Chronic activation of Toll-like receptor 2 induces an ichthyotic skin phenotype

Background Ichthyosis defines a group of chronic conditions that manifest phenotypically as a thick layer of scales, often affecting the entire skin. While the gene mutations that lead to ichthyosis are well documented, the actual signalling mechanisms that lead to scaling are poorly characterized; however, recent publications suggest that common mechanisms are active in ichthyotic tissue and in analogous models of ichthyosis. Objectives To determine common mechanisms of hyperkeratosis that may be easily targeted with small-molecule inhibitors. Methods We combined gene expression analysis of gene-specific short hairpin RNA (shRNA) knockdowns in rat epidermal keratinocytes (REKs) of two genes mutated in autosomal recessive congenital ichthyosis (ARCI), Tgm1 and Alox12b, and proteomic analysis of skin scale from patients with ARCI, as well as RNA sequencing data from rat epidermal keratinocytes treated with the Toll-like receptor 2 (TLR2) agonist Pam3CSK4. Results We identified common activation of the TLR2 pathway. Exogenous TLR2 activation led to increased expression of important cornified envelope genes and, in organotypic culture, caused hyperkeratosis. Conversely, blockade of TLR2 signalling in keratinocytes from patients with ichthyosis and our shRNA models reduced the expression of keratin 1, a structural protein overexpressed in ichthyosis scale. A time course of TLR2 activation in REKs revealed that although there was rapid initial activation of innate immune pathways, this was rapidly superseded by widespread upregulation of epidermal differentiation-related proteins. Both nuclear factor kappa B phosphorylation and GATA3 upregulation was associated with this switch, and GATA3 overexpression was sufficient to increase keratin 1 expression. Conclusions Taken together, these data define a dual role for TLR2 activation during epidermal barrier repair that may be a useful therapeutic modality in treating diseases of epidermal barrier dysfunction. Using an integrated analysis of proteomics data from patient scales and datasets from our autosomal recessive congenital ichthyosis (ARCI) in vitro models, we identified upregulation of the Toll-like receptor 2 (TLR2) pathway. TLR2 activation caused scaling in rat epidermal keratinocyte organotypic models. TLR2 inhibition reduced expression of the terminal differentiation marker keratin 1 in ARCI models. RNA sequencing analysis of a time course of TLR2 activation showed a biphasic gene expression pattern, a short-term innate immune response and a longer term, GATA3-mediated hyperkeratotic response.