The transmembrane protein claudin-1 is critical for formation of the epidermal barrier structure called tight junctions (TJ) and has been shown to be important in multiple disease states. These include neonatal ichthyosis and sclerosing cholangitis syndrome, atopic dermatitis and various viral infections. To develop a model to investigate the role of claudin-1 in different disease settings, we used CRISPR/Cas9 to generate human immortalized keratinocyte (KC) lines lacking claudin-1 (CLDN1 KO). We then determined whether loss of claudin-1 expression affects epidermal barrier formation/function and KC differentiation/stratification. The absence of claudin-1 resulted in significantly reduced barrier function in both monolayer and organotypic cultures. CLDN1 KO cells demonstrated decreases in gene transcripts encoding the barrier protein filaggrin and the differentiation marker cytokeratin-10. Marked morphological differences were also observed in CLDN1 KO organotypic cultures including diminished stratification and reduced formation of the stratum granulosum. We also detected increased proliferative KC in the basale layer of CLDN1 KO organotypic cultures. These results further support the role of claudin-1 in epidermal barrier and suggest an additional role of this protein in appropriate stratification of the epidermis.

Three-dimensional human epidermal equivalents (HEEs) are a state-of-the-art organotypic culture model in preclinical investigative dermatology and regulatory toxicology. In this study, we investigated the utility of electrical impedance spectroscopy (EIS) for noninvasive measurement of HEE epidermal barrier function. Our setup comprised a custom-made lid fit with 12 electrode pairs aligned on the standard 24-transwell cell culture system. Serial EIS measurements for 7 consecutive days did not impact epidermal morphology, and readouts showed comparable trends with HEEs measured only once. We determined 2 frequency ranges in the resulting impedance spectra: a lower frequency range termed EISdiff correlated with keratinocyte terminal differentiation independent of epidermal thickness and a higher frequency range termed EISSC correlated with stratum corneum thickness. HEEs generated from CRISPR/Cas9-engineered keratinocytes that lack key differentiation genes FLG, TFAP2A, AHR, or CLDN1 confirmed that keratinocyte terminal differentiation is the major parameter defining EISdiff. Exposure to proinflammatory psoriasis- or atopic dermatitis-associated cytokine cocktails lowered the expression of keratinocyte differentiation markers and reduced EISdiff. This cytokine-associated decrease in EISdiff was normalized after stimulation with therapeutic molecules. In conclusion, EIS provides a noninvasive system to consecutively and quantitatively assess HEE barrier function and to sensitively and objectively measure barrier development, defects, and repair.

ABSTRACT 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.

Tissue chip (TC) devices, also known as microphysiological systems (MPS) or organ chips (OCs or OoCs), seek to mimic human physiology on a small scale. They are intended to improve upon animal models in terms of reproducibility and human relevance, at a lower monetary and ethical cost. Virtually all TC systems are analyzed at an endpoint, leading to widespread recognition that new methods are needed to enable sensing of specific biomolecules in real time, as they are being produced by the cells. To address this need, we incorporated photonic biosensors for inflammatory cytokines into a model TC. Human bronchial epithelial cells seeded in a microfluidic device were stimulated with lipopolysaccharide, and the cytokines secreted in response sensed in real time. Sensing analyte transport through the TC in response to disruption of tissue barrier was also demonstrated. This work demonstrates the first application of photonic sensors to a human TC device, and will enable new applications in drug development and disease modeling.

ABSTRACT 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.

Skin diseases are characterized by cutaneous cytokine profiles. This includes lupus (IFNγ), atopic dermatitis ([AD], IL-4, IL-13 and IL-22) and psoriasis (IL-17A and IL-22). Janus kinase inhibitors (JAKi) are in the clinic (i.e. abrocitinib, upadacitinib and topical ruxolitinib for AD and deucravacitinib for psoriasis) or in development to treat these diseases. Of these skin diseases, only AD is associated with cutaneous viral infections. Notably, some JAKi (tofacitinib, baracitinib and ruxolitinib) have an increased risk of viral infections. These two observations suggest that specific cytokines alone or in combination with selective JAKi may alter keratinocytes' susceptibility to viral infections. To evaluate this hypothesis, immortalized (N/TERT2G) and primary human keratinocytes were treated with the aforementioned cytokines alone or in combination with JAKi of varying selectivities and viral susceptibility was assessed by vaccinia virus and herpes simplex virus-1 plaque assays. Only exposure to IL-4+IL-13 or IL-22 significantly (p<0.01) increased keratinocyte susceptibility to both viruses with no effect from IL-17A treatment. Specifically, there was a peak increase of 12.2±3.1-fold (IL-4+IL-13) or a 7.7±2.8-fold (IL-22) in vaccinia virus infection. Conversely, IFNγ significantly reduced viral susceptibility (63.1±64.4-fold, p<0.05). JAK1 selective inhibition reduced IL-4+IL-13 induced viral susceptibility by 44±16% (p<0.05) while IL-22-enhanced viral susceptibility was only inhibited by TYK2 blockade (76±19%, p<0.05). Additionally, IFNγ-mediated viral protection was reversed by JAK2 inhibition (366±294% increase in infection, p=0.056). Cytokines found in AD lesions (IL-4, IL-13 and IL-22) enhance epidermal viral susceptibility and highlight pathways that may make subjects at risk for eczema herpeticum. The increased viral infections observed after use of FDA approved JAKi, especially with JAK2 selectivity, may be due to their inhibitory actions on the antiviral IFN response in KC.

Little is known about whether type 1 (IFNγ), 2 (IL-4/IL-13), or 3 (IL-17A/IL-22) cytokines affect the susceptibility of keratinocytes (KC) to viruses. These immune pathways predominate in various skin diseases: lupus, atopic dermatitis (AD), and psoriasis, respectively. Janus kinase inhibitors (JAKi) are approved to treat both AD and psoriasis, and are in clinical development for lupus. We evaluated whether these cytokines alter viral susceptibility of KC and determined if this effect is modulated by treatment with JAKi. Viral susceptibility to vaccinia virus (VV) or herpes simplex virus-1 (HSV-1) ± JAKi was assessed in immortalized and primary human KC pretreated with cytokines. Exposure to type 2 (IL-4 + IL-13) or the type 3 (IL-22) cytokines significantly increased KC viral susceptibility. Specifically, there was a peak increase of 12.2 ± 3.1-fold (IL-4 + IL-13) or 7.7 ± 2.8-fold (IL-22) in VV infection as measured by plaque number. Conversely, IFNγ significantly reduced susceptibility to VV (63.1 ± 64.4-fold). The IL-4 + IL-13-induced viral susceptibility was reduced (44 ± 16%) by JAK1 inhibition, while the IL-22-enhanced viral susceptibility was diminished (76 ± 19%) by TYK2 inhibition. IFNγ-mediated resistance to viral infection was reversed by JAK2 inhibition (366 ± 294% increase in infection). Cytokines expressed in AD skin (IL-4, IL-13, IL-22) increase KC viral susceptibility while IFNγ is protective. JAKi that target JAK1 or TYK2 reversed cytokine-enhanced viral susceptibility, while JAK2 inhibition reduced the protective effects of IFNγ.

The skin barrier protects against our environment. Three-dimensional human epidermal equivalent (HEE) models are invaluable to monitor in vitro epidermal development and to assess the effect of cosmetics and pharmaceutical compounds. Most studies rely heavily on end-point analysis for which HEE cultures are terminated and harvested. However, it is advantageous to monitor epidermal development to optimize the window for intervention studies. Using a custom-made 12-channel lid fitting a 24-well cell culture system, static and labor-intensive transepithelial electrical resistance (TEER) analysis using conventional voltohmmeters was replaced by electrical impedance spectroscopy (EIS). This technology measures impedance following an electrical current of 10Hz – 100.000Hz and was used to monitor epidermal development and skin barrier functionality over consecutive days, which was also validated by histological readout. Interfering with proliferation (i.e., EGFR inhibition) or differentiation (knockout of FLG, TFAP2A, or CLDN1 expression by CRISPR/Cas9) resulted in 35-65% reduced impedance spectra in contrast to wildtype HEEs. In addition, exposure to Th2 pro-inflammatory cytokines reduced impedance by 35%, an effect that was diminished by Th2 inflammation dampening therapeutics (i.e., AHR activating ligands). In conclusion, EIS provides a high-throughput, standardized system for measuring in vitro epidermal development and monitoring skin barrier function. Being non-intrusive, EIS allows for longitudinal and repetitive measuring, minimizing batch effects, increasing study reproducibility, and maximizing experimental throughput.

The epidermis, and keratinocytes (KC) in particular, are important for skin immunity. To identify which epidermal layer is most susceptible to viral infection, we utilized an immortalized KC line that differentiates in the presence of high calcium and a replication-competent, fluorescently-tagged herpes simplex virus (HSV)-1. Human KC were infected with HSV-1 at different stages of differentiation: undifferentiated (low calcium), at the initiation of differentiation (high calcium), and at 24-hour intervals post-differentiation (high calcium). Three days later, infection and replication were quantified via fluorescent imaging. Cytopathic effect was quantified by viral plaques and monolayer clearance. KC infected at the initiation of differentiation showed significantly (p<0.01) increased fluorescence (n=6) and monolayer clearance (n=7) relative to undifferentiated cells or cells differentiated for ≥ 24 hours. This suggests that KC are most susceptible to viral infection during the first 24 hours of differentiation, which is analogous to KC in the lower stratum spinosum. To determine if these findings held true in vivo, we evaluated the histopathology of 6 HSV and 12 varicella zoster virus (VZV) skin lesions. In sections where the edge of the viral immunoreactivity was visible, 100% of VZV (5/5) and 66% of HSV (4/6) cases had viral staining between the stratum basale and granulosum. Collectively, these data suggest that KC that have differentiated to the stratum spinosum level are particularly susceptible to viral infection. This result has implications for dermatological diseases (i.e. atopic dermatitis, Darier disease) with impaired skin barrier function where viral infections cause significant comorbidities.

3D human epidermal equivalents (HEEs) are a state-of-the-art in vitro organotypic model system in investigative dermatology yet heavily rely on end-points analysis to assess barrier development and functionality. Non-intrusive, quantitative methods that enable longitudinal analysis increase study reproducibility and are preferred as an objective readout. Here we investigated the utility of a electrical impedance spectroscopy (EIS) device equipped with a customized smart-lid fitting a 24-transwell cell culture system. EIS measured for seven consecutive days during epidermal development did not impact morphology and readouts were comparable to end-point measurements. Interfering with terminal differentiation (e.g., knockout of FLG, TFAP2A, CLDN1 or AHR) showed clear defects in barrier formation and reduced impedance values. Exposure to Th2 pro-inflammatory cytokines reduced the impedance significantly which was rescued by treatment using anti-inflammatory therapeutics (i.e., AHR activating ligands). Impedance values at lower frequencies (102-103 Hz) correlated to protein levels of keratinocyte differentiation markers FLG and IVL whereas impedance at higher frequencies (104-105 Hz) correlated to stratum corneum thickness. In conclusion, EIS provides a high-throughput standardized system where HEE barrier formation and function can non-invasively be monitored over time. Future potential applications include the assessment of intrinsic or extrinsic factors influencing the skin barrier as well as the pre-clinical screening of skin barrier-promoting agents.

Dendritic cells (DCs) are unique cell populations that link the innate and adaptive immune responses. However, how Cutibacterium acnes strains regulate DC function in acne remains unclear. We stimulated three DC cell-types [Langerhans cell-derived (LCDC), monocyte-derived (moDC), and myeloid (mDC)] with "healthy (CH)" or "acne (CA)" associated C. acnes strains and performed RNAseq to define DC immune signatures. Further, we conducted Differential Gene Expression (DEG) followed by Gene Set Enrichment Analysis (GSEA), Gene Ontology (GO) and pathway deconvolution. Gene expression analysis in DC cell-types exposed to C. acnes compared to the untreated controls revealed 4004 DEG in LCDC, 4438 in moDC and 5004 in mDC. GSEA identified unique genes and inflammatory pathways in acne, whereas, pathway deconvolution, demonstrated that LCDCs had the lowest response following C. acnes stimulation. Focusing on genes differentially expressed in each DC cell-type, we found 1301 genes exclusively upregulated after CA stimulation of LCDC, with GO analysis revealing genes involved in cytokine-cytokine receptor interactions, IL-17 and MAPK signaling. In moDC, we observed similar GO results with 19 uniquely upregulated genes. In mDC, we found 109 uniquely downregulated genes, including H2A, H4, PAD4, C5a, and PLC that are involved in extracellular trap formation pathways. Our findings support the hypothesis that C. acnes strains differentially regulate downstream DC functions. Future studies should focus on developing therapies that enhance DC function and abrogate the inflammatory pathways in acne.

The human epidermis is composed of four visually distinct layers: the stratum basale, stratum spinosum, stratum granulosum, and stratum corneum, which result from a complex differentiation process. This study utilized in vitro viral infection assays with cultured human keratinocytes (KCs), bioinformatic analysis of epidermal single-cell RNA-sequencing datasets, and histopathologic assessment of human viral skin infections (herpes simplex virus [HSV] and varicella-zoster virus [VZV]) to address whether specific KC layers and/or stages of differentiation are more susceptible to viral infection and/or spread.