Keratinocytes activated by IL-4/IL-13 express IL-2Rγ with consequences on epidermal barrier function.

Atopic dermatitis (AD) is a Th2-type inflammatory disease characterized by an alteration of epidermal barrier following the release of IL-4 and IL-13. These cytokines activate type II IL-4Rα/IL-13Rα1 receptors in the keratinocyte. Whilst IL-2Rγ, that forms type I receptor for IL-4, is only expressed in haematopoietic cells, recent studies suggest its induction in keratinocytes, which questions about its role. We studied expression of IL-2Rγ in keratinocytes and its role in alteration of keratinocyte function and epidermal barrier. IL-2Rγ expression in keratinocytes was studied using both reconstructed human epidermis (RHE) exposed to IL-4/IL-13 and AD skin. IL-2Rγ induction by type II receptor has been analyzed using JAK inhibitors and RHE knockout (KO) for IL13RA1. IL-2Rγ function was investigated in RHE KO for IL2RG. In RHE, IL-4/IL-13 induce expression of IL-2Rγ at the mRNA and protein levels. Its mRNA expression is also visualized in keratinocytes of lesional AD skin. IL-2Rγ expression is low in RHE treated with JAK inhibitors and absent in RHE KO for IL13RA1. Exposure to IL-4/IL-13 alters epidermal barrier, but this alteration is absent in RHE KO for IL2RG. A more important induction of IL-13Rα2 is reported in RHE KO for IL2RG than in not edited RHE. These results demonstrate IL-2Rγ induction in keratinocytes through activation of type II receptor. IL-2Rγ is involved in the alteration of the epidermal barrier and in the regulation of IL-13Rα2 expression. Observation of IL-2Rγ expression by keratinocytes inside AD lesional skin suggests a role for this receptor subunit in the disease.

Deletion of TNFAIP6 Gene in Human Keratinocytes Demonstrates a Role for TSG-6 to Retain Hyaluronan Inside Epidermis.

TSG-6 is a soluble protein secreted in the extracellular matrix by various cell types in response to inflammatory stimuli. TSG-6 interacts with extracellular matrix molecules, particularly hyaluronan (HA), and promotes cutaneous wound closure in mice. Between epidermal cells, the discrete extracellular matrix contains HA and a tiny amount of TSG-6. However, challenges imposed to keratinocytes in reconstructed human epidermis revealed strong induction of TSG-6 expression, after exposure to T helper type 2 cytokines to recapitulate the atopic dermatitis phenotype or after fungal infection that causes secretion of cytokines and antimicrobial peptides. After both types of challenge, enhanced release of TSG-6 happens simultaneously with increased HA production. TSG-6 deficiency in N/TERT keratinocytes was created by inactivating TNFAIP6 using CRISPR/Cas9. Some TSG-6 -/- keratinocytes analyzed through scratch assays tend to migrate more slowly but produce reconstructed human epidermis that exhibits normal morphology and differentiation. Few significant alterations were noticed by transcriptomic analysis. Nevertheless, reduced HA content in TSG-6 -/- reconstructed human epidermis was observed, along with enhanced HA release into the culture medium, and this phenotype was even more pronounced after the challenging conditions. Reintroduction of cells producing TSG-6 in reconstructed human epidermis reduced HA leakage. Our results show a role for TSG-6 in sequestering HA between epidermal cells in response to inflammation.

Responses of Reconstructed Human Epidermis to Trichophyton rubrum Infection and Impairment of Infection by the Inhibitor PD169316.

Despite the threatening incidence of dermatophytosis, information is still lacking about the consequences of infection on epidermal barrier functions and about the keratinocyte responses that alert immune components. To identify the mechanisms involved, arthroconidia of the anthropophilic dermatophyte Trichophyton rubrum were prepared to infect reconstructed human epidermis (RHE) in vitro. Integrity of the barrier was monitored during infection by measurements of transepithelial electrical resistance and dye-permeation through the RHE. Expression and release of pro-inflammatory cytokines and antimicrobial peptides by keratinocytes inserted into the RHE were assessed, respectively, by quantitative reverse transcriptase-PCR (to analyze mRNA content in tissue extracts) and by ELISA (to detect proteins in culture media). Results reveal that infection by T. rubrum is responsible for disruption of the epidermal barrier, including loss of functional tight junctions. It additionally causes simultaneous expression and release of cytokines and antimicrobial peptides by keratinocytes. Potential involvement of the p38 mitogen-activated protein kinase signaling pathway was evaluated during infection by targeted inhibition of its activity. Intriguingly, among several p38 mitogen-activated protein kinase inhibitors, PD169316 alone was able to inhibit growth of T. rubrum on Sabouraud agar and to suppress the process of infection on RHE. This suggests that PD169316 acts on a specific target in dermatophytes themselves.

Atopic Dermatitis Studies through In Vitro Models.

Atopic dermatitis (AD) is a complex inflammatory skin condition that is not fully understood. Epidermal barrier defects and Th2 immune response dysregulations are thought to play crucial roles in the pathogenesis of the disease. A vicious circle takes place between these alterations, and it can further be complicated by additional genetic and environmental factors. Studies investigating in more depth the etiology of the disease are thus needed in order to develop functional treatments. In recent years, there have been significant advances regarding in vitro models reproducing important features of AD. However, since a lot of models have been developed, finding the appropriate experimental setting can be difficult. Therefore, herein, we review the different types of in vitro models mimicking features of AD. The simplest models are two-dimensional culture systems composed of immune cells or keratinocytes, whereas three-dimensional skin or epidermal equivalents reconstitute more complex stratified tissues exhibiting barrier properties. In those models, hallmarks of AD are obtained, either by challenging tissues with interleukin cocktails overexpressed in AD epidermis or by silencing expression of pivotal genes encoding epidermal barrier proteins. Tissue equivalents cocultured with lymphocytes or containing AD patient cells are also described. Furthermore, each model is placed in its study context with a brief summary of the main results obtained. In conclusion, the described in vitro models are useful tools to better understand AD pathogenesis, but also to screen new compounds in the field of AD, which probably will open the way to new preventive or therapeutic strategies.

Methyl-ß-cyclodextrin treatment combined to incubation with interleukin-4 reproduces major features of atopic dermatitis in a 3D-culture model.

Atopic dermatitis (AD) skin is characterized by over-expression of interleukin (IL)-4, IL-13 and IL-25. When methyl-ß-cyclodextrin (MßCD) treatment preceded exposure to these interleukins, combination of both treatments was found to mimic hallmarks of AD in vitro, such as barrier weakening, histological alterations and typical signaling responses in a reconstructed human epidermis (RHE). However, the respective role of each IL and whether any of them is critical when combined with MßCD treatment was unknown. Therefore, this work aimed to distinguish RHE responses after exposure to MßCD and each one of the three IL reported to mimic typical features of AD. IL-4 incubation preceded by MßCD was found responsible for altered histology, as well as for barrier alterations, evidenced by electrical resistance and dye permeation measurements. This combination further decreased loricrin (LOR) immunoreactivity, whereas mainly IL-25, combined to MßCD treatment, was able to downregulate filaggrin (FLG) mRNA level. Carbonic anhydrase II (CA2) and hyaluronan synthase 3 (HAS3), two other markers up-regulated in AD, were also induced when MßCD treatment was followed by IL-4, whilst the expression of neural epidermal growth factor-like 2 (NELL2) was up-regulated by paired IL-4 and IL-13. In conclusion, multiple features of AD were found in this in vitro model mainly when treatment of RHE by IL-4 was conducted after preliminary MßCD incubation.

The activation of cultured keratinocytes by cholesterol depletion during reconstruction of a human epidermis is reminiscent of monolayer cultures.

Transient cholesterol depletion from plasma membranes of human keratinocytes has been shown to reversibly activate signalling pathways in monolayer cultures. Consecutive changes in gene expression have been characterized in such conditions and were interestingly found to be similar to transcriptional changes observed in keratinocytes of atopic dermatitis (AD) patients. As an inflammatory skin disease, AD notably results in altered histology of the epidermis associated with a defective epidermal barrier. To further investigate whether the activation of keratinocytes obtained by cholesterol depletion could be responsible for some epidermal alterations reported in AD, this study was undertaken to analyse cholesterol depletion in stratified cultures of keratinocytes, i.e. a reconstructed human epidermis (RHE). RHE contains heterogeneous populations of keratinocytes, either proliferating or progressively differentiating and stratifying towards the creation of a cornified barrier. Cholesterol depletion induced in this model was found reversible and resulted in activation of signalling pathways similar to those previously identified in monolayers. In addition, selected changes in the expression of several genes suggested that keratinocytes in RHE respond to cholesterol depletion as monolayers. However, preserved histology and barrier function indicate that some additional activation, likely from the immune system, is required to obtain epidermal alterations such as the ones found in AD.

Hyaluronan metabolism in human keratinocytes and atopic dermatitis skin is driven by a balance of hyaluronan synthases 1 and 3.

Hyaluronan (HA) is a glycosaminoglycan synthesized directly into the extracellular matrix by three hyaluronan synthases (HAS1, HAS2, and HAS3). HA is abundantly synthesized by keratinocytes but its epidermal functions remain unclear. We used culture models to grow human keratinocytes as autocrine monolayers or as reconstructed human epidermis (RHE) to assess HA synthesis and HAS expression levels during the course of keratinocyte differentiation. In both the models, epidermal differentiation downregulates HAS3 mRNA expression while increasing HAS1 without significant changes in hyaluronidase expression. HA production correlates with HAS1 mRNA expression level during normal differentiation. To investigate the regulation of HAS gene expression during inflammatory conditions linked to perturbed differentiation, lesional and non-lesional skin biopsies of atopic dermatitis (AD) patients were analyzed. HAS3 mRNA expression level increases in AD lesions compared with healthy and non-lesional skin. Simultaneously, HAS1 expression decreases. Heparin-binding EGF-like growth factor (HB-EGF) is upregulated in AD epidermis. An AD-like HAS expression pattern is observed in RHE incubated with HB-EGF. These results indicate that HAS1 is the main enzyme responsible for HA production by normal keratinocytes and thus, must be considered as an actor of normal keratinocyte differentiation. In contrast, HAS3 can be induced by HB-EGF and seems mainly involved in AD epidermis.

Reconstruction of normal and pathological human epidermis on polycarbonate filter.

This chapter provides methods suitable for the culture of primary human keratinocytes in serum-free culture conditions, starting from very small skin biopsies. It also explains procedures required for reconstruction of a stratified epidermis on polycarbonate filter, starting from keratinocytes cultured in serum-free conditions. Tissues reconstructed according to this method have been proven suitable for characterization of epidermal morphogenesis and for in vitro studies of the epidermal barrier. Utilization of the same method for successful isolation of keratinocytes from a patient suffering from Darier's disease and the reconstruction of a pathological epidermis which displays the same histological features as in vivo are also presented.

Epidermal morphogenesis during progressive in vitro 3D reconstruction at the air-liquid interface.

Keratinocyte monolayers, cultured in immersed conditions, constitute a frequently used in vitro model system to study keratinocytes behaviour in response to environmental assaults. However, monolayers lack the keratinocyte terminal differentiation and the organization of the epidermal tissue, which are observed in vivo. Advancements of in vitro techniques were used to reconstruct three-dimensional equivalents that mimic human epidermis in terms of layering, differentiation and barrier function. Here, we update a published method and illustrate the progressive morphogenesis responsible for in vitro reconstruction. The analysis of cell proliferation, expression of differentiation markers and barrier efficacy demonstrate the excellent similarity of the reconstructed tissue with normal human epidermis. Availability of epidermal tissue during its reconstruction phase in culture appears crucial for studies intending to challenge the barrier function.