Response Of Keratinocytes Research Articles

Pathophysiological Responses of Oral Keratinocytes After Exposure to Flavored E-Cigarette Liquids

Background: Electronic cigarettes (ECIGs) have grown in popularity, particularly among adolescents and young adults. Flavored ECIG-liquids (E-liquids) are aerosolized by these ECIGs and inhaled into the respiratory system. Several studies have shown detrimental effects of E-liquids in airway tissues, revealing that flavoring agents may be the most irritating component. However, research on the effects of E-liquids on biological processes of the oral cavity, which is the first site of aerosol contact, is limited. Hence, this study focuses on the effects of E-liquid flavors on oral epithelial cells using the OKF6/TERT-2 cell line model. Methodology: E-liquid was prepared with and without flavors (tobacco, menthol, cinnamon, and strawberry). OKF6/TERT-2 oral epithelial cells, cultured at 37 °C and 5% CO2, were exposed to 1% E-liquid ± flavors for 24 h. Outcomes determined include cell morphology, media pH, wound healing capability, oxidative stress, expression of mucin and tight junction genes, glycoprotein release, and levels of inflammatory cytokines (TNFα, IL-6, and IL-8). Results: Exposure to 1% flavored E-liquids negatively affect cellular confluency, adherence, and morphology. E-liquids ± flavors, particularly cinnamon, increase oxidative stress and production of IL-8, curtail wound healing recovery, and decrease glycoprotein release. Gene expression of muc5b is downregulated after exposure to E-liquids. In contrast, E-liquids upregulate occludin and claudin-1. Conclusions: This study suggests that ECIG use is not without risk. Flavored E-liquids, particularly cinnamon, result in pathophysiological responses of OKF6/TERT-2 cells. The dysregulation of inflammatory responses and cellular biology induced by E-liquids may contribute to various oral pathologies.

Anti-inflammatory and antioxidant properties of Camellia sinensis L. extract as a potential therapeutic for atopic dermatitis through NF-κB pathway inhibition

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by immune dysregulation and excessive cytokine production. This study aimed to explore the potential of Camellia sinensis L. water extract (CSE) as a treatment for AD by the impact of CSE on inflammatory responses in keratinocytes, particularly concerning the production of inflammatory cytokines and the modulation of signaling pathways relevant to AD pathogenesis. CSE was obtained via hot water extraction from Camellia sinensis L. Ultra-high-performance liquid chromatography (UPLC) analyzed catechin and caffeine content. Cell viability was assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), polyphenol and flavonoid content were determined. 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay measured antioxidant activity. Enzyme-Linked Immunosorbent Assay (ELISA), western blotting, and Immunofluorescence (IF) assays examined cytokines, pathways, and protein localization, respectively. Molecular docking assessed compound binding with inflammation-related proteins. UPLC identified six CSE components including epigallocatechin (EGC) epicatechin (EC), caffeine (CF), catechin (C), epigallocatechin gallate (EGCG), and epicatechin gallate (ECG). CSE demonstrated a significant reduction in the production of inflammatory cytokines interleukin (IL)-2 and IL-6 in TNF-α/IFN-γ activated keratinocytes. Treatment with CSE inhibited the mitogen-activated protein kinase (MAPK) pathway, which resulted in decreased phosphorylation of p38, Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK). Exposure of TNF-α/IFN-γ- stimulated human keratinocytes (HaCaT) cells to CSE resulted in a 200 µg/mL dependent inhibition of p65 and signal transducer and activator of transcription 1 (STAT-1) translocation from the cytosol to the nucleus, as confirmed through immunofluorescence (IF) staining. Molecular docking simulations provided insights into the underlying mechanisms of CSE action, which supported its potential as a therapeutic agent for AD. CSE might be a potential candidate for its therapeutic efficacy for inflammatory skin conditions like AD. Thus, based on this evidence, the authors suggest that CSE should be studied further for its anti-inflammatory activities and topical application in the treatment of AD.

Fluoxetine exerts anti-proliferative effect in human epidermal keratinocytes.

We have recently shown that fluoxetine (FX) suppressed polyinosinic-polycytidylic acid-induced inflammatory response and endothelin release in human epidermal keratinocytes, via the indirect inhibition of the phosphoinositide 3-kinase (PI3K)-pathway. Because PI3K-signaling is a positive regulator of the proliferation, in the current, highly focused follow-up study, we assessed the effects of FX (14 µM) on the proliferation and differentiation of human epidermal keratinocytes. We found that FX exerted anti-proliferative actions in 2D cultures (HaCaT and primary human epidermal keratinocytes [NHEKs]; 48- and 72-h; CyQUANT-assay) as well as in 3D reconstructed epidermal equivalents (48-h; Ki-67 immunohistochemistry). Importantly, FX did not influence epidermal thickness (hematoxylin-eosin staining), and it did not have a major impact on the differentiation-associated alteration of the gene expression pattern (24-h treatments; RNA-Seq). Moreover, neither keratin (K)-1, nor K10 expression was altered by FX in NHEKs (RT-qPCR) or in 3D epidermal equivalents (semi-quantitative immunohistomorphometry). FX did not influence differentiation-induced up-regulation of occludin (RT-qPCR; NHEKs), and did not alter differentiation-associated barrier forming capacity of epidermal keratinocytes (electrical impedance; Lucifer Yellow penetration assay). Our data indicate that, besides the previously reported combined anti-inflammatory and putative anti-pruritic effects, FX may also suppress proliferation of human epidermal keratinocytes without impairing their differentiation and barrier-forming capacity.

IL-22-mediated microRNA-124-3p/GRB2 axis regulates hyperproliferation and inflammatory response of keratinocytes in psoriasis.

Psoriasis is an inflammatory dermatosis that features overproliferation and inflammatory reaction of keratinocytes. A study reported that IL-22 is involved in the pathogenesis of psoriasis by mediating miR-124 to regulate the expression of fibroblast growth factor receptor 2 in keratinocytes. A microRNA may target multiple target genes. Therefore, we speculate that miR-124-3p may also target other downstream genes to affect IL -22-induced keratinocyte function. A possible target gene of miR-124-3p, growth factor receptor-bound protein 2 (GRB2), was screened by analyzing the target gene databases. GRB2 expression was elevated and miR-124-3p expression was decreased in psoriatic lesions compared to psoriatic adjacent normal skins and healthy controls. We performed the following cell experiments in the IL-22-stimulated HaCaT cell model. In keratinocytes transfected with the miR-124-3p mimics, GRB2 expression was significantly lower. We analyzed the regulation of keratinocyte proliferation by GRB2 and miR-124-3p. High levels of GRB2 promoted keratinocyte proliferation and expression of Ki67, PCNA, and K16, which were inhibited by low expression of GRB2. In addition, we found that the effect of GRB2 inhibitors on the proliferation and inflammatory response of keratinocytes was dose-dependent. Finally, we investigated the influence of GRB2 on inflammatory mediators in keratinocytes with the ELISA. After low expression of GRB2, the mRNA expression and secretion of the pro-inflammatory factor were suppressed. When both GRB2 and miR-124-3p were overexpressed, the cellular overproliferation and inflammation caused by GRB2 overexpression were significantly reversed by miR-124-3p. In summary, IL-22-mediated miR-124-3p regulates keratinocyte hyperproliferation and inflammatory response by suppressing GRB2 expression in psoriasis.

In vitro modulatory effect of nanoencapsulated tocotrienol-rich fraction, ascorbyl tetraisopalmitate, and carotenes on atopic dermatitis-like model of human epidermal keratinocytes

The aim of the study was to investigate the regulatory effects of mixed antioxidants, including tocotrienol-rich fraction, ascorbyl tetraisopalmitate, and carotenes, on inflammatory response and epidermal integrity of normal human epidermal keratinocytes (NHEK) stimulated with cytokines to mimic atopic dermatitis (AD). Nano encapsulated mixed antioxidants (NMA) were prepared by encapsulating tocotrienol-rich fraction, ascorbyl tetraisopalmitate and carotenes into nanoemulsion. The interleukin (IL)-4 (50 ng/mL), interferon (IFN)-γ (50 ng/mL), and tumor necrosis factor (TNF)-α (20 ng/mL) individually stimulated NHEK cells were treated with NMA (2.1-2100 ng/mL). Then, cytokines, chemokines and structural proteins expressions were determined. Cell monolayer integrity was assessed using dispase-based keratinocyte dissociation and wound-healing assays.The key findings of the study showed that NMA treatment (2100 ng/mL) resulted in a complete abolishment of upmodulation thymic stromal lymphopoietin (TSLP) expression induced by IL-4, IFN-γ, and TNF-α. Upregulated expression of thymus and activation regulated chemokine (TARC) following IL-4 stimulation was attenuated by NMA (2.1-2100 ng/mL). The increment of interferon gamma-induced protein 10 (IP-10) and monocyte chemoattractant protein-1 (MCP-1) expressions resulting from IL-4, IFN-γ, and TNF-α stimulations was significantly suppressed by NMA treatment. The downregulated filaggrin expression provoked by IL-4 and TNF-α stimulations was abrogated with NMA treatment (2.1-2100 ng/mL). Treatment with NMA significantly improved the production of cornified envelope proteins, decreased the dissociation scores and accelerated wound healing of IL-4, IFN-γ, and TNF-α stimulated NHEK.

Atractylodin mitigates UVB radiation-induced oxidative stress and photoaging responses by enhancing NrF2 signaling in human epidermal keratinocytes.

This study explores the protective role of Atractylodin (ATN) on ultraviolet-B (UVB) radiation-exposed oxidative damage and photoaging responses in human epidermal keratinocytes (HaCaT). In vitro, experiments involved subjecting HaCaT cells to UVB radiation (50 mJ/cm2) for a 24 h incubation period, leading to cell death, increased reactive oxygen species (ROS), and DNA damaged lesion (8-Oxo Gunosine). ATN treatment effectively mitigated cell toxicity, ROS generation, and 8-Oxo Gunosine in UVB-exposed HaCaT cells. Furthermore, ATN demonstrated its ability to counteract UVB radiation-exposed oxidative stress by inhibiting the activation of phosphorylated-extracellular signal-regulated kinase-1 (Erk-1), phosphorylated-c-Jun N-terminal kinase (p-Jnk), and phosphorylated p38 Mitogen-Activated Protein Kinase (p-p38) in HaCaT cells. Nuclear factor erythroid 2-related factor 2 (NrF2), recognized for its antioxidant properties, emerged as a key player in protecting against oxidative damage. ATN was observed to inhibit the depletion of NrF2 expression, thereby preventing the depletion of superoxide dismutase (SOD), and glutathione (GSH) in UVB-exposed HaCaT cells. Additionally, ATN inhibited activator protein-1 (AP-1) and matrix metalloproteinases such as MMP-1 and MMP-9 in UVB-exposed HaCaT cells. In conclusion, our findings highlight that ATN effectively prevents UVB-exposed skin oxidative damage and photoaging by modulating NrF2 expression.

Pilot study of topical ruxolitinib demonstrates efficacy and blunting of heterogeneous inflammatory processes in mild hidradenitis suppurativa.

Therapeutic options for mild hidradenitis suppurativa (HS) represent a significant gap in the current treatment landscape, with no FDA approved therapies for early stage HS. Topical JAnus Kinase inhibitors (JAKi) are a compelling option due to the known upregulation of inflammatory JAK signaling in HS lesions and the recent success of systemic JAKi for moderate to severe HS. This is a pilot, single-site, open-label, prospective 24-week clinical trial with topical ruxolitinib (NCT04414514). The goals of this study were to assess clinical efficacy in a pilot cohort and investigate the underlying biological mechanisms associated with clinical response. Male and female patients with mild HS (Hurley stage I or II), with active inflammatory nodules, were recruited. All subjects were observed for 8 weeks to monitor lesion counts (observational phase), then proceeded with active therapy (treatment phase) for 16 weeks. 1.5% topical ruxolitinib cream was applied twice daily, covering the entirety of each HS-affected body site. Clinician and patient reported outcome measures were recorded throughout the study. Lesional skin punch biopsies were obtained at the start and end of therapy for downstream mechanistic RNA-seq and histological analyses. Ten subjects enrolled in this study, four subjects dropped out of the study before the treatment phase of the trial. Six individuals with Hurley stage I (no tunnels) completed the entire study, with five of six successfully achieving HiSCR through 16 weeks of therapy. In this interim analysis, differential gene expression and gene set enrichment analyses revealed reduced activation of JAK-dependent IFN, IL-6, IL-2, and EGFR signaling, antimicrobial responses, and keratinocyte responses. In contrast, signatures of wound healing and lipid metabolism were increased following JAKi treatment, indicating a return toward homeostasis. Histological analyses revealed that clinically-responsive patients had significantly reduced epidermal and dermal inflammation. Affected inflammatory infiltrate included neutrophils, T cells, and plasma cells, with the predominantly affected cell types specific to the patient. Collectively, the broad activity of topical ruxolitinib on inflammatory signaling processes resulted in promising efficacy, even with heterogeneity in baseline inflammation, in this pilot cohort. Importantly, topical treatment not only resolved epidermal inflammation, but also cleared deeper inflammatory infiltrate. The efficacy observed in this trial provides rationale to further investigate topical JAKi and other novel topical therapies in HS.

Effects of Salvia microphylla Extract on the Skin Wound and Barrier Function in Human Keratinocytes

Purpose: Salvia microphylla (SM) has variety bioactivities including antimutagenic, lipase-inhibitory and insecticidal effects. However, its biological activities on skin remain unclear. In this study, we examined the effect of SM water extract (SMW) of SM on skin wound healing- and skin barrier-linked responses in human keratinocytes (HaCaT cells).Methods: The antioxidant activities were measured using 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. Proliferation was confirmed by MTT assay, and migration was measured using a scratch wound healing assay. In order to confirm the proliferation and migration-related signal pathway, it was confirmed by Western blot. Type Ⅰ collagen synthesis and MMP-1 production were confirmed by enzyme-linked immunosorbent assay (ELISA). In addition, the expression of filaggrin, involucurin, and loricin were measured by RT-qPCR.Results: DPPH and ABTS radical scavenging activity of SMW increased a concentration-dependent manner. SMW induced proliferation and migration of HaCaTs, especially at 400 μg/mL(proliferation-inducing activity=133.03%; migration-inducing activity=213.43%). Furthermore, SMW significantly enhanced the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, p38 MAPK and serine/threonine-specific protein kinase (AKT) in HaCaTs, stimulated Type I collagen synthesis and reduced MMP-1 production. It was confirmed that mRNA expression of filaggrin and involucrin, proteins that play important roles in the structure and function of the skin barrier, was significantly increased.Conclusion: These results demonstrate that SMW has skin regeneration and wound healing activity and skin barrier improvement in HaCaTs. In the future, SMW was verified for its applicability as a material for skin regeneration and skin barrier improvement material in the cosmetics industry.

P74 Guselkumab clinical super-response is associated with faster normalization of blood and skin pathology: data from the Phase 3b GUIDE trial

Abstract The ongoing GUIDE trial (NCT03818035) examines the clinical and immunological impact of an extended dosing interval of guselkumab [an interleukin (IL)-23p19 subunit inhibitor] in patients with moderate-to-severe plaque-type psoriasis. A mechanistic substudy was conducted to explore the molecular differences in the skin and blood of ‘super-responder’ (SRe) and non-SRe patients. SRes were defined as patients achieving a psoriasis area and severity index (PASI) = 0 at both Week [W]20 and W28 following guselkumab treatment. RNA-sequencing was performed on non-lesional (at W0) and lesional skin samples (at W0, W4, W28, and W68) along with serum analyses of matching timepoints. Published gene sets related to psoriasis skin and cytokine-stimulated keratinocytes were used for gene set variation analysis. At W0, ∼1100 protein-coding transcripts were upregulated in lesional vs. non-lesional skin, and ∼3000 protein-coding transcripts were downregulated in lesional vs. non-lesional skin. Following treatment with guselkumab, robust pharmacodynamic changes were observed for immune- and psoriasis inflammation-related gene signatures. Faster changes were observed in SRes vs. non-SRes. Signatures related to psoriasis skin inflammation, IL-17-stimulated keratinocyte response, T-helper (Th)17 cells, and many keratinocyte subtypes (differentiated, proliferating and inflamed keratinocytes) were more significantly reduced in SRes vs. non-SRes as early as W4. By W28, all signatures that were upregulated at baseline in lesional vs. non-lesional skin were normalized in SRes, including molecular scar skin and Th17 pathway signatures. In contrast, among non-SRes, molecular scar and immune-cell-regulated signatures (including macrophage, Th22, mucosal-associated invariant T cells, and CD4+ T cells) were <75% normalized at W28. In both SRes and non-SRes, signatures initially downregulated in lesional vs. non-lesional skin at W0, including those for adipocytes, smooth muscles, meta-analysis-derived psoriasis downregulated genes, and IL-17-stimulated KC downregulated genes, represent the few signatures that did not return to non-lesional skin levels after 68 weeks of guselkumab treatment. Consistent with the effect of guselkumab on inflammatory processes in the skin as observed via RNA sequencing, guselkumab treatment decreased the serum levels of several inflammatory mediators as early as W4. The levels of IL-17A, IL-17F and IL-22 were comparable in SRes and non-SRes, while the reduction in beta defensin-2 level was more pronounced in SRes at W4 and W28. These reductions were maintained to W68. In summary, molecular analyses of SRes and non-SRes in the GUIDE clinical trial show kinetic and quantitative differences in the response to guselkumab treatment.

FC21 Sexual dimorphism in keratinocyte response to IL-36 cytokines

Abstract Pustular psoriasis is a devastating subtype of psoriasis associated with significant morbidity and mortality. Pustular psoriasis is the only form of psoriasis that shows prominent sex bias, with the majority of cases found in women. Pustular psoriasis, and to a lesser extent plaque psoriasis, is characterized by the prominent involvement of the IL-36 family of cytokines, which consists of three pro-inflammatory cytokines: IL-36A, IL-36B and IL-36G, and the IL-36 receptor antagonist (IL-36RA/IL36RN). All three IL-36 members and the IL-36 receptor antagonist are increased (5- to 10-fold) in psoriatic skin. Single-cell and spatial-seq data demonstrated that IL-36 responses are primarily localized to the supraspinous compartment of the epidermis and strongly correlate with and act downstream of both IL-17A and TNF responses. CRISPR/Cas9 targeted knocking out of each IL-36 family member in keratinocytes demonstrates that both IL36G and IL36R KOs, but not IL36A KO, suppress both IL-17A and TNF responses (P < 0.001). Notably, the suppressive role of IL36G KO occurred in the absence of neutrophil proteases, which have been considered primary activators of the IL-36 axis in the skin. Bulk RNA-seq data from primary keratinocytes (n = 47) showed marked sex bias in IL-36G response, with female keratinocytes having a more robust pro-inflammatory response to IL-36G compared with male keratinocytes (P < 0.001). Furthermore, female keratinocytes showed higher expression of the type I IFN, IFNK (P < 0.001; FC-3.84), and IFN signature genes, such as MX1 (P < 0.001; FC-4.78), indicating an association between IFN-κ and the IL-36 axis in female keratinocytes. These data provide novel insights into IL-36 biology, demonstrate its role in amplifying IL-17 and TNF responses in the epidermis, and suggest that the sex bias of IL-36 response may contribute to the marked female bias of pustular forms of psoriasis.

360 Nutritional and Environmental Factors Determine Staphylococcus aureus Ability to Form Biofilm, and Induce an Inflammatory Response in Primary Human Keratinocytes

360 Nutritional and Environmental Factors Determine Staphylococcus aureus Ability to Form Biofilm, and Induce an Inflammatory Response in Primary Human Keratinocytes

320 The implication of RNA modification m6Am in the regulation of cellular response in human primary keratinocytes suggests its potential importance for the development of psoriasis

320 The implication of RNA modification m6Am in the regulation of cellular response in human primary keratinocytes suggests its potential importance for the development of psoriasis

346 MicroRNA-378a-3p may enhance inflammatory responses of human primary keratinocytes in atopic dermatitis and during Staphylococcus Aureus infection

346 MicroRNA-378a-3p may enhance inflammatory responses of human primary keratinocytes in atopic dermatitis and during Staphylococcus Aureus infection

TOP2A Promotes Proliferation, Migration, and Inflammatory Response in M5-Treated Keratinocytes by Binding CTBP1 to Activate Wnt/β-Catenin Signaling.

Psoriasis is a chronic cutaneous disease, affecting a significant portion of the global population. Topoisomerase II alpha (TOP2A) is upregulated in psoriasis samples, but the precise molecular mechanism remains unclear. We aimed to clarify the biological contribution of TOP2A in psoriasis progression. An in vitro psoriasis model was established on M5-induced keratinocytes (HaCaT cells) to simulate the psoriasis-like alterations. Following TOP2A knockdown without or with c terminal binding protein 1 (CTBP1) overexpression, CCK-8 and EDU staining were employed to analyze the viability and proliferation of HaCaT cells under M5 conditions. The capacities of HaCaT cell migration and invasion were examined with wound healing- and transwell assays. RT-qPCR and immunoblotting were adopted for evaluation of the inflammation and differentiation of M5-stimualted HaCaT cells. Additionally, the binding between TOP2A and CTBP1 was predicated using bioinformatics tools and detected by Co-IP. Finally, the expression of proteins in Wnt/β-catenin signaling was analyzed with the application of immunoblotting. Results suggested that TOP2A was upregulated in psoriasis skin lesions and M5-induced HaCaT cells. Interference with TOP2A attenuated the proliferation, migration, invasion, and inflammatory response in M5-treated HaCaT cells. In particular, TOP2A bound to CTBP1 and upregulated CTBP1 expression in HaCaT cells. Remarkably, CTBP1 upregulation blocked the impacts of TOP2A depletion on the biological behaviors of M5-treated HaCaT cells. Besides, TOP2A deficiency upregulated DKK1 expression as well as downregulated Wnt1, β-catenin, and c-Myc expression in HaCaT cells exposed to M5, which was restored by further CTBP1 overexpression. In summary, TOP2A binds CTBP1 to activate Wnt/β-catenin signaling, thereby promoting the progression of psoriasis.

Cell penetrable peptide nucleic acids targeting PDZK1IP1 with anti-inflammatory potential in human keratinocytes

PDZK1-interacting protein 1 (PDZK1IP1) has emerged as a potential therapeutic target for skin inflammatory diseases and epithelial tumors. This study investigates the modulation of PDZK1IP1 gene expression using peptide nucleic acids (PNAs), a class of oligonucleotide therapeutics known for their robust binding affinity to complementary nucleic acid sequences and their resistance to degradation by nucleases. To enhance water solubility and cellular permeability, modified PNA oligomers were synthesized by conjugating nucleobases with primary amine chains. A study using a fluorescein-labeled modified PNA oligomer demonstrated significantly enhanced cellular permeability in HaCaT cells compared to the unmodified PNA. These modified PNA oligomers effectively suppressed PDZK1IP1 gene expression and alleviated interferon γ (IFNγ)-induced inflammatory responses in normal human keratinocytes. These findings suggest the potential application of modified PNAs targeting PDZK1IP1 in the treatment of skin inflammatory diseases.

Systemic Sclerosis Dermal Fibroblast Exosomes Trigger Type 1 Interferon Responses in Keratinocytes via a TBK/JAK/STAT Signaling Axis.

Activation of type I interferon (IFN) response has been shown to correlate with disease activity in systemic sclerosis (SSc). It is currently unknown whether the tissue-specific type I IFN activation is a consequence of the response observed in blood or rather its source. Exosomes from SSc fibroblasts were recently shown to activate macrophages in vitro. Here, we aimed to determine the source of type I IFN signature in SSc skin biopsies and the potential role of exosomes from SSc dermal fibroblasts in the process. Skin biopsies were obtained from the forearms of healthy patients and of those with SSc and processed for dermal fibroblasts and keratinocytes. Exosomes were isolated from healthy and SSc dermal fibroblast supernatants by ultracentrifugation and added to human skin keratinocytes. Keratinocyte transcriptome was analyzed by RNA sequencing (RNA-seq) analysis. TANK-binding kinase (TBK) and JAK were inhibited using a small molecule inhibitor (GSK8612) and tofacitinib, respectively. SSc skin biopsies showed the highest levels of type I IFN response in the epidermal layer. RNA-seq analysis of keratinocytes transcriptome following exposure to dermal fibroblast exosomes showed strong up-regulation of IFN signature genes induced by SSc exosomes compared to healthy control. Inhibition of TBK or JAK activity suppressed the up-regulation of the IFN signature induced by SSc exosomes. IFN activation of SSc keratinocytes is dependent on their crosstalk with dermal fibroblasts and inducible by extracellular exosomes. Our data indicate that SSc fibroblast exosomes contribute to the type I IFN activation in SSc skin through activation of pattern recognition receptors upstream of TBK.

IgG autoantibodies in bullous pemphigoid directly induce a pathogenic MyD88-dependent pro-inflammatory response in keratinocytes.

-IgG antibodies from bullous pemphigoid (BP) patients induce significant upregulation of several inflammatory markers in keratinocytes including cytokines (IL-6, IL-24, TGF-β1), chemokines (CXCL16, CTACK, MIP-3β, RANTES), C1s, DPP4, and MMP9. Several of these markers, including IL-8, IL-24, and MMP9 are regulated by MyD88.-Spatial transcriptomics reveals that BP patient blistered skin demonstrated similar transcriptomic profiles to BP-IgG-treated keratinocytes. With attached skin demonstrating a comparable transcriptome to that seen in spongiotic dermatitis.-In a mouse BP model, keratinocyte-specific MyD88 deficiency results in decreased disease severity with a subsequent decrease in serum IL-1β, IL-4, and IL-9 levels. IgG from patients with bullous pemphigoid (BP) induces a pro-inflammatory response in keratinocytes, indicating their direct role in driving the inflammatory response in BP.

Human keratinocyte response to 4,4’-methylene diphenyl diisocyanate-glutathione conjugate exposure

Workplace exposure to diisocyanates like 4,4’-methylene diphenyl diisocyanate can cause occupational asthma (MDI-OA), and the underlying biological pathways are still being researched. Although uncertainty remains, evidence supports the hypothesis that dermal exposure to MDI plays an important role in the development of MDI-OA. Gene expression, proteomics, and informatics tools were utilised to characterise changes in expression of RNA and protein in cultured human HEKa keratinocyte cells following exposure to conjugates of MDI with glutathione (MDI-GSH). RT-qPCR analysis using a panel of 39 candidate primers demonstrated 9 candidate genes upregulated and 30 unchanged. HPLC-MS/MS analysis of HEKa cell lysate identified 18 540 proteins across all samples 60 proteins demonstrate statistically significant differential expression in exposed cells, some of which suggest activation of immune and inflammatory pathways. The results support the hypothesis that dermal exposures have the potential to play an important role in the development of MDI-OA. Furthermore, proteomic and gene expression data suggest multiple immune (adaptive and innate) and inflammatory pathways may be involved in the development of MDI-OA.

Radiofrequency Currents Modulate Inflammatory Processes in Keratinocytes.

Keratinocytes play an essential role in the inflammatory phase of wound regeneration. In addition to migrating and proliferating for tissue regeneration, they produce a large amount of cytokines that modulate the inflammatory process. Previous studies have shown that subthermal treatment with radiofrequency (RF) currents used in capacitive resistive electric transfer (CRET) therapy promotes the proliferation of HaCat keratinocytes and modulates their cytokine production. Although physical therapies have been shown to have anti-inflammatory effects in a variety of experimental models and in patients, knowledge of the biological basis of these effects is still limited. The aim of this study was to investigate the effect of CRET on keratinocyte proliferation, cytokine production (IL-8, MCP-1, RANTES, IL-6, IL-11), TNF-α secretion, and the expression of MMP9, MMP1, NF-κB, ERK1/2, and EGFR. Human keratinocytes (HaCat) were treated with an intermittent 448 kHz electric current (CRET signal) in subthermal conditions and for different periods of time. Cell proliferation was analyzed by XTT assay, cytokine and TNF-α production by ELISA, NF-κB expression and activation by immunofluorescence, and MMP9, MMP1, ERK1/2, and EGF receptor expression and activation by immunoblot. Compared to a control, CRET increases keratinocyte proliferation, increases the transient release of MCP-1, TNF-α, and IL-6 while decreasing IL-8. In addition, it modifies the expression of MMPs and activates EGFR, NF-κB, and ERK1/2 proteins. Our results indicate that CRET reasonably modifies cytokine production through the EGF receptor and the ERK1/2/NF-κB pathway, ultimately modulating the inflammatory response of human keratinocytes.

TLR3 activation mediates partial epithelial-to-mesenchymal transition in human keratinocytes.

TLR3 is expressed in human skin and keratinocytes, and given its varied role in skin inflammation, development, and regeneration, we sought to determine the cellular response in normal human keratinocytes to TLR3 activation. We investigated this mechanism by treating primary human keratinocytes with both UVB, an endogenous and physiologic TLR3 activator, and poly(I:C), a synthetic and selective TLR3 ligand. TLR3 activation with either UVB or poly(I:C) altered keratinocyte morphology, coinciding with the key features of epithelial-to-mesenchymal transition: increased epithelial-to-mesenchymal transition gene expression, enhanced migration, and increased invasion properties. These results confirm and extend previous studies demonstrating that in addition to its classical role in the innate immune response, TLR3 signaling also regulates stem cell-like properties and developmental programs.