Skin barrier lipid enzyme activity in Netherton patients is associated with protease activity and ceramide abnormalities.

Individuals with Netherton syndrome (NTS) have increased serine protease activity, which strongly impacts the barrier function of the skin epidermis and leads to skin inflammation. Here, we investigated how serine protease activity in NTS correlates with changes in the stratum corneum (SC) ceramides, which are crucial components of the skin barrier. We examined two key enzymes involved in epidermal ceramide biosynthesis, β-glucocerebrosidase (GBA) and acid-sphingomyelinase (ASM). We compared in situ expression levels and activities of GBA and ASM between NTS patients and controls and correlated the expression and activities with i) SC ceramide profiles, ii) in situ serine protease activity, and iii) clinical presentation of patients. Using activity-based probe labeling, we visualized and localized active epidermal GBA, and a newly developed in situ zymography method enabled us to visualize and localize active ASM. Reduction in active GBA in NTS patients coincided with increased ASM activity, particularly in areas with increased serine protease activity. NTS patients with scaly erythroderma exhibited more pronounced anomalies in GBA and ASM activities than patients with ichthyosis linearis circumflexa. They also displayed a stronger increase in SC ceramides processed via ASM. We conclude that changes in the localization of active GBA and ASM correlate with i) altered SC ceramide composition in NTS patients, ii) local serine protease activity, and iii) the clinical manifestation of NTS.


INTRODUCTION
Netherton syndrome (NTS) is a severe autosomal recessive disorder related to uncontrolled serine protease activity caused by mutations in the SPINK5 gene (serine protease inhibitor Kazal-type 5) that encodes for the protease inhibitor LEKTI (Lympho-epithelial Kazal-type-related inhibitor). This protein is crucial for a proper skin desquamation (shedding of the skin). Increased epidermal serine protease activity in NTSpatients results in scaling and superficial peeling of the skin and skin inflammation (1, 2). Clinical manifestation varies to a high extent: some subjects demonstrate extensive and severe scaly erythroderma whereas others develop ichthyosis linearis circumflexa (ILC) with variable severity. The origin for this variation is not fully understood (3). Newborns are susceptible to life-threatening dehydration caused by increased water loss resulting from a defective skin barrier function (4). This barrier is primarily located in the stratum corneum (SC), and formed by terminally differentiated keratinocytes (corneocytes) embedded in a lipid matrix (5,6). This matrix is composed of different lipid classes, like ceramides, cholesterol and fatty acids. Whereas in other tissues ceramides are usually involved in metabolism or cell signalling, ceramides in the stratum corneum mainly function as skin barrier components. SC Ceramides have very unique features compared to those present in other tissues: i) their carbon chains are much longer and ii) there is a large variation in their molecular architecture (subclass overview in Supplemental Figure S1) (7,8). Ceramides are not synthesized in the SC, but their precursors are synthesized by keratinocytes located in the viable epidermal layers (9,10). These ceramide precursors (glucosylceramides and sphingomyelins) are subsequently stored in lamellar bodies. These lamellar bodies contain also the enzymes necessary for the final conversion once the lamellar bodies extrude their content into the extracellular environment. This  Figure S1) (11,12).
Previously, we reported an altered SC ceramide composition in NTS-patients (14). However, it is unknown whether this change in ceramide composition is caused by a disbalance of epidermal GBA and ASM enzyme activities in SC of NTS. Besides, the relation between expression/activity of both enzymes and how this relates to the ceramide composition and the clinical manifestation are not understood. Our aim was therefore to localize both expression and activity of ASM and GBA in the epidermis of 10 NTS-patients ( -) and 5 healthy controls. The results provide mechanistic insight how changes in localization of ASM and GBA associate with increased [AS] and [NS] SC ceramides, and whether this correlates with the localization of protease activity and patient clinical manifestations.

Subject inclusion and skin processing
The study was conducted according to Declaration of Helsinki principles, with written informed consent from patients (or parents in case of minors). Study approval was obtained from the Comité de Protection des personnes in France, (number 101-13). Registration was performed at the French national regulatory agency (ANSM, Agence National de sécurité du Médicament, number 131066B42). Ten NTS-patients (details in Supplemental Table S2 and Supplemental Figure S4) were compared to 5 healthy controls. SC ceramides were obtained by harvesting SC of the ventral forearm with 10 poly(phenylene sulfide) tapestrips (Nichiban, Tokyo, Japan) prior to ceramide extraction. Besides, 4 mm biopsies were taken for immunohistochemical staining and in-situ enzyme activity assays. Concerning NTS-patients, all biopsies were from lesional skin sites except for NTS . Subsequently, biopsies were snap-frozen in liquid nitrogen with matrix specimen (TissueTek O.C.T., Sakura Finetek Europe, Alphen a/d Rijn, Netherlands) and cut to enzyme studies (see below for more detail, and Figure 2 for an overview of the staining procedures).

Immunohistochemical staining of ASM and GBA
Frozen skin sections were washed in PBS, pH=7.4, blocked with horse serum, incubated overnight at 4°C with primary antibody for GBA (ab125065, Abcam Cambridge, UK) and ASM (NBP2-45889, Novus Biologicals, Littleton, CO). Sections were washed in PBS and labeled with secondary antibody for GBA (711-295-152, Jackson ImmunoResearch Laboratories, West Grove, PA) or ASM (ab97035, Abcam). After 1h incubation period, sections were washed twice in PBS and once in demineralized water and mounted using Vectashield with diamidino-phenylindole solution (DAPI, Vector Laboratories, Burlingame, CA).

In-situ zymography of active ASM
A new method was developed to visualize active ASM in human skin sections by in-situ zymography using 6-hexadecanoyl-4-methylumbelliferylphosphorylcholine (6-HMU-PC) as ASM specific substrate

In-situ Activity Based Probe (ABP)-labeling of active GBA
Active GBA was visualized using the recently developed ABP labeling method (15,16

Fluorescence microscopy
Protease activity was visualized with a Leica TCS SP8 SMD confocal microscope and analyzed with ImageJ software. All other stainings were imaged using a Zeiss Imager.D2 microscope connected to a Zeiss AxioCam Mrm camera (Zeiss, Göttingen, Germany). Images were taken at objective lens magnifications of 20x and 63x (+10x ocular lens magnification). Activity of ASM was visualized by 6-ex=380nm, em=460nm. Active GBA was visualized with ABPex em=610nm.

SC ceramide extraction and analysis
A liquid/liquid extraction protocol was used to extract the SC lipids, including the ceramides. This procedure is based on the common methods to extract SC lipids, the Folch extraction and the Bligh and Dyer extraction (17,18). Briefly, each SC sample was extracted using three successive extraction steps with different ratios LC/MS ceramide data is plotted as relative abundance of internal standard corrected peak areas (%).

Statistics
For statistics on two independent means, p-values were calculated using unpaired t-tests with Welch correction (for unequal variances). A Two-way ANOVA with a Sidak's multiple comparison test was performed to determine significant differences between the 12 ceramide subclasses for both groups (Healthy vs NTS). Correlations and the corresponding p-values are described with the Pearson correlation coefficient.

Abnormal ASM-activity and expression in NTS
We developed an in-situ zymography method using selective ASM substrate that results in fluorogenic product 6-HMU (20). Figure 3a demonstrates that in control skin tissue, active ASM is predominantly localized at the interface between the stratum granulosum (SG) and SC, as well as the innermost SC layers.
Fluorescent signal was also observed to a less extent in more superficial SC layers and in the viable epidermis. At higher magnifications, individual 'striations' of fluorescent signal are observed, illustrating ASM-activity in the lipid matrix surrounding the corneocytes. ASM-activity is not homogenously distributed among the striations, indicated by the high regional variance in fluorescence intensity. Figure   3b,c illustrate that ASM is predominantly expressed at the SG/SC interface, and thus resembles to a large extent the results of the ASM-activity assay. However, the local variation in intensity that was observed for ASM-activity is not observed for the expression, implying that not all expressed ASM is active. Besides, ASM is also de novo expressed in the viable epidermis near the cell nuclei ( Figure 3c).

ASM-expression and activity in the epidermis of NTS demonstrated large variations between subjects and
even within a single skin section (Overview of all individuals in Supplemental Figure S2). NTS-patients demonstrate the following differences compared to control skin: i) areas with intense staining of active ASM

Altered GBA-activity and expression in NTS
We compared the GBA-activity pattern with the enzyme expression in NTS and controls. In control skin, active GBA is not observed in the dermis or viable epidermis, but is evident throughout the entire SC, with NTS subjects -GBA-expression appeared (partially) in the intracellular space throughout the viable epidermis, either with or without a more intense expression at the SG and SC transition area (Figure 4g).
This expression pattern was particularly seen at skin areas with substantial parakeratosis,

Inverse correlation between GBA activity and ASM-activity in single NTS skin sections
When analyzing complete skin sections from each NTS-patient, six out of ten patients demonstrate a varying expression and/or activity profile along each section (multiple skin sections per subject were analyzed).  Figure S2). In general, there was strong evidence for an inverse relationship between the activity of GBA and ASM, rather then with the expression of both enzymes.

Localization of active GBA and ASM coincide with serine protease activity
Next, we compared the localization of the lipid enzymes with the localization of serine protease activity, generally not abundantly present in control skin (Supplemental Figure S2). In contrast, NTS-subjects had a large variation in serine protease activity, as observed for the activity of ASM and GBA. For individual at Walaeus Library / BIN 299, on May 12, 2020 www.jlr.org subjects, areas with increased serine protease activity matched areas with enhanced ASM-activity and decreased/absent GBA-activity. This is illustrated for NTS in Figure 5.

Altered SC ceramide composition in NTS
Metabolic processes of GBA and ASM directly affect the SC ceramide composition. Figure 6a shows the ceramide profile (expressed as relative peak areas) of the twelve most prominent ceramide subclasses from SC of controls versus NTS (individual data in Supplemental Figure S3). SC ceramide data of NTS was excluded due to an extremely low amount of detected lipid content, leading to data that could be easily

DISCUSSION
This study is the first to assess and localize the expression and activity of both GBA and ASM in NTS. The new zymography method that we applied, uses 6-HMU-PC. This is, according to literature, a more robust and selective substrate-alternative than the standard Amplex Red Peroxidase/choline oxidase assay (20,23).
The developed method is also less labor intensive and enabled us to visualize with high spatial resolution active ASM in human epidermis. Together with GBA-activity labelling, we could localize both active enzymes in the SC lipid layers of NTS-patients and controls. This allowed us to relate activity of key lipid processing enzymes GBA and ASM to the ceramide profile.

GBA-activity is abnormal in NTS.
NTS-patients demonstrated abnormal GBA-expression and activity, particularly at areas with parakeratosis.
GBA was still expressed in most NTS-patients, but only minimally active at the SC/SG interface where lipid synthesis and metabolism are crucial for optimal formation of the lamellar layers (24). A change in the local cellular environment (e.g. local pH, discussed below) or the absence of activator protein saposin-C (25,26) may be underlying factors. Reduced/inactive GBA will lead to a cell-mediated response in which GBAexpression is upregulated by the cell to maintain homeostasis (27), explaining GBA overexpression in several NTS-subjects.

The increase in ASM-activity can (at least in part) explain the increment in subclasses [AS] and [NS] in
NTS, as those ceramides are reported to be the only subclasses that originate from conversion of sphingomyelins by ASM (besides by GBA) (11,12). The strong correlation between ceramides [NS] and [AS] (and between no other ceramides; Supplemental Table S1)

Protease activity matches GBA and ASM activity
Another key finding from this study is the mutual relationship between GBA, ASM and serine protease activities. Although NTS-patients demonstrated an extensive variation in the localization of active enzyme, a decrease in GBA-activity coincided with an increase in both ASM-activity and serine protease activity. Particularly for NTS , , , , this colocalization is most apparent at heavily nucleated at Walaeus Library / BIN 299, on May 12, 2020 www.jlr.org SC areas (Supplemental Figure S2). In contrast, the absence of serine protease activity correlated with the presence of active GBA. This implies a direct or indirect link between epidermal proteases and these lipid enzymes. One such common factor could be the local skin-pH. The acidic environment of the SC (between 4-6) is crucial for epidermal barrier integrity, lipid enzymes function, and serine protease activity (43).
Changes in skin-pH directly affect enzyme activity of lipid enzymes like GBA and ASM, which may lead to incompletely processed lamellar membranes and a disruptive skin barrier (23). Moreover, an increase in local skin-pH in NTS will lead to further increased protease activity of kallikreins 5 and 7 (besides the increment due to LEKTI deficiency). These proteases are involved in the desquamation process and degradation of lipid processing enzymes like GBA and ASM (23,44). Indeed, NTS-patients suffer from defective Kallikrein 5/7 inhibition, which may contribute to the defective skin barrier in these patients (42,45). The increase in ceramides [AS] and [NS] will contribute to a more permeable barrier, as demonstrated with lipid membranes in which these ceramide subclasses were increased (46). ceramides. In contrast, NTS , who had a minor form of NTS, displayed a ceramide composition that was (of all NTS-subjects) most comparable to control skin. and scalp structure evaluation), which may sometimes prove difficult or could -even today -lead to missed cases, in which misdiagnosis occurred for many years (47). Current DNA screening tests for SPINK5 mutations are, in practice, not feasible for daily diagnostic confirmation. Therefore, analysis of the ceramides and the respective enzyme expression/activity localization can be useful as a complementary method that may assist in diagnosing these patients.

Correlation with clinical form of NTS
Overall, the introduction of a new method to analyze both expressed and active ASM and GBA in-situ enabled us to reveal the relation between these lipid enzymes, the protease activity, and the SC ceramide composition in NTS patients. In addition, we demonstrate that differences in these enzyme activities relate to the clinical form of NTS.

DATA AVAILABILITY
Original datasets related to this article can be found at: http://dx.doi.org/10.17632/wr9nw7vhm9.1, hosted at Mendeley Data (van Smeden, 2019). All other data is included in the manuscript.  1h incubation period. a3) After a second washing procedure, samples were analyzed by fluorescent microscopy. b) Activity of GBA was visualized by Activity Based Probe (ABP) labeling. b1) Skin sections are exposed to a solution of ABP-MDW941, followed by a 1h incubation period in which the ABP binds with high affinity and specificity to active GBA only. b2) Subsequently, Samples are washed and active GBA is localized via fluorescent microscopy. c) Both protease activity and the new method to visualize ASM activity is established by in-situ zymography. c1) First, skin sections are exposed to a solution with substrate that can specifically bind to the enzyme of interest (6-HMU-PC to visualize active ASM, and BODIPY FL casein for serine protease activity). c2) Then, an incubation period is maintained in which the substrate is converted into a product that is fluorescent (1h for ASM activity, overnight for protease activity).