Comparative analyses of Netherton syndrome patients and Spink5 conditional knock-out mice uncover disease-relevant pathways.

Netherton syndrome (NS) is a rare skin disease caused by loss-of-function mutations in the serine peptidase inhibitor Kazal type 5 (SPINK5) gene. Disease severity and the lack of efficacious treatments call for a better understanding of NS mechanisms. Here we describe a novel and viable, Spink5 conditional knock-out (cKO) mouse model, allowing to study NS progression. By combining transcriptomics and proteomics, we determine a disease molecular profile common to mouse models and NS patients. Spink5 cKO mice and NS patients share skin barrier and inflammation signatures defined by up-regulation and increased activity of proteases, IL-17, IL-36, and IL-20 family cytokine signaling. Systemic inflammation in Spink5 cKO mice correlates with disease severity and is associated with thymic atrophy and enlargement of lymph nodes and spleen. This systemic inflammation phenotype is marked by neutrophils and IL-17/IL-22 signaling, does not involve primary T cell immunodeficiency and is independent of bacterial infection. By comparing skin transcriptomes and proteomes, we uncover several putative substrates of tissue kallikrein-related proteases (KLKs), demonstrating that KLKs can proteolytically regulate IL-36 pro-inflammatory cytokines. Our study thus provides a conserved molecular framework for NS and reveals a KLK/IL-36 signaling axis, adding new insights into the disease mechanisms and therapeutic targets.

Netherton syndrome subtypes share IL-17/IL-36 signature with distinct IFN-α and allergic responses.

BACKGROUND: Netherton syndrome (NS) is a rare recessive skin disorder caused by loss-of-function mutations in SPINK5 encoding the protease inhibitor LEKTI (lymphoepithelial Kazal-type-related inhibitor). NS patients experience severe skin barrier defects, display inflammatory skin lesions, and have superficial scaling with atopic manifestations. They present with typical ichthyosis linearis circumflexa (NS-ILC) or scaly erythroderma (NS-SE). OBJECTIVE: We used a combination of several molecular profiling methods to comprehensively characterize the skin, immune cells, and allergic phenotypes of NS-ILC and NS-SE patients. METHODS: We studied a cohort of 13 patients comprising 9 NS-ILC and 4 NS-SE. RESULTS: Integrated multiomics revealed abnormal epidermal proliferation and differentiation and IL-17/IL-36 signatures in lesion skin and in blood in both NS endotypes. Although the molecular profiles of NS-ILC and NS-SE lesion skin were very similar, nonlesion skin of each disease subtype displayed distinctive molecular features. Nonlesion and lesion NS-SE epidermis showed activation of the type I IFN signaling pathway, while lesion NS-ILC skin differed from nonlesion NS-ILC skin by increased complement activation and neutrophil infiltration. Serum cytokine profiling and immunophenotyping of circulating lymphocytes showed a TH2-driven allergic response in NS-ILC, whereas NS-SE patients displayed mainly a TH9 axis with increased CCL22/MDC and CCL17/TARC serum levels. CONCLUSIONS: This study confirms IL-17/IL-36 as the predominant signaling axes in both NS endotypes and unveils molecular features distinguishing NS-ILC and NS-SE. These results identify new therapeutic targets and could pave the way for precision medicine of NS.

Secukinumab Therapy for Netherton Syndrome.

Importance: Netherton syndrome (NS) is a rare, severe genetic disorder of cornification with high morbidity. Treatment for NS has been notoriously difficult. Recent studies showed an upregulated helper T cell (TH) 17/interleukin 23 (IL-23) pathway in NS, suggesting the possibility of treatment strategies that target IL-17. Objective: To evaluate the clinical response of NS to treatment with the IL-17 antagonist secukinumab. Design, Setting, and Participants: This case series study reports the experience of compassionate use therapy with secukinumab in 4 patients with severe NS, including 2 children, from December 1, 2018, to December 1, 2019, with 3 patients still undergoing treatment at the time of final analysis. Data were analyzed from December 1, 2018, to December 1, 2019. Main Outcomes and Measures: Expression of IL-17 in the skin was evaluated by immunohistochemical analysis, and serum cytokine concentrations were measured using a commercially available assay. Treatment response was assessed using the Ichthyosis Area and Severity Index (IASI) total score, including measures of erythema and scaling, the Dermatology Life Quality Index (DLQI), and the 5-D itch scale. Results: In all 4 patients (age range, 9-27 years; 3 male and 1 female), immunostaining with an IL-17A antibody showed an increased number of positive cells in lesional skin. Cytokine assessment in serum samples revealed increased levels of CCL20. Treatment duration with secukinumab was 3 to 12 months at the time of this report. After 3 months of therapy, IASI scores were reduced by 44% to 88%, DLQI scores were reduced by 40% to 76%, and 5-D itch scale scores were reduced by 27% to 62%. This outcome was sustained at the 6-month follow-up. Two patients with an erythrodermic phenotype showed marked improvement of all parameters. A refractory palmoplantar eczematous eruption occurred in 2 patients, and a candidal nail infection developed in 2 patients. No severe adverse events were reported. Conclusions and Relevance: This initial case series reporting the use of anti-IL-17 therapy in NS demonstrated marked cutaneous improvement, particularly in 2 pediatric patients with erythrodermic phenotypes. Further studies are needed to evaluate the long-term benefit of this potential treatment modality.

Effects of Bioactive Marine-Derived Liposomes on Two Human Breast Cancer Cell Lines.

Breast cancer is the leading cause of death from cancer among women. Higher consumption of dietary marine n-3 long-chain polyunsaturated fatty acids (LC-PUFAs) is associated with a lower risk of breast cancer. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are two n-3 LC-PUFAs found in fish and exert anticancer effects. In this study, natural marine- derived lecithin that is rich in various polyunsaturated fatty acids (PUFAs) was extracted from salmon heads and transformed into nanoliposomes. These nanoliposomes were characterized and cultured with two breast cancer lines (MCF-7 and MDA-MB- 231). The nanoliposomes decreased the proliferation and the stiffness of both cancer cell types. These results suggest that marine-derived lecithin possesses anticancer properties, which may have an impact on developing new liposomal delivery strategies for breast cancer treatment.

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

Netherton syndrome is a rare autosomal recessive skin disease caused by loss-of-function mutations in SPINK5 encoding LEKTI protein that results in unopposed activity of epidermal kallikrein-related peptidases (KLKs), mainly KLK5, KLK7, and KLK14. Although the function of KLK5 and KLK7 has been previously studied, the role of KLK14 in skin homeostasis and its contribution to Netherton syndrome pathogenesis remains unknown. We generated a transgenic murine model overexpressing human KLK14 (TghKLK14) in stratum granulosum. TghKLK14 mice revealed increased proteolytic activity in the granular layers and in hair follicles. Their hair did not grow and displayed major defects with hyperplastic hair follicles when hKLK14 was overexpressed. TghKLK14 mice displayed abnormal epidermal hyperproliferation and differentiation. Ultrastructural analysis revealed cell separation in the hair cortex and increased thickness of Huxley's layer. Desmoglein (Dsg) 2 staining was increased, whereas Dsg3 and Dsg4 were markedly reduced. In vitro studies showed that hKLK14 directly cleaves recombinant human DSG3 and recombinant human DSG4, suggesting that their degradation contributes to hair abnormalities. Their skin showed an inflammatory signature, with enhanced expression of IL-36 family members and their downstream targets involved in innate immunity. This in vivo study identifies KLK14 as an important contributor to hair abnormalities and skin inflammation seen in Netherton syndrome.

DDB2 (damaged-DNA binding 2) protein: a new modulator of nanomechanical properties and cell adhesion of breast cancer cells.

DDB2, known for its role in DNA repair, was recently shown to reduce mammary tumor invasiveness by inducing the transcription of IκBα, an inhibitor of NF-κB activity. Since cellular adhesion is a key event during the epithelial to mesenchymal transition (EMT) leading to the invasive capacities of breast tumor cells, the aim of this study was to investigate the role of DDB2 in this process. Thus, using low and high DDB2-expressing MDA-MB231 and MCF7 cells, respectively, in which DDB2 expression was modulated experimentally, we showed that DDB2 overexpression was associated with a decrease of adhesion abilities on glass and plastic areas of breast cancer cells. Then, we investigated cell nanomechanical properties by atomic force microscopy (AFM). Our results revealed significant changes in the Young's Modulus value and the adhesion force in MDA-MB231 and MCF7 cells, whether DDB2 was expressed or not. The cell stiffness decrease observed in MDA-MB231 and MCF7 expressing DDB2 was correlated with a loss of the cortical actin-cytoskeleton staining. To understand how DDB2 regulates these processes, an adhesion-related gene PCR-Array was performed. Several adhesion-related genes were differentially expressed according to DDB2 expression, indicating that important changes are occurring at the molecular level. Thus, this work demonstrates that AFM technology is an important tool to follow cellular changes during tumorigenesis. Moreover, our data revealed that DDB2 is involved in early events occurring during metastatic progression of breast cancer cells and will contribute to define this protein as a new marker of metastatic progression in this type of cancer.

Interaction of Iron II Complexes with B-DNA. Insights from Molecular Modeling, Spectroscopy, and Cellular Biology.

We report the characterization of the interaction between B-DNA and three terpyridin iron II complexes. Relatively long time-scale molecular dynamics (MD) is used in order to characterize the stable interaction modes. By means of molecular modeling and UV-vis spectroscopy, we prove that they may lead to stable interactions with the DNA duplex. Furthermore, the presence of larger π-conjugated moieties also leads to the appearance of intercalation binding mode. Non-covalent stabilizing interactions between the iron complexes and the DNA are also characterized and evidenced by the analysis of the gradient of the electronic density. Finally, the structural deformations induced on the DNA in the different binding modes are also evidenced. The synthesis and chemical characterization of the three complexes is reported, as well as their absorption spectra in presence of DNA duplexes to prove the interaction with DNA. Finally, their effects on human cell cultures have also been evidenced to further enlighten their biological effects.

Manganese superoxide dismutase in breast cancer: from molecular mechanisms of gene regulation to biological and clinical significance.

Breast cancer is one of the most common malignancies of all cancers in women worldwide. Many difficulties reside in the prediction of tumor metastatic progression because of the lack of sufficiently reliable predictive biological markers, and this is a permanent preoccupation for clinicians. Manganese superoxide dismutase (MnSOD) may represent a rational candidate as a predictive biomarker of breast tumor metastatic progression, because its gene expression is profoundly altered between early and advanced breast cancer, in contrast to expression in the normal mammary gland. In this review, we report the characterization of some gene polymorphisms and molecular mechanisms of SOD2 gene regulation, which allows a better understanding of how MnSOD is decreased in early breast cancer and increased in advanced breast cancer. Several studies display the biological significance of MnSOD level in proliferation as well as in invasive and angiogenic abilities of breast tumor cells by controlling superoxide anion radical (O2(•-)) and hydrogen peroxide (H2O2). Particularly, they report how these reactive oxygen species may activate some signaling pathways involved in breast tumor growth. Emerging understanding of these findings provides an interesting framework for guiding translational research and suggests a way to define precisely the clinical interest of MnSOD as a prognostic and/or predicting marker in breast cancer, by associating with some regulators involved in SOD2 gene regulation and other well-known biomarkers, in addition to the typical clinical parameters.

DDB2: a novel regulator of NF-κB and breast tumor invasion.

The DNA repair protein damaged DNA-binding 2 (DDB2) has been implicated in promoting cell-cycle progression by regulating gene expression. DDB2 is selectively overexpressed in breast tumor cells that are noninvasive, but not in those that are invasive. We found that its overexpression in invasive human breast tumor cells limited their motility and invasiveness in vitro and blocked their ability to colonize lungs in vivo, defining a new function for DDB2 in malignant progression. DDB2 overexpression attenuated the activity of NF-κB and the expression of its target matrix metalloprotease 9 (MMP9). Mechanistic investigations indicated that DDB2 decreased NF-κB activity by upregulating expression of IκBα by binding the proximal promoter of this gene. This effect was causally linked to invasive capacity. Indeed, knockdown of DDB2-induced IκBα gene expression restored NF-κB activity and MMP9 expression, along with the invasive properties of breast tumor cells overexpressing DDB2. Taken together, our findings enlighten understanding of how breast cancer cells progress to an invasive phenotype and underscore potential clinical interest in DDB2 as a prognostic marker or therapeutic target in this setting.