Fig 1.
Loss of Klk5 expression reverses skin and whisker anomalies in Spink5-/- mice.
(A) Macroscopic appearance of wt, Spink5-/-, Klk5-/-, Spink5-/-Klk5-/- mice. Photos were taken 30 h after birth for wt, Klk5-/- and Spink5-/-Klk5-/- mice and 5 h for Spink5-/- mice; (B) Microscopic appearance of muzzle area (scale bar 1mm) and (C) whisker ultrastructure by scanning electron microscopy (SEM). Scale bar, 10μm.
Fig 2.
Skin barrier function is restored by Klk5 knockout.
(A) Hematoxylin/eosin/safranin (HES) stained skin sections show no stratum corneum detachment and normal thickness of the living layer in the epidermis of Spink5-/-Klk5-/- (taken post-natally at 30 h) as opposed to Spink5-/- (at 5 h) characterized by extensive stratum corneum/stratum granulosum separation and acanthosis; Scale bar: 50μm (B) Skin barrier defect is remarkably improved in Spink5-/-Klk5-/- as demonstrated by the toluidine blue dye penetration assay. The dye is excluded from entering the body of the Spink5-/-Klk5-/- mouse in sharp contrast to the deep blue stain of Spink5-/-; (C) Transepidermal water loss (TEWL) assay. TEWL values are significantly lower in Spink5-/-Klk5-/- as compared to Spink5-/- and similar to wt animals, reflecting normalization of the skin barrier defect on the Lekti-deficient background solely by Klk5 deletion. *p<0.05 (Mann-Whitney U test); (D) TEWL values over time in wt (n = 9), Spink5-/- (n = 9), Klk5-/- (n = 10) and combined Spink5-/-Klk5-/- deficient (n = 8) newborn mice at 37°C. Data are shown as mean ± s.e.m. (standard error of the mean). Stars on the graph represent statistics between Spink5-/- and Spink5-/-Klk5-/- at the different time points **p<0.01 (Mann-Whitney U test). Note that values for Spink5-/-Klk5-/- and Klk5-/- are not significantly different from wt whereas Spink5-/- values are significantly different from wt for all time points.
Fig 3.
Klk5 is a key regulator of epidermal proteolysis.
In situ zymography using fluorescence-quenched casein (A) or elastin (B). Skin tissue sections were prepared from wt, Spink5-/-, Klk5-/-, and Spink5-/-Klk5-/- mice. Ablation of Klk5 expression highly reduced both caseinolytic and elastinolytic activities in the stratum corneum of Spink5-/-Klk5-/- as compared to Spink5-/-, respectively. Fluorescence intensity data was transformed into a color gradient (as shown) using ImageJ software. Dashed white lines represent the dermal-epidermal junction; The asterisk (*) indicates the epidermis, scale bar: 25μm (C) Changes in proteolytic activity using colorimetric substrates that target different proteases. Activity in wt, Spink5-/- and Spink5-/-Klk5-/- was detected by measuring absorbance at 405 nm after overnight incubation with substrates for either KLK7 or KLK14. Data are shown as the mean ± SEM of duplicates for four mice per genotype; (D) Analysis of mRNA expression in skin by RT-qPCR of Klk7 and Klk14. Results show high expression of both Klks in Spink5-/-. In Spink5-/-Klk5-/- skin, expression of Klk14 is equal to wt while Klk7 remained slightly higher. Data are shown as the mean ± s.e.m. of triplicate amplification for at least three mice per genotype. Results are normalized to wt mean (set as 1.0).
Fig 4.
The ultrastructural architecture of Spink5-/- epidermis is normalized by Klk5 knockout.
(A) Immunostaining on skin section shows restored Dsg1 in Spink5-/-Klk5-/- compared to Spink5-/- consistent with diminished pathological proteolysis observed in Fig 3, scale bar: 25μm (B) Immunostaining on skin section shows partial restoration of Dsc1 in Spink5-/-Klk5-/- compared to Spink5-/-, scale bar: 25μm. (C) Transmission electron micrographs of skin ultrasections from: wt (C1-2), Spink5-/- (D1-3), Klk5-/- (E1-3), and Spink5-/-Klk5-/- (F1-3) mice. Nuclei (depicted with star in D2) present in the stratum corneum of Spink5-/- denote incomplete differentiation (parakeratosis). Separation of desmosomes in Spink5-/- is evident, as indicated by arrows (D3). Of note, also the well-organized compact desmosomes in Klk5-/- and Spink5-/-Klk5-/- skin sections (E2-3, F3).
Fig 5.
Spink5-/-Klk5-/- skin does not show evidence of abnormal epidermal differentiation.
(A, B) IHF analyses show normalized Involucrin and Loricrin expression in Spink5-/-Klk5-/- skin compared to Spink5-/- as indicated by arrows; scale bar: 25μm (C) Deletion of Klk5 blocks Flg degradation. Immunostaining of Flg extends to the granular layer and the stratum corneum in wt, Klk5-/-, Spink5-/-Klk5-/- while it is drastically reduced in the granular layer and restricted to the detached stratum corneum in Spink5-/- mice as indicated by arrows; scale bar: 25μm.
Fig 6.
Klk5 deletion results in remarkable suppression of the inflammatory phenotype of NS.
(A-E). Results show remarkable abrogation of pro-allergic and pro-inflammatory cytokines quantified by RT-qPCR. (A) Il-1β, Tslp, Tnf-α; (B) Il-6, Il-18, the type 17 promoting cytokine Il-23p19; (C) Ifn-γ, Il-4, Il-17A, Il-22; (D) Th17 regulated genes Defb4, Slpi, Cxcl1 and Ccl20; (E) Th17/22 regulated genes s100a7, s100a8, s100a9. Data are shown as the mean ± s.e.m. of triplicate amplification for at least five mice per genotype. Results are normalized to wt mean (set as 1.0). *p<0.05, **p<0.005, ***p<0.001 (Mann-Whitney U test). (F) Quantification of mast cells based on toluidine blue staining of skin tissue (mast cells stain purple). An increased number of mast cells was detected in Spink5-/- dermis; (G) IHC analysis shows abolishment of Tslp expression in Spink5-/-Klk5-/- skin as compared to Spink5-/-; Scale bar: 50μm.