KLK5 Inactivation Reverses Cutaneous Hallmarks of Netherton Syndrome

<div><p>Netherton Syndrome (NS) is a rare and severe autosomal recessive skin disease which can be life-threatening in infants. The disease is characterized by extensive skin desquamation, inflammation, allergic manifestations and hair shaft defects. NS is caused by loss-of-function mutations in <i>SPINK5</i> encoding the LEKTI serine protease inhibitor. LEKTI deficiency results in unopposed activities of kallikrein-related peptidases (KLKs) and aberrantly increased proteolysis in the epidermis. <i>Spink5</i>^<i>-/-</i> mice recapitulate the NS phenotype, display enhanced epidermal Klk5 and Klk7 protease activities and die within a few hours after birth because of a severe skin barrier defect. However the contribution of these various proteases in the physiopathology remains to be determined. In this study, we developed a new murine model in which <i>Klk5</i> and <i>Spink5</i> were both knocked out to assess whether Klk5 deletion is sufficient to reverse the NS phenotype in <i>Spink5</i>^<i>-/-</i> mice. By repeated intercrossing between <i>Klk5</i>^<i>-/-</i> mice with <i>Spink5</i>^<i>-/-</i> mice, we generated <i>Spink5</i>^<i>-/-</i><i>Klk5</i>^<i>-/-</i> animals. We showed that <i>Klk5</i> knock-out in Lekti-deficient newborn mice rescues neonatal lethality, reverses the severe skin barrier defect, restores epidermal structure and prevents skin inflammation. Specifically, using <i>in situ</i> zymography and specific protease substrates, we showed that <i>Klk5</i> knockout reduced epidermal proteolytic activity, particularly its downstream targets proteases KLK7, KLK14 and ELA2. By immunostaining, western blot, histology and electron microscopy analyses, we provide evidence that desmosomes and corneodesmosomes remain intact and that epidermal differentiation is restored in <i>Spink5</i>^<i>-/-</i><i>Klk5</i>^<i>-/-</i>. Quantitative RT-PCR analyses and immunostainings revealed absence of inflammation and allergy in <i>Spink5</i>^<i>-/-</i><i>Klk5</i>^<i>-/-</i> skin. Notably, Il-1β, Il17A and Tslp levels were normalized. Our results provide <i>in vivo</i> evidence that KLK5 knockout is sufficient to reverse NS-like symptoms manifested in <i>Spink5</i>^<i>-/-</i> skin. These findings illustrate the crucial role of protease regulation in skin homeostasis and inflammation, and establish KLK5 inhibition as a major therapeutic target for NS.</p>