Alain Hovnanian, MD, PhD, Departments of Genetics and Dermatology, Necker Hospital for Sick Children, Paris, France

A severe ichthyosis with constant allergy
Netherton syndrome (NS) was first described by Comel and Netherton in 1949 and 1958, respectively. It is a rare disease, with an incidence estimated around 1 in 100,000 births, distributed worldwide. NS is among the most severe genetic diseases of keratinization, which has the very specific feature to be always associated with severe allergy. It is transmitted in an autosomal recessive manner, i.e. parents are healthy carriers of a mutated copy of the disease gene which they both transmit to their affected child.

NS is characterized by the clinical triad of redness and scaling of the skin, a specific hair shaft abnormality and severe eczema. Affected babies present at birth with generalized redness (erythroderma) and scaling of the skin. Complications are frequent during the neonatal period and can be life-threatening. These include severe hypernatraemic dehydration, systemic infections, failure to thrive, growth retardation, and sometimes lung and gastro-intestinal complications. Hair, eyebrows and eyelashes are often sparse, fragile, short, and grow slowly. By the age of one year, they show a highly specific feature named "bamboo hair" (or trichorrhexis invaginata) which is seen under light microscopy examination (the distal part of the hair shaft is invaginated within the proximal part, resulting in a "bamboo-like hair shaft." Later, affected children constantly develop severe allergy which manifests as atopic dermatits (eczema of the skin with itching and elevated IgE levels in the serum), often associated with hay fever, multiple food allergies and sometimes asthma.

NS is caused by defective expression of a protease inhibitor
The underlying cause of NS remained elusive until 2000 when our group identified SPINK5 (Serine Protease INhibitor of Kazal Type 5) as the defective gene in NS(1). This came as a surprise, since SPINK5 encodes the protease inhibitor LEKTI (Lympho Epithelial Kazal type inhibitor), a family of proteins which has not previously been implicated in any disease. This discovery not only was a breakthrough in the understanding of the disease mechanism, but it immediately benefited patients and their families for diagnosis and genetic counselling. Indeed, no diagnostic test for NS was available prior to the identification of the causative gene. Disclosure of the NS gene allowed molecular diagnosis of NS by SPINK5 analysis to be performed in each family. This also led to the development of LEKTI antibodies to design the first quick, easy and reliable diagnostic test of the disease by LEKTI immunodetection of skin sections from patient biopsies.
Immunohistochemical and molecular diagnosis of NS
We and others have now studied a large number of patients from different cohorts worldwide. The vast majority of patients harbour mutations leading to the loss of LEKTI expression(2,3). This allows rapid and unambiguous diagnosis of the disease by immunohistological examination of a skin biopsy of the patient which shows complete absence of LEKTI in the most superficial living layer of the epidermis (granular layer).
Genetic counselling and prenatal diagnosis
The identification of SPINK5 mutations in NS families allows to confirm or to establish the diagnosis of NS, which is essential for genetic counselling. Indeed, due to its recessive mode of inheritance, a couple with an affected child is at 25% risk of recurrence at each pregnancy. The identification of the causative SPINK5 mutations in a given family allows to offer early prenatal diagnosis of the disease(4). This is now performed at 10 weeks of gestation by fetal DNA analysis isolated from chorionic villus sampling. This is an outdoor procedure which is available in most specialized obstetric centers. This represents a major progress since prenatal diagnosis of NS was not possible prior to the identification of the disease gene.
What do we know on this protease inhibitor?
LEKTI is a "super" serine protease inhibitor made of 15 domains separated by linker regions. Each domain, except domain 1, contains a rigid inhibitory loop which blocks the catalytic site of the target protease(s). LEKTI is produced as a precursor molecule which is quickly cleaved within linker regions to generate multidomains. These proteolytic forms are secreted by keratinocytes into the extracellular space between the last living layer of the epidermis (granular layer) and the stratum corneum, where they inhibit their target proteases.(5,6) LEKTI is expressed in all stratified human epithelia, including the skin, the oral and genital mucosa where it is expressed in the most superficial layers.(3) LEKTI is thus expressed at the front side of the body defense. Interestingly, LEKTI is expressed in the thymus, a lymphoïd organ which is essential for T cell education and immune tolerance. LEKTI is not detectable in haematopoietic cells, nor in the lung and the gastrointestinal track, but proteoltic fragments are detected in the blood circulation, suggesting a possible role at distance. We have also studied how LEKTI affects skin cells in culture. LEKTI is a protein that strongly inhibits enzymes that break down proteins (proteases). There is one area of the LEKTI protein, in the middle of the protein (domains 8-11) that is most effective. This fragment strongly inhibits kallikrein (KLK) proteases 5,7 and 14, particularly KLK5 (7). Kallikreins are proteases which play important roles in the epidermis, including in the integrity of the skin barrier, and in preventing skin peeling (desquamation) and inflammation. Remarkably, the binding of this proteolytic fragment to KLK5 depends on how the acid-base balance of the skin environment; is it strongly active at neutral pH (pH7, in the deep layers of the stratum corneum), and weak at acidic pH (pH5, in the superficial layers of the stratum corneum or outermost area of the skin). Thus, the Ph gradient of the stratum corneum allows the progressive release of KLK5 from most superficial layers of the skin. This fine regulation of desquamation is totally absent in NS due to the lack of LEKTI, resulting in fully active KLK5 in the deep layers of the statum corneum. This leads to premature detachment of the outermost layers of skin.
KLK5 at the center of the disease mechanism in NS

To further understand LEKTI function in vivo, we and others have generated a mouse model in which the murine spink5 gene is invalidated. Lack of lekti expression in mice recapitulates NS features and causes a major skin barrier defect.(8) Newborn mice show severe peeling of the skin resulting from premature detachment of the stratum corneum. They display a drastic skin barrier defect which leads to death of the pups by dehydation within 10 hours. Biochemical analysis of skin extracts of these animals identified KLK5 and KLK7 as two major proteases which are overactive in this mouse model. KLK5 hyperactivity leads to the degradation of components of the adhesion complexes ((corneo)desmosomes) which attach the last living layer of the epidermis to the stratum corneum, resulting in cleavage of these structures and premature detachment of the stratum corneum. Terminal differentiation markers and lipids of the stratum corneum are also abnormal, showing that protease dysregulation also affects these important processes of skin homeostasis. The SPINK5 knock-out mouse model disclosed a major mechanism of the disease, i.e. hyperactive KLK5 degrades (corneo)desmosomal components leading to premature stratum corneum detachment and a severe skin barrier defect. This was initially thought to underlie the development of allergy in NS through increased penetration of allergens and microbes. However, the fact that other ichthyoses with a skin barrier defects do not develop allergy prompt us to search for a specific cause for the allergic manifestations seen in NS patients. We recently demonstrated that another mechanism, also driven by KLK5 hyperactivity, was underlying inflammation and allergy in NS.(9) This pathway involves the activation of specific receptors (Protease activated receptor-2, or PAR-2) which are present at the cell surface of keratinocytes, and are activated upon cleavage by KLK5. Since Spink5 knock-out mice die shortly after birth, we used a graft model of Spink5 deficient skin onto immunodeficient mice. We showed that in grafted lekti deficient skin, KLK5 hyperactivity leads to enhanced PAR-2 activity, resulting in the production of several pro-inflammatory cytokines, including TSLP (Thymus Stromal Lymphopoïein), a major molecule which is strongly expressed in atopic dermatitis. TSLP is known to activate Langerhans cells, which are antigen presenting cells present in the epidermis. Langerhans cells activate dby TSLP migrate into lymp nodes where they induce the differentiation of T cells into pro-allergic T cells (called Th2 cells). In addition to TSLP, other important cytokines ar produced by Lekti deficient epidermis, such as TARC (Thymus and activation-regulated chemokine), MDC (Macrophage-derived cytokine), TNF-alpha and interleukin 8 (IL8) which contribute to the recruitment and activation of inflammatory cells such as eosinophils and mast cells. These cells also produce major pro-inflammatory cytokines which contribute to a pro-inflammatory environment which favors the development of allergic reactions. This adds to the release of IL-1beta by keratinocytes after mechanical stress induced by sratum corneum detachment. A remarkable feature is that this signalling pathway is initiated in the absence of any challenge by allergens or microbes, since it is detectable during embryonic life of SPINK5 knock-out mice as soon as 19.5 days. In addition, this biological cascade is maintained in lekti deficient keratinocytes (epidermal cells) in culture, which demonstrates that it is an intrinsic property of these cells.(9)
To what extent are these findings relevant to NS patients?
We subsequently tested patient skin samples to see whether these observations were relevant to patients with NS. Detailed histological, ultrastructural, and immunohistological analyses showed that this was indeed the case. In patient skin, all features of these two pathways were present : one involved stratum corneum detachment associated with increased KLK5 activity, degradation of (corneo)desmosomal components and (corneo)desmosomal cleavage;(10) the other implicated PAR-2 activation, NF-KB signaling (a major ativation pathway in keratinocytes), production of TSLP, TNF-alpha, IL-8 and IL-1beta. All these abnormalities were maintained in patient keratinocytes in culture, demonstrating that they are intrinsic properties of NS human epidermis.(9)
What have we learned on the disease mechanism?
From these results, we have learned that LEKTI inhibits major epidermal proteases involved in the desquamation and the inflammation processes of the skin. They have pointed to two major biological cascades involved in NS, both initiated by KLK5 hyperactivity. On one side, KLK5 hyperactivity leads to (corneo)desmosomal cleavage and stratum corneum detachment. In parallel, and even prior to stratum corneum detachment, KLK5 hyperactivity triggers the secretion of major pro-inflammatory and pro-allergic molecules, the first of which is TSLP, a key molecule in atopic dermatitis and in asthma. For these reasons, although NS is a rare monogenic disease, it serves as a disease model to study the links between skin barrier defects and allergy, and to a further extent, for the study of atopic dermatitis in which the implication of several genes makes the genetic investigation more difficult. Our results are in agreement with the current notion that epithelia play a major role in the initiation of skin allergy, as shown by the role of Filaggrin stop mutations in atopic dermatitis and in ichthyosis vulgaris. These data describe a new pathway, which directly links LEKTI deficiency to allergy, and may provide a link with asthma and food allergies. Further work will focus on the search for immunological abnormalities T cells from NS patients, the study of the role of LEKTI in the thymus, and the development of new treatments based on these new breakthroughs.
Do these results lead to new treatments for NS?
There is currently no specific treatment for NS. During the neonatal period, prevention and treatment of dehydration and infection, correct food intake are essential and may require a hospitalization in a neonatal care unit. Subsequently, the use of topical steroids and calcineurin inhibitors (Protopic) is restricted to limited skin areas because of their secondary effects. Emollients and moisturizing to protect from the skin barrier defect, as well as antiseptic treatment to prevent skin infection are key elements, but a specific treatment is still missing. Our work has identified several potent target molecules for therapeutic intervention. These include KLK5, a direct LEKTI target which initiates the biological cascades causing skin barrier defect, inflammation and allergy. Although KLK5 appears as the first target, due to its role in initiating both stratum corneum detachment and PAR-2 activation, more downstream actors include PAR-2, TSLP, TNF-alpha, IL8, all actors whose inhibition has the potential to diminish the inflammatory and allergic cascade. Some therapeutic agents such as antibodies which specifically block each of these molecules already exist and are commercialized, but the benefit/risk ratio should be considered and their use in NS requires pre-clinical studies in animal models. Other therapeutic strategies involve pharmacological approaches (identification of new KLK5 inhibitors through intensive high throughput screening efforts), gene therapy approaches aiming at degrading specific mRNAs, or replacement therapy using the most potent LEKTI fragments whose size is compatible with skin penetration. While these different approaches are being currently developed, the prevention of the consequences of the skin barrier defect by the regular use of moistering agents and emollients, and the prevention of infections are important elements of the treatment to pursue.

References :

1.Chavanas S, Bodemer C, Rochat A, Hamel-Teillac D, Ali M, Irvine AD, Bonafe JL, Wilkinson J, Taieb A, Barrandon Y, Harper JI, de Prost Y and Hovnanian A. Mutations in SPINK5, encoding a serine protease inhibitor, cause Netherton syndrome. Nat Genet. 2000. 25:141-2.

2.Bitoun E, Chavanas S, Irvine AD, Lonie L, Bodemer C, Paradisi M, Hamel-Teillac D, Ansai Si, Mitsuhashi Y, Taïeb A, de Prost Y, Zambruno G, Harper JI and Hovnanian A. Netherton Syndrome : disease expression and spectrum of SPINK5 mutations in 21 families. J Invest Dermatol. 2002. 118:352-361

3.Bitoun E, Micheloni A, Lamant L, Bonnart C, Tartaglia-Polcini A, Cobbold C, Saati TA, Mariotti F, Mazereeuw-Hautier J, Boralevi F, Hohl D, Harper J, Bodemer C, D'Alessio M, Hovnanian A. LEKTI proteolytic processing in human primary keratinocytes, tissue distribution, and defective expression in Netherton syndrome. Hum Mol Genet. 2003. 12:2417-2430.

4.Bitoun E, Bodemer C, Amiel J, de Prost Y, Stoll C, Calvas P, Hovnanian A. Prenatal diagnosis of a lethal form of Netherton syndrome by SPINK5 mutation analysis. Prenat Diagn. 2002. 22:121-6.

5.Ishida-Yamamoto A, Deraison C, Bonnart C, Bitoun E, Robinson R, O'brien TJ, Wakamatsu K, Ohtsubo S, Takahashi H, Hashimoto Y, Dopping-Hepenstal PJ, McGrath JA, Iizuka H, Richard G, Hovnanian A. LEKTI Is Localized in Lamellar Granules, Separated from KLK5 and KLK7, and Is Secreted in the Extracellular Spaces of the Superficial Stratum Granulosum. J Invest Dermatol. 2005. 124:360-6.

6.Bonnart C, Tartaglia-Porcini A, Micheloni A, Cianfarani F, Andrè A, Zambruno G, Hovnanian A, D'Alessio M. The human SPINK5 gene encodes multiple LEKTI isoforms derived from alternative pre-mRNA processing. J Invest Dermatol. 2006. 126:315-24.

7.Deraison C and Bonnart C, Lopez F, Besson C, Robinson R, Jayakumar A, Wagberg F, Brattsand M, Hachem JP, Leonardsson G, Hovnanian A. LEKTI fragments specifically inhibit KLK5, KLK7 and KLK14 and control desquamation through a pH-dependent interaction. Mol Biol Cell. 2007. 18:3607-3619.

8.Descargues P, Deraison C, Bonnart C, Kreft M, Kishibe M, Ishida-Yamamoto A, Elias P, Barrandon Y, Zambruno G, Sonnenberg A, Hovnanian A. Spink5-deficient mice mimic Netherton syndrome through degradation of desmoglein 1 by epidermal protease hyperactivity. Nat Genet. 2005. 37:56-65.

9.Briot A, Deraison C, Lacroix M, Bonnart C, Robin A, Besson C, Dubus P, Hovnanian A. Kallikrein 5 induces atopic dermatitis-like lesions through PAR2-mediated thymic stromal lymphopoietin expression in Netherton syndrome. J Exp Med. 2009, 206(5):1135-47.

10.Descargues P, Deraison C, Prost , Fraitag S, Mazereeuw-Hautier J, D'Alessio, M, Ishida-Yamamoto A, Bodemer C, Zambruno G, Hovnanian A. Corneodesmosomal cadherins are preferential targets of Stratum Corneum Trypsin-and Chymotrypsin-like hyperactivity in Netherton syndrome. J Invest Dermatol. 2006. 126:1622-32.

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