Epidermal cell models are critical for studying skin biology. The gold standard used by the scientific community has historically been primary cell cultures from discarded tissue, typically from neonates (foreskin). Although directly applicable to humans, this system suffers from multiple issues, including substantial donor-to-donor variability and a finite number of divisions in culture. As such, we have identified a faithful alternative called N/TERT2G cells. These cells show many of the characteristics of primary cells, including barrier formation, differentiation kinetics and/or protein expression, and pathogenesis. From our observations, N/TERT2G cells can serve as a reproducible and genetically manipulatable platform in studying skin biology.

Atopic dermatitis (AD) is the most common chronic and relapsing inflammatory skin disease. AD is typically characterized by skewed T helper (Th) 2 inflammation, yet other inflammatory profiles (Th1, Th17, Th22) have been observed in human patients. How cytokines from these different Th subsets impact barrier function in this disease is not well understood. As such, we investigated the impact of the canonical Th17 cytokine, IL-17A, on barrier function and protein composition in primary human keratinocytes and human skin explants. These studies demonstrated that IL-17A enhanced tight junction formation and function in both systems, with a dependence on STAT3 signaling. Importantly, the Th2 cytokine, IL-4 inhibited the barrier-enhancing effect of IL-17A treatment. These observations propose that IL-17A helps to restore skin barrier function, but this action is antagonized by Th2 cytokines. This suggests that restoration of IL-17/IL-4 ratio in the skin of AD patients may improve barrier function and in so doing improve disease severity.

Individuals with atopic dermatitis (AD) are highly colonized by Staphylococcus aureus and are more susceptible to severe viral complications. We hypothesized that S. aureus secreted virulence factors may alter keratinocyte biology to enhance viral susceptibility through disruption of the skin barrier, impaired keratinocyte differentiation, and/or inflammation. To address this hypothesis, human keratinocytes were exposed to conditioned media from multiple S. aureus strains that vary in virulence factor production (USA300, HG003, and RN4220) or select purified virulence factors. We have identified the S. aureus enterotoxin- like superantigen SE l Q, as a virulence factor of interest, since it is highly produced by USA300 and was detected on the skin of 53% of AD subjects ( n = 72) in a study conducted by our group. Treatment with USA300 conditioned media or purified SE l Q resulted in a significant increase in keratinocyte susceptibility to infection with vaccinia virus, and also significantly decreased barrier function. Importantly, we have previously demonstrated that keratinocyte differentiation influences susceptibility to viral infection, and our qPCR observations indicated that USA300 S. aureus and SE l Q alter differentiation in keratinocytes. CRISPR/Cas9 was used to knock out CD40, a potential enterotoxin receptor on epithelial cells. We found that CD40 expression on keratinocytes was not completely necessary for SE l Q-mediated responses, as measured by proinflammatory cytokine expression and barrier function. Together, these findings support that select S. aureus virulence factors, particularly SE l Q, enhance the susceptibility of epidermal cells to viral infection, which may contribute to the increased cutaneous infections observed in individuals with AD. Importance: Staphylococcus aureus skin colonization and infection are frequently observed in individuals with atopic dermatitis. Many S. aureus strains belong to the clonal group USA300, and these strains produce superantigens including the staphylococcal enterotoxin- like Q (SE l Q). Our studies highlight that SE l Q may play a key role by altering keratinocyte differentiation and reducing barrier function; collectively, this may explain the AD-specific enhanced infection risk to cutaneous viruses. It is unclear what receptor mediates SE l Q’s effects on keratinocytes. We have shown that one putative surface receptor, CD40, was not critical for its effects on proinflammatory cytokine production or barrier function.

Staphylococcus aureus is a leading cause of skin and soft tissue infections. Colonization by this bacterium is increased in individuals with chronic cutaneous diseases such as atopic dermatitis, psoriasis, and bullous pemphigoid. The greater abundance of S. aureus on the skin of subjects with atopic dermatitis in particular has been linked to recurrent cutaneous infections. The primary cell type of the epidermal layer of the skin is the keratinocyte, and it is thought that S. aureus internalized in keratinocytes associates with an increased incidence of skin infections. This study addresses whether keratinocyte differentiation and/or inflammation, two important characteristics altered in cutaneous diseases, influence bacterial internalization. To do this, S. aureus internalization was measured in immortalized and primary keratinocytes that were differentiated using high Ca ²⁺ -containing media and/or exposed to cytokines characteristic of atopic dermatitis (IL-4 and IL-13) or psoriasis (IL-17A and IL-22) skin. Our results indicate that S. aureus internalization is uniquely decreased upon keratinocyte differentiation, since this was not observed with another skin-resident bacterium, S. epidermidis . Additionally, treatment with IL-4 + IL-13 diminished bacterial internalization. We interpret this decrease as a mechanism of keratinocyte-based bacterial killing since a similar number of bacterial genomes were detected in cytokine-treated cells, but less viable internalized S. aureus was recovered. Finally, of the receptors reported for S. aureus binding/internalizing into keratinocytes, expression of the α ₅ component of the α ₅ β ₁ integrin was in greatest accordance with the number of internalized bacteria in the context of keratinocyte differentiation. IMPORTANCE: Individuals with chronic cutaneous diseases demonstrate heightened susceptibility for severe and recurrent infections from Staphylococcus aureus . What drives this altered susceptibility remains poorly understood. Previous publications have detected S. aureus as deep as the dermal layer of skin in subjects with atopic dermatitis, suggesting that the cutaneous environment of this disease enables deeper bacterial infiltration than occurs in healthy individuals. This observation indicates that S. aureus has greater opportunity to interact with multiple skin cell types in individuals with chronic inflammatory skin diseases. Identifying the characteristics of the skin that influence bacterial internalization, a common method to establish reservoirs and evade the immune response, is critical for our understanding of S. aureus pathogenesis. The significance of this research is the novel identification of epidermal characteristics that influence S. aureus internalization. With this knowledge, methods can be developed to identify patient populations at greater risk for cutaneous infections.