Akt-dependent Pp2a activity is required for epidermal barrier formation during late embryonic development

Acquisition of epidermal barrier function occurs late in mouse gestation. Several days before birth a wave of barrier acquisition sweeps across murine fetal skin, converging on dorsal and ventral midlines. We investigated the molecular pathways active during epidermal barrier formation. Akt signaling increased as the barrier wave crossed epidermis and Jun was transiently dephosphorylated. Inhibitor experiments on embryonic explants showed that the dephosphorylation of Jun was dependent on both Akt and protein phosphatase 2A (Pp2a). Inhibition of Pp2a and Akt signaling also caused defects in epidermal barrier formation. These data are compatible with a model for developmental barrier acquisition mediated by Pp2a regulation of Jun dephosphorylation, downstream of Akt signaling. Support for this model was provided by siRNA-mediated knockdown of Ppp2r2a (Pr55α or B55α), a regulatory subunit of Pp2a expressed in an Akt-dependent manner in epidermis during barrier formation. Ppp2r2a reduction caused significant increase in Jun phosphorylation and interfered with the acquisition of barrier function, with barrier acquisition being restored by inhibition of Jun phosphorylation. Our data provide strong evidence that Ppp2r2a is a regulatory subunit of Pp2a that targets this phosphatase to Jun, and that Pp2a action is necessary for barrier formation. We therefore describe a novel Akt-dependent Pp2a activity that acts at least partly through Jun to affect initial barrier formation during late embryonic epidermal development

CD161(+) Tconv and CD161(+) Treg Share a Transcriptional and Functional Phenotype despite Limited Overlap in TCR beta Repertoire

Human regulatory T cells (Treg) are important in immune regulation, but can also show plasticity in specific settings. CD161 is a lectin-like receptor and its expression identifies an effector-like Treg population. Here, we determined how CD161+ Treg relate to CD161+ conventional T cells (Tconv). Transcriptional profiling identified a shared transcriptional signature between CD161+ Tconv and CD161+ Treg, which is associated with T helper (Th)1 and Th17 cells, and tissue homing, including high expression of gut-homing receptors. Upon retinoic acid (RA) exposure, CD161+ T cells were more enriched for CCR9+ and integrin α4+β7+ cells than CD161− T cells. In addition, CD161+ Tconv and CD161+ Treg were enriched at the inflamed site in autoimmune arthritis, and both CD161+ and CD161− Treg from the inflamed site were suppressive in vitro. CD161+ T cells from the site of autoimmune arthritis showed a diminished gut-homing phenotype and blunted response to RA suggesting prior imprinting by RA in the gut or at peripheral sites rather than during synovial inflammation. TCRβ repertoires of CD161+ and CD161− Tconv and Treg from blood showed limited overlap whereas there was clear overlap between CD161+ and CD161− Tconv, and CD161+ and CD161− Treg from the inflamed site suggesting that the inflamed environment may alter CD161 levels, potentially contributing to disease pathogenesis

A mechanistic target of rapamycin complex 1/2 (mTORC1)/V-Akt murine thymoma viral oncogene homolog 1 (AKT1)/cathepsin H axis controls filaggrin expression and processing in skin, a novel mechanism for skin barrier disruption in patients with atopic dermatitis

BACKGROUND: Filaggrin, encoded by the FLG gene, is an important component of the skin’s barrier to the external environment and genetic defects in FLG strongly associate with Atopic Dermatitis (AD). However, not all AD patients have FLG mutations. OBJECTIVE: We hypothesised that these patients may possess other defects in filaggrin expression and processing, contributing to barrier disruption and AD, and therefore present novel therapeutic targets for this disease. RESULTS: We describe the relationship between the mTORC1 protein subunit RAPTOR, the serine/threonine kinase AKT1 and the protease cathepsin H, for which we establish a role in filaggrin expression and processing. Increased RAPTOR levels correlated with decreased filaggrin expression in AD. In keratinocyte cell culture, RAPTOR up-regulation or AKT1 shRNA knockdown reduced the expression of the protease cathepsin H. Skin of cathepsin H-deficient mice and CTSH shRNA knockdown keratinocytes showed reduced filaggrin processing and the mouse showed both impaired skin barrier function and a mild proinflammatory phenotype. CONCLUSION: Our findings highlight a novel, potentially treatable, signalling axis controlling filaggrin expression and processing which is defective in AD

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 and often affects the entire skin. While the gene mutations that lead to ichthyosis are well documented, the actual signalling mechanisms that lead to scaling are poorly characterised, however recent publications suggest that there are common mechanisms active in ichthyotic tissue, and in analogous models of ichthyosis. OBJECTIVE: To determine common mechanisms of hyperkeratosis that may be easily targeted with small molecule inhibitors. METHODS: We combined gene expression analysis of gene-specific shRNA knockdowns in rat epidermal keratinocytes of two genes mutated in autosomal recessive congenital ichthyosis (ARCI), Transglutaminase 1 (TGM1) and arachidonate 12-lipoxygenase, 12R type (ALOX12B), and proteomic analysis of skin scale from ARCI patients.as well as RNAseq data from rat epidermal keratinocytes treated with the Toll-like receptor-2 agonist PAM3CSK. RESULTS: we identified a common activation of the Toll-like receptor (TLR) 2 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 ichthyosis patient keratinocytes and our shRNA models reduced the expression of keratin 1, a structural protein over-expressed in ichthyosis scale. A time-course of Tlr2 activation in rat epidermal keratinocytes revealed that although there was rapid initial activation of innate immune pathways, this was rapidly superseded by widespread up-regulation of epidermal differentiation related proteins. Both NFκβ phosphorylation and Gata3 up-regulation was associated with this switch and Gata3 overexpression was sufficient to increase Keratin 1 expression. CONCLUSION: Taken together, these data define a dual role for Toll-like receptor 2 activation during epidermal barrier repair, that may be a useful therapeutic modality in treating diseases of epidermal barrier dysfunction

Loss of Function Filaggrin Mutations are Associated with Reduced History of Acne Vulgaris in a Cohort of Bangladeshi Atopic Eczema Patients in East London.

Acne vulgaris (AV) is the eighth most common non-fatal disease globally. Previous work identified an association between AV and increased Filaggrin (FLG) expression in the follicular epidermis, but further work did not find a clear link between loss of function (LoF) Filaggrin gene (FLG) mutations and protection from AV. In this work we aimed to explore any association between AV and FLG LoF mutations using a cohort of genotyped Bangladeshi patients with atopic eczema (AE) in East London. Retrospective notes review was performed on 245 patients who had been genotyped for FLG LoF mutations and undergone clinical assessment. The Chi squared or Fisher's exact test was used to determine differences between groups. We found a significant reduction in history of AV in AE patients with FLG LoF mutations relative to AE patients without FLG mutations (p = 0.02). We showed a non-significant reduction in AV diagnosis in patients with impaired barrier function (measured by trans epidermal water loss) and palmar hyperlinearity. We found that patients with severe AE were less likely to have a history of AV only if they had an existing FLG LoF mutation (p = 0.02). In the context of AE, our work suggests that FLG LoF mutations protect patients from developing AV

Kallikrein-Mediated Cytokeratin 10 Degradation Is Required for Varicella Zoster Virus Propagation in Skin.

Varicella zoster virus (VZV) is a skin-tropic virus that infects epidermal keratinocytes and causes chickenpox. Although common, VZV infection can be life-threatening, particularly in the immunocompromized. Therefore, understanding VZV-keratinocyte interactions is important to find new treatments beyond vaccination and antiviral drugs. In VZV-infected skin, kallikrein 6 and the ubiquitin ligase MDM2 are upregulated concomitant with keratin 10 (KRT10) downregulation. MDM2 binds to KRT10, targeting it for degradation via the ubiquitin-proteasome pathway. Preventing KRT10 degradation reduced VZV propagation in culture and prevented epidermal disruption in skin explants. KRT10 knockdown induced expression of NR4A1 and enhanced viral propagation in culture. NR4A1 knockdown prevented viral propagation in culture, reduced LC3 levels, and increased LAMP2 expression. We therefore describe a drug-able pathway whereby MDM2 ubiquitinates and degrades KRT10, increasing NR4A1 expression and allowing VZV replication and propagation.CT and RO were funded by Great Ormond Street Children’s Charity. JB re-ceives funding from the UCL/UCLH Biomedical Resource Centre. DPD wassupported by a New Investigator Award from the Medical Research Council(UK MRC). Sequencing was performed by the UCL Pathogen Genomics Unitfunded by the MRC&UCL/UCLH BRC