The impact of irritant challenge on the skin barrier and myeloid resident immune cells in post-menopausal women is modulated by hormone replacement therapy.

BACKGROUND: Sex hormone changes during menopausal transition contribute to declining skin health. However, how menopause and its treatment by hormone replacement therapy (HRT) impact the skin barrier and immune system is unclear. Therefore, we examined how menopause and HRT affect skin barrier and immune cell composition in post-menopausal women following irritant challenge. METHODS: Two cohorts of post-menopausal women were recruited to the study, one untreated (HRT-; n = 10; mean age 56.5 yrs [range 48-63 yrs]) and the other receiving HRT (n = 8; mean age 54 yrs [range 48-63 yrs]). Skin irritation was induced by applying 1.25% topical Sodium Lauryl Sulfate (SLS) to occluded buttock skin for 48 hours. Clinical assessment was conducted after 24 hours, followed by biopsy of both SLS-challenged and unchallenged skin for analysis of skin barrier proteins and immune cell distribution using immunofluorescence. RESULTS: Clinically, there were no significant differences in skin irritant responses between those taking or not taking HRT (including increased skin redness and blood flow). In response to SLS challenge a significant increase in trans-epidermal water loss (p<0.05), filaggrin deposition and keratin-10-positive cell layers (p<0.01) was observed in individuals receiving HRT compared to the HRT- group. Following SLS challenge in individuals taking HRT, a significant (p<0.01) reduction of CD207+ cells in the epidermis was observed, accompanied by an increase of CD207+ cells in the dermis, indicative of migrating Langerhans' cells (LCs). Significantly fewer migrating LCs were observed in those not receiving HRT (p<0.01). Furthermore, the number of dermal dendritic cells (DCs), macrophages, and CD11c+CD206- and CD68+CD206- subsets were found to be significantly (p<0.05) higher in those taking HRT following SLS challenge. CONCLUSION: Individuals receiving HRT displayed enhanced skin barrier response to SLS challenge with thicker filaggrin and increased keratin-10-positive epidermal cell layers. Following challenge, HRT users exhibited elevated counts of LCs, inflammatory DCs, and macrophages in the dermis. These may render skin both, more prone to inflammation and more capable of resolving it, while also promoting skin repair.

Menopause induces changes to the stratum corneum ceramide profile, which are prevented by hormone replacement therapy.

The menopause can lead to epidermal changes that are alleviated by hormone replacement therapy (HRT). We hypothesise that these changes could relate to altered ceramide production, and that oestrogen may have a role in keratinocyte ceramide metabolism. White Caucasian women were recruited into three groups: pre-menopausal (n = 7), post-menopausal (n = 11) and post-menopausal taking HRT (n = 10). Blood samples were assessed for hormone levels, transepidermal water loss was measured to assess skin barrier function, and stratum corneum lipids were sampled from photoprotected buttock skin. Ceramides and sphingomyelins were analysed by ultraperformance liquid chromatography with electrospray ionisation and tandem mass spectrometry. Post-menopausal stratum corneum contained lower levels of ceramides, with shorter average length; changes that were not evident in the HRT group. Serum oestradiol correlated with ceramide abundance and length. Ceramides had shorter sphingoid bases, indicating altered de novo ceramide biosynthesis. Additionally, post-menopausal women had higher sphingomyelin levels, suggesting a possible effect on the hydrolysis pathway. Treatment of primary human keratinocytes with oestradiol (10 nM) increased production of CER[NS] and CER[NDS] ceramides, confirming an effect of oestrogen on cutaneous ceramide metabolism. Taken together, these data show perturbed stratum corneum lipids post-menopause, and a role for oestrogen in ceramide production.

Role of distinct fibroblast lineages and immune cells in dermal repair following UV radiation-induced tissue damage.

Solar ultraviolet radiation (UVR) is a major source of skin damage, resulting in inflammation, premature ageing, and cancer. While several UVR-induced changes, including extracellular matrix reorganisation and epidermal DNA damage, have been documented, the role of different fibroblast lineages and their communication with immune cells has not been explored. We show that acute and chronic UVR exposure led to selective loss of fibroblasts from the upper dermis in human and mouse skin. Lineage tracing and in vivo live imaging revealed that repair following acute UVR is predominantly mediated by papillary fibroblast proliferation and fibroblast reorganisation occurs with minimal migration. In contrast, chronic UVR exposure led to a permanent loss of papillary fibroblasts, with expansion of fibroblast membrane protrusions partially compensating for the reduction in cell number. Although UVR strongly activated Wnt signalling in skin, stimulation of fibroblast proliferation by epidermal ß-catenin stabilisation did not enhance papillary dermis repair. Acute UVR triggered an infiltrate of neutrophils and T cell subpopulations and increased pro-inflammatory prostaglandin signalling in skin. Depletion of CD4- and CD8-positive cells resulted in increased papillary fibroblast depletion, which correlated with an increase in DNA damage, pro-inflammatory prostaglandins, and reduction in fibroblast proliferation. Conversely, topical COX-2 inhibition prevented fibroblast depletion and neutrophil infiltration after UVR. We conclude that loss of papillary fibroblasts is primarily induced by a deregulated inflammatory response, with infiltrating T cells supporting fibroblast survival upon UVR-induced environmental stress.

Inflammaging and the Skin.

As global life expectancy continues to rise, we are challenged with maintaining health into old age. One strategy is to target the chronic low-level inflammation associated with aging, termed inflammaging. This is characterized by increased levels of circulating proinflammatory cytokines and a shift toward cellular senescence, changes that are believed to drive many age-associated conditions, including dementia, arthritis, and type 2 diabetes. As with other organs, the skin undergoes functional decline during aging, becoming more fragile and susceptible to infection; however, the contribution of inflammaging is not well-understood. This review article describes the evidence for inflammaging in the skin, its relationship with senescence, and how this relates to declining skin structure and function.

UV radiation recruits CD4+GATA3+ and CD8+GATA3+ T cells while altering the lipid microenvironment following inflammatory resolution in human skin in vivo.

OBJECTIVES: Solar ultraviolet radiation (UVR) has major adverse effects on human health. While the mechanisms responsible for induction of UVR-induced inflammation are well-documented, the mediation of its resolution and longer-term adaptive homeostasis is unknown. Therefore, we examined the skin immune and lipid profile over time following UVR inflammation. METHODS: To investigate the self-resolving events of UVR inflammation in vivo, human skin was exposed to a single pro-inflammatory dose of UVR. Skin biopsies and suction blister fluid were taken at intervals up to 2 weeks post-UVR. The immune infiltrate was quantified by immunohistochemistry, and lipid mediators were profiled by liquid chromatography/mass spectrometry. RESULTS: We identified that cellular resolution events including switching of macrophage phenotype apply to human sunburn. However, UVR-induced inflammation in humans involves a post-resolution phase that differs from other experimental models. We demonstrate that 2 weeks after the initiating UVR stimulus, there is considerable immune activity with CD8+GATA3+ T cells maintained in human skin. Our results challenge the dogma of CD4+FOXP3+ T cells being the main effector CD4+ T-cell population following UVR, with CD4+GATA3+ T cells the dominant phenotype. Furthermore, lipid mediators are elevated 14 days post-UVR, demonstrating the skin lipid microenvironment does not revert to the tissue setting occurring prior to UVR exposure. CONCLUSION: We have identified for the first time that CD4+GATA3+ and CD8+GATA3+ T-cell subpopulations are recruited to UVR-inflamed human skin, demonstrating discrepancies between the adaptive UVR response in mice and humans. Future strategies to abrogate UVR effects may target these T-cell subpopulations and also the persistent alteration of the lipid microenvironment post-UVR.

Dynamics of the human skin mediator lipidome in response to dietary ω-3 fatty acid supplementation.

Nutritional supplementation with fish oil or ω-3 (n-3) polyunsaturated fatty acids (PUFAs) has potential benefits for skin inflammation. Although the differential metabolism of the main n-3PUFA eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) could lead to distinct activities, there are no clinical studies comparing their relative efficacy in human skin. Following a 10-wk oral supplementation of healthy volunteers and using mass spectrometry-based lipidomics, we found that n-3PUFA mainly affected the epidermal mediator lipidome. EPA was more efficient than DHA in reducing production of arachidonic acid-derived lipids, and both n-3PUFA lowered N-acyl ethanolamines. In UV radiation-challenged skin (3 times the minimum erythemal dose), EPA attenuated the production of proinflammatory lipids, whereas DHA abrogated the migration of Langerhans cells, as assessed by immunohistochemistry. Interestingly, n-3PUFA increased the infiltration of CD4+ and CD8+ T cells but did not alter the erythemal response, either the sunburn threshold or the resolution of erythema, as assessed by spectrophotometric hemoglobin index readings. As EPA and DHA differentially impact cutaneous inflammation through changes in the network of epidermal lipids and dendritic and infiltrating immune cells, they should be considered separately when designing interventions for cutaneous disease.-Kendall, A. C., Pilkington, S. M., Murphy, S. A., Del Carratore, F., Sunarwidhi, A. L., Kiezel-Tsugunova, M., Urquhart, P., Watson, R. E. B., Breitling, R., Rhodes, L. E., Nicolaou, A. Dynamics of the human skin mediator lipidome in response to dietary ω-3 fatty acid supplementation.

Differential reorganisation of cutaneous elastic fibres: a comparison of the in vivo effects of broadband ultraviolet B versus solar simulated radiation.

Long-term exposure of human skin to ultraviolet radiation (UVR) in sunlight negatively impacts its appearance and function with photoaged skin having a characteristic leathery, rough appearance, with deep wrinkles. These clinical features of photodamage are thought to result from UVR-induced remodelling of the dermal extracellular matrix, particularly the elastic fibre system. There are few in vivo human data on the impact of acute UVR exposure on this fibre system and particularly solar-simulated radiation (SSR)-mediated effects. We examined the differential effect of broadband UVB and SSR on the human dermal elastic fibre system, and specifically the microfibrillar components fibrillin-1, fibulin-2 and fibulin-5. Healthy white Caucasian adults (skin type II-III) were recruited and irradiated with 3× their minimal erythema dose of broadband UVB (n = 6) or SSR (n = 6) on photoprotected buttock skin. Punch biopsies were taken 24 h after irradiation and from unirradiated control skin. Overall, histological assessment of elastic fibres revealed significantly less elastic fibre staining in broadband UVB (P = 0.004) or SSR (P = 0.04) irradiated skin compared to unirradiated control skin. Significantly less staining of fibrillin-1-positive microfibrils was also observed in the papillary dermis of UVB irradiated skin (P = 0.02) but not skin exposed to SSR. Conversely, immunohistochemistry for fibulin-5-positive microfibrils revealed significantly less expression in skin exposed to SSR (P = 0.04) but not to broadband UVB. There was no significant change in fibulin-2-positive microfibrils following either broadband UVB or SSR irradiation. Thus, broadband UVB and SSR mediate differential effects on individual components of the dermal elastic fibre network in human skin. Further human studies are required to explore the mechanisms underlying these findings and the impact of potential photoprotective agents.

Lower levels of interleukin-1ß gene expression are associated with impaired Langerhans' cell migration in aged human skin.

Langerhans' cells (LC) play pivotal roles in skin immune responses, linking innate and adaptive immunity. In aged skin there are fewer LC and migration is impaired compared with young skin. These changes may contribute to declining skin immunity in the elderly, including increased skin infections and skin cancer. Interleukin-1ß (IL-1ß) and tumour necrosis factor-α (TNF-α) are mandatory signals for LC migration and previous studies suggest that IL-1ß signalling may be dysregulated in aged skin. Therefore, we sought to explore the mechanisms underlying these phenomena. In skin biopsies of photoprotected young (< 30 years) and aged (> 70 years) human skin ex vivo, we assessed the impact of trauma, and mandatory LC mobilizing signals on LC migration and gene expression. Biopsy-related trauma induced LC migration from young epidermis, whereas in aged skin, migration was greatly reduced. Interleukin-1ß treatment restored LC migration in aged epidermis whereas TNF-α was without effect. In uncultured, aged skin IL-1ß gene expression was lower compared with young skin; following culture, IL-1ßmRNA remained lower in aged skin under control and TNF-α conditions but was elevated after culture with IL-1ß. Interleukin-1 receptor type 2 (IL1R2) gene expression was significantly increased in aged, but not young skin, after cytokine treatment. Keratinocyte-derived factors secreted from young and aged primary cells did not restore or inhibit LC migration from aged and young epidermis, respectively. These data suggest that in aged skin, IL-1ß signalling is diminished due to altered expression of IL1B and decoy receptor gene IL1R2.

Effect of oral eicosapentaenoic acid on epidermal Langerhans cell numbers and PGD2 production in UVR-exposed human skin: a randomised controlled study.

Langerhans cells (LCs) are sentinels of skin's immune system, their loss from epidermis contributing to UVR suppression of cell-mediated immunity (CMI). Omega-3 polyunsaturated fatty acids show potential to reduce UVR suppression of CMI in mice and humans, potentially through modulation of LC migration. Our objectives were to examine whether eicosapentaenoic acid (EPA) ingestion influences UV-mediated effects on epidermal LC numbers and levels of immunomodulatory mediators including prostaglandin (PG)D2 , which is expressed by LC. In a double-blind randomised controlled study, healthy individuals took 5-g EPA-rich (n=40) or control (n=33) lipid for 12 weeks; UVR-exposed and unexposed skin samples were taken pre- and postsupplementation. Epidermal LC numbers were assessed by immunofluorescence for CD1a, and skin blister fluid PG and cytokines were quantified by LC-MS/MS and Luminex assay, respectively. Presupplementation, UVR reduced mean (SEM) LC number/mm2 from 913 (28) to 322 (40) (P<.001), and mean PGD2 level by 37% from 8.1 (11.6) to 5.1 (5.6) pg/µL; P<.001), while IL-8 level increased (P<.001). Despite confirmation of EPA bioavailability in red blood cells and skin in the active group, no between-group effect of EPA was found on UVR modulation of LC numbers, PGD2 or cytokine levels postsupplementation. Thus, no evidence was found for EPA reduction of photoimmunosuppression through an impact on epidermal LC numbers. Intriguingly, UVR exposure substantially reduced cutaneous PGD2 levels in humans, starkly contrasting with reported effects of UVR on other skin PG. Lowered PGD2 levels could reflect LC loss from the epidermis and/or altered dendritic cell activity and may be relevant for phototherapy of skin disease.

Distribution of bioactive lipid mediators in human skin.

The skin produces bioactive lipids that participate in physiological and pathological states, including homeostasis, induction, propagation, and resolution of inflammation. However, comprehension of the cutaneous lipid complement, and contribution to differing roles of the epidermal and dermal compartments, remains incomplete. We assessed the profiles of eicosanoids, endocannabinoids, N-acyl ethanolamides, and sphingolipids, in human dermis, epidermis, and suction blister fluid. We identified 18 prostanoids, 12 hydroxy-fatty acids, 9 endocannabinoids and N-acyl ethanolamides, and 21 non-hydroxylated ceramides and sphingoid bases, several demonstrating significantly different expression in the tissues assayed. The array of dermal and epidermal fatty acids was reflected in the lipid mediators produced, whereas similarities between lipid profiles in blister fluid and epidermis indicated a primarily epidermal origin of suction blister fluid. Supplementation with omega-3 fatty acids ex vivo showed that their action is mediated through perturbation of existing species and formation of other anti-inflammatory lipids. These findings demonstrate the diversity of lipid mediators involved in maintaining tissue homeostasis in resting skin and hint at their contribution to signaling, cross-support, and functions of different skin compartments. Profiling lipid mediators in biopsies and suction blister fluid can support studies investigating cutaneous inflammatory responses, dietary manipulation, and skin diseases lacking biomarkers and therapeutic targets.

Impact of EPA ingestion on COX- and LOX-mediated eicosanoid synthesis in skin with and without a pro-inflammatory UVR challenge--report of a randomised controlled study in humans.

SCOPE: Eicosapentaenoic acid (EPA), abundant in oily fish, is reported to reduce skin inflammation and provide photoprotection, potential mechanisms include competition with arachidonic acid (AA) for metabolism by cyclooxygenases/lipoxygenases to less pro-inflammatory mediators. We thus examine impact of EPA intake on levels of AA, EPA and their resulting eicosanoids in human skin with or without ultraviolet radiation (UVR) challenge. METHODS AND RESULTS: In a double-blind randomised controlled study, 79 females took 5 g EPA-rich or control lipid for 12 wk. Pre- and post-supplementation, red blood cell and skin polyunsaturated fatty acids were assessed by GC, and eicosanoids from unexposed and UVR-exposed skin by LC-MS/MS. Active supplementation increased red blood cell and dermal EPA versus control (both p < 0.001), lowering relative AA:EPA content (4:1 versus 15:1 and 5:1 versus 11:1, respectively; both p < 0.001). Pre-supplementation, UVR increased PGE2, 12-hydroxyeicosatetraenoic acids, 12-HEPE (all p < 0.001) and PGE3 (p < 0.05). Post-EPA, PGE2 was reduced in unchallenged skin (p < 0.05) while EPA-derived PGE3 (non-sign) and 12-HEPE (p < 0.01) were elevated post-UVR. Thus, post-EPA, PGE2 :PGE3 was lower in unchallenged (12:1 versus 28:1; p < 0.05) and UVR exposed (12:1 versus 54:1; p < 0.01) skin; 12-hydroxyeicosatetraenoic acids:12-HEPE was lower in UVR-exposed skin (3:1 versus 11:1; p < 0.001). CONCLUSION: Dietary EPA augments skin EPA:AA content, shifting eicosanoid synthesis towards less pro-inflammatory species, and promoting a regulatory milieu under basal conditions and in response to inflammatory insult.

Nutritional abrogation of photoimmunosuppression: in vivo investigations.

Skin cancer is a major public health concern, and the primary aetiological factor in the majority of skin cancers is ultraviolet radiation (UVR) exposure. UVR not only induces potentially mutagenic DNA damage but also suppresses cell-mediated immunity (CMI), allowing cancerous cells to escape destruction and progress to tumours. A considerable proportion of an individual's annual sun exposure is obtained outside the vacation period when topical and physical measures for photoprotection are irregularly used. Certain nutrients could provide an adjunctive protective role, and evidence is accruing from experimental studies to support their use in abrogation of photoimmunosuppression. Moreover, developments in clinical research methods to evaluate impact of solar-simulated radiation on cutaneous CMI allow the immune protective potential of nutritional agents to be examined in humans in vivo. This article summarises the mediation of CMI and its suppression by UVR, evaluates the methodology for quantitative assessment in vivo, reviews the human studies reported on nutritional abrogation of photoimmunosuppression including recent randomized controlled trials and discusses the mechanisms of photoprotection by the nutrients. This includes, in addition to antioxidants, novel studies of omega-3 polyunsaturated fatty acids and nicotinamide.

Randomized controlled trial of oral omega-3 PUFA in solar-simulated radiation-induced suppression of human cutaneous immune responses.

BACKGROUND: Skin cancer is a major public health concern, and the majority of cases are caused by solar ultraviolet radiation (UVR) exposure, which suppresses skin immunity. Omega-3 (n-3) PUFAs protect against photoimmunosuppression and skin cancer in mice, but the impact in humans is unknown. OBJECTIVES: We hypothesized that EPA-rich n-3 PUFA would abrogate photoimmunosuppression in humans. Therefore, a nutritional study was performed to assess the effect on UVR suppression of cutaneous cell-mediated immunity (CMI) reflected by nickel contact hypersensitivity (CHS). DESIGN: In a double-blind, randomized controlled study, 79 volunteers (nickel-allergic women, 22-60 y old, with phototype I or II) took 5 g n-3 PUFA-containing lipid (70% EPA plus 10% DHA) or a control lipid daily for 3 mo. After supplementation, nickel was applied to 3 skin sites preexposed on 3 consecutive days to 1.9, 3.8, or 7.6 J/cm(2) of solar-simulated radiation (SSR) and to 3 unexposed control sites. Nickel CHS responses were quantified after 72 h and the percentage of immunosuppression by SSR was calculated. Erythrocyte [red blood cell (RBC)] EPA was measured by using gas chromatography. RESULTS: SSR dose-related suppression of the nickel CHS response was observed in both groups. Photoimmunosuppression appeared less in the n-3 PUFA group than in the control group (not statistically significant [mean difference (95% CI): 6.9% (-2.1%, 15.9%)]). The difference was greatest at 3.8 J/cm(2) SSR [mean difference: 11% (95% CI: 0.5%, 21.4%)]. Postsupplementation RBC EPA was 4-fold higher in the n-3 PUFA group than in the control group (mean difference: 2.69% (95% CI: 2.23%, 3.14%), which confirmed the EPA bioavailability. CONCLUSION: Oral n-3 PUFAs appear to abrogate photoimmunosuppression in human skin, providing additional support for their chemopreventive role; verification of study findings is required. This trial was registered at clinicaltrials.gov as NCT01032343.

Three-way assessment of long-chain n-3 PUFA nutrition: by questionnaire and matched blood and skin samples.

The long-chain n-3 PUFA, EPA, is believed to be important for skin health, including roles in the modulation of inflammation and protection from photodamage. FFQ and blood levels are used as non-invasive proxies for assessing skin PUFA levels, but studies examining how well these proxies reflect target organ content are lacking. In seventy-eight healthy women (mean age 42·8, range 21-60 years) residing in Greater Manchester, we performed a quantitative analysis of long-chain n-3 PUFA nutrition estimated from a self-reported FFQ (n 75) and correlated this with n-3 PUFA concentrations in erythrocytes (n 72) and dermis (n 39). Linear associations between the three n-3 PUFA measurements were assessed by Spearman correlation coefficients and agreement between these measurements was estimated. Average total dietary content of the principal long-chain n-3 PUFA EPA and DHA was 171 (SD 168) and 236 (SD 248) mg/d, respectively. EPA showed significant correlations between FFQ assessments and both erythrocyte (r 0·57, P< 0·0001) and dermal (r 0·33, P= 0·05) levels, as well as between erythrocytes and dermis (r 0·45, P= 0·008). FFQ intake of DHA and the sum of n-3 PUFA also correlated well with erythrocyte concentrations (r 0·50, P< 0·0001; r 0·27, P= 0·03). Agreement between ranked thirds of dietary intake, blood and dermis approached 50% for EPA and DHA, though gross misclassification was lower for EPA. Thus, FFQ estimates and circulating levels of the dietary long-chain n-3 PUFA, EPA, may be utilised as well-correlated measures of its dermal bioavailability.

Omega-3 polyunsaturated fatty acids: photoprotective macronutrients.

Ultraviolet radiation (UVR) in sunlight has deleterious effects on skin, while behavioural changes have resulted in people gaining more sun exposure. The clinical impact includes a year-on-year increase in skin cancer incidence, and topical sunscreens alone provide an inadequate measure to combat overexposure to UVR. Novel methods of photoprotection are being targeted as additional measures, with growing interest in the potential for systemic photoprotection through naturally sourced nutrients. Omega-3 polyunsaturated fatty acids (n-3 PUFA) are promising candidates, showing potential to protect the skin from UVR injury through a range of mechanisms. In this review, we discuss the biological actions of n-3 PUFA in the context of skin protection from acute and chronic UVR overexposure and describe how emerging new technologies such as nutrigenomics and lipidomics assist our understanding of the contribution of such nutrients to skin health.

Ultraviolet-radiation induced skin inflammation: dissecting the role of bioactive lipids.

Acute exposure of human skin to the ultraviolet radiation (UVR) in sunlight results in the sunburn response. This is mediated in part by pro-inflammatory eicosanoids and other bioactive lipids, which are in turn produced via mechanisms including UVR-induction of oxidative stress, cell signalling and gene expression. Sunburn is a self-limiting inflammation offering a convenient and accessible system for the study of human cutaneous lipid metabolism. Recent lipidomic applications have revealed that a wider diversity of eicosanoids may be involved in the sunburn response than previously appreciated. This article reviews the effects of UVR on cutaneous lipids and examines the contribution of bioactive lipid mediators in the development of sunburn. Since human skin is an active site of polyunsaturated fatty acid (PUFA) metabolism, and these macronutrients can influence the production of eicosanoids/bioactive lipids, as well as modulate cell signalling, gene expression and oxidative stress, the application of PUFA as potential photoprotective agents is also considered.