Detrimental Effects of IFN-γ on an Epidermolysis Bullosa Simplex Cell Model and Protection by a Humanized Anti-IFN-γ Monoclonal Antibody.

Epidermolysis bullosa is a group of severe skin blistering disorders, which currently have no cure. The pathology of epidermolysis bullosa is recognized as having an inflammatory component, but the role of inflammation in different epidermolysis bullosa disorders is unclear. Epidermolysis bullosa simplex (EBS) is primarily caused by sequence variants in keratin genes; its most severe form, EBS generalized severe, is characterized by aggregates of keratin proteins, and cell models of EBS generalized severe show constitutively elevated stress. IFN-γ is a major mediator of inflammation, and we show that the addition of IFN-γ alone to disease model keratinocytes promotes keratin aggregation, decreases cell-cell junctions, delays wound closure, and reduces cell proliferation. IFN-γ exposure weakens the intercellular cohesion of monolayers on mechanical stress, with IFN-γ-treated EBS monolayers more fragmented than IFN-γ-treated wild-type monolayers. A humanized monoclonal antibody to IFN-γ neutralized the detrimental effects on keratinocytes, restoring cell proliferation, increasing cell-cell adhesion, accelerating wound closure in the presence of IFN-γ, and reducing IFN-γ-mediated keratin aggregation in EBS cells. These suggest that treatment with IFN-γ blocking antibodies may constitute a promising new therapeutic strategy for patients with EBS and may also have ameliorating effects on other inflammatory skin diseases.

A cell-based drug discovery assay identifies inhibition of cell stress responses as a new approach to treatment of epidermolysis bullosa simplex.

In the skin fragility disorder epidermolysis bullosa simplex (EBS), mutations in keratin 14 (K14, also known as KRT14) or keratin 5 (K5, also known as KRT5) lead to keratinocyte rupture and skin blistering. Severe forms of EBS are associated with cytoplasmic protein aggregates, with elevated kinase activation of ERK1 and ERK2 (ERK1/2; also known as MAPK3 and MAPK1, respectively), suggesting intrinsic stress caused by misfolded keratin protein. Human keratinocyte EBS reporter cells stably expressing GFP-tagged EBS-mimetic mutant K14 were used to optimize a semi-automated system to quantify the effects of test compounds on keratin aggregates. Screening of a protein kinase inhibitor library identified several candidates that reduced aggregates and impacted on epidermal growth factor receptor (EGFR) signalling. EGF ligand exposure induced keratin aggregates in EBS reporter keratinocytes, which was reversible by EGFR inhibition. EBS keratinocytes treated with a known EGFR inhibitor, afatinib, were driven out of activation and towards quiescence with minimal cell death. Aggregate reduction was accompanied by denser keratin filament networks with enhanced intercellular cohesion and resilience, which when extrapolated to a whole tissue context would predict reduced epidermal fragility in EBS patients. This assay system provides a powerful tool for discovery and development of new pathway intervention therapeutic avenues for EBS.

Assays to Study Consequences of Cytoplasmic Intermediate Filament Mutations: The Case of Epidermal Keratins.

The discovery of the causative link between keratin mutations and a growing number of human diseases opened the way for a better understanding of the function of the whole intermediate filament families of cytoskeleton proteins. This chapter describes analytical approaches to identification and interpretation of the consequences of keratin mutations, from the clinical and diagnostic level to cells in tissue culture. Intermediate filament pathologies can be accurately diagnosed from skin biopsies and DNA samples. The Human Intermediate Filament Database collates reported mutations in intermediate filament genes and their diseases, and can help clinicians to establish accurate diagnoses, leading to disease stratification for genetic counseling, optimal care delivery, and future mutation-aligned new therapies. Looking at the best-studied keratinopathy, epidermolysis bullosa simplex, the generation of cell lines mimicking keratinopathies is described, in which tagged mutant keratins facilitate live-cell imaging to make use of today's powerful enhanced light microscopy modalities. Cell stress assays such as cell spreading and cell migration in scratch wound assays can interrogate the consequences of the compromised cytoskeletal network. Application of extrinsic stresses, such as heat, osmotic, or mechanical stress, can enhance the differentiation of mutant keratin cells from wild-type cells. To bring the experiments to the next level, 3D organotypic human cultures can be generated, and even grafted onto the backs of immunodeficient mice for greater in vivo relevance. While development of these assays has focused on mutant K5/K14 cells, the approaches are often applicable to mutations in other intermediate filaments, reinforcing fundamental commonalities in spite of diverse clinical pathologies.