Keratins as an Inflammation Trigger Point in Epidermolysis Bullosa Simplex

Abstract

Epidermolysis bullosa simplex (EBS) is a group of inherited keratinopathies that, in most cases, arise due to mutations in keratins and lead to intraepidermal ruptures. The cellular pathology of most EBS subtypes is associated with the fragility of the intermediate filament network, cytolysis of the basal layer of the epidermis, or attenuation of hemidesmosomal/desmosomal components. Mutations in keratins 5/14 or in other genes that encode associated proteins induce structural disarrangements of different strengths depending on their locations in the genes. Keratin aggregates display impaired dynamics of assembly and diminished solubility and appear to be the trigger for endoplasmic reticulum (ER) stress upon being phosphorylated by MAPKs. Global changes in cellular signaling mainly occur in cases of severe dominant EBS mutations. The spectrum of changes initiated by phosphorylation includes the inhibition of proteasome degradation, TNF-α signaling activation, deregulated proliferation, abnormal cell migration, and impaired adherence of keratinocytes. ER stress also leads to the release of proinflammatory danger-associated molecular pattern (DAMP) molecules, which enhance avalanche-like inflammation. Many instances of positive feedback in the course of cellular stress and the development of sterile inflammation led to systemic chronic inflammation in EBS. This highlights the role of keratin in the maintenance of epidermal and immune homeostasis.

Keywords: aggregation; basal layer; blistering; chemokine; cytokine; epidermis; epidermolysis bullosa simplex; inflammation; injury; keratin; keratinocyte; mutation; phosphorylation; proinflammatory cascade; skin; stress; wound healing.

Figure 1

Scheme of key regulatory factors of healthy epidermal keratinocytes under normal and inflammatory conditions. Transforming growth factor-α (TGF-α), heparin-binding epidermal growth factor (HB-EGF), and insulin-like growth factor 1 (IGF-1) were shown to be essential for KC migration [21]. Different neurotrophins, with the most abundant being nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and glial-cell-line-derived neurotrophic factor (GDNF), are involved in the regulation of KC proliferation, migration, and apoptosis [22]. The specific expression of the cytokines and chemokines C-C motif ligand 2, 20, and 22 (CCL2, 20, and 22) and CXCL1, 8, and 12 by KCs determines the immune response [23]. KCs manage the recruitment of the T-cell skin population to sites of damage through TNFα and IL-1α expression. They also realize a systemic shift in the T-cell response type through the thymic stromal lymphopoietin (TSLP) stimulation of Langerhans cells [24]. Neural calcitonin-gene-related peptide (CGRP) was shown to enhance keratinocyte proliferation and, together with substance P (SP), induce the inflammatory response of KCs [25]. Thus, the whole network of KCs involved in these processes is multifaceted.

Figure 2

Clinical pictures of patients affected by epidermolysis bullosa simplex (I and II), generalized severe (EBS-sev.). (A) Blistering of the palm at 6 years of age with surrounding subtle hyperkeratosis. Bullae of the fingers with visible desquamation. (B) The same patient at 11 years of age, where amelioration of symptoms is seen. (C) Circinate blistering seen on the inner thigh. (D) Hemorrhagic blistering of the large toe, with focal hyperkeratosis and subtle onycholysis at 2 years of age. (E) Grouped hemorrhagic blisters on the lateral aspect of the foot of the same patient. (F) Generalized plantar keratoderma at 15 years of age. Images were adopted from [42].

Figure 3

Keratin turnover and regulation of intracellular trafficking. Blue arrows indicate keratin circuit in healthy conditions; red arrows indicate the balance of mutant forms. The increase and decrease in color intensities reflect the relative strength of a process. Site-specific phosphorylation of keratins plays an important role in epidermal KCs [91]. The phosphorylated and sumoylated state of keratin determines its solubility and protects keratins from ubiquitination and proteasomal degradation [92]. As a result, a total decrease in cell adherence and hemidesmosomal/desmosomal interactions in the case of mutant keratins was demonstrated [93,94]. Aggregation of keratins is observed in EBS cells in severe types of the disease. The basal level of SAPKs (stress-activated protein kinases) is higher in cells derived from severe EBS cells than in healthy cells [95]. Aggregation of nonmutated keratin is connected with mitotic activity in healthy cells.

Figure 4

Effect of hypo-osmotic shock on the keratin cytoskeleton. (A). Normal immortalized cell line, NEB-1. (B). EBS-derived cell line, KEB-7. Both cell lines were subjected to hypo-osmotic shock and then fixed, permeabilized, and stained for K14 intermediate filaments. Clear peripheral aggregates were seen in KEB-7 cells 30 min after hypo-osmotic shock and remained for at least 3 h (seen in the picture). No filament fragmentation or aggregates were observed for NEB-1 cells. Scale bar = 22 µm. Images are adopted from [102].

Figure 5

Wild-type and mutant HACAT D7 clone with disrupted KIF. (A). HACAT wild type. (B). Mutant HACAT D7. CRISPR/Cas9 technology was applied to modify KRT5 in HaCaT cells. Paired gRNAs specific to exon 7 of the KRT5 gene were used. WT—HACAT with wildtype K5; D7—mutation K5 L473P. Aggregates were observed in nondividing cells. Confocal images of immunofluorescence analysis of cells with antibodies against KRT5 (ab207351). Images are adopted from [110].

Figure 6

Scheme of key inflammatory participants in EBS skin. Aberrations in the keratin network of EBS keratinocytes activate stress signaling and trigger inflammatory and apoptotic signaling pathways, which lead to the secretion of pro-inflammatory agents. Acting through positive feedback mechanisms, proinflammatory agents contribute to the infiltration of different immune cells in the areas of EBS skin blistering [11,125]. This results in increased cell death, pruritus, and pain and induces chronic inflammation. IL-6/1β/1α—interleukins 6/1β/1α [126,127]; CXCL1/5/8—CXC chemokine ligand 1/5/8 [125]; S100—S100 proteins; HMGB—high-mobility group box [9]; MAPKs—mitogen-activated protein kinases [95]; NF-kB—nuclear factor kappa-light-chain-enhancer of activated B cells; TNF-a—tumor necrosis factor-alpha [7]; TRADD—tumor necrosis factor receptor type 1-associated DEATH domain protein [10,119]; p120—catenin delta-1 [128]; ERK1/2—extracellular signal-regulated protein kinase ½ [129]; TSLP—thymic stromal lymphopoietin [130]; CCL2/19/20—C-C motif ligand 2/19/20 [126]; Th17—T helper 17 cells [131]; CD4 +T-cells—T lymphocytes with CD4 receptors; CD8+T-cells—T lymphocytes with CD8 receptors [132]. Arrows indicate activation of an expression or process.