Oleic acid‐induced interleukin‐36γ: A possible link between facial skin redness and sebum

Redness of the facial skin is an important cosmetic concern. Although qualitative and quantitative modifications of sebum on the skin surface are major pathogenic factors of chronic inflammatory skin conditions, the relationship between skin redness, sebum, and mild inflammation on the cheeks of healthy subjects remains elusive.


| INTRODUC TI ON
Human sebum almost coats the entire skin surface, and the secretion is particularly high on the face, scalp, chest, and back. Sebum is a complex mixture of free fatty acids (FFAs) and triacylglycerols (TAGs) (up to 57.5% of total lipids), wax esters (WEs) (26%), squalene (SQ) (12%), and cholesterol and cholesterol esters (ChEs) (4.5%). 1 The sebum TAGs secreted from sebaceous glands are hydrolyzed into FFAs, diacylglycerols (DAGs), and monoglycerides by lipases secreted from bacteria such as Cutibacterium acnes and Staphylococcus epidermidis. 2 The resulting FFAs are composed of both saturated and unsaturated FFAs with chains predominantly 16 and 18 carbons long (stearic acid, C18:0; oleic acid, C18:1, cis-9; linoleic acid, C18:2, cis-9, cis-12; palmitic acid, C16:0; sapienic acid, C16:1, cis-6, and palmitoleic acid, C16:1, cis-9). 3 The primary functions of sebum are to soften the skin, regulate the water content of the epidermis, inhibit the growth of Gram-positive bacteria, and prevent the invasion of external organisms. 4 It also contributes to transporting antioxidants including vitamin E to the skin surface, generating body odor and pheromones, and protecting from ultraviolet (UV) irradiation. 1,5 Thus, sebum on the skin surface plays an important physiological role in the maintenance of skin homeostasis. In contrast, increased sebum excretion rate and qualitative and quantitative modifications of sebum are major pathogenic factors involved in chronic inflammatory dermatologic conditions including rosacea, acne vulgaris, and seborrheic dermatitis, leading to erythema (redness) in areas of the body with sebaceous glands. 3,6,7 The redness of the cheeks plays an important role in perceptions of health and attractiveness for both genders, 8 and having a healthy appearance is universally desired. When redness is caused by oxygenated blood, the perceivers judge redder faces as healthier; thus, facial redness may reflect the cardiovascular health of individuals. 8 However, redness of the facial skin beyond a certain degree can be perceived as unhealthy and induces self-embarrassment. Therefore, facial skin redness is an important cosmetic concern. Until now, rosacea, which is a chronic inflammatory condition of the facial skin and affects the health-related quality of life in patients, has been the most commonly reported underlying condition of excessive facial redness. Importantly, a link has been proposed between the pathogenesis of rosacea and skin sebum. Specifically, rosacearelated facial redness is primarily distributed in sebum-rich areas, including the cheeks, nose, chin, and forehead. Among the affected individuals, there are changes in the relative composition of sebum, such as elevated levels of myristic acid (C14:0) and reduced levels of saturated long-chain fatty acids. 9 Additionally, isotretinoin, which exerts a strong sebosuppressive activity, was effective in reducing erythema in rosacea. 7 However, not much research has been focused on healthy subjects; therefore, no information is available on the relationship between cheek redness, skin sebum, and mild inflammation in healthy subjects.
Recently, in a parallel study, we conducted a proteomic analysis of tape-stripped stratum corneum (SC) to identify novel indicators that correlate with the degree of facial skin redness, and discovered that the ratio of the pro-inflammatory cytokine interleukin (IL)-36γ to the anti-inflammatory cytokine IL-37 in the SC is positively correlated with erythema index in healthy Japanese females (Kuwano et al., article submitted). Therefore, we proposed the IL-36γ/IL-37 ratio as a novel skin inflammatory indicator that can be measured minimally invasively. In this study, to explore a skincare strategy for mitigating unfavorable increase in skin redness, we investigated the relationship between facial skin redness, the amount and composition of skin surface sebum, and inflammation, specifically the IL-36γ/ IL-37 ratio in the SC, in healthy Japanese males and females. To investigate the relationship between representative sebum lipids and the inflammatory indicator, we also examined the effects of monounsaturated FFAs (C16:1 and C18:1) on the IL-36γ and IL-37 mRNA expression levels in cultured normal human keratinocytes. Our data provide evidence that the quantity of total lipids in sebum, particularly the proportion of monounsaturated fatty acids (C16:1 and C18:1) in sebum, is positively correlated with skin redness in healthy subjects, and oleic acid (C18:1, cis-9)-mediated IL-36γ may be a link between them. Therefore, targeting the facial skin sebum, particularly oleic acid, would be a promising strategy for skin redness care.

| Human clinical study
For this study, 198 healthy Japanese subjects (age range: 20-55 years; mean age: 38.55 ± 9.83 years; 129 females and 69 males) were recruited. Prior to analysis, each participant was acclimated for at least between them. Our study provides a possible skincare strategy for mitigating unfavorable increase in skin redness by targeting the facial skin sebum, particularly oleic acid.

K E Y W O R D S
erythema, interleukin-36γ, oleic acid, skin redness, skin sebum 10 min in a room with constant humidity (40%) and temperature (24°C).
The study was approved by the Ethical Committee of Kao Corporation and was conducted in accordance with the Declaration of Helsinki. The participants received adequate explanation of the study and provided written informed consent. Individuals were excluded if they had any skin diseases, wounds, eczema in the face, or any allergies that could influence skin redness.

| Measurements of skin redness parameters
The a* value (for the D65 illuminant) and erythema index (EI) at the cheek were obtained from the spectral reflectance measured using a spectrophotometer (CM-2600d, Konica-Minolta Inc., Tokyo, Japan), and the EI was calculated using the formula proposed by Dawson et al. 10 Facial images of each participant were acquired using a VISIA-CR skin analysis imaging system (Canfield Scientific).

| Measurements of IL-36γ, IL-37, IL-1 receptor antagonist (ra), and IL-1α in the SC
To quantify the inflammatory cytokines present in the SC, tape stripping was performed on the cheek skin of the participants by pressing and stripping D-Squame (Promotool). Three sequentially stripped tapes were obtained from a single individual. Proteins were extracted from the SC tapes by shaking for overnight at 4°C in phosphate-buffered saline containing 0.1% Triton ×-100. After sonication at 15°C for 10 min, the solution was centrifuged at 15000 × g for 15 min at 4°C, and the resulting supernatants were collected and used as the SC extracts for further experiments. The protein concentration was quantified using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). IL-36γ, IL-37, IL-1ra, and IL-1α levels were determined using Quantikine® enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Inc.).

| Sebum analysis
The face was washed and 90 min later, the sebum was collected from the cheek with cigarette paper (1.7 cm × 1. The raw data were analyzed using TraceFinder 4.1 software (Thermo Fisher Scientific). The amount of total lipids in the sebum was calculated as the sum of the amounts of each lipid constituents.

| Cell culture
Normal human epidermal keratinocytes (Kurabo) were routinely grown in EpiLife medium containing 60 μM Ca 2+ (Thermo Fisher Scientific) and human keratinocyte growth supplement (Kurabo) in a humidified atmosphere containing 5% CO 2 at 37°C. Upon reaching confluence, the cells were cultured in EpiLife medium containing 60 μM Ca 2+ without keratinocyte growth supplement, and treated with lipids dissolved in ethanol.

| Quantitative real-time PCR
Total RNA was isolated using the RNeasy Mini Kit (Qiagen) and cDNA synthesis was performed using a High Capacity RNA-to-cDNA kit

| Statistical analysis
Data were compared using unpaired Student's t test for twogroup comparisons and one-way analysis of variance, followed by Tukey's test or Dunnett's test for multiple comparisons. Correlations were examined using Pearson's correlation coefficient analysis.
Differences were considered statistically significant at p < 0.05. All statistical analyses were performed using Microsoft Excel (Office 365) (Microsoft) or IBM SPSS Statistics 25.0 (IBM).

| RE SULTS
3.1 | Total lipid quantity and percentage composition of monounsaturated FFAs in the sebum are positively correlated with redness parameters of the cheek skin of healthy Japanese subjects We first examined the correlation between the total lipid quantity in the sebum and skin redness parameters (EI and a* values) on the cheek skin of healthy Japanese subjects. As shown in Table 1 and  (Table 1). Figure 1E shows a representative image of the increased skin redness on the cheek of a participant who had a relatively high total lipid amount in the sebum. These results indicate that the total lipid quantity in the sebum is related to skin redness on the cheek of healthy subjects, and among the lipid constituents examined, FFAs (C16:1 and C18:1) may particularly contribute to the skin redness.

| IL-36γ/IL-37 ratios in the SC positively correlated with skin redness parameters of the cheek skin of healthy Japanese subjects
To examine the involvement of inflammation in cheek redness, we assessed the correlation between skin redness parameters (EI and a* values) corresponding to the cheek of the subjects and the inflammatory indicators in the SC such as the IL-36γ/IL-37 ratio (Kuwano et al., article submitted) and the IL-1ra/IL-1α ratio, which was elevated in a variety of inflammatory conditions such as atopic dermatitis (AD), psoriasis, and chronically sun-exposed skin. 11,12 As shown in Figure 2A,B, IL-1ra/IL-1α ratios significantly but modestly correlated with EI and a* values (r = 0.259, ***p < 0.001 and r = 0.202, **p = 0.006, respectively). IL-36γ/IL-37 ratios also positively correlated with EI and a* values (r = 0.336, ***p < 0.001 and r = 0.307, ***p < 0.001, respectively) ( Figure 2C,D), but the degree of correlation between IL-36γ/IL-37 ratio and skin redness parameters was higher than that between IL-1ra/IL-1α ratio and skin redness parameters.  (Table 1); hence, glyceryl trioleate was used as a negative control. As shown in Figure 3, palmitoleic acid and oleic acid, but not palmitic acid and stearic acid, significantly upregulated IL1A mRNA expression ( Figure 3A) and slightly increased IL1RN mRNA expression ( Figure 3B), resulting in a significant decrease in the IL1RN/IL1A ratio in keratinocytes ( Figure 3C). In contrast, glyceryl trioleate significantly increased the IL1RN/IL1A ratio by slightly downregulating IL1A mRNA expression ( Figure 3A,C). Oleic acid strongly induced IL36G mRNA expression and slightly increased IL37 mRNA expression, whereas palmitoleic acid slightly induced IL36G mRNA expression and significantly increased IL37 mRNA expression ( Figure 3D,E), thereby resulting in a sharp increase in the IL36G/IL37 ratio following oleic acid treatment ( Figure 3F). Palmitic acid, stearic acid, and glyceryl trioleate had negligible effects on the IL36G and IL37 mRNA expression ( Figure 3D,E) and IL36G/IL37 ratio ( Figure 3F).

| Oleic acid increases the IL36G/IL37 ratio in a dose-and time-dependent manner mainly through upregulation of IL36G mRNA expression in cultured normal human epidermal keratinocytes
We further examined the dose-and time-dependent effects of oleic acid on IL36G and IL37 mRNA expression in cultured keratinocytes. As shown in Figure 4A

| Involvement of NMDA receptors in oleic acidinduced IL36G and IL37 expression in cultured normal human epidermal keratinocytes
Previously, the NMDA-type glutamate receptor antagonist MK801 suppressed oleic acid-induced IL1A mRNA expression by inhibiting the increase in intracellular Ca 2+ concentration in cultured human keratinocytes. 13 To examine the involvement of NMDA-type glutamate receptors in the oleic acid-mediated IL36G and IL37 expression, we assessed the effects of MK801 on the mRNA expression of IL36G and IL37 in oleic acid-treated keratinocytes. As shown in Figure 5A,B, MK801 decreased the oleic acid-induced increase in IL36G and IL37 mRNA expression in keratinocytes in a dose-dependent manner. Consequently, the oleic acid-mediated enhancement in the IL36G/IL37 ratio was attenuated in a dosedependent manner by MK801 ( Figure 5C). These results indicate that oleic acid regulates IL36G/IL37 ratio by utilizing the NMDAtype glutamate receptor for both IL36G and IL37 mRNA expression in keratinocytes.

| DISCUSS ION
In this study, we demonstrated that the total lipid content and the IL-1ra is a cytokine that blocks the IL-1 receptor to antagonize IL-1α and IL-1β, and IL-1ra/IL-1α ratio in the SC has been considered a classical hallmark of various kinds of inflammation. 11 Previous studies reported that the IL-1ra/IL-1α ratio in the SC temporarily AD, and regular sun exposure. 11,12 In this study, the IL-1ra/IL-1α ratio in the SC positively correlated with skin redness parameters, suggesting that cheek skin with elevated levels of redness may be under low-grade, chronic inflammatory conditions. However, since our in vitro studies revealed that palmitoleic acid and oleic acid reduced and glyceryl trioleate increased the IL1RN/IL1A ratio, respectively, in cultured human epidermal keratinocytes, FFAs (C16:1 and C18:1) are unlikely to contribute to skin redness in an IL-1ra/IL-1α-dependent manner. One of the interesting findings of this study was that the ratio of IL-36γ to IL-37 in the SC was more strongly correlated with skin redness parameters than the IL-1ra/ IL-1α ratio. These results suggest that both IL-1ra/IL-1α and IL-36γ/ IL-37 ratios in the SC are related to cheek skin redness in healthy subjects; however, the IL-36γ/IL-37-mediated pathway may contribute more to skin redness than the IL-1ra/IL-1α-mediated pathway. Also, oleic acid significantly elevated the IL36G/IL37 ratio by strongly enhancing IL36G mRNA expression in the cultured keratinocytes, suggesting that oleic acid in sebum may play, at least in part, a role in skin redness through the IL36G/IL37-dependent pathway. Currently, IL-36γ is recognized as a key mediator in psoriatic pathology and is known to be elevated in skin lesion of acne. 17  conditions, and an imbalance between them could trigger skin redness. As previously observed with oleic acid-induced IL1A mRNA expression, 13  pro-inflammatory oleic acid increased the number of neutrophils in the wound healing area and stimulated the neutrophils to release IL-1β and VEGFα. 24 Together with the fact that palmitoleic acid did not increase the IL1RN/IL1A ratio in keratinocytes, the mechanisms underlying the relationship between FFAs (C16:1) and the cheek redness remain elusive. Further detailed analyses would be beneficial in investigating the influence of sapienic acid, the most abundant FFA (C16:1) in human sebum, 3 on the IL-36γ/IL-37 and IL-1ra/IL-1α ratios in cultured keratinocytes.
In conclusion, our study provides evidence that oleic acidinduced IL-36γ may be a link between facial skin redness and sebum, improving our understanding of the diverse functions of skin sebum and its potential to regulate skin redness and inflammatory skin conditions. These findings could lead to a new skincare strategy for mitigating the unfavorable increase in skin redness by targeting facial skin sebum and/or C16 or C18 monounsaturated FFAs, particularly oleic acid.

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors have no conflict of interest to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

E TH I C S S TATEM ENT
The study was approved by the Ethical Committee of Kao