Epidermal Nbn deletion causes premature hair loss and a phenotype resembling psoriasiform dermatitis

Abstract

Nijmegen Breakage Syndrome is a disease caused by NBN mutations. Here, we report a novel function of Nbn in skin homeostasis. We found that Nbn deficiency in hair follicle (HF) progenitors promoted increased DNA damage signaling, stimulating p16Ink4a up-regulation, Trp53 stabilization and cytokines secretion leading to HF-growth arrest and hair loss. At later stages, the basal keratinocytes layer exhibited also enhanced DNA damage response but in contrast to the one in HF progenitor was not associated with pro-inflammatory cytokines expression, but rather increased proliferation, lack of differentiation and immune response resembling psoriasiform dermatitis. Simultaneous Nbn and Trp53 inactivation significantly exacerbated this phenotype, due to the lack of inhibition of pro-inflammatory cytokines secretion by Trp53. Altogether, we demonstrated novel functions of Nbn in HF maintenance and prevention of skin inflammation and we provide a mechanistic explanation that links cell intrinsic DNA maintenance with large scale morphological tissue alterations.

Keywords: Gerotarget; Nbn; inflammation; psoriasiform dermatitis; skin.

Figure 1. Nbn depletion leads to premature hair loss due to depletion of Krt15+ and Cd34+ HF progenitors

A. Hair loss in Nbn^Egr2-Cre skins from P35 to P90. Representative Nbn^Ctrl and Nbn^Egr2-Cre mice at different age, P18, P35 and P90. The hair loss can be observed from P35. B. Histological analysis of Nbn^Egr2-Cre skin reveals progressive thickening of the epidermis and absence of HFs by P90. Scale bar 100 μm. C. Progressive depletion of Krt15+ HF progenitors and increase number of Krt14+ keratinocytes in Nbn^Egr2-Cre skins. Scale bar 20 μm. D. Sox9 immunostaining in Nbn^Ctrl and Nbn^Egr2-Cre skins. E. Quantification of Krt15+, Sox9+ and Cd34+ cells in P18, P35 and P90 Nbn^Egr2-Cre skins. The Krt15+, Sox9+ and Cd34+ cells were count per skin length (mm). For Krt15: Nbn^Ctrl P18 (N = 4), Nbn^Egr2-Cre P18 (N = 4), Nbn^Ctrl P35 (N = 4), Nbn^Egr2-Cre P35 (N = 4), Nbn^Ctrl P90 (N = 2), Nbn^Egr2-Cre P90 (N = 4). ***: p = 0.0003, **: p = 0.0018. For Sox9: Nbn^Ctrl P18 (N = 2), Nbn^Egr2-Cre P18 (N = 3), Nbn^Ctrl P35 (N = 3), Nbn^Egr2-Cre P35 (N = 3), Nbn^Ctrl P90 (N = 3), Nbn^Egr2-Cre P90 (N = 3). **: p = 0.0021. For Cd34: Nbn^Ctrl P18 (N = 2), Nbn^Egr2-Cre P18 (N = 3), Nbn^Ctrl P35 (N = 3), Nbn^Egr2-Cre P35 (N = 2), Nbn^Ctrl P90 (N = 2), Nbn^Egr2-Cre P90 (N = 4).*: p < 0.05.

Figure 2. Nbn deficiency in HF progenitors triggers DNA damage response and secretion of pro-inflammatory cytokines

A. Increase of γ-H2afx foci and Trp53 stabilization in Nbn^Egr2-Cre HFs at P18. Scale bar 20 μm. B. Quantification of cleaved caspase 3 (Casp3+) in hair sections. Nbn^Ctrl P18 (N = 3), Nbn^Egr2-Cre P18 (N = 4). C. Real-time RT-PCR analysis of Bax and Bbc3 (Puma) expression in Nbn^Egr2-Cre P18 skins. Nbn^Ctrl P18 (N = 4), Nbn^Egr2-Cre P18 (N = 4). ***: p < 0.0001. D. Reduction of BrdU incorporation in Nbn^Egr2-Cre HF sections. Nbn^Ctrl P18 (N = 3), Nbn^Egr2-Cre P18 (N = 4). *: p = 0.0108. E. Reduction of Ki67 positive (Ki67+) cells in Nbn^Egr2-Cre HF sections. Nbn^Ctrl P18 (N = 3), Nbn^Egr2-Cre P18 (N = 4). *: p = 0.0108. Real-time RT-PCR analysis of Cdkn2a (p16^INK4A) F. and Cdkn1a (p21^Cip1/Waf1) G. expressions in Nbn^Egr2-Cre P18 and P35 skins. Nbn^Ctrl (N = 4), Nbn^Egr2-Cre (N = 4). ***: p < 0.0001, **: p = 0.0077. Real-time RT-PCR analysis of pro-inflammatory cytokines: Ccl2 H., Nbn^Ctrl P18 (N = 2), Nbn^Egr2-Cre P18 (N = 3), Nbn^Ctrl P35 (N = 6), Nbn^Egr2-Cre P35 (N = 6); Il6 I. Nbn^Ctrl P18 (N = 2), Nbn^Egr2-Cre P18 (N = 2), Nbn^Ctrl P35 (N = 5), Nbn^Egr2-Cre P35 (N = 5); Tnf J., Nbn^Ctrl P18 (N = 2), Nbn^Egr2-Cre P18 (N = 3), Nbn^Ctrl P35 (N = 5), Nbn^Egr2-Cre P35 (N = 5) and Il1b K., Nbn^Ctrl P18 (N = 2), Nbn^Egr2-Cre P18 (N = 3), Nbn^Ctrl P35 (N = 6), Nbn^Egr2-Cre P35 (N = 6). ***: p < 0.0001, **: p = 0.0085, *: p < 0.05.

Figure 3. Nbn-deficiency promotes psoriasiform dermatitis

A. Characterization of the psoriasiform dermatitis and inflammatory response using various cell type markers: Krt14, Krt10 and Loricrin; proliferation marker: Pcna (scale bar 20 μm). Immunohistochemistry of P90 Nbn^Egr2-Cre skins indicates inflammatory response with T-cell marker (Cd3+, scale bar 20 μm), coloration of mast cells (blue) with GIEMSA (scale bar 40 μm) and macrophages (F4/80+). B. P90 Nbn^Egr2-Cre skins highly expressed typical human psoriasiform dermatitis interleukins Il17c, Il23 and Il33. Nbn^Ctrl (N = 4), Nbn^Egr2-Cre (N = 4). **** : p < 0.0001, *** : p = 0.0002, ** : p = 0.0077. C. Up-regulation of p-Stat3, p-S6, p-p38 and p-p44 in P90 Nbn^Egr2-Cre skins. D. γ-H2afx foci in keratinocytes trigger Trp53 stabilization and E. increased Cdkn2a (p16^INK4A) and Cdkn1a (p21^Cip1/Waf1) expression in P90 Nbn^Egr2-Cre skins. Nbn^Ctrl (N = 4), Nbn^Egr2-Cre (N = 4). ****: p < 0.0001.

Figure 4. Trp53 inactivation triggers worsening of Nbn^Egr2-Cre phenotype

A. The hair loss in Nbn/Trp53^Egr2-Cre skins from P18 to P90 is similar to the Nbn^Egr2-Cre ones B. Histological analysis of Nbn/Trp53^Egr2-Cre skins reveals progressive aggravation of Nbn^Egr2-Cre skins lesions by Trp53 inactivation at P90. Scale bar 100 μm. C. Characterization of Nbn/Trp53^Egr2-Cre skins using Krt15, Krt10, Krt14, Cd3+ and γ-H2afx staining. Remark the lack of Krt15+ cells, the ectopic localization of Krt10- and Krt14-positive cells and the high number of DSBs revealed by γ-H2afx foci accumulation in Nbn/Trp53^Egr2-Cre. Scale bar 20 μm.D. Dramatic increase of Il17c, Il23 and Il33 expression in Nbn/Trp53^Egr2-Cre skins (Nbn^Ctrl (N = 2), Nbn^Egr2-Cre (N = 2) and Nbn/Trp53^Egr2-Cre (N = 2). E. Strong nuclear localization/activation of p-p44, p-p38 and p-Stat3 in Nbn/Trp53^Egr2-Cre skins lesions. S6 phosphorylation occurs also in the basal keratinocytes layer. Scale bar 20 μm. F. Inactivation of Trp53 is associated with increase of Cdkn2a expression in Nbn/Trp53^Egr2-Cre skins. Nbn^Ctrl (N = 2), Nbn^Egr2-Cre (N = 2) and Nbn/Trp53^Egr2-Cre (N = 2). ***: p < 0.001. Scale bar 20 μm.