Olivier Gouin

Claire Barbieux

Florent Leturcq

Mathilde Bonnet

Des Claustres

Evgeniya Petrova

MD Alain Hovnanian
email: alain.hovnanian@inserm.fr

Mathilde Bonnet Des Claustres

Transgenic Kallikrein 14 Mice Display Major Hair Shaft Defects Associated with Desmoglein 3 and 4 Degradation, Abnormal Epidermal Differentiation, and IL-36 Signature

Keywords: Netherton Syndrome, KLK14, skin inflammation, hair shaft defects, Desmogleins, Il-36a

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Netherthon syndrome (NS) is a rare autosomal recessive skin disease that affects approximately one in 200,000 persons and can be life threatening in infants [START_REF] Hovnanian | Netherton syndrome: skin inflammation and allergy by loss of protease inhibition[END_REF]. Newborns affected with NS exhibit congenital scaling erythroderma, develop a hair shaft defect and atopic manifestations with elevated serum IgE levels [START_REF] Hannula-Jouppi | IgE allergen component-based profiling and atopic manifestations in patients with Netherton syndrome[END_REF]. NS is caused by loss-of-function mutations in SPINK5 encoding lymphoepithelial Kazal-type-related inhibitor (LEKTI). LEKTI deficiency results in unopposed protease activity in the epidermis [START_REF] Chavanas | Mutations in SPINK5, encoding a serine protease inhibitor, cause Netherton syndrome[END_REF][START_REF] Descargues | Spink5-deficient mice mimic Netherton syndrome through degradation of desmoglein 1 by epidermal protease hyperactivity[END_REF][START_REF] Kasparek | A viable mouse model for Netherton syndrome based on mosaic inactivation of the Spink5 gene[END_REF]. The main targets of LEKTI are the kallikrein-related peptidases (KLKs), mainly KLK5, KLK7 and KLK14 [START_REF] Deraison | LEKTI fragments specifically inhibit KLK5, KLK7, and KLK14 and control desquamation through a pH-dependent interaction[END_REF][START_REF] Egelrud | hK5 and hK7, two serine proteinases abundant in human skin, are inhibited by LEKTI domain 6[END_REF][START_REF] Hovnanian | Netherton syndrome: skin inflammation and allergy by loss of protease inhibition[END_REF][START_REF] Meyer-Hoffert | Identification of lympho-epithelial Kazal-type inhibitor 2 in human skin as a kallikrein-related peptidase 5-specific protease inhibitor[END_REF]. Unrestrained KLK5 and KLK7 proteolytic activities are currently considered as playing a major role in NS pathogenesis [START_REF] Furio | Transgenic kallikrein 5 mice reproduce major cutaneous and systemic hallmarks of Netherton syndrome[END_REF][START_REF] Furio | KLK5 Inactivation Reverses Cutaneous Hallmarks of Netherton Syndrome[END_REF][START_REF] Kasparek | KLK5 and KLK7 Ablation Fully Rescues Lethality of Netherton Syndrome-Like Phenotype[END_REF].

Spink5 knock out (Spink5KO) mice were the first in vivo model that faithfully reproduced all clinical features of NS patients, but these animals died from dehydration only a few hours after birth [START_REF] Descargues | Spink5-deficient mice mimic Netherton syndrome through degradation of desmoglein 1 by epidermal protease hyperactivity[END_REF][START_REF] Hewett | Lethal, neonatal ichthyosis with increased proteolytic processing of filaggrin in a mouse model of Netherton syndrome[END_REF][START_REF] Kasparek | A viable mouse model for Netherton syndrome based on mosaic inactivation of the Spink5 gene[END_REF][START_REF] Kasparek | KLK5 and KLK7 Ablation Fully Rescues Lethality of Netherton Syndrome-Like Phenotype[END_REF][START_REF] Yang | Epidermal detachment, desmosomal dissociation, and destabilization of corneodesmosin in Spink5-/-mice[END_REF]. Despite neonatal Spink5KO lethality, this model was instrumental in disclosing the involvement of KLKs in NS pathogenesis. Spink5KO pups revealed unopposed KLK5 proteolytic activity and its downstream targets (KLK7, KLK14, and elastase 2), resulting in the degradation of desmosomal proteins, increased profilaggrin processing (Flg) and lipid anomalies, leading to stratum corneum (SC) detachment and a profound skin barrier defect with severe skin inflammation [START_REF] Bonnart | Elastase 2 is expressed in human and mouse epidermis and impairs skin barrier function in Netherton syndrome through filaggrin and lipid misprocessing[END_REF][START_REF] Descargues | Spink5-deficient mice mimic Netherton syndrome through degradation of desmoglein 1 by epidermal protease hyperactivity[END_REF][START_REF] Furio | Transgenic kallikrein 5 mice reproduce major cutaneous and systemic hallmarks of Netherton syndrome[END_REF][START_REF] Furio | KLK5 Inactivation Reverses Cutaneous Hallmarks of Netherton Syndrome[END_REF]. While KLK5 can cleave desmoglein 1 (DSG1), desmocollin 1 (DSC1) and corneodesmosin (CDSN), KLK7 and KLK14 were shown to cleave CDSN and DSG1, respectively [START_REF] Caubet | Degradation of Corneodesmosome Proteins by Two Serine Proteases of the Kallikrein Family, SCTE/KLK5/hK5 and SCCE/KLK7/hK7[END_REF][START_REF] Borgoño | A Potential Role for Multiple Tissue Kallikrein Serine Proteases in Epidermal Desquamation[END_REF]. To gain further insight into the role of KLK5 in NS, several murine models were developed including a transgenic mouse overexpressing human KLK5 (TghKLK5) in the stratum granulosum (SG) and a double knockout mouse for Spink5 and Klk5 (Spink5/Klk5KO) [START_REF] Furio | Transgenic kallikrein 5 mice reproduce major cutaneous and systemic hallmarks of Netherton syndrome[END_REF][START_REF] Furio | KLK5 Inactivation Reverses Cutaneous Hallmarks of Netherton Syndrome[END_REF]. TghKLK5 mice display major NS clinical features including SC detachment, scaling, skin inflammation, hair shaft defects and exhibit a prolonged survival [START_REF] Furio | Transgenic kallikrein 5 mice reproduce major cutaneous and systemic hallmarks of Netherton syndrome[END_REF]. Conversely, Klk5 deletion in Spink5/Klk5KO mice normalizes major hallmarks of NS in newborn pups, confirming the predominant role of KLK5 in NS pathophysiology. Nevertheless, KLK5 deficiency does not rescue the lethality of Spink5KO mice, suggesting that other actors directly or indirectly cause NS [START_REF] Furio | KLK5 Inactivation Reverses Cutaneous Hallmarks of Netherton Syndrome[END_REF]. KLK7 and KLK14 proteolytic activities are increased in the epidermis of Spink5KO and TghKLK5, and are normalized in Spink5Klk5KO mice, supporting the notion that KLK5 activates KLK7 and KLK14 in vivo [START_REF] Furio | Transgenic kallikrein 5 mice reproduce major cutaneous and systemic hallmarks of Netherton syndrome[END_REF][START_REF] Furio | KLK5 Inactivation Reverses Cutaneous Hallmarks of Netherton Syndrome[END_REF][START_REF] Prassas | Unleashing the therapeutic potential of human kallikrein-related serine proteases[END_REF]. Double knockout mice for Spink5 and Klk7 (Spink5/Klk7KO) displayed numerous epidermal lesions and died within 12 hours after birth, indicating that lack of Klk7 does not prevent the development of the NS phenotype [START_REF] Kasparek | KLK5 and KLK7 Ablation Fully Rescues Lethality of Netherton Syndrome-Like Phenotype[END_REF]. In contrast, triple knockout mice for Spink5, Klk5 and Klk7 (Spink5/Klk5/Klk7KO) fully rescued lethality after birth and the animals survived until adulthood with no major skin alteration, although they displayed hair abnormalities until 20 days after birth [START_REF] Kasparek | KLK5 and KLK7 Ablation Fully Rescues Lethality of Netherton Syndrome-Like Phenotype[END_REF]. While the involvement of both KLK5 and KLK7 in the epidermis has been extensively studied in these murine models, the role of KLK14 in skin homeostasis and its involvement in NS pathogenesis remained relatively unexplored probably due to the lack of animal model available.

In this study, we developed and characterized a transgenic murine model overexpressing human KLK14 (TghKLK14) in the SG of the epidermis in order to gain further insight into the role of KLK14 in NS pathogenesis. This study demonstrates that KLK14, to our knowledge previously unreported, plays a major causal role in hair shaft defects seen in NS murine models and also contributes to skin inflammation.

Results

hKLK14 is overexpressed and active in the stratum granulosum of TghKLK14 mice

To analyse the consequences of excessive KLK14 proteolytic activity, a transgenic mouse model overexpressing hKLK14 under the control of the human involucrin (IVL) promoter was generated to target KLK14 expression in the SG of murine epidermis. Animals carrying the full-length cDNA encoding hKLK14 (Figure 1A) were identified using a qPCR genotyping strategy targeting the hKLK14 transgene and the IVL promoter. Using a hKLK14-specific antibody, the correct localisation of hKLK14 in the SG of interfollicular epidermis and in hair follicles of TghKLK14 mice was confirmed (Figure 1B). Western-blot analysis of skin protein lysates from TghKLK14 mice showed that hKLK14 was overexpressed, while hKLK14 remained undetectable in WT mice (Figure 1C). In situ zymography of skin cryosections from TghKLK14 mice using casein-BODIPY-FL substrate revealed increased proteolytic activity mainly localized in the SG and in hair follicles (Figure 1D andE).

TghKLK14 mice display major hair abnormalities and a skin barrier defect

TghKLK14 newborns were indistinguishable from WT controls. Before the 5 th day after birth, while hair growth led to skin coloration in WT pups, the skin of TghKLK14 pups remained pink without visible hair (Figure 2A). At this age, no significant difference in transepidermal water loss (TEWL) was observed (Figure 2A). From day 5 to 20, TghKLK14 mice displayed no visible hair except on their face (Figure 2B), where whiskers and hair around their eyes and muzzle were observed (Figure S1A). At the same time, TEWL was increased, although their skin showed no visible redness or scaling (Figure 2B). TghKLK14 mice between 20 and 30 days of age showed progressive recovery of hair growth on their entire body, although their fur remained sparse throughout their body (Figure 2C). TEWL of residual nude skin was still significantly increased in comparison to WT skin, whereas hairy skin areas trended to present increased TEWL values with no statistical significance (Figure 2C).

From 30 days of life, no difference could be observed in the hair shaft structure, including the hair tip, between TghKLK14 and WT mice (Figure S1B). Analysis of transgene expression showed that reversion of hair abnormalities was associated with loss of hKLK14 protein expression in the skin from TghKLK14 older than 30 days. (Figure S1C). Histological analysis was performed on skin from TghKLK14 mice at age 1 day, 20 days, 30 days and 50 days (Figure 2A-C, Figure S1D). The epidermis of TghKLK14 mice between 1 and 20 days of age was hyperkeratotic and acanthotic with a 2-fold increase in epidermal thickness compared to WT mice, while at 30 days of age the epidermal thickness was similar to WT mice (Figure 2D). Between 1 and 20 days of age, the SC was disrupted and detached from the epidermis. Mice younger than 5 days displayed comparable hair follicle numbers than WT mice but hair shafts were fragmented in approximately 50% of the hair follicles. At the age of 20 days, hair follicles became hyperplastic although their number was not significantly decreased compared to WT mice. At the age of 20 days, hair follicles in the anagen phase were hyperkeratotic with ingrown hair shafts, which were abnormally structured, curled up, broken and fragmented without normal alternation of black and white bands (Figure 2B). In contrast, TghKLK14 mice older than 30 days showed normal hair growth although their fur remained sparse. Histological analysis showed hair follicles with normal appearance but reduced in number (Figure 2C, Figure S1D).

Histological analysis of TghKLK14 skin discloses hair shaft defects and epidermal abnormalities

In order to analyse the impact of hKLK14 overexpression on skin homeostasis, mice at 20 days of age, prior to the loss of transgene expression, were further investigated. Ki67 and keratin 14 (Krt14) immunostaining was increased in the stratum basal (SB) of TghKLK14 mice compared to WT controls (Figure 3A), indicating epidermal hyperproliferation. Increased protein expression of Krt14 was confirmed by Western blotting of skin extracts from TghKLK14 mice (Figure 3B andC).

Expression of terminal epidermal differentiation markers such as Ivl and Flg, was strongly increased in the SG of TghKLK14 mice compared to WT mice, as determined by immunofluorescence staining of skin sections, Western blotting of skin extracts (Figure 3) and by RT-qPCR (Figure S2A). Assessment of Flg expression by immuno-blotting revealed an increase of the dimer form with no significant variation in trimer and monomer forms in comparison to WT mice. Flg fragments with a molecular weight between 15kDa and 25kDa, were enhanced in TghKLK14 skin extracts (Figure3B).

hKLK14 hyperactivity leads to Dsg3 and Dsg4 degradation associated with hair shaft defects

Transmission electron microscopy of hair follicle cross sections was performed on 20-day-old TghKLK14 mice (Figure 4A). The hair shaft structure was abnormal as evidenced by cortex alterations. Cells from the hair shaft cortex were separated from each other, leading to gap formation where melanin granules could be seen in the cell interspace in the cortex (Figure 4A). While the thickness of Henle's layer (He) of the inner root sheath (IRS) and the outer root sheath (ORS) remained comparable to WT mice, the Huxley's layer (Hx) thickness of IRS was increased by 2-fold in TghKLK14 mice (Figure 4A and B). Since desmogelin (Dsg) and Cdsn proteins are particularly important in hair shaft structure and organisation, Dsg and Cdsn abnormalities were investigated in the skin of TghKLK14 mice.

Here, we hypothesized that KLK14 could cleave Dsg and Cdsn, involved in hair formation. The pattern of Dsg1 expression in TghKLK14 skin was comparable to that of WT mice and was localised in the inter-follicular epidermis and the ORS/IRS of hair follicles (Figure 4C). Dsg2 staining displayed a similar distribution but was increased in TghKLK14 mice (Figure 4C), as confirmed by immunoblotting (Figure 4C). In contrast, Dsg3 and Dsg4, which localized to hair follicles and to interfollicular epidermis in WT mice, were drastically reduced in TghKLK14 mice compared to WT controls (Figure 4C). Immunoblotting of skin extracts confirmed that Dsg3 and Dsg4 protein levels were reduced in comparison with WT mice (Figure 4D andE). Increased Dsg4 and Dsg2 transcript levels were detected in skin lysates from TghKLK14 mice, while those of Dsg1 and Dsg3 remained unchanged (Figure S2B). Digestion time-course analysis provided evidence for direct in vitro proteolytic degradation of rhDSG1, rhDSG2, rhCDSN (Figure S2C), rhDSG3, rhDSG4, and rmCdsn by thermolysin-activated rhKLK14 (Figure 4F, Figure S2D).

hKLK14 hyperactivity triggers skin inflammation and immune cell infiltration

To address the potential role of KLK14 in impacting gene expression in the skin, mRNA expression levels of several pro-inflammatory interleukins and chemokines (Il-36a, Il-36b, Il-36g, Il-17c, chemokine C-X-C motif ligand 20; Ccl20 and interferon-γ; Ifn-g), antimicrobial peptides (S100a8, S100a9 and Defensin b4; Defb4) and receptors triggering neutrophil-and monocyte-mediated inflammatory responses (Triggering Receptor Expressed On Myeloid Cells 1;Trem1) were analysed in skin extracts from TghKLK14 mice and compared to WT mice (Figure 5A). The expression of Il-36 family members was up-regulated in skin extracts from TghKLK14 mice by 23-, 3-and 2-fold-change (FC) for Il-36a, Il-36b and Il-36g, respectively. Ccl20 (3.5-FC) and Ifn-g (12.5-FC) were also significantly increased in TghKLK14 mice. Enhanced expression of Trem1 (17-FC) and antimicrobial and cytotoxic peptides such as S100a8 (12-FC), S100a9 (43-FC), and Defb4 (9-FC) indicated that innate immunity against viral and bacterial infections was stimulated in TghKLK14 (Figure 5A).

Immunofluorescence staining of Il-36a and Il-17c showed a significant increase in the epidermis of TghKLK14 mice compared to WT mice confirming the differences seen at the transcript level.

Increased expression of Il-36a and Il-17c was more pronounced in the SG but also extended across most epidermal layers (Figure 5B). The increased protein level of Il-17c and Il-36a was further validated by immunoblotting of skin extracts from TghKLK14 and WT mice (Figure 5C). Il-17c and Il-36 family cytokines are known to be involved in immune cell chemo-attraction. HES and toluidine blue coloration analyses revealed an increased number of eosinophils and mast cells, respectively (Figure 5D andF). Immunofluorescence staining showed a strong infiltrate of Cd3 + T cells in the epidermis of TghKLK14 mice, while neutrophils (NIMP-R14) could not be detected neither in TghKLK14 skin sections nor in WT (Figure 5E andF). Immunostaining for Cd4 and Cd8 revealed that Cd4 + cells were mainly localised in the dermis whereas Cd8 + cells were found in the epidermis, similarly to Cd3 + cells (Figure 5E). The immune cell infiltrates were quantified by counting the amount of positive cells in 2 different fields of view (Figure 5F).

Discussion

While the function of KLK5 and KLK7 in NS pathophysiology has been previously studied in several in vivo murine models [START_REF] Furio | Transgenic kallikrein 5 mice reproduce major cutaneous and systemic hallmarks of Netherton syndrome[END_REF][START_REF] Furio | KLK5 Inactivation Reverses Cutaneous Hallmarks of Netherton Syndrome[END_REF][START_REF] Kasparek | A viable mouse model for Netherton syndrome based on mosaic inactivation of the Spink5 gene[END_REF][START_REF] Kasparek | KLK5 and KLK7 Ablation Fully Rescues Lethality of Netherton Syndrome-Like Phenotype[END_REF], the role of KLK14 remains poorly understood. In this study, we developed and characterized a transgenic NS model overexpressing hKLK14 in the granular layers of the epidermis in order to investigate the contribution of KLK14 hyperactivity in NS pathogenesis in vivo.

Lekti deficiency in Spink5KO mice or KLK5 overexpression in TghKLK5 mice results in unopposed serine protease activity leading to increased proteolytic activity in the SG and SC [START_REF] Descargues | Spink5-deficient mice mimic Netherton syndrome through degradation of desmoglein 1 by epidermal protease hyperactivity[END_REF][START_REF] Furio | Transgenic kallikrein 5 mice reproduce major cutaneous and systemic hallmarks of Netherton syndrome[END_REF][START_REF] Furio | KLK5 Inactivation Reverses Cutaneous Hallmarks of Netherton Syndrome[END_REF][START_REF] Kasparek | A viable mouse model for Netherton syndrome based on mosaic inactivation of the Spink5 gene[END_REF]. In this study, KLK14 overexpression in the epidermis led to enhanced proteolytic activity that mainly co-localized with hKLK14 detection in the SG and hair follicles. Despite the improvement of NS clinical features in Spink5/Klk5KO and prolonged survival of Spink5/Klk5/Klk7KO mice, KLK5 and/or KLK7 deletion does not prevent hair abnormalities to develop as shown by delayed hair growth up to 20 days after birth in both murine models [START_REF] Furio | KLK5 Inactivation Reverses Cutaneous Hallmarks of Netherton Syndrome[END_REF][START_REF] Kasparek | KLK5 and KLK7 Ablation Fully Rescues Lethality of Netherton Syndrome-Like Phenotype[END_REF]. While TghKLK14 mice did not display clinical skin inflammation, they exhibited major hair anomalies when the transgene was expressed, supporting the notion that KLK14 could be directly involved in hair defects in NS. Between 20 and 30 days of age, the fur of TghKLK14 mice grew progressively and hair had normal appearance but remained sparse over the whole body. Reversal of hair shaft anomalies was associated with loss of epidermal hKLK14 expression, likely resulting from silencing of the hKLK14 transgene. Histological analysis of skin from 20-day-old TghKLK14 mice showed hyperplastic hair follicles and hair shaft degeneration, confirming that KLK14 plays a central role in hair integrity. These mice also exhibited acanthosis and hyperkeratosis with increased expression of proliferation markers (Ki67, Krt14) and epidermal differentiation markers (Ivl, Flg), as seen in TghKLK5 mice [START_REF] Furio | Transgenic kallikrein 5 mice reproduce major cutaneous and systemic hallmarks of Netherton syndrome[END_REF]. Unlike Spink5KO mice which show Ivl and Flg over-degradation, TghKLK14 mice display increased expression of both markers [START_REF] Descargues | Spink5-deficient mice mimic Netherton syndrome through degradation of desmoglein 1 by epidermal protease hyperactivity[END_REF][START_REF] Furio | KLK5 Inactivation Reverses Cutaneous Hallmarks of Netherton Syndrome[END_REF], suggesting that increased degradation of Ivl and Flg in NS involves additional proteases and/or factors. In TghKLK14, the dimer form of Flg and Flg fragments were increased at the protein level, suggesting that in contrast to caspase-14, KLK14 is likely to be involved in the proteolytic processing of Flg but not in Flg fragment degradation [START_REF] Hoste | Caspase-14 is required for filaggrin degradation to natural moisturizing factors in the skin[END_REF]. Previous works have shown that deregulated expression of proliferation and differentiation markers was normalized in Spink5/Kkl5KO and Spink5/Kkl5/Klk7KO mice and that epidermal thickness was strongly improved, indicating that Spink5KO-mediated epidermal abnormalities are KLK5-and KLK7-dependent [START_REF] Furio | Transgenic kallikrein 5 mice reproduce major cutaneous and systemic hallmarks of Netherton syndrome[END_REF][START_REF] Kasparek | KLK5 and KLK7 Ablation Fully Rescues Lethality of Netherton Syndrome-Like Phenotype[END_REF]. Here we provide evidence that KLK14 is also an inducer of acanthosis and hyperkeratosis. KLK14 is involved in KLK proteolytic cascades in the skin by cleaving and activating pro-KLK1, pro-KLK3, pro-KLK5 and pro-KLK11, leading to global enhancement of KLKs proteolytic activities [START_REF] Emami | Human kallikrein-related peptidase 14 (KLK14) is a new activator component of the KLK proteolytic cascade. Possible function in seminal plasma and skin[END_REF]. It is therefore likely that KLK14 overexpression in TghKLK14 mice activates pro-KLK5, leading to a phenotype, which overlaps with TghKLK5 mice. Another feature of NS is SC detachment caused by KLK5-and KLK7-mediated desmosomal cleavage through Dsg1, Dsc1, CDSN degradation [START_REF] Caubet | Degradation of Corneodesmosome Proteins by Two Serine Proteases of the Kallikrein Family, SCTE/KLK5/hK5 and SCCE/KLK7/hK7[END_REF][START_REF] Descargues | Spink5-deficient mice mimic Netherton syndrome through degradation of desmoglein 1 by epidermal protease hyperactivity[END_REF][START_REF] Descargues | Corneodesmosomal cadherins are preferential targets of stratum corneum trypsin-and chymotrypsin-like hyperactivity in Netherton syndrome[END_REF][START_REF] Furio | Transgenic kallikrein 5 mice reproduce major cutaneous and systemic hallmarks of Netherton syndrome[END_REF].

Except for DSG1 [START_REF] Borgoño | A Potential Role for Multiple Tissue Kallikrein Serine Proteases in Epidermal Desquamation[END_REF], it was unknown whether KLK14 could directly cleave other DSG family members. DSGs are distributed in a specific manner through the hair follicle and epidermal structures : DSG1 and 4 are expressed in suprabasal layers, while DSG2 and 3 localize in basal layers [START_REF] Wu | Desmoglein isotype expression in the hair follicle and its cysts correlates with type of keratinization and degree of differentiation[END_REF]. Of all DSGs members, Dsg4 is the only one expressed in the hair shaft cortex where hair abnormalities predominate in TghKLK14 mice [START_REF] Bazzi | Desmoglein 4 is expressed in highly differentiated keratinocytes and trichocytes in human epidermis and hair follicle[END_REF][START_REF] Bazzi | Desmoglein 4 is Regulated by Transcription Factors Implicated in Hair Shaft Differentiation[END_REF]. Increased IRS thickness and gaps in the hair shaft cortex observed in TghKLK14 mice suggest alterations in Dsg3 and/or Dsg4. In support of this possibility, the expression level of Dsg3 and Dsg4 was drastically reduced in TghKLK14 skin and we provided evidence that KLK14 can directly cleave DSG3 and DSG4. Besides the degradation of Dsg family members, SC detachment observed in TghKLK14 could also partly result from the ability of KLK14 to cleave Cdsn, although no evidence for Cdsn degradation in vivo could be provided in the absence of specific antibodies to murine Cdsn available. Cdsn deficient mice exhibit not only inflammatory features but also a skin barrier defect leading to postnatal death. In addition, grafts of skin from Cdsn -/-mice develop sparse hair in the first weeks of the graft, and hair is completely lost after 9 weeks of grafting [START_REF] Leclerc | Corneodesmosin gene ablation induces lethal skin-barrier disruption and hair-follicle degeneration related to desmosome dysfunction[END_REF]. It is therefore possible that the combined proteolytic degradation of Dsg4, Dsg3 and Cdsn contributes to hair loss seen in TghKLK14, although complete and specific lack of each desmosomal component separately leads to different hair abnormalities.

Indeed, loss-of-function mutations in DSG4 cause autosomal recessive hypotrichosis (LAH) in humans and the lanceolate hair (lah) phenotype in rodents, which are characterized by abnormal epidermal hyperproliferation, impaired hair shaft differentiation, and hair loss [START_REF] Kljuic | Desmoglein 4 in hair follicle differentiation and epidermal adhesion: evidence from inherited hypotrichosis and acquired pemphigus vulgaris[END_REF][START_REF] Montagutelli | Lanceolate hair (lah): a recessive mouse mutation with alopecia and abnormal hair[END_REF]. TgKLK14 mice share several epidermal and hair abnormalities with the lanceolate hair mice, but they did not develop the specific lance-head shaped hair shaft abnormality [START_REF] Jahoda | The lanceolate hair rat phenotype results from a missense mutation in a calcium coordinating site of the desmoglein 4 gene[END_REF][START_REF] Kljuic | Desmoglein 4 in hair follicle differentiation and epidermal adhesion: evidence from inherited hypotrichosis and acquired pemphigus vulgaris[END_REF]. Dsg3KO mice also display a hair loss phenotype resulting from defective anchorage of telogen hairs to the follicular epithelium which was not seen in TgKLK14 mice [START_REF] Koch | Desmoglein 3 anchors telogen hair in the follicle[END_REF]. Finally, dominant negative mutations in CDSN lead to hypotrichosis simplex type 2, revealing the role of CDSN in the maintenance of hair in humans [START_REF] Levy-Nissenbaum | Hypotrichosis simplex of the scalp is associated with nonsense mutations in CDSN encoding corneodesmosin[END_REF].

In vitro enzymatic digestion of rhDSG1 by rhKLK14 is consistent with previous data showing that KLK14 mediates DSG1 degradation [START_REF] Borgoño | A Potential Role for Multiple Tissue Kallikrein Serine Proteases in Epidermal Desquamation[END_REF][START_REF] Emami | Human kallikrein-related peptidase 14 (KLK14) is a new activator component of the KLK proteolytic cascade. Possible function in seminal plasma and skin[END_REF]. However, neither Dsg1 protein nor transcript expression were increased in TghKLK14, suggesting that additional mechanisms prevent over-degradation of Dsg1 by hKLK14 in vivo. TghKLK14 mice display increased Dsg2 transcript and protein levels in their skin. Transgenic mice overexpressing Dsg2 in SG show a histological signature similar to TghKLK14 mice, with acanthosis and keratinocyte hyperproliferation associated with Krt14, Ivl and Flg overexpression [START_REF] Brennan | Suprabasal Dsg2 expression in transgenic mouse skin confers a hyperproliferative and apoptosis-resistant phenotype to keratinocytes[END_REF]. Dsg2 overexpression could therefore contribute to epidermal deregulation seen in TghKLK14 mice. The mechanism by which KLK14 hyperactivity leads to increased Dsg2 remains uncertain. However, a previous study reported that loss of Dsg3 is compensated by enhanced Dsg2 expression in human keratinocytes [START_REF] Hartlieb | Desmoglein 2 compensates for desmoglein 3 but does not control cell adhesion via regulation of p38 mitogen-activated protein kinase in keratinocytes[END_REF], suggesting that increased Dsg2 expression in TghKLK14 could be a compensatory mechanism to extensive degradation of Dsg3.

Recent transcriptomic profiling studies in skin extracts from NS patients revealed an increase of both IL-36A and IL-17C [START_REF] Malik | Ichthyosis molecular fingerprinting shows profound TH17 skewing and a unique barrier genomic signature[END_REF][START_REF] Paller | An IL-17-dominant immune profile is shared across the major orphan forms of ichthyosis[END_REF]. Besides affecting genes involved in innate immunity (S100a8, S100a9, Defb4, and Trem1) previously identified as deregulated in NS [START_REF] Furio | Transgenic kallikrein 5 mice reproduce major cutaneous and systemic hallmarks of Netherton syndrome[END_REF][START_REF] Furio | KLK5 Inactivation Reverses Cutaneous Hallmarks of Netherton Syndrome[END_REF], KLK14 overexpression also deregulated inflammatory cytokines such as Ifn-g, Il-17c, Ccl20 and Il-36 family members. Interestingly, TghKLK14 mice and transgenic mice overexpressing Il-36a [START_REF] Blumberg | Opposing activities of two novel members of the IL-1 ligand family regulate skin inflammation[END_REF][START_REF] Blumberg | IL-1RL2 and its ligands contribute to the cytokine network in psoriasis[END_REF] or Il-17c [START_REF] Johnston | Keratinocyte overexpression of IL-17C promotes psoriasiform skin inflammation[END_REF] share histological skin features including acanthosis with increased expression of inflammatory cytokines and chemokines (Cxcl2, Cxcl8 and Ccl20) and antimicrobial peptides (Defb4, S100a7, S100a8 and S100a9). This is consistent with the notion that IL-36 and IL-17 family members are powerful inducers of anti-microbial peptides such as DEFb4 and S100 peptides, but also of inflammatory cytokines such as IFN-g [START_REF] Carrier | Inter-regulation of Th17 cytokines and the IL-36 cytokines in vitro and in vivo: implications in psoriasis pathogenesis[END_REF]. CCL20 is also and important chemokine induced by inflammatory cytokines and IL-17 pathway activation [START_REF] Harper | Th17 cytokines stimulate CCL20 expression in keratinocytes in vitro and in vivo: implications for psoriasis pathogenesis[END_REF]. The expression of IL-36, IL-17c and CCL20 is triggered by Toll like Receptor (TLR) activation, including TLR-3, -4, -5 [START_REF] Boutet | IL-36, IL-37, and IL-38 Cytokines in Skin and Joint Inflammation: A Comprehensive Review of Their Therapeutic Potential[END_REF][START_REF] Johnston | Keratinocyte overexpression of IL-17C promotes psoriasiform skin inflammation[END_REF][START_REF] Lebre | Human Keratinocytes Express Functional Toll-Like Receptor 3, 4, 5, and 9[END_REF]. Therefore, increased expression of these genes is likely to be initiated by the skin barrier observed in TghKLK14 mice.

The cytokine expression profile related with the analysis of immune cells infiltrates. Transgenic mice overexpressing Il-36a [START_REF] Blumberg | Opposing activities of two novel members of the IL-1 ligand family regulate skin inflammation[END_REF][START_REF] Blumberg | IL-1RL2 and its ligands contribute to the cytokine network in psoriasis[END_REF] and Il-17c [START_REF] Johnston | Keratinocyte overexpression of IL-17C promotes psoriasiform skin inflammation[END_REF] displayed eosinophil and mast cell infiltrates as well as Cd3 + T cell and Cd4 + cell infiltration, which were also detected in TghKLK14 skin. In TghKLK14, Cd4 + cells were exclusively observed in the dermis whereas Cd3 + Cd8 + T cells were detected only in the epidermis, suggesting that Cd4 + cells were plasmacytoid-like dendritic cells (pDC) and not T lymphocytes [START_REF] Palamara | Identification and Characterization of pDC-Like Cells in Normal Mouse Skin and Melanomas Treated with Imiquimod[END_REF]. The cytotoxic T cell infiltration might result from enhanced expression of IFN-g since this cytokine activates CD8 + T cells motility [START_REF] Bhat | Interferon-γ derived from cytotoxic lymphocytes directly enhances their motility and cytotoxicity[END_REF]. In psoriasis, IL-36 activates pDC that play a critical role in the initiation of skin inflammation by producing type-I IFN [START_REF] Catapano | Interleukin-36 promotes systemic Type-I IFN responses in severe forms of psoriasis[END_REF]. Therefore, infiltration of pDC-like in the skin from TghKLK14 mice could be an early sign of skin inflammation. Taken together, this study identifies KLK14 as a potential inducer of skin inflammation and immune cell infiltration through the production of epidermal cytokines such as Il-36a and Il-17c, which might also contribute to hair follicle abnormalities.

In summary, overexpression of KLK14 in the epidermis results in major hair defects and epidermal abnormalities. Marked decreased expression of Dsg3 and Dsg4 together with the demonstration that rhKLK14 degrades these desmosomal components in vitro, support the possibility that KLK14mediated Dsg3 and Dsg4 degradation contributes to the NS phenotype in TghKLK14 mice.

Furthermore, these results establish a role of KLK14 in inducing expression of Il-36a and Il-17c and identify KLK14 as a potential therapeutic target to improve skin and hair abnormalities in NS. This study provides evidence that KLK14 overexpression in the epidermis elicits major hair shaft defects, but also skin barrier anomalies, skin inflammation and immune cell infiltration, establishing KLK14 as an important player in NS pathogenesis.

Materials and methods

Transgenic mouse line establishment

The IVL-TghKLK14 mouse lines were established at the Mouse Clinical Institute -(Illkirch, France).

The full length cDNA encoding human KLK14 (OriGene clone SC317386; NM_022046) was cloned into the human IVL promoter vector, pH3700-pL2 [START_REF] Carroll | Characterization of the human involucrin promoter using a transient beta-galactosidase assay[END_REF] (Figure S3). The transgene (comprising the IVL promoter, IVL exon 1, intron 1, a SV40 intron and human KLK14 cDNA followed by a SV40 pA sequence) was excised from pH3700-pL2 by digestion with SalI and injected into the pronuclei of C57BL/6N fertilized oocytes, and finally implanted into pseudo-pregnant females. Founder mice were genotyped and crossed with wild-type C57BL/6N animals to establish the transgenic line. Genotyping was performed using the listed primer sequences in Table S1.

Experiments were carried out according to Institution guidelines and EU legislations (agreement #15833-2018011017165115).

Transepidermal Water Loss measurement. TEWL was measured using Tewameter TM300 apparatus (Courage + Khazaka, Cologne, Germany).

RNA isolation and Quantitative polymerase chain reaction

Total mRNA was extracted using RNeasy® Fibrous Tissue Mini Kit (Qiagen, Redwood, CA, USA).

Reverse transcription was carried out with 100 ng of total RNA using Super Script IV Reverse transcription kit (Life Technologies, Carlsbad, CA, USA). cDNA was amplified using Mesa Green qPCR kits for SYBR® Assay (Eurogentec, Liège, Belgium) and Applied Biosystems 7300 Real Time PCR System (Life Technologies, Carlsbad, CA, USA). Primers for each target gene are listed in Table S1. Relative gene expression was normalized to Hprt and calculated using the 2 ^-ΔΔCt method.

Immunofluorescence staining

Formalin-fixed paraffin or snap frozen O.C.T.-embedded skin sections (5-μm thickness) from TghKLK14 and WT mice were used. Hematoxylin/eosin/safranin staining and immunofluorescence staining were performed on paraffin-embedded skin sections using standard histological procedures.

Dsg3, Dsg4, Cd3, Cd4, Cd8 and NIMP-R14 stainings were performed on skin cryosections.

Antibodies used in this study are listed in Table S2. Isotype controls were used for each experiment.

Nuclei were stained in blue with 1 μg/ml DAPI. Image acquisition was performed using Leica TCS SP8 SMD confocal microscope and pictures were further processed using ImageJ software.

Immunoblotting

Tissue samples were lysed in RIPA buffer (Life Technologies, Carlsbad, CA, USA) containing Protease Inhibitor Cocktail Tablets (Roche, Basel, Switzerland). Proteins were loaded on a 4-12% precast gel (Invitrogen, Carlsbad, CA, USA) for SDS-PAGE separation. Proteins were transferred to nitrocellulose membranes using Trans-Blot Turbo system (Bio-Rad, Hercules, CA, USA). The antibodies used are listed in Table S2.

Proteolytic degradation of DSG1, 2, 3 and 4 and CDSN by KLK14 10 ng rhKLK14 were activated with 1 ng thermolysin during 1 hour at 37°C and rhKLK14 activity was tested using 100 μM Boc-VPR-AMC Fluorogenic peptide substrate, following the manufacturer's recommendations (R&D systems, Lille, France) (Figure S2E). 10 ng activated rhKLK14 was incubated with 1.5 μg rhDSG1, 2, 3 or 4 or rh or rmCDSN during 5, 15, 30, 60, 120 and 180 min at 37°C (R&D systems, Lille, France). Incubation with 1.5 μg pro-HGF was used as a positive control [START_REF] Reid | In vitro evidence that KLK14 regulates the components of the HGF/Met axis, pro-HGF and HGF-activator inhibitor 1A and 1B[END_REF] (Figure S2F). Digestion products of DSG 1, 2, 3 and 4 were revealed by immunoblotting whereas CDSN digestion gel were silver-stained according to manufacturer's instructions (Pierce Silver Stain Kit, ThermoFisher).

In situ zymography Skin cryosections (5 μm thickness) were washed with 2% Tween 20 in PBS and incubated overnight at 37°C with 100 μg/ml BODIPY FL casein (Life Technologies, Carlsbad, CA, USA). Proteolytic activity was visualized and analyzed using Leica SP8 SMD confocal microscope and ImageJ software, respectively. (A) mRNA expression of Trem1,Defb4 S100a8,S100a9,Ccl20 and ng of activated rhKLK14 during 0, 5, 15, 30, 60 120, and 180 min at 37°C. Assay buffer and 1 ng thermolysin alone were used as negative control. (D) Negative control showed that assay buffer and thermolysin alone did not digest rhDSG4, rhDSG3 and rmCdsn. 1.5 μg of rhDSG3, rhDSG4 and rmCdsn were incubated with assay buffer or 1 ng thermolysin or 10 ng of rhKLK14 activated with 1 ng thermolysin during 180 min at 37°C. (E) Kinetics of rhKLK14 proteolytic activity showed that rhKLK14 was biologically active. 10 ng activated rhKLK14 by 1 ng thermolysin was incubated with 100 µM substrate Boc-VPR-AMC substrate. Thermolysin or Boc-VPR-AMC substrate alone was used as a negative control. (F) Positive control for rhKLK14 activity showed that pro-HGF was cleaved by rhKLK14 into HGF αand βchains. 1.5 μg of rhpro-HGF were incubated with 10 ng of activated rhKLK14 during 0, 5, 15, 30, 60 120, and 180 min at 37°C. Negative control showed that assay buffer and 1 ng thermolysin alone did not digest pro-HGF. Data are representative at least from 3 independent experiments.

Figure S3: Sequence of the

Figure 1 :

 1 Figure 1: Generation of TghKLK14 mice.

Figure 2 :

 2 Figure 2: TghKLK14 mice display hair abnormalities and a skin barrier defect.

Figure 4 :

 4 Figure 4: TghKLK14 mice display defective hair shaft structure with Dsg3 and Dsg4 drastic

Figure 5 :

 5 Figure 5: TghKLK14 display skin inflammation with increased expression of Il-36a and Il-17c in

  Ifn-g normalized to Hprt in skin extracts from TghKLK14 (Tg) and WT mice at the age of 20 days. (C) Immuno-detection of Il-36a and Il-17c on skin sections and skin lysates from 20-day-old TghKLK14 and WT mice. Protein level measurement of Il-36a and Il-17c by immunoblotting was normalized to Hsc70 and expressed as fold change relative to WT controls. (D) Eosinophil and Mast cells detection in Hematoxylin/eosin/safranin (HES) and toluidine blue stained skin section from TghKLK14 mice and WT at the age of 20 days, respectively. (E) Immunostaining of CD3 + cells, CD4 + cells, CD8a + cells and neutrophils (NIMP-R14) on skin sections from 20-day-old TghKLK14 and WT mice. (F) Eosinophil and Mast cell counts per mm 2 of skin section as well as CD3 + cells, CD4 + cells, CD8a + cell and neutrophil counts per field in the skin. For immunostaining, pictures are representative of 3 independent experiments performed on skin sections from at least 4 mice. Scale bar, 50 μm; blue, Dapi; red, targets; the white line delineates the dermal-epidermal junction. Data are mean values ± SEM from skin extracts or lysates of at least 4 mice. P-values are calculated using Mann-Whitney test. *, P < 0.05; **, P < 0,01; ***, P < 0.001; ****, P < 0.0001.

Figure S1 :

 S1 Figure S1: Loss of transgene expression leads to normalization of skin anomalies with a decrease

Figure S2 :

 S2 Figure S2: Validation of rhDSGs proteolytic degradation by rhKLK14.

Acknowledgments

We are grateful to the Mouse Clinical Institute (Institut Clinique de la Souris, MCI/ICS, Illkirch, France, department of Genetic Engineering and Model Validation), for generating the TghKLK14 mice. The pH3700-pL2 plasmid containing the involucrin promoter was a gift from Dr. Julia Segre (National Human Genome Research Institute, Bethesda, Maryland, USA). We thank Mr. Alain Schmitt for the access to the electronic microscopy platform at Cochin hospital. We acknowledge Pierre Bonnesoeur for his help for silver staining. This project has received funding from the Fondation des Maladies Rares and the European Union's Horizon 2020 research and innovation program under the ERA-NET Cofund action N° 643578" (E-Rare-3 JTC 2015) (KLKIN project, coord. A.H.).

Data Availability Statement

Data sharing is not applicable to this article as no datasets were generated or analysed during the current study. However, all data can be shared upon request to the authors.

Transmission electron microscopy

Skin samples were fixed in 4% paraformaldehyde and 2% glutaraldehyde, washed in Sorenesen buffer, post fixed in 2% OsO4, dehydrated in graded ethanol solution and embedded in epon. Skin sections were counterstained with uranyl acetate and lead citrate before imaging using a 1011 transmission electron microscope (Jeol Inc., Japan) coupled to an Erlangshen CCD camera and a Digital Micrograph software (Gatan Inc.)

Statistical analysis

Experiments were performed three times using at least 4 mice. Significance level was assessed using a Student t test, or Mann-Whitney test: * p < 0.05, ** p < 0.005, *** p < 0.0005, **** p < 0.0001.

Competing interests

The authors declare no conflict of interest.

Author Contributions

Conceptualization