Topical N-palmitoyl serinol, a commensal bacterial metabolite, prevents the development of epidermal permeability barrier dysfunction in a murine model of atopic dermatitis-like skin

Abstract

Recent studies have demonstrated that commensal bacterial metabolites benefit human health. Because of the crucial role of the epidermal permeability barrier in cutaneous and extracutaneous function, we assessed whether the topical applications of N-palmitoyl serinol (NPS) would improve the epidermal permeability barrier in murine skin. Our results show that the topical application of 0.5% NPS in ethanol twice daily for 1 week lowered basal transepidermal water loss rates and accelerated barrier recovery in normal mice. Moreover, topical NPS prevented the emergence of epidermal permeability barrier dysfunction in a murine model of allergic contact dermatitis. These results suggest that topical NPS could be used to prevent or treat skin disorders characterized by inflammation and an abnormal epidermal permeability barrier.

Introduction

Atopic dermatitis is a common inflammatory dermatosis. Its prevalence in canines and felines is as high as 15% and 12%, respectively (1,2). Treatment of eczematous dermatitis has been a big challenge for veterinarians, due in part to the uncertainty of its pathogenesis. However, a line of evidence suggests that epidermal permeability barrier dysfunction plays a pathogenic role in the development of dermatitis.

When the epidermal permeability barrier is compromised, it provokes cutaneous inflammation and increases cutaneous inflammatory responses to external stimuli (3), but when it is improved, it prevents and/or alleviates contact dermatitis (4). Identifying ingredients that benefit the epidermal permeability barrier can lead to the development of products for the management of inflammatory skin disorders.

Evidence indicates that commensal bacterial metabolites benefit human health (5). Previous studies show that commensal bacterial metabolites can activate G protein-coupled receptors, which can promote the formation of tight junction barriers in the epidermis (6,7). Because tight junction barriers can affect the epidermal permeability barrier (8), we assessed whether the topical applications of N-palmitoyl serinol (NPS), a commensal bacterial metabolite, would benefit the epidermal permeability barrier in mice.

Materials and methods

Six- to 8-week-old female C57BL/6J mice were purchased from the Guangdong Animal Center (Guangzhou, Guangdong, China). They were fed a standard diet and had water ad libitum. N-palmitoyl serinol (Neuromide) (Dr. Raymond Laboratories, Englewood Cliffs, New Jersey, USA), which is registered with the United States Food and Drug Administration as a Type IV Drug Master File, was gifted by Dr. Byeong Deog Park. 1-fluoro-2,4-dinitrobenzene (DNFB) was purchased from Aladdin (Shanghai, China).

Experimental protocols

All animal procedures were approved by the Animal Study Subcommittee of the Southern Medical University and performed in accordance with their guidelines.

The backs of mice were shaved with an electric shaver 24 h before the experiment. In normal skin, both flanks of C57BL/6J mice were treated topically with either ethanol (control group) or 0.2% or 0.5% NPS in ethanol, twice daily for 1 wk. Eighteen hours after the last application, epidermal biophysical properties, including transepidermal water loss (TEWL), stratum corneum hydration, and skin surface pH, were measured by their respective probes connected to an MPA580 monitor (Courage + Khazaka Electronic, Köln, Germany). Transepidermal water loss rates and stratum corneum hydration were measured with TM300 and CM825, respectively. For barrier recovery, barrier disruption was achieved by repeated applications of cellophane tape until TEWL increased threefold. TEWL was measured immediately and 3.5 h after tape application (9).

Atopic dermatitis-like skin was achieved by applying 50 μL of 0.08% DNFB in acetone to both flanks of 6- to 8-week-old C57BL/6J mice twice daily for 4 wk, following sensitization with 25 μL of 0.5% DNFB (10). Thirty min after each DNFB application, 100 μL of either 0.5% NPS in ethanol or ethanol alone was applied to both flanks of the DNFB-treated area. Eighteen hours after the last NPS application, epidermal biophysical properties, including TEWL, stratum corneum hydration, and skin surface pH, were measured with their respective probes connected to an MPA580 monitor, followed by 6 mm full skin biopsies for histology.

Statistics

Data are expressed as the mean + standard error of the mean (SEM). GraphPad Prism 5 Software (San Diego, California, USA) was used for all statistical analyses. One-way analysis of variance (ANOVA) with Newman-Keuls multiple comparison was used to determine significances when 3 or more groups were compared. Unpaired 2-tailed Student’s t-test with Mann-Whitney U-test correction was used to determine the statistical significances between 2 groups.

Results

We first assessed epidermal permeability barrier function in normal C57BL/6J mice following topical application of either 0.2% or 0.5% NPS in ethanol twice daily for 1 wk. As seen in Figure 1, topical application of 0.5% NPS significantly lowered TEWL rates, while accelerating permeability barrier recovery 3.5 h after barrier disruption. Barrier recovery was also accelerated following treatment with 0.2% NPS [20.8% ± 2.89% (vehicle) versus 42.36% ± 0.27% (NPS), P < 0.01], although basal TEWL remained unchanged. These results demonstrate that topical commensal bacterial metabolites such as NPS improve the epidermal permeability barrier in mice.

Figure 1.

Topical application of N-palmitoyl serinol (NPS) improves the epidermal permeability barrier in mice. Data are expressed as mean ± SEM.

Since NPS improves the epidermal permeability barrier in normal mouse skin, we determined next whether topical NPS could prevent the emergence of epidermal permeability barrier dysfunction in a mouse model of atopic dermatitis-like skin, which displays elevated TEWL rates and reduced stratum corneum hydration (10).

As shown in Figure 2A, mice which received DNFB alone had significantly increased TEWL rates compared to those which did not (i.e., normal controls) (P < 0.001). In mice which received DNFB, those treated with topical applications of 0.5% NPS displayed markedly lower TEWL rates (P < 0.05). A marked increase in the stratum corneum hydration level was also observed in DNFB mice following treatment with 0.5% NPS (P < 0.001) (Figure 2B). However, levels of skin surface pH were comparable among the 3 groups (F = 1.369). In parallel with the improvements in the epidermal permeability barrier and stratum corneum hydration, NPS also partially prevented epidermal hyperproliferation and inflammatory infiltration in the atopic dermatitis-like model (Figure 3). Taken together, these results indicate that NPS prevents the emergence of dysfunctions in the epidermal permeability barrier and in the level of stratum corneum hydration in a murine model of atopic dermatitis-like skin.

Figure 2.

Topical applications of N-palmitoyl serinol (NPS) prevent the emergence of epidermal permeability barrier dysfunction and the reduction in stratum corneum hydration in a murine model of atopic dermatitislike skin. Data are expressed as mean ± SEM.

Figure 3.

Topical N-palmitoyl serinol (NPS) attenuates epidermal hyperproliferation and inflammatory infiltration in a murine model of atopic dermatitislike skin. Skin biopsies were taken following 4 wk of daily applications of 1-fluoro-2,4-dinitrobenzene (DNFB) with or without NPS. A — Normal mouse skin. B — Mouse skin treated with DNFB + ethanol. C — Mouse skin treated with DNFB + 0.5% NPS.

Discussion

Recently, evidence showed that commensal bacterial metabolites have health benefits. We show here that topical applications of a commensal bacterial metabolite, NPS, improve the epidermal permeability barrier in both normal and inflamed skin. Although the exact mechanisms by which NPS benefits the epidermal permeability barrier are unclear, evidence points to at least 3 potential mechanisms. First, NPS can activate G protein-coupled receptors (6), leading to the accelerated formation of tight junctions (7); the latter are determinants of the epidermal permeability barrier (11). Second, NPS can increase the production of ceramide (12), which is a key component of the stratum corneum lipids required to form the membrane bilayers, a structure of the epidermal permeability barrier (13). Third, NPS can activate cannabinoid receptors, resulting in the inhibition of cutaneous inflammation (14); the TEWL rate correlates positively with the severity of cutaneous inflammation (15). It is worth noting that improvement of the epidermal permeability barrier can alleviate cutaneous inflammation. Thus, NPS benefits epidermal function through multiple mechanisms.

In summary, topical applications of NPS, a metabolite of commensal bacteria, improve epidermal permeability barrier function in both normal and inflamed skin. Because defective epidermal permeability barrier function can cause both cutaneous and systemic inflammation, topical SNP could potentially be used to mitigate and/or prevent the development of eczematous dermatitis in felines and canines. However, whether topical SNP benefits the epidermal permeability barrier and eczematous dermatitis in a clinical setting remains to be determined.