxytamoxifen. (B) DSS-treated iECre;Krit1fl/fl mice exhibit colitis of varying severity associated with thinned mucus layer (red bracket), crypt dilation and abscesses (red arrows), and an inflamed (middle) or entirely eroded (right) epithelium. Scale bars, 100 μm. Results are representative of n > 3 animals per treatment group. H&E, hematoxylin and eosin. (C) CCM lesion burden in susceptible iECre;Krit1fl/fl mice was measured after DSS or vehicle exposure assessed visually (left) and using microCT imaging (right). Scale bars, 1 mm. (D) Blinded microCT quantitation of CCM lesion burden in P21 iECre;Krit1fl/fl littermates after gavage with vehicle or DSS. n > 18 per treatment group and 8 distinct litters. (E) CCMs form in resistant iECre;Ccm2fl/fl mice after DSS exposure. CCMs were assessed visually (left) and using microCT imaging (right). Scale bars, 1 mm. (F) Blinded microCT quantitation of CCM lesion burden in resistant P21 iECre;Ccm2fl/fl littermates after gavage with vehicle or DSS. n ≥ 8 per treatment group and 4 distinct litters. Error bars are shown as SEM, and significance is determined by unpaired, two-tailed Welch’s t test (D) or unpaired, two-tailed Student’s t test (F). **P < 0.01.

  • Fig. 3 Effect of brain endothelial and gut epithelial loss of PDCD10 or KRIT1 on CCM formation.

    (A) CCM formation in iBECre;Pdcd10fl/fl and iBECre;VilCre;Pdcd10fl/fl littermates assessed visually (left and middle) and using microCT imaging (right). Scale bars, 1 mm. (B) Blinded microCT quantitation of CCM lesion burden in P21 iBECre;Pdcd10fl/fl and iBECre;VilCre;Pdcd10fl/fl littermates. n ≥ 19 animals per genotype and 10 distinct litters. (C) CCM formation in iECre;Krit1fl/fl and iECre;VilCre;Krit1fl/fl littermates was assessed visually (left and middle) and using microCT imaging (right). Scale bars, 1 mm. (D) Blinded microCT quantitation of CCM lesion burden in P21 iECre;Krit1fl/fl and iECre;VilCre;Krit1fl/fl littermates. n ≥ 16 animals per genotype and 7 distinct litters. (E to G) H&E staining of hindbrain sections from P21 iBECre;VilCre;Pdcd10fl/fl, iBECre;Pdcd10fl/fl, and iBECre;VilCre;Pdcd10fl/+ littermates. Arrows indicate extravascular hemorrhage that was observed in iBECre;VilCre;Pdcd10fl/fl animals. Boxes in left images denote area of magnified image on the right. Scale bars, 500 μm (left images) and 100 μm (right images). (H and I) H&E staining of hindbrain sections from P21 iECre;Krit1fl/fl and iECre;VilCre;Krit1fl/fl littermates. Boxes in left images denote area of magnified image on the right. Scale bars, 500 μm (left images) and 100 μm (right images). Results are representative of n ≥ 3 animals per genotype and 4 distinct litters. Error bars are shown as SEM, and significance is determined by unpaired, two-tailed Welch’s t test. ***P < 0.001; n.s., P > 0.05.

  • Fig. 4 Gut barrier and blood TLR4 ligand after gut epithelial loss of PDCD10 or KRIT1.

    (A and B) H&E histology of methacarn-fixed colons from iBECre;Pdcd10fl/fl, iBECre;VilCre;Pdcd10fl/+, and iBECre;VilCre;Pdcd10fl/fl animals (A) or iECre;Krit1fl/fl and iECre;VilCre;Krit1fl/fl animals (B) at P21. Low-magnification images of the colonic fecal pellet (top) and higher-magnification images of the colonic crypts (bottom). The brains of these animals with CCM lesions are shown in Fig. 3. Brackets denote the thickness of the mucus layer. Arrows indicate goblet cells. Asterisks highlight colonic crypts. Scale bars, 100 μm (top) and 50 μm (bottom). Results are representative of n ≥ 16 animals per genotype and at least 7 distinct litters. (C and D) Quantitation of colonic mucus layer from the indicated H&E-stained, methacarn-fixed, tissue sections in (A) and (B). Each point represents measurements around one fecal pellet, n ≥ 16 animals per genotype and at least 7 distinct litters. (E) Fecal lipocalin-2 (LCN2) concentration measured by enzyme-linked immunosorbent assay (ELISA) in VilCre;Pdcd10fl/fl, VilCre;Pdcd10fl/+, or littermate controls (Pdcd10fl/+ or fl/fl). n ≥ 12 animals per genotype from 4 distinct litters. (F) Fecal LCN2 concentration measured by ELISA in VilCre;Krit1fl/fl and littermate Krit1fl/fl animals. n ≥ 11 animals per genotype and 3 distinct litters. (G and H) Immunostaining for the neutrophil marker Ly6G is shown for P21 colons from Pdcd10fl/fl, VilCre;Pdcd10fl/+, and VilCre;Pdcd10fl/fl littermates as well as Krit1fl/fl and VilCre;Krit1fl/fl littermates. Scale bars, 50 μm. (I and J) Quantification of Ly6G-positive crypt abscesses per colonic section analyzed. Each point represents a distinct animal. Results are representative of n ≥ 4 animals per genotype and at least 3 distinct litters. (K and L) TLR4 agonist activity detected in the blood of VilCre;Pdcd10fl/fl animals and VilCre;Krit1fl/fl animals. n ≥ 13 animals per genotype and at least 7 distinct litters. Error bars are shown as SEM, and significance is determined by Kruskal-Wallis one-way ANOVA with Dunn’s correction for multiple comparisons (C, E, and I) or unpaired, two-tailed Student’s t test (D, F, and J). ****P < 0.0001, **P < 0.01; n.s., P > 0.05.

  • Fig. 5 Relationship of the colonic mucus barrier and CCM formation.

    (A) H&E staining of methacarn-fixed colon samples from iBECre;Pdcd10fl/fl, iBECre;Pdcd10fl/fl;Muc2+/−, and iBECre;Pdcd10fl/fl;Muc2−/− animals. The mucus barrier is indicated with brackets. Scale bars, 100 μm. (B) Quantitation of the area of the mucus layers shown in (A). Each point represents measurement around one fecal pellet. n ≥ 10 animals per genotype and 9 distinct litters. (C) CCM formation in representative P21 iBECre;Pdcd10fl/fl, iBECre;Pdcd10fl/fl;Muc2+/−, and iBECre;Pdcd10fl/fl;Muc2−/− animals is shown visually in the hindbrain (top), hindbrain and forebrain (middle), and with microCT imaging (bottom). Scale bars, 1 mm. (D) Blinded microCT quantification of CCM lesion volumes in P21 iBECre;Pdcd10fl/fl, iBECre;Pdcd10fl/fl;Muc2+/−, and iBECre;Pdcd10fl/fl;Muc2−/− littermates. n ≥ 25 animals per genotype and 17 distinct litters. Error bars are shown as SEM, and significance is determined by Kruskal-Wallis one-way ANOVA with Dunn’s correction for multiple comparisons (B and D). ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.

  • Fig. 6 Analysis of the gut microbiome after loss of gut epithelial PDCD10 or MUC2.

    (A) Experimental design in which females were mated, individually housed before natural delivery of pups, and fecal pellets collected from each pup at P21 before weaning for 16S rRNA bacterial gene sequencing. (B to E) PCoA of weighted UniFrac bacterial composition distances from the feces of the indicated animals. Each box shows analysis of littermate animals while accounting for caging differences. P values compare bacterial compositions in the indicated groups using PERMANOVA also considering individual cage differences and corrected for multiple comparisons using the Bonferroni method. (B to D) n ≥ 10 animals per genotype and at least 4 distinct cages/litters. Overall P values comparing homozygous animals to wild-type or heterozygous controls are shown in plot, as well as the pairwise comparisons (q values) in legend. (F) Mean difference of logit-transformed relative abundance of the indicated bacteria taxa estimated with linear mixed-effects models in VilCre;Krit1fl/fl, VilCre;Pdcd10fl/fl, iBECre;VilCre;Pdcd10fl/fl, and iBECre;Pdcd10fl/fl;Muc2−/− animals compared to their respective VilCre-negative or Muc2+/+ littermate controls. (G) Mean difference of logit-transformed relative abundance of the indicated bacteria taxa estimated with linear mixed-effects models in VilCre;Pdcd10fl/+, iBECre;VilCre;Pdcd10fl/+, and iBECre;Pdcd10fl/fl;Muc2+/− animals compared to their respective VilCre-negative or Muc2+/+ littermate controls. Significance (false discovery rate, q) determined by linear mixed-effects models with Benjamini-Hochberg correction for multiple comparisons.

  • Fig. 7 Effects of chronic P80 dietary emulsifier intake on the colonic mucosal barrier and CCM formation.

    (A) H&E staining of methacarn-fixed colon samples from vehicle and Pdcd10+/− animals administered 1% P80 in drinking water for 2 weeks. The mucus barrier is indicated with brackets. Scale bars, 100 μm. (B) Quantitation of the area of the mucus layers shown in (A). Each point represents measurement around one fecal pellet. n = 7 animals per genotype. (C and D) PCoA of unweighted and weighted UniFrac bacterial composition distances from the feces of the indicated animals administered 1% P80 for 4 months is shown. P values compare bacterial compositions in the indicated groups using PERMANOVA also considering individual cage differences and corrected for multiple comparisons using the Bonferroni method. n = 12 animals per genotype and 5 distinct cages per treatment. (E to G) Spontaneous CCM formation was analyzed using microCT imaging (E and F) and lesion volume measurement normalized to total brain volume (G) in animals of the indicated genotypes administered 1% P80 for 4 months. n ≥ 16 animals per treatment group. Error bars are shown as SEM, and significance is determined by Kruskal-Wallis one-way ANOVA with Dunn’s correction for multiple comparisons (B and D) or Mann-Whitney nonparametric U test (G). ****P < 0.0001, **P < 0.01, *P < 0.05.

  • Fig. 8 Demonstration of a therapeutic gut-brain axis using dexamethasone.

    (A) Visual assessment of CCM formation in 10-day-old (P10) iECre;Krit1fl/fl littermates treated at P5 with vehicle or the indicated doses of dexamethasone. A dose of 0.8 mg/kg was used for all following experiments. Scale bars, 1 mm. Images are representative of n ≥ 3 animals per treatment group from at least 2 distinct litters. (B) Visual assessment of CCM formation in iECre;Krit1fl/fl littermates treated with vehicle or dexamethasone. Scale bars, 1 mm. (C) Blinded microCT quantitation of CCM lesion burden in P10 iCre;Krit1fl/fl littermates treated with vehicle or dexamethasone. n ≥ 8 animals per treatment group and 3 distinct litters. (D) Relative amounts of Krit1, Map3k3, Klf2, Klf4, Tlr4, and Cd14 expression in cerebellar endothelial cells freshly isolated from P10 iECre;Krit1fl/fl littermates treated with P5 vehicle or dexamethasone. n ≥ 4 animals per treatment group and 3 distinct litters. (E) Visual assessment of CCM formation in iECre;Krit1fl/fl;Nr3c1fl/fl littermates treated with vehicle or dexamethasone. Scale bars, 1 mm. Results are representative of n ≥ 5 animals per group and 5 independent experiments. (F) Blinded microCT quantitation of CCM lesion burden in P10 iECre;Krit1fl/fl;Nr3c1fl/fl littermates treated with vehicle or dexamethasone. n ≥ 8 animals per treatment group and 3 distinct litters. (G) H&E staining of P10 colon sections from iECre;Krit1fl/fl littermates treated with P5 vehicle or dexamethasone. Note the goblet cells in the dexamethasone-treated colon (arrows). Scale bars, 50 μm. (H) Immunoblot analysis of MUC2 expression in P10 colons from iECre;Krit1fl/fl littermates treated with P5 vehicle or dexamethasone (top; anti-MUC2). Total protein loading detected using REVERT is shown below. Molecular weight markers (in kilodalton) on left. Each lane is whole colonic lysate from one distinct animal. Results are representative of n ≥ 5 animals per group and 2 distinct litters. (I and J) Visual assessment of CCM formation in iBECre;Pdcd10fl/fl;Muc2+/− (I) and iBECre;Pdcd10fl/fl;Muc2−/− (J) animals treated with P5 vehicle or dexamethasone. Scale bars, 1 mm. (K) Blinded microCT quantitation of CCM lesion burden in P10 iBECre;Pdcd10fl/fl;Muc2+/− and iBECre;Pdcd10fl/fl;Muc2−/− littermates treated with vehicle or dexamethasone. n ≥ 6 animals per treatment group and ≥3 distinct litters. (L and M) Visual assessment of CCM formation in iECre;Krit1fl/fl;Nr3c1fl/fl (L) and iECre;VilCre;Krit1fl/fl;Nr3c1fl/fl (M) littermates at P10 after treatment with P5 vehicle or dexamethasone. Scale bars, 1 mm. (N) Blinded microCT quantitation of CCM lesion burden in P10 iECre;Krit1fl/fl;Nr3c1fl/fl and iECre;VilCre;Krit1fl/fl;Nr3c1fl/fl littermates treated with vehicle or dexamethasone. n ≥ 8 animals per treatment group and 3 distinct litters. Error bars are shown as SEM, and significance is determined by unpaired, two-tailed Student’s t test. ****P < 0.0001, ***P < 0.001, **P < 0.01.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/520/eaaw3521/DC1

    Materials and Methods

    Fig. S1. PDCD10 deficiency in brain endothelial cell signaling in mice and the gut microbiome in humans compared to KRIT1 and CCM2.

    Fig. S2. Single-cell RNA sequencing of the intestinal epithelium shows broad expression of Krit1, Ccm2, and Pdcd10 across cell types.

    Fig. S3. Analysis of the hindbrain upon deletion of Pdcd10 solely in IECs.

    Fig. S4. Quantification of the colonic mucus layer.

    Fig. S5. Gut epithelial loss of CCM2 or MAP3K3 and subsequent effects on the colonic mucus barrier and fecal LCN2.

    Fig. S6. Measurement of Krit1, Ccm2, and Pdcd10 mRNA in the colonic epithelium.

    Fig. S7. Analysis of goblet cell numbers, MUC2 expression, and colonic crypt morphology after intestine epithelial deletion of Krit1, Ccm2, or Pdcd10.

    Fig. S8. E-cadherin (CDH1) expression and localization in colons lacking PDCD10, KRIT1, or MUC2.

    Fig. S9. EpCAM expression and localization in colons lacking PDCD10, KRIT1, or MUC2.

    Fig. S10. Intestinal junctions in PDCD10-deficient embryonic zebrafish.

    Fig. S11. Heatmaps of 16S rRNA bacterial gene sequencing results for each animal by cage/litter.

    Fig. S12. Unweighted principal component plots of 16S rRNA bacterial gene sequencing.

    Fig. S13. Relative abundance boxplots of changed bacterial taxa as detected by 16S rRNA bacterial gene sequencing.

    Fig. S14. Heatmaps of 16S rRNA bacterial gene sequencing results for the P80 emulsifier experiment by treatment group and cage.

    Fig. S15. Dexamethasone effects on CCMs and colonic goblet cells.

    Fig. S16. Model of the role of PDCD10 in the gut epithelium and its effect on CCM formation in the brain.

    Table S1. Clinical characteristics of patients with CCM recruited to participate in gut microbiome study.

    Data file S1. Raw data.

    References (5569)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. PDCD10 deficiency in brain endothelial cell signaling in mice and the gut microbiome in humans compared to KRIT1 and CCM2.
    • Fig. S2. Single-cell RNA sequencing of the intestinal epithelium shows broad expression of Krit1, Ccm2, and Pdcd10 across cell types.
    • Fig. S3. Analysis of the hindbrain upon deletion of Pdcd10 solely in IECs.
    • Fig. S4. Quantification of the colonic mucus layer.
    • Fig. S5. Gut epithelial loss of CCM2 or MAP3K3 and subsequent effects on the colonic mucus barrier and fecal LCN2.
    • Fig. S6. Measurement of Krit1, Ccm2, and Pdcd10 mRNA in the colonic epithelium.
    • Fig. S7. Analysis of goblet cell numbers, MUC2 expression, and colonic crypt morphology after intestine epithelial deletion of Krit1, Ccm2, or Pdcd10.
    • Fig. S8. E-cadherin (CDH1) expression and localization in colons lacking PDCD10, KRIT1, or MUC2.
    • Fig. S9. EpCAM expression and localization in colons lacking PDCD10, KRIT1, or MUC2.
    • Fig. S10. Intestinal junctions in PDCD10-deficient embryonic zebrafish.
    • Fig. S11. Heatmaps of 16S rRNA bacterial gene sequencing results for each animal by cage/litter.
    • Fig. S12. Unweighted principal component plots of 16S rRNA bacterial gene sequencing.
    • Fig. S13. Relative abundance boxplots of changed bacterial taxa as detected by 16S rRNA bacterial gene sequencing.
    • Fig. S14. Heatmaps of 16S rRNA bacterial gene sequencing results for the P80 emulsifier experiment by treatment group and cage.
    • Fig. S15. Dexamethasone effects on CCMs and colonic goblet cells.
    • Fig. S16. Model of the role of PDCD10 in the gut epithelium and its effect on CCM formation in the brain.
    • Table S1. Clinical characteristics of patients with CCM recruited to participate in gut microbiome study.
    • References (5569)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

Stay Connected to Science Translational Medicine

Navigate This Article