Novel transgenic models for the delineation of mechanisms regulating proteolysis and inflammation in skin diseases
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Χάρλα, Ελένη
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PART I: Netherton syndrome (NS) and peeling skin disease (PSD) are genetic rare skin disorders sharing clinical characteristics including allergies, epidermal overdesquamation, and skin inflammation. Kallikrein-related peptidases (KLKs), which are proteolytic enzymes, were reported to be hyperactivated both in NS and PSD skin. Specifically, published studies have proposed that hyperactivity of KLK5 and KLK7 are the main contributors of epidermal overdesquamation observed in NS (Furio et al., 2015; Kasparek et al., 2017; Bisyris et al., 2021a and 2021b; Zingkou et al., 2021). Furthermore, elevated expression, as well as activity of KLK5, were reported in PSD (Oji et al., 2010; Komatsu et al., 2006). In terms of this thesis, Part I will investigate the role of KLK6 in NS, while the role of KLK5 in PSD will be assessed in Part II. NS is a severe rare disease caused by inherited homozygous mutations in Spink5, encoding the endogenous serine protease inhibitor LEKTI. NS is characterized by atopic manifestations, allergies, inflammation, and potentially lethal skin overdesquamation and inflammation associated with highly elevated epidermal proteolysis. The role of KLK6 in NS pathology remains unknown. A skin biopsy obtained from a patient with NS was analyzed and KLK6 expression was found increased along with increased nuclear localization of the NF-κB transcription factor when compared to normal specimens obtained from three healthy donors. This thesis addresses the hypothesis whether deletion of Klk6 in Spink5 null-epidermis could suppress overdesquamation and/or inflammation associated with NS. To experimentally test this hypothesis, we bred the double knockout (DKO) mouse model Spink5-/-Klk6-/- by crossing Klk6-/- mice with heterozygous Spink5+/– mice that display a rather normal phenotype, since Spink5-/- mice die homogeneously within 4-5 hours from birth (Zingkou et al., 2019). We found that deletion of Klk6 in Lekti-deficient epidermis could not reverse the neonatal lethal NS phenotype (Zingkou et al., 2019). Extensive detachment of the stratum corneum, and impaired skin barrier function was observed in Spink5-/-Klk6-/- mice by eosin/hematoxylin staining and toluidine blue dye penetration assay, respectively. Gel zymography and in situ zymography showed that overall proteolytic activities in the epidermis of Spink5-/-Klk6-/- mice were elevated similarly to Spink5-/-. In contrast, Spink5-/-Klk6-/- mice displayed normalized keratinocyte differentiation, confirmed by IHC staining, and normalized expression of (coreno)desmosomal proteins including keratin 5, involucrin, loricrin, desmoglein-1, desmocollin-1 and corneodesmosin (Zingkou et al., 2019). In addition, ablation of Klk6 suppressed abnormal keratinocyte proliferation associated with NS since Ki67 expression was significantly reduced in the epidermis of DKO mice. This could be correlated with suppressed relative expression of Tslp and other inflammatory cytokines such as Tnf-α, Il-1β, Il-6, Il-17α, Il-23, Il-13 and Il-4 in DKO skin, as well as reduced numbers of infiltrating mast cells in the dermis. Par-2 expression, nuclear localization of NF-κB and Erk1/2 phosphorylation was reduced in DKO compared to Spink5-/- mice, indicative of suppressed activation of the Erk1/2-NF-κB signaling. Klk6 deletion could suppress skin inflammation via the KLK6-ERK1/2-NF-kB-TSLP axis. Cumulatively, elimination of Klk6 in the Lekti-deficient epidermis was not sufficient to ameliorate overdesquamation, a consequence of aberrantly elevated proteolysis, and did not restore the defective skin barrier leading to neonatal lethality. Nevertheless, ablation of Klk6 resulted in remarkable repression of epidermal inflammation, indicated by reduced Tslp expression via repression of the Erk1/2-NF-κB pathway. In addition, epidermal differentiation was normalized (Zingkou et al., 2019). Most importantly, our findings demonstrate for the first time that KLK6 is implicated in skin inflammation and may represent a novel druggable target for NS and other more common skin inflammatory diseases, such as atopic dermatitis.
PART II: Homozygous mutations in CDSN gene, encoding corneodesmosin, cause type B peeling skin disease, an autosomal recessive skin disorder with ichthyosiform erythroderma and exfoliation (Bowden, 2011). Cdsn-/- mice recapitulate PSD clinical symptoms showing aberrant overdesquamation, which leads to neonatal lethality due to severe dehydration (Leclerc et al., 2009). Since KLK5 hyperactivation has been reported in severe ichthyoses such as the PSD, we hypothesized that elimination of Klk5 could reverse the cutaneous hallmarks of PSD. Thus, we bred Cdsn-/-Klk5-/- mice previously generated (Sotiropoulou et al., personal communication) by crossing Klk5-/- mice with heterozygous Cdsn+/– mice that display a normal phenotype, since Cdsn-/- mice die homogeneously 30 min after birth. Macroscopic and histological examination of newborn Cdsn-/-Klk5-/- mice and isolated skin biopsies, showed that ablation of Klk5 in the Cdsn-null background does not reverse the lethal phenotype of PSD and could not restore a normal structure of the epidermis. Toluidine blue dye penetration assay revealed that deletion of Klk5 could not restore skin barrier function, since Cdsn-/-Klk5-/- pups stained blue, indicative of extensive skin barrier disruption. Gel and in situ zymography showed that elevated proteolysis was not suppressed in the epidermis. In conclusion, targeted deletion of Klk5 alone was not sufficient to reduce aberrantly elevated proteolytic activities and associated overdesquamation in the epidermis of Cdsn-/-. Consequently, the defective skin barrier and associated neonatal lethality could not be rescued. Nonetheless, ablation of Klk5 in the Cdsn-null background restored normal epidermal differentiation. Integrated transcriptomics and degradomics analyses are ongoing to identify alternative proteases that are active in PSD epidermis.
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Kallikrein-related peptidase 6, Skin inflammation, Netherton syndrome, Peeling skin syndrome