Biological effects of air pollution on the function of human skin equivalents.

The World Health Organization reports that 99% of the global population are exposed to pollution levels higher than the recommended air quality guidelines. Pollution-induced changes in the skin have begun to surface; however, the effects require further investigation so that effective protective strategies can be developed. This study aimed to investigate some of the aging-associated effects caused by ozone and particulate matter (PM) on human skin equivalents. Full-thickness skin equivalents were exposed to 0.01 µg/µL PM, 0.05 µg/µL PM, 0.3 ppm ozone, or a combination of 0.01 µg/µL PM and 0.3 ppm ozone, before skin equivalents and culture medium were harvested for histological/immunohistochemical staining, gene and protein expression analysis using qPCR, Western blotting, and ELISA. Markers include MMP-1, MMP-3, COL1A1, collagen-I, 4-HNE, HMGCR, and PGE2. PM was observed to induce a decrease in epidermal thickness and an enhanced matrix building phenotype, with increases in COL1A1 and an increase in collagen-I protein expression. By contrast, ozone induced an increase in epidermal thickness and was found to induce a matrix-degrading phenotype, with decreases in collagen-I gene/protein expression and increases in MMP-1 and MMP-3 gene/protein expression. Ozone was also found to induce changes in lipid homeostasis and inflammation induction. Some synergistic damage was also observed when combining ozone and 0.01 µg/µL PM. The results presented in this study identify distinct pollutant-induced effects and show how pollutants may act synergistically to augment damage; given individuals are rarely only exposed to one pollutant type, exposure to multiple pollutant types should be considered to develop effective protective interventions.

Low-level red plus near infrared lights combination induces expressions of collagen and elastin in human skin in vitro.

OBJECTIVE: Light therapy has attracted medical interests as a safe, alternative treatment for photo-ageing and photo-damaged skin. Recent research suggested the therapeutic activity of red and infrared (IR) lights may be effective at much lower energy levels than those used clinically. This study was to evaluate the efficacy of low-level red plus near IR light emitting diode (LED) combination on collagen and elastin and ATP production. METHODS: Human dermal fibroblasts or skin tissues were irradiated daily by red (640 nm) plus near IR (830 nm) LED lights combination at 0.5 mW/cm2 for 10 minutes (0.3 J/cm2 ). qPCR, ELISAs or histology were used to determine the gene and protein expressions. Fluorescent measurement was used to assess crosslinks of collagen and elastic fibres. ATP production was evaluated by ATP assay. RESULTS: Treatment of human fibroblast cell cultures with low-level red plus near IR lights combination was found to significantly increase LOXL1, ELN and COL1A1 and COL3A1 gene expressions as well as the synthesis of the procollagen type I and elastin proteins. Treating human skin explants with low-level red plus near IR lights combination similarly induced significant increases in the same gene expressions, type III collagen and elastic fibre formation and crosslinks. ATP production was increased in human dermal fibroblasts after red plus near IR lights combination treatment. CONCLUSION: Low-level red plus near IR lights combination stimulated the production of collagen and elastin production associated with anti-ageing benefits. These findings suggest that low-level red plus near IR LED light combination may provide an effective treatment opportunity for people with photo-aged skin.

OBJECTIF: La luminothérapie a suscité des intérêts médicaux en tant que traitement alternatif sûr pour la photo-vieillissement et la peau endommagée. Des recherches récentes ont suggéré que L'activité thérapeutique des feux rouges et infrarouges (IR) pourrait être efficace à des niveaux d'énergie beaucoup plus faibles que ceux utilisés en clinique. Cette étude avait pour but d'évaluer l'efficacité de la combinaison de diodes électroluminescentes (DEL) rouges de faible intensité et de diodes électroluminescentes (IR) sur la production de collagène, d'élastine et d'ATP. MÉTHODES: Les fibroblastes dermiques humains ou les tissus cutanés ont été irradiés quotidiennement par une combinaison de feux rouges (640nm) et de feux à DEL proches de l'IR (830nm) à 0,5mW/cm2 pendant 10minutes (0,3J/cm2). qPCR, ELISA ou histologie ont été utilisés pour déterminer les expressions géniques et protéiques. Des mesures fluorescentes ont été utilisées pour évaluer les liens croisés du collagène et des fibres élastiques. La production d'ATP a été évaluée au moyen d'un essai ATP. RÉSULTATS: Le traitement de cultures de cellules de fibroblastes humaines avec une combinaison rouge de faible intensité et proche des lumières IR a permis d'augmenter significativement les expressions des gènes LOXL1, ELN et COL1A1 et COL3A1, ainsi que la synthèse des protéines de procollagène de type I et d'élastine. Le traitement des explants de peau humaine avec une combinaison rouge de bas niveau et proche des lumières IR a également induit des augmentations significatives dans les mêmes expressions géniques, la formation de collagène de type III et de fibres élastiques et les liaisons croisées. La production d'ATP a augmenté dans les fibroblastes dermiques humains après le traitement combiné rouge et proche des feux IR. CONCLUSION: L'association du rouge de bas niveau et des lumières infrarouges a stimulé la production de collagène et d'élastine associée aux bienfaits de l'antivieillissement. Ces résultats suggèrent que la combinaison de faible intensité de rouge plus proche de la lumière IR LED peut fournir une opportunité de traitement efficace pour les personnes ayant la peau photo-âgée.

Propionibacterium acnes susceptibility to low-level 449 nm blue light photobiomodulation.

BACKGROUND AND OBJECTIVE: Recent advances in low-level light devices have opened new treatment options for mild to moderate acne patients. Light therapies have been used to treat a variety of skin conditions over the years but were typically only available as treatments provided by professional clinicians. Clinical application of blue light has proven to be effective for a broader spectral range and at lower fluences than previously utilized. Herein, we tested the hypothesis that sub-milliwatt/cm2 levels of long-wave blue light (449 nm) effectively kills Propionibacterium acnes, a causative agent of acne vulgaris, in vitro. MATERIALS AND METHODS: Two types of LED light boards were designed to facilitate in vitro blue light irradiation to either six-well plates containing fluid culture or a petri plate containing solid medium. P. acnes. Survival was determined by counting colony forming units (CFU) following irradiation. P. acnes was exposed in the presence and absence of oxygen. Coproporphyrin III (CPIII) photoexcitation was spectrophotometrically evaluated at 415 and 440 nm to compare the relative photochemical activities of these wavelengths. RESULTS: 422 and 449 nm blue light killed P. acnes in planktonic culture. Irradiation with 449 nm light also effectively killed P. acnes on a solid agar surface. Variation of time or intensity of light exposure resulted in a fluence-dependent improvement of antimicrobial activity. The presence of oxygen was necessary for killing of P. acnes with 449 nm light. CPIII displayed clear photoexcitation at both 415 and 440 nm, indicating that both wavelengths are capable of initiating CPIII photoexcitation at low incident light intensities (50 uW/cm2 ). CONCLUSION: Herein we demonstrate that sub-milliwatt/cm2 levels of long-wave blue light (449 nm) effectively kill P. acnes. The methods and results presented allow for deeper exploration and design of light therapy treatments. Results from these studies are expanding our understanding of the mode of action and functionality of blue light, allowing for improved options for acne patients. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

Low-level red LED light inhibits hyperkeratinization and inflammation induced by unsaturated fatty acid in an in vitro model mimicking acne.

BACKGROUND AND OBJECTIVE: Acne vulgaris is a chronic inflammatory disease of the pilosebaceous units (PSU), associated with increased sebum production, abnormal follicular keratinization (hyperkeratinization), follicular overgrowth of Propionibacterium acnes (P. acnes), and increased inflammatory mediator release. Light therapy has attracted medical interests as a safe alternative treatment for acne. Both blue and red light therapies at high doses >10 J/cm2 have demonstrated marked effects on inflammatory acne lesions. However, few studies have investigated the effects of lower doses of light. The aim of this study is to investigate the biological effects of lower doses of red light at 0.2-1.2 J/cm2 for acne using an in vitro model previously developed to mimic the inflammation and hyperkeratinization observed clinically in acne. MATERIALS AND METHODS: Human epidermal equivalents were topically exposed to an unsaturated fatty acid, oleic acid (OA), followed by red light-emitting diode (LED) light treatments (light-plus-OA treatments). Endpoints evaluated included the proinflammatory cytokine IL-1α, epidermal barrier integrity, as measured by transepithelial electrical resistance (TEER), and stratum corneum (SC) thickness to monitor hyperkeratinization. RESULTS: OA-induced IL-1α release was significantly (P < 0.05) reduced following red LED light at 0.2, 0.5, and 1.2 J/cm2 , from 266 ± 11 pg/ml of no-light-plus-OA-treated (OA treatment without light) controls to 216 ± 9, 231 ± 8, and 212 ± 7 pg/ml, respectively. Histological examination showed that SC thickening following OA treatment was reduced from 43% of total epidermis for no-light-plus-OA treatment to 37% and 38% of total epidermis following 0.5 and 1.1 J/cm2 red light plus OA treatment, respectively (P < 0.05). Moreover, 1.1 J/cm2 red-light-plus-OA treatment improved OA-induced TEER changes from 29% of baseline for no-light-plus-OA treatment, to 36% of baseline. CONCLUSION: Low level red LED light therapy could provide beneficial effects of anti-inflammation, normalizing pilosebaceous hyperkeratinization, and improving barrier impairment in Acne vulgaris. Lasers Surg. Med. 50:158-165, 2018. © 2017 Wiley Periodicals, Inc.

In vitro modeling of unsaturated free fatty acid-mediated tissue impairments seen in acne lesions.

Acne vulgaris is a disease of pilosebaceous units with multifactorial pathogenesis, including hyperkeratinization, increased sebum secretion, and inflammation. Recently, it was suggested that acne subjects may have also impaired skin barrier. We hypothesized that excess unsaturated free fatty acids (UFFA) present in the sebum may cause barrier impairment associated with increased follicular stratum corneum (SC) thickening and inflammation seen in acne. Therefore, epidermal and sebaceous lipid profiles from acne and healthy subjects were analyzed and an in vitro epidermal tissue model was developed to validate this hypothesis. Significantly increased levels of free fatty acids (p < 0.05) were observed in skin lipids of human acne vs. healthy subjects. Exposure of human epidermal equivalents (HEEs) to the UFFA oleic acid (OA), also present in sebum, led to barrier impairment associated with increased SC lipid disorder, increased secretion of interleukin-1α (IL-1α), and excessive SC thickening. Furthermore, the expression of genes encoding for inflammatory cytokines and epidermal differentiation proteins was also increased both in acne lesions and in OA-treated HEEs. Taken together, these data are in agreement with the hypothesis that excess UFFAs in sebum of acne subjects may contribute to impaired skin barrier associated with the increased follicular SC thickness and inflammation seen in acne. Moreover, OA induces similar molecular and phenotypic changes in HEEs as those seen in acne lesions and suggests that an UFFA-treated epidermal tissue model can be used to study the UFFA-mediated pathways involved in the pathogenesis of inflammatory acne and for the development of appropriate therapies.

Topical stabilized retinol treatment induces the expression of HAS genes and HA production in human skin in vitro and in vivo.

Skin Aging manifests primarily with wrinkles, dyspigmentations, texture changes, and loss of elasticity. During the skin aging process, there is a loss of moisture and elasticity in skin resulting in loss of firmness finally leading to skin sagging. The key molecule involved in skin moisture is hyaluronic acid (HA), which has a significant water-binding capacity. HA levels in skin decline with age resulting in decrease in skin moisture, which may contribute to loss of firmness. Clinical trials have shown that topically applied ROL effectively reduces wrinkles and helps retain youthful appearance. In the current study, ROL was shown to induce HA production and stimulates the gene expression of all three forms of hyaluronic acid synthases (HAS) in normal human epidermal keratinocytes monolayer cultures. Moreover, in human skin equivalent tissues and in human skin explants, topical treatment of tissues with a stabilized-ROL formulation significantly induced the gene expression of HAS mRNA concomitant with an increased HA production. Finally, in a vehicle-controlled human clinical study, histochemical analysis confirmed increased HA accumulation in the epidermis in ROL-treated human skin as compared to vehicle. These results show that ROL increases skin expression of HA, a significant contributing factor responsible for wrinkle formation and skin moisture, which decrease during aging. Taken together with the activity to increase collagen, elastin, and cell proliferation, these studies establish that retinol provides multi-functional activity for photodamaged skin.

p38 MAP Kinase Inhibition Reduces Propionibacterium acnes-Induced Inflammation in Vitro.

INTRODUCTION: Propionibacterium acnes, a ubiquitous skin bacterium, stimulates keratinocytes to produce a number of proinflammatory cytokines and may contribute to inflammatory acne. The aim of the study was to investigate whether P. acnes-induced proinflammatory cytokine release is mediated by P. acnes-induced activation of p38 mitogen-activated protein kinase (p38 MAPK or p38) in human keratinocytes. METHODS: Immunohistochemistry was used to evaluate p38 phosphorylation in human skin samples with or without acne. Primary human keratinocytes and epidermal skin equivalents were exposed to viable P. acnes. Phosphorylation of MAPKs without or with p38 inhibitors was examined by Western blot and cytokine secretion was detected by Enzyme-Linked Immunosorbent Assay (ELISA). RESULTS: Increased levels of phospho-p38 were observed in human acne lesions, predominantly in follicular and perifollicular keratinocytes. Exposure of cultured human keratinocytes to viable P. acnes resulted in phosphorylation of multiple members of the MAPK family, including rapid and transient activation of p38 and extracellular signal-related kinase (ERK1/2) and relatively slow but sustained activation of c-Jun N-terminal kinases (JNK1/2). Viable P. acnes induced the secretion of interleukin-1α (IL-1α), tumor necrosis factor-α (TNF-α), and IL-8 from human keratinocytes. The phosphorylation of p38 (phospho-p38) and the secretion of cytokines induced by P. acnes in cultured keratinocytes were inhibited by SB203580, a p38α/ß inhibitor. Furthermore, SCIO-469, a selective inhibitor of p38α, showed similar effects in cultured keratinocytes. Topical treatment of SCIO-469 inhibited the P. acnes-induced phospho-p38 and cytokine secretion in human epidermal equivalents. CONCLUSION: The data demonstrate that P. acnes induces p38-dependent inflammatory responses in keratinocytes, and suggest that p38 may play an important role in the pathogenesis of inflammatory acne. FUNDING: Johnson & Johnson.

IL-11, IL-1α, IL-6, and TNF-α are induced by solar radiation in vitro and may be involved in facial subcutaneous fat loss in vivo.

BACKGROUND: The loss of subcutaneous (sc) fat is associated with aging. Inflammatory cytokines, such as interleukin-1 α (IL-1α), interleukin-11 (IL-11) and tumor necrosis factor-α (TNF-α), are known to inhibit the differentiation of preadipocytes. OBJECTIVE: This study investigated the potential role of inflammatory cytokines in solar-radiation-induced facial fat loss. METHODS: Cultured fibroblasts, keratinocytes, and skin equivalents were exposed to various doses of radiation from a solar simulator. Inflammatory cytokines' mRNA production and protein secretion were examined by qRT-PCR and ELISA, respectively. In some experiments, epidermal-dermal equivalents were pretreated topically with a broad-spectrum sunscreen prior to solar simulated radiation (SSR). Human facial preadipocytes treated with recombinant IL-11 or with conditioned media from solar-irradiated equivalents were evaluated for the level of adipocyte differentiation by image analyses, Oil red O staining, and the expression of adipocyte differentiation markers. RESULTS: IL-11, IL-1α, IL-6, and TNF-α protein secretion were induced from epidermal-dermal equivalents by exposure to SSR. A sunscreen prevented SSR-induced inflammatory cytokines production from such equivalents. Exposure of facial preadipocytes to conditioned medium from solar-irradiated epidermal-dermal equivalents inhibited their differentiation into mature adipocytes. Consequently, conditioned medium from sunscreen-pretreated, solar-irradiated equivalents did not inhibit differentiation of preadipocytes. A cocktail of neutralizing antibodies to IL-11, IL-1α, IL-6 and TNF-α significantly reduced the SSR-induced inhibition of preadipocyte differentiation. CONCLUSION: These results support the hypothesis that SSR-induced inflammatory cytokine may be involved in the photoaging-induced loss of facial subcutaneous fat. Inhibition of this process, e.g. by sunscreens, might slow or prevent photoaging-induced changes in facial contouring.

A melanocortin receptor 1 and 5 antagonist inhibits sebaceous gland differentiation and the production of sebum-specific lipids.

BACKGROUND: The melanocortin receptor-5 (MC5R) is present in human sebaceous glands, where it is expressed in differentiated sebocytes only. The targeted disruption of MC5R in mice resulted in reduced sebaceous lipid production and a severe defect in water repulsion. OBJECTIVE: To investigate the physiological function of MC5R in human sebaceous glands. METHODS: A novel MC1R and MC5R antagonist (JNJ-10229570) was used to treat primary human sebaceous cells or human skins grafted onto severe combined immunodeficient (SCID) mice. Transcription profiling, lipid analyses, and histological and immunohistochemical staining were used to analyze the effect of MC5R inhibition on sebaceous gland differentiation and sebum production. RESULTS: JNJ-10229570 dose dependently inhibited the production of sebaceous lipids in cultured primary human sebocytes. Topical treatment with JNJ-10229570 of human skins transplanted onto SCID mice resulted in a marked decrease in sebum-specific lipid production, sebaceous gland's size and the expression of the sebaceous differentiation marker epithelial-membrane antigen (EMA). Treatment with flutamide, a known inhibitor of sebum production, gave similar results, validating the human skin/SCID mouse experimental system for sebaceous secretion studies. CONCLUSION: Our data suggest that antagonists of MC1R and MC5R could be effective sebum suppressive agents and might have a potential for the treatment of acne and other sebaceous gland pathologies.

Melanocortin-5 receptor and sebogenesis.

The melanocortins (α-MSH, ß-MSH, γ-MSH, and ACTH) bind to the melanocortin receptors and signal through increases in cyclic adenosine monophosphate to induce biological effects. The melanocortin MC(5) and MC(1) receptors are expressed in human sebaceous glands, which produce sebum, a lipid mixture of squalene, wax esters, triglycerides, cholesterol esters, and free fatty acids that is secreted onto the skin. Excessive sebum production is one of the major factors in the pathogenesis of acne. The expression of melanocortin MC(5) receptor has been associated with sebocyte differentiation and sebum production. Sebaceous lipids are down-regulated in melanocortin MC(5) receptor-deficient mice, consistent with the observation that α-MSH acts as a sebotropic hormone in rodents. These findings, which suggest that melanocortins stimulate sebaceous lipid production through the MC(5) receptor, led to our search for MC(5) receptor antagonists as potential sebum-suppressive agents. As predicted, an antagonist was shown to inhibit sebocyte differentiation in vitro, and to reduce sebum production in human skin transplanted onto immunodeficient mice. The melanocortin MC(5) receptor antagonists may prove to be clinically useful for the treatment of sebaceous disorders with excessive sebum production, such as acne.

The role of keratinocyte growth factor in melanogenesis: a possible mechanism for the initiation of solar lentigines.

Solar lentigines (SLs) are hyperpigmentary lesions presented on sun-exposed areas of the skin and associated with ageing. The molecular mechanism of SL initiation is not completely understood. Ultraviolet B (UVB) stimulates keratinocytes to produce interlukin-1 alpha (IL-1α), which then induces keratinocyte growth factor (KGF) secretion; therefore, we examined their possible roles in the induction of SLs. We found that KGF increases pigment production in both pigmented epidermal equivalents and human skin explants. In addition, UVB exposure increases KGF expression, and KGF treatment induces tyrosinase (TYR) expression in primary melanocytes. The KGF-induced pigmentary changes were confirmed using pigmented Yucatan swine, and human skins grafted onto immuno-deficient mice. In both model systems, the topical treatment with KGF, alone or in combination with IL-1α, resulted in the in vivo formation of hyperpigmentary lesions with increased pigment deposition and elongated rete ridges, which resemble the histological features of human SLs. Preliminary immunohistochemical analysis of human skins showed a moderate increase in KGF, and a strong induction in KGF receptor (KGFR) in SL lesions. In summary, KGF increases pigment production and deposition in vitro and in vivo. Moreover, we show for the first time the in vivo generation of hyperpigmentary lesions with histological resemblance to human SLs and indicate the involvement of KGF/KGFR in the molecular pathology of human SLs.

LIGR, a protease-activated receptor-2-derived peptide, enhances skin pigmentation without inducing inflammatory processes.

The protease-activated receptor-2 (PAR-2) is a seven transmembrane G-protein-coupled receptor that could be activated by serine protease cleavage or by synthetic peptide agonists. We showed earlier that activation of PAR-2 with Ser-Leu-Ile-Gly-Arg-Leu-NH(2) (SLIGRL), a known PAR-2 activating peptide, induces keratinocyte phagocytosis and increases skin pigmentation, indicating that PAR-2 regulates pigmentation by controlling phagocytosis of melanosomes. Here, we show that Leu-Ile-Gly-Arg-NH(2) (LIGR) can also induce skin pigmentation. Both SLIGRL and LIGR increased melanin deposition in vitro and in vivo, and visibly darkened human skins grafted onto severe combined immuno-deficient (SCID) mice. Both SLIGRL and LIGR stimulated Rho-GTP activation resulting in keratinocyte phagocytosis. Interestingly, LIGR activates only a subset of the PAR-2 signaling pathways, and unlike SLIGRL, it does not induce inflammatory processes. LIGR did not affect many PAR-2 signaling pathways, including [Ca(2+)] mobilization, cAMP induction, the induction of cyclooxgenase-2 (COX-2) expression and the secretion of prostaglandin E2, interleukin-6 and -8. PAR-2 siRNA inhibited LIGR-induced phagocytosis, indicating that LIGR signals via PAR-2. Our data suggest that LIGR is a more specific regulator of PAR-2-induced pigmentation relative to SLIGRL. Therefore, enhancing skin pigmentation by topical applications of LIGR may result in a desired tanned-like skin color, without enhancing inflammatory processes, and without the need of UV exposure.

Melanocortin-5 receptor: a marker of human sebocyte differentiation.

Melanocortin receptors (MC1R-MC5R) and their ligands (melanocyte-stimulating hormone (MSH) and adrenocorticotrophin hormone (ACTH)) have been shown to influence physiological functions of cells and organs, including exocrine glands. Since relatively little is known about MC5R expression and function in the human sebaceous gland, we examined expression of MC5R by immunohistochemistry and RT-PCR in human sebaceous cells in vivo and in vitro. In human skin, MC5R was detected only in differentiating, lipid-laden sebaceous cells but not in basal, undifferentiated sebaceous cells. Similarly, in cultured human sebocytes MC5R was only detectable at the onset of differentiation and in fully differentiated cells displaying prominent lipid granules. The lipid profile of the cultured and differentiated human sebocytes was shown to be human sebum-specific using (14)C-acetate labeling and high performance thin layer chromatography. Our studies suggest that MC5R is a marker of human sebocyte differentiation.

Proopiomelanocortin peptides and sebogenesis.

Previous animal studies have demonstrated that alpha-melanocyte-stimulating hormone (alpha-MSH) is a sebotropic hormone in rats and that targeted disruption of melanocortin 5 receptor (MC5-R) can down-regulate sebum output in mice. To study the role of proopiomelanocortin (POMC) peptides in the regulation of human sebaceous lipid production and sebocyte differentiation, we established a primary human sebocyte culture system. Sebocytes were derived from normal human facial skin. Differentiation of sebocytes, induced by POMC-derived peptides such as MSH, adrenocorticotropic hormone (ACTH), or bovine pituitary extract (BPE), resulted in the appearance of prominent cytoplasmic lipid droplets. Partial induction of sebocyte differentiation also was observed in serum-depleted cultures, but there was very limited spontaneous differentiation in serum-containing medium. Analysis by high-performance thin-layer chromatography (HPTLC) of (14)C-acetate-labeled lipids showed a dose-dependent increase in synthesis of sebaceous-specific lipid (i.e., squalene) induced by NDP alpha-MSH. Molecular studies using RT-PCR showed a low level of human MC5-R expression under serum-free condition but a substantial increase after treatment with NDP alpha-MSH or BPE. In contrast, MC1-R expression remained the same, independent of treatment. Our data indicate that expression of MC5-R correlates with sebocyte differentiation and suggest a regulatory role for MC5-R in human sebaceous lipid production.