Barrier Function Of Keratinocytes Research Articles

Keratinocytes use FPR2 to detect Staphylococcus aureus and initiate antimicrobial skin defense.

Keratinocytes form a multilayer barrier that protects the skin from invaders or injuries. The barrier function of keratinocytes is in part mediated by the production of inflammatory modulators that promote immune responses and wound healing. Skin commensals and pathogens such as Staphylococcus aureus secrete high amounts of phenol-soluble modulin (PSM) peptides, agonists of formyl-peptide receptor 2 (FPR2). FPR2 is crucial for the recruitment of neutrophils to the sites of infection, and it can influence inflammation. FPR1 and FPR2 are also expressed by keratinocytes but the consequences of FPR activation in skin cells have remained unknown. Since an inflammatory environment influences S. aureus colonization, e. g. in patients with atopic dermatitis (AD), we hypothesized that interference with FPRs may alter keratinocyte-induced inflammation, proliferation, and bacterial colonization of the skin. To assess this hypothesis, we investigated the effects of FPR activation and inhibition in keratinocytes with respect to chemokine and cytokine release as well as proliferation and skin wound gap closure. We observed that FPR activation induces the release of IL-8, IL-1α and promotes keratinocyte proliferation in a FPR-dependent manner. To elucidate the consequence of FPR modulation on skin colonization, we used an AD-simulating S. aureus skin colonization mouse model using wild-type (WT) or Fpr2-/- mice and demonstrate that inflammation enhances the eradication of S. aureus from the skin in a FPR2-dependent way. Consistently, inhibition of FPR2 in the mouse model or in human keratinocytes as well as human skin explants promoted S. aureus colonization. Our data indicate that FPR2 ligands promote inflammation and keratinocyte proliferation in a FPR2-dependent manner, which is necessary for eliminating S. aureus during skin colonization.

The Effects of Mucopolysaccharide Polysulfate on Steroid-Induced Tight Junction Barrier Dysfunction in Human Epidermal Keratinocytes and a 3D Skin Model

Introduction: The long-term use of topical corticosteroids (TCS) is associated with side effects such as skin atrophy and barrier deterioration. Moisturizers, such as mucopolysaccharide polysulfate (MPS), have been reported to prevent relapses in atopic dermatitis (AD) when used in combination with TCS. However, the mechanisms underlying the positive effects of MPS in combination with TCS in AD are poorly understood. In the present study, we investigated the effects of MPS in combination with clobetasol 17-propionate (CP) on tight junction (TJ) barrier function in human epidermal keratinocytes (HEKa) and 3D skin models. Methods: The expression of claudin-1, which is crucial for TJ barrier function in keratinocytes, and transepithelial electrical resistance (TEER) was measured in CP-treated human keratinocytes incubated with and without MPS. A TJ permeability assay, using Sulfo-NHS-Biotin as a tracer, was also conducted in a 3D skin model. Results: CP reduced claudin-1 expression and TEER in human keratinocytes, whereas MPS inhibited these CP-induced effects. Moreover, MPS inhibited the increase in CP-induced TJ permeability in a 3D skin model. Conclusion: The present study demonstrated that MPS improved TJ barrier impairment induced by CP. The improvement of TJ barrier function may partially be responsible for the delayed relapse of AD induced by the combination of MPS and TCS.

Evaluation of the Efficacy of Xyloglucan, Pea Protein and Opuntia ficus-indica Extract in a Preclinical Model of Psoriasis

Psoriasis is a chronic inflammatory skin disease characterized by epidermal gene abnormalities, epidermal barrier defects and inflammation. Corticosteroids are considered to be standard treatments, but often come with side effects and lose efficacy with long-term use. Alternative treatments targeting the epidermal barrier defect are needed to manage the disease. Film-forming substances such as xyloglucan, pea protein and Opuntia ficus-indica extract (XPO) have generated interest for their ability to restore skin barrier integrity and may pose an alternative approach to disease management. Thus, the aim of this two-part study was to evaluate the barrier-protective properties of a topical cream containing XPO on the membrane permeability of keratinocytes exposed to inflammatory conditions and compare its efficacy to dexamethasone (DXM) in an in vivo model of psoriasis-like dermatitis. XPO treatment significantly reduced S. aureus adhesion, subsequent skin invasion and restored epithelial barrier function in keratinocytes. Furthermore, the treatment restored the integrity of keratinocytes, reducing tissue damage. In mice with psoriasis-like dermatitis, XPO significantly reduced erythema, inflammatory markers and epidermal thickening with a superior efficacy to dexamethasone. Given the promising results, XPO may represent a novel steroid-sparing therapeutic for epidermal-related diseases such as psoriasis, thanks to its ability to preserve skin barrier function and integrity.

Improvements in Human Keratinocytes and Antimicrobial Effect Mediated by Cell-Free Supernatants Derived from Probiotics

The skin acts as a physical and physiological barrier, thereby protecting the body from various environmental components and stimuli. Cell-free supernatants (CFS) derived from probiotics can improve skin functions and retain moisture. In this study, to assess the efficacy of CFS derived from Ligilactobacillus salivarius and Limosilactobacillus fermentum, we investigated the barrier strengthening and moisturizing effects of CFS in keratinocytes along with their antibacterial effects. We also determined the adhesive effects of probiotics on colorectal cells. To confirm improvements in moisturization and barrier function mediated by CFS in keratinocytes, hyaluronic acid (HA) production, and mRNA expression of HA synthases (HAS)2, HAS3, and FLG were measured. The results showed that CFS from L. salivarius MG242 and L. fermentum MG901 increased the expression of these genes along with the production of HA (2.40- and 1.95-fold of control). Additionally, CFS derived from L. salivarius MG242 and L. fermentum MG901 inhibited the growth of S. aureus and E. coli, thereby demonstrating inhibitory effects against harmful pathogens observed on the skin. These results indicate that the use of CFS derived from L. salivarius MG242 and L. fermentum MG901 may increase moisturization in the skin and improve barrier function of keratinocytes along with elimination of potential pathogens.

Protective effect of Aquaphilus dolomiae extract‐G1, ADE‐G1, on tight junction barrier function in a Staphylococcus aureus‐infected atopic dermatitis model

Atopic dermatitis (AD) is a common skin disease characterized by recurrent pruritic inflammatory skin lesions and defects of the skin barrier. Bacterial infection with Staphylococcus aureus contributes to increased severity of AD by compromising the barrier further. A microorganism component of Avène Thermal Spring Water, Aquaphilus dolomiae, is thought to contribute to some of its beneficial effects to skin, eg AD alleviation. Here, we have investigated the effects of an extract of A.dolomiae, A.dolomiae extract-G1 (ADE-G1), on the structural barrier function of keratinocytes, tight junction (TJ) protein expression and the expression of several genes altered in AD patients. An epidermal cell culture model mimicking the AD environment and phenotype was used, in which S.aureus-infected cell cultures of normal human epidermal keratinocytes were exposed to a proinflammatory environment. Endpoints measured included the transepithelial electrical resistance (TER) and immunohistological staining of the epidermal TJ proteins, claudin and occludin. Additional analysis was made of several genes known to be differentially regulated in skin from AD patients (C-C motif chemokine ligand 20 (CCL20), interleukin-8 (IL-8), S100 calcium binding protein A7 (S100A7), defensin beta 4 (DEFB4) and filaggrin). Aquaphilus dolomiae extract-G1 strongly increased TER in non-infected cells and provided protection against infection by overcoming the decrease in TER induced by the infection with S.aureus. In infected cells exposed to a pro-inflammatory environment - depicting AD-like conditions - TER protection by ADE-G1 was still observed. Gene expression analysis of infected and pro-inflammatory stimulated cells indicated that ADE-G1 modulated the inflammatory response (induced IL-8 and attenuated CCL20 expression), increased antimicrobial activities (induced DEFB4 and A100A7) and strengthened barrier function (restored filaggrin expression). ADE-G1 reinforces barrier function and strongly protects TJ barrier disruption induced by bacterial infection and inflammation.

Organic osmolytes increase expression of specific tight junction proteins in skin and alter barrier function in keratinocytes.

The epidermal barrier is important for water conservation, failure of which is evident in dry-skin conditions. Barrier function is fulfilled by the stratum corneum, tight junctions (TJs, which control extracellular water) and keratinocyte mechanisms, such as organic osmolyte transport, which regulate intracellular water homeostasis. Organic osmolyte transport by keratinocytes is largely unexplored and nothing is known regarding how cellular and extracellular mechanisms of water conservation may interact. We aimed to characterize osmolyte transporters in skin and keratinocytes, and, using transporter inhibitors, to investigate whether osmolytes can modify TJs. Such modification would suggest a possible link between intracellular and extracellular mechanisms of water regulation in skin. Immunostaining and quantitative polymerase chain reaction of organic osmolyte-treated organ-cultured skin were used to identify changes to organic osmolyte transporters, and TJ protein and gene expression. TJ functional assays were performed on organic osmolyte-treated primary human keratinocytes in culture. Immunostaining demonstrated the expression of transporters for betaine, taurine and myo-inositol in transporter-specific patterns. Treatment of human skin with either betaine or taurine increased the expression of claudin-1, claudin-4 and occludin. Osmolyte transporter inhibition abolished this response. Betaine and taurine increased TJ function in primary human keratinocytes invitro. Treatment of skin with organic osmolytes modulates TJ structure and function, which could contribute to the epidermal barrier. This emphasizes a role for organic osmolytes beyond the maintenance of intracellular osmolarity. This could be harnessed to enhance topical therapies for diseases characterized by skin barrier dysfunction.

A Dual Zinc plus Arginine formulation attenuates the pathogenic properties of Porphyromonas gingivalis and protects gingival keratinocyte barrier function in an in vitro model

ABSTRACT Background and objectives Porphyromonas gingivalis, a late colonizer of the periodontal biofilm, has been strongly associated with the chronic form of periodontitis. The aim of this study was to investigate the effects of a Dual Zinc plus Arginine formulation (aqueous solution and dentifrice) on the pathogenic properties of P. gingivalis and the barrier function of an in vitro gingival epithelium model. Results The Dual Zinc plus Arginine aqueous solution and dentifrice inhibited the hemolytic and proteolytic activities of P. gingivalis. The Dual Zinc plus Arginine aqueous solution and dentifrice enhanced the barrier function of an in vitro gingival epithelium model as determined by a time-dependent increase in transepithelial electrical resistance and decrease in paracellular permeability. This was associated with an increased immunolabeling of two important tight junction proteins: zonula occludens-1 and occludin. The deleterious effects of P. gingivalis on keratinocyte barrier function as well as the ability of the bacterium to translocate through a gingival epithelium model were attenuated in the presence of either Dual Zinc plus Arginine aqueous solution or dentifrice. Conclusions The Dual Zinc plus Arginine formulation may offer benefits for patients affected by periodontal disease through its ability to attenuate the pathogenic properties of P. gingivalis and promote keratinocyte barrier function.

Enolase-1 expression in the stratum corneum is elevated with parakeratosis of atopic dermatitis and disrupts the cellular tight junction barrier in keratinocytes.

Previous studies have shown that enolase-1 (ENO1) in the stratum corneum (SC) is more highly expressed in patients with atopic dermatitis (AD) than in healthy individuals, suggesting that it is a novel biomarker for evaluating skin condition in patients with AD. However, the mechanism underlying high ENO1 expression in the SC and its pathological relevance in AD are unclear. In this study, the relationship between ENO1 expression and keratinization of epidermis was investigated, and the role of high ENO1 expression in keratinocytes was characterized. ENO1 expression and morphological characteristics were examined in SC from the cheeks of 24 patients with AD. Additionally, the localization of ENO1 in the excised human epidermis was observed. Moreover, to analyse the role of ENO1 in cellular barrier function, tight junction proteins (TJs) and transepithelial electrical resistance (TEER) in keratinocytes with ENO1 overexpression were evaluated. Furthermore, the localization of ENO1 and plasminogen in keratinocytes was evaluated by immunostaining, and the cellular barrier function in keratinocytes was examined after treatment with tranexamic acid (TXA). ENO1 expression was substantially correlated with the rate of nucleated corneocytes in AD. In addition, ENO1 localized in the basal to spinous layers, but was its expression dramatically decreased in healthy human SC. ENO1 overexpression in human epidermal keratinocytes reduced the expression of TJs (claudin-4, E-cadherin, tricellulin, and occludin) and TEER, and treatment with anti-ENO1 IgG reversed these effects. ENO1 colocalized with plasminogen in keratinocytes. Treatment with TXA rescued the ENO1-induced reductions in TJ and TEER expression. We found a substantial correlation between ENO1 expression and the rate of nucleated corneocytes in AD and decreased ENO1 expression with nuclear disappearance. These results suggest that high ENO1 expression in the SC of AD is caused by deficient keratinization, which is an AD characteristic. Moreover, ENO1 overexpression in keratinocytes promoted dysfunction of TJ dynamics, leading to reduced integrity of the cellular barrier, and these effects might be mediated by plasmin activity. We propose that ENO1 is a useful indicator of parakeratosis and might have a potential role in cellular TJ barrier function in the epidermis.

Host Defense (Antimicrobial) Peptide, Human β-Defensin-3, Improves the Function of the Epithelial Tight-Junction Barrier in Human Keratinocytes

Human β-defensins (hBDs) are host defense peptides that not only exhibit microbicidal properties but also stimulate various cellular activities, including keratinocyte proliferation, migration, and wound healing. hBDs are overexpressed in the skin in cases of psoriasis but are downregulated in atopic dermatitis skin, although both diseases are associated with stratum corneum barrier defects. Because the tight-junction (TJ) barrier is also dysfunctional in both atopic dermatitis and psoriasis patients, we hypothesized that hBDs may regulate the TJ barrier function in keratinocytes. We observed that, among the hBDs tested, only hBD-3 increased the expression of several claudins and their localization along the cell-cell borders. In addition, hBD-3 elevated the transepithelial electrical resistance and reduced the paracellular permeability of keratinocyte layers, and this effect was reversed by the claudin inhibitor ochratoxin A, CCR6 antibody, and CCR6 small interfering RNA. Moreover, hBD-3 enhanced the activation of Rac1, atypical protein kinase C, glycogen synthase kinase-3, and phosphatidylinositol 3 kinase, which are required for the hBD-3-mediated regulation of the TJ barrier function, as evidenced by the effects of their respective inhibitors. Collectively, our findings provide evidence regarding the contribution of host defense peptides to the innate immunity of skin by regulating TJ barrier function, in addition to their antimicrobial and other immunomodulatory activities.

The reconstructed skin micronucleus assay in EpiDerm™: Reduction of false-positive results – a mechanistic study with epigallocatechin gallate

The high rate of false-positive or misleading results in in vitro mammalian genotoxicity testing is a hurdle in the development of valuable chemicals, especially those used in cosmetics, for which in vivo testing is banned in the European Union. The reconstructed skin micronucleus (RSMN) assay in EpiDerm™ (MatTek Corporation, USA) has shown promise as a follow-up for positive in vitro mammalian genotoxicity tests. However, few studies have explored its better predictive performance compared with existing in vitro assays. In the present study, we followed the protocol of the RSMN assay and used eight chemicals to compare micronucleus (MN) induction with EpiDerm™ with that in normal human epidermal keratinocytes (NHEKs), both derived from human skin. The assessments of EpiDerm™ conformed to those of in vivo MN assay, whereas those of NHEKs did not. The effect of cell differentiation status on MN induction was further addressed using a model compound, epigallocatechin gallate (EGCG), which is a major component of green tea extract that shows positive results in in vitro mammalian genotoxicity assays via oxidative stress and negative results in in vivo MN studies. RSMN assay in an underdeveloped epidermal model, EpiDerm-201™ (MatTek Corporation), showed a negative result identical to that in EpiDerm™, indicating that the barrier function of keratinocytes has limited impact. Analysis of the gene expression profile of both EpiDerm™ and NHEKs after EGCG treatment for 12h revealed that the expression of genes related to genotoxic response was significantly induced only in NHEKs. Conversely, antioxidative enzyme activities (catalase and glutathione peroxidase) in EpiDerm™ were higher than those in NHEKs. These results indicate that EpiDerm™ has antioxidant properties similar to those of a living body and is capable of eliminating oxidative stress that may be caused by EGCG under in vitro experimental conditions.

The broad spectrum of interepithelial junctions in skin and lung

The broad spectrum of interepithelial junctions in skin and lung

Fibroblast Growth Factor-Peptide Improves Barrier Function and Proliferation in Human Keratinocytes After Radiation

Epidermal keratinocytes, which can be severely damaged after ionizing radiation (IR), are rapid turnover cells that function as a barrier, protecting the host from pathogenic invasion and fluid loss. We tested fibroblast growth factor-peptide (FGF-P), a small peptide derived from the receptor-binding domain of FGF-2, as a potential mitigator of radiation effects via proliferation and the barrier function of keratinocytes. Keratinocytes isolated from neonatal foreskin were grown on transwells. After being exposed to 0, 5, or 10 Gy IR, the cells were treated with a vehicle or FGF-P. The permeability of IR cells was assessed by using transepithelial electrical resistance (TEER) and a paracellular tracer flux of fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA) with Ussing chambers. The cell proliferation was measured with yellow tetrazolium salt (MTT) and tritiated thymidine ([3H]-TdR) assays. The phosphorylation of extracellular signal-regulated kinases (ERK) was measured in an enzyme-linked immunosorbent (ELISA)-like assay, and the proteins related to tight junctions (TJ) and adherens junctions (AJ) were examined with Western blotting. We used a mouse model to assess the ability of FGF-P to promote the healing of skin β burns created with a strontium applicator. We found (1) FGF-P reduced the permeability of irradiated keratinocytes, as evidenced by increased TEER and decreased diffusion of FITC-BSA, both associated with the regulation of different proteins and levels of TJ and AJ; and (2) FGF-P enhanced the proliferation of irradiated keratinocytes, as evidenced by increased MTT activity and [3H]-TdR incorporation, which was associated with activation of the ERK pathway; and (3) FGF-P promoted the healing of skin β burns. FGF-P enhances the barrier function, including up-regulation of TJ proteins, increases proliferation of human keratinocytes, and accelerates the healing of skin β burns. FGF-P is a promising mitigator that improves the proliferation and barrier function of keratinocytes after IR.

Glutamate receptors on human melanocytes regulate the expression of MiTF

Glutamate is the major excitatory neurotransmitter in the central nervous system but has also important functions in the epidermis. It is involved in keratinocyte barrier function and in re-epithelialization processes after wounding. Recently, glutamate signalling has been suggested to be implicated in the development of melanoma. The present study examined the expression and functionality of metabotropic and ionotropic glutamate receptors on normal human melanocytes. We found that cultured melanocytes expressed the ionotropic glutamate receptors GluR2 and 4 [alpha-amino-3-hydroxy-5-methyl-4-isoxsazolepropionic acid (AMPA) receptors] and N-methyl-d-aspartate (NMDA) receptors 2A and 2C and possibly the metabotropic glutamate receptor 1. Melanocytes were also found to express specific glutamate transporters and decarboxylases, but appeared neither to produce nor to release l-glutamate. Stimulation with 10 or 100 microM AMPA or NMDA elevated intracellular calcium concentrations in melanocytes, and thus demonstrated the functionality of the glutamate receptors. Millimolar concentrations of l-glutamate did not induce melanocyte toxicity and had no stimulating effect on melanin production. However, blockage of AMPA and NMDA receptors with CFM-2, memantine or MK801 caused a rapid and reversible change in melanocyte morphology, which was associated with disorganisation of actin and tubulin microfilaments. After 24 h of treatment with the AMPA receptor inhibitor CFM-2, there was a sharp reduction in the expression of the crucial melanocyte differentiation and proliferation factor MiTF. The results of this study demonstrate a role for glutamate in melanocyte regulation that may have implications in melanocyte associated disorders.

Permeability Barrier Disruption Coordinately Regulates mRNA Levels for Key Enzymes of Cholesterol, Fatty Acid, and Ceramide Synthesis in the Epidermis

The extracellular lipids of the stratum corneum, which are comprised mainly of cholesterol, fatty acids, and ceramides, are essential for epidermal permeability barrier function. Moreover, disruption of the permeability barrier results in an increased cholesterol, fatty acid, and ceramide synthesis in the underlying epidermis. This increase in lipid synthesis has been shown previously to be due to increased activities of HMG-CoA reductase, acetyl-CoA carboxylase, fatty acid synthase and serine palmitoyl transferase, key enzymes of cholesterol, fatty acid, and ceramide synthesis, respectively. In the present study, we determined whether the mRNA levels for the key enzymes required for synthesis of these three classes of lipids increase coordinately during barrier recovery. By northern blotting, the steady-state mRNA levels for HMG-CoA reductase, HMG-CoA synthase, farnesyl pyrophosphate synthase, and squalene synthase, key enzymes for cholesterol synthesis, all increased significantly after barrier disruption by either acetone or tape stripping. Additionally, the steady-state mRNA levels of acetyl-CoA carboxylase and fatty acid synthase, required for fatty acid synthesis, as well as serine palmitoyl transferase, the rate-limiting enzyme of de novo ceramide synthesis, also increased. Furthermore, artificial restoration of the permeability barrier by occlusion after barrier disruption prevented the increase in mRNA levels for all of these enzymes, except farnesyl pyrophosphate synthase, indicating a specific link of the increase in mRNA levels to barrier requirements. The parallel increase in epidermal mRNA levels for the enzymes required for cholesterol, fatty acid, and ceramide synthesis may be due to one or more transcription factors that regulate lipid requirements for permeability barrier function in keratinocytes.