Fig 2: Chromatin landscape associated with Nrp1/Flt1/Vegfa signaling in psoriasis.(A) Percentage of total up-regulated peaks in K14-Vegfa and depending either on Nrp1/Flt1 (=Nrp1/Flt1 dependent) or specifically on Nrp1 (=Nrp1 dependent) or Flt1 (Flt1 dependent) or independent of Nrp1 or Flt1 expression (=Nrp1/Flt1 independent). (B) Percentage of down-regulated peaks and their distribution in the different categories. (C) Enriched TF motifs found in the peaks that were up- or down-regulated (D) in an Nrp1/Flt1-dependent manner. P value of enrichment of the motif in peaks compared with background and percentage of peaks containing the motif. (E) Percentage of total up-regulated peaks in K14-Vegfa overlapping with up-regulated genes and depending either on Nrp1/Flt1 expression (=Nrp1/Flt1 dependent) or specifically on Nrp1 (=Nrp1 dependent) or Flt1 (Flt1 dependent) or independent of Nrp1 or Flt1 expression (=Flt1/Nrp1 independent). (F) mRNA expression of up-regulated genes by RNA-seq in FACS-isolated basal keratinocytes (n = 2) (means ± SEM). (G) Enriched transcription TF motifs in the peaks up-regulated in an Nrp1/Flt1-dependent manner. (H) Percentage of total down-regulated genes and their distribution in the different categories. (I) mRNA expression of down-regulated genes by RNA-seq in FACS-isolated basal keratinocytes (n = 2) (means ± SEM). (J) Enriched TF motifs in the peaks down-regulated in an Nrp1/Flt1-dependent manner.

Fig 3: Transcriptional landscape associated with Nrp1/Flt1/Vegfa signaling in psoriasis.(A) Pie chart showing the percentage of total up-regulated genes in K14-Vegfa and depending either on common Nrp1/Flt1 (=Nrp1/Flt1 dependent) or specifically on Nrp1 (=Nrp1 dependent) or Flt1 (Flt1 dependent) expression. Genes with unchanged expression after Nrp1or Flt1 epidermal ablation were also represented (=Nrp1/Flt1-independent up genes). (B) GO analysis of up-regulated genes in Vegfa overexpression in an Nrp1- and Flt1-dependent manner. (C) mRNA relative expression of up-regulated genes by RNA-seq in Vegfa overexpression in FACS-isolated basal keratinocytes (n = 2) (means ± SEM). (D) Pie chart showing the percentage of total down-regulated genes in K14-Vegfa and depending either on common Nrp1/Flt1 expression (=Nrp1/Flt1 dependent) or specifically on Nrp1 (=Nrp1 dependent) or Flt1 (Flt1 dependent) expression. Genes with unchanged expression after Nrp1 or Flt1 epidermal ablation were also represented (=Nrp1/Flt1-independent down genes). (E) GO analysis of down-regulated genes in Vegfa overexpression in an Nrp1- and Flt1-dependent manner. (F) mRNA relative expression of down-regulated genes by RNA-seq in Vegfa-overexpressing mice and depending on Nrp1 or Flt1 expression in FACS-isolated keratinocytes (n = 2) (means ± SEM).

Fig 4: Cell autonomous function of Nrp1 in the epidermis is critical for Vegfa-induced psoriasis.(A) Strategy to constitutively activate Vegfa and delete Nrp1 expression in epidermis. (B) Vegfa and Nrp1 mRNA expression by qRT-PCR on FACS-isolated keratinocytes (n = 3) (means ± SEM, Mann-Whitney test). (C) Nrp1 expression assessed by Western blot performed on FACS-isolated basal keratinocytes. (D) Nrp1 protein expression (n = 3) (means ± SEM, Mann-Whitney test). (E) Naso-oral region, ear, and tail. (F) H&E on tail skin. Scale bars, 50 µm. (G) Epidermal tail thickness measured microscopically (n = 10) (mean ± SEM, Student’s t test). (H) K14/EdU staining. Scale bars, 50 µm. (I) Percentage of EdU-positive BCs [n = 507 (Ctrl), n = 487 (K14-Vegfa), n = 490 (K14-Vegfa/Nrp1 cKO) total BCs; n = 10 mice] (means ± SEM, Student’s t test). (J) K14/CD45 staining. Scale bars, 50 µm. (K) Density of CD45-positive cells in the dermal IFE area of 344,965 µm2 (Ctrl), 449,687 µm2 (K14-Vegfa), and 423,876 µm2 (K14-Vegfa/Nrp1 cKO); n = 10 mice. CD45-positive cells per 10,000 µm2 (means ± SEM, Student’s t test). (L) K14/CD31 staining. Scale bars, 50 µm. (M) Number of CD31-positive cells calculated in a dermal IFE area of 409,560 µm2 (Ctrl), 432,890 µm2 (K14-Vegfa), and 428,532 µm2 (K14-Vegfa/Nrp1 cKO); n = 10 mice. Number of CD31-positive cells per 10000 µm2 (means ± SEM, Student’s t test). Photo credit: Benhadou Farida, Laboratory of Stem Cells and Cancer.

Fig 5: Function-blocking anti-Nrp1 antibodies improve Vegfa-induced psoriasis.(A to C) Naso-oral region, ear, and tail, (D) H&E on tail skin. Scale bars, 50 µm. (E) Measures of epidermal thickness (n = 3) (means ± SEM, Mann-Whitney). (F) K14/EdU staining. Scale bars, 50 µm. (G) Percentage of EdU-positive BCs [n = 387 (isotype, day 0), n = 354 (isotype, day 15), n = 424 (Nrp1b AB, day 0), n = 409 (Nrp1b AB, day 15), n = 391 (Nrp1a AB, day 0), n = 421 (Nrp1a AB, day 15) total BCs, n = 3] (means ± SEM, Mann-Whitney). (H) K14/CD45 staining. Scale bars, 50 µm. (I) Density of CD45-positive cells in the dermal IFE area of 254,342 µm2 (isotype, day 0), 298,567 µm2 (isotype, day 15), 267,890 µm2 (Nrp1b AB, day 0), 287,908 µm2 (Nrp1b AB, day 15), 257,560 µm2 (Nrp1a AB, day 0), 294,901 µm2 (Nrp1a AB, day 15); n = 10. Number of CD45-positive cells per 10,000 µm2 (means ± SEM, Mann-Whitney). (J) K14/CD31 staining. Scale bar, 50 µm. (K) Microvascular density in dermal IFE area of 267,980 µm2 (isotype, day 0), 234,589 µm2 (isotype, day 15), 222,370 µm2 (Nrp1b AB, day 0), 223,456 µm2 (Nrp1b AB, day 15), 200,154 µm2 (Nrp1a AB, day 0), and 212,980 µm2 (Nrp1a AB at day 15). Number of CD31-positive cells per 10,000 µm2 (means ± SEM, Mann-Whitney). Photo credit: Benhadou Farida, Laboratory of Stem Cells and Cancer.