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

Abstract

Background

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.

Aims

We aimed to explore the correlation between cheek redness and sebum and inflammatory cytokines in the stratum corneum (SC) of healthy subjects. We also examined the effects of representative sebum lipids on the gene expression of inflammatory cytokines in cultured keratinocytes.

Patients/Methods

This study included 198 healthy participants. Skin sebum was analyzed using flow injection analysis, and skin redness was assessed using a spectrophotometer. Inflammatory cytokines in tape‐stripped SC were measured using enzyme‐linked immunosorbent assay.

Results

Cheek redness parameters positively correlated with the amount of skin sebum and the proportion of monounsaturated free fatty acids (C16:1 and C18:1) in the sebum. They also positively correlated with the interleukin (IL)‐36γ/IL‐37 ratio in the SC. Among the representative sebum lipids examined, oleic acid (C18:1, cis‐9) dose‐ and time‐dependently regulated the mRNA expression of IL‐36γ and IL‐37 in cultured keratinocytes, and this effect was attenuated by the N‐methyl‐D‐aspartate (NMDA)‐type glutamate receptor antagonist, MK801.

Conclusions

Skin surface sebum may be related to cheek redness in healthy subjects, and oleic acid‐induced IL‐36γ through NMDA‐type glutamate receptors may be a link 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.

Abbreviations

• AD
• atopic dermatitis
• CCL
• chemokine (C-C motif) ligand
• ChE
• cholesterol ester
• CXCL
• chemokine (C-X-C motif) ligand
• DAG
• diacylglycerol
• EI
• erythema index
• ELISA
• enzyme-linked immunosorbent assay
• FFA
• free fatty acid
• FIA-MS
• flow injection analysis coupled to mass spectrometry
• FTMS
• Fourier transform mass spectrometry
• HPLC
• high-performance liquid chromatography
• IL
• interleukin
• IL-1ra
• IL-1 receptor antagonist
• NMDA
• N-methyl-D-aspartate
• RPLP0
• ribosomal protein lateral stalk subunit P0
• SC
• stratum corneum
• SQ
• squalene
• SQOOH
• SQ monohydroperoxide
• TAG
• triacylglycerol
• UV
• ultraviolet
• VEGF
• vascular endothelial growth factor
• WE
• wax ester

INTRODUCTION

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%). 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. 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). 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. It also contributes to transporting antioxidants including vitamin E to the skin surface, generating body odor and pheromones, and protecting from ultraviolet (UV) irradiation. 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, , and seborrheic dermatitis, leading to erythema (redness) in areas of the body with sebaceous glands.

The redness of the cheeks plays an important role in perceptions of health and attractiveness for both genders, 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. 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, rosacea-related 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. Additionally, isotretinoin, which exerts a strong sebosuppressive activity, was effective in reducing erythema in rosacea. 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.

MATERIALS AND METHODS

Reagents

Oleic acid, palmitic acid, palmitoleic acid, and glyceryl trioleate were obtained from Sigma-Aldrich (MO, USA). Stearic acid was purchased from Tokyo Kasei Co. (Tokyo, Japan). The N-methyl-D-aspartate (NMDA)-type glutamate receptor antagonist, MK801 (dizocilpine; (5S, 10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine), was obtained from Fujifilm Wako Pure Chemicals.

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 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. 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 × 1.7 cm, RIZLA: RIZLA BLUE DOUBLE). The paper was immersed in ethanol (high-performance liquid chromatography [HPLC] grade > 99.8%, Kanto Chemical Co., Inc.) in a screw tube, and the solvent was evaporated under nitrogen flow. The sebum was then extracted with a 1 mL mixture of chloroform (HPLC grade > 99.7%, Kanto Chemical Co., Inc.) and methanol (1:1, vol/vol) by sonication for 5 min. An internal standard solution containing 1 μM tritridecanoin (TAG, C39:0, Larodan Fine Chemicals AB), 10 μM ditridecanoin (DAG, C26:0, Larodan Fine Chemicals AB), 100 μM lauric acid (methyl-d3) (FFA, C12:0-d3, Cambridge Isotope Laboratories), 10 μM lauryl palmitoleate (WE, C28:1, Santa Cruz Biotechnology, Inc.), 10 μM cholesteryl acetate (ChE, C2:0, Olbracht Serdary Research Laboratories), and 5 μM cholesteryl caprate (ChE, C10:0, Nu-Chek Prep, Inc.) was prepared in chloroform. An aliquot (10 μL) of the internal standard solution was dried by evaporation, and the residue was dissolved in 50 μL of the sebum solution. Mass spectrometric analysis of sebum was performed using flow injection analysis coupled with mass spectrometry (FIA-MS) using a Vanquish UHPLC system (Thermo Fisher Scientific) coupled to a Q-Exactive Focus (Thermo Fisher Scientific) equipped with a heated electrospray source. For the mobile phase, methanol/chloroform (1:1, vol/vol) containing 15 mM ammonium acetate (Guaranteed Reagent, Fujifilm Wako Pure Chemicals) was used at a flow rate of 0.1 mL/min. The optimized experimental design parameters were as follows: voltages in the positive and negative ion modes 3.0 and 2.5 kV, respectively; heated capillary temperature, 275 C; sheath gas pressure, 60 psi; auxiliary gas, 20 psi; and heated vaporizer temperature, 350°C. The Fourier transform mass spectrometry (FTMS) scan type used was full MS, and the parameters were as follows: resolution, 70 000; autogain control target, under 3 × 10; and m/z range, 180–1200. The system was controlled using Xcalibur 4.1.31.9 (Thermo Fisher Scientific). 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 Ca²⁺ (Thermo Fisher Scientific) and human keratinocyte growth supplement (Kurabo) in a humidified atmosphere containing 5% CO₂ at 37°C. Upon reaching confluence, the cells were cultured in EpiLife medium containing 60 μM Ca²⁺ 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 (Applied Biosystems). Quantitative real-time PCR was performed using TaqMan Fast Universal PCR Master Mix (Thermo Fisher Scientific). TaqMan probes were purchased from Applied Biosystems, and the relative quantification values of IL36G (IL-36γ) (Hs00219742_m1), IL37 (IL-37) (Hs00367201_m1), IL1A (IL-1α) (Hs00174092_m1), and IL1RN (IL-1ra) (Hs00893626_m1) were normalized against RPLP0 (ribosomal protein lateral stalk subunit P0) (Hs99999902_m1) using QuantStudio™ 12 K Flex Systems (Applied Biosystems).

Statistical analysis

Data were compared using unpaired Student's t test for two-group 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).

RESULTS

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  and Figure , the quantity of total lipids in the sebum positively correlated with EI (r = 0.430, ***p < 0.001) (Figure ) and a* values (r = 0.372, ***p < 0.001) (Figure ). We further assessed the correlation of each lipid constituent in the sebum with skin redness, and found that the percentage composition of FFA (C16:1), FFA (C18:1), WE, and SQ in the sebum showed positive correlations with the EI (r = 0.403, ***p < 0.001; r = 0.324, ***p < 0.001; r = 0.253, ***p < 0.001; and r = 0.229, ***p < 0.001, respectively) and a* values (r = 0.364, ***p < 0.001; r = 0.337, ***p < 0.001; r = 0.193, **p = 0.006; and r = 0.181, *p = 0.011, respectively) (Table  and Figure ). Notably, FFAs levels (C16:1 and C18:1) were more strongly correlated than were WE and SQ. In contrast, the proportions of other lipid constituents such as FFA (C16:0), FFA (C18:0), TAG, DAG, ChE, SQ epoxide, and SQ monohydroperoxide (SQOOH) in the sebum did not positively correlate with the EI and a* values (Table ). Figure  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.

TABLE 1 Correlation of skin redness parameters with total lipid and lipid composition in sebum from healthy cheek skin.

	EI (A.U.)	a* (A.U.)
r	p	r	p
Total lipid (μg/cm2)	0.430	< 0.001***	0.372	< 0.001***
FFA (C16:0) (% of total lipid)	0.049	0.490	0.073	0.305
FFA (C16:1) (% of total lipid)	0.403	< 0.001***	0.364	< 0.001***
FFA (C18:0) (% of total lipid)	−0.216	0.022*	−0.189	0.078
FFA (C18:1) (% of total lipid)	0.324	< 0.001***	0.337	< 0.001***
TAG (% of total lipid)	−0.240	< 0.001***	−0.248	< 0.001***
DAG (% of total lipid)	−0.150	0.036*	−0.082	0.263
WE (% of total lipid)	0.253	< 0.001***	0.193	0.006**
ChE (% of total lipid)	0.172	0.016*	0.094	0.190
SQ (% of total lipid)	0.229	< 0.001**	0.181	0.011*
SQ epoxide (% of total lipid)	−0.009	0.897	−0.047	0.509
SQOOH (% of total lipid)	−0.271	< 0.001***	−0.196	0.006**

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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. As shown in Figure , 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 ), 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.

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Oleic acid specifically increases the IL36G/IL37 ratio, but not the IL1RN/IL1A ratio, in cultured normal human epidermal keratinocytes

We examined the effects of palmitic acid (C16:0), palmitoleic acid (C16:1, cis-9), stearic acid (C18:0), and oleic acid (C18:1, cis-9) on IL1RN/IL1A and IL36G/IL37 ratios in cultured normal human epidermal keratinocytes. Although sapienic acid (C16:1, cis-6) has the highest amount of FFA among the FFAs (C16:1) in skin sebum, we used palmitoleic acid (C16:1, cis-9) instead as a representative FFA (C16:1) in our in vitro studies, because palmitoleic acid is another abundant FFA (C16:1) in sebum and easily available commercially. TAG in sebum was negatively correlated with skin redness parameters (Table ); hence, glyceryl trioleate was used as a negative control. As shown in Figure , palmitoleic acid and oleic acid, but not palmitic acid and stearic acid, significantly upregulated IL1A mRNA expression (Figure ) and slightly increased IL1RN mRNA expression (Figure ), resulting in a significant decrease in the IL1RN/IL1A ratio in keratinocytes (Figure ). In contrast, glyceryl trioleate significantly increased the IL1RN/IL1A ratio by slightly downregulating IL1A mRNA expression (Figure ). 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 ), thereby resulting in a sharp increase in the IL36G/IL37 ratio following oleic acid treatment (Figure ). Palmitic acid, stearic acid, and glyceryl trioleate had negligible effects on the IL36G and IL37 mRNA expression (Figure ) and IL36G/IL37 ratio (Figure ).

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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 , oleic acid dose-dependently enhanced IL36G and IL37 mRNA expression, showing the highest upregulation at a concentration of 50 μM, which is a 56- and 2.3-fold increase, respectively, compared with that under control condition. Since oleic acid elicited a stronger mRNA expression for IL36G than for IL37, the IL36G/IL37 ratio was 26- and 25-fold upregulated at concentrations of 25 μM and 50 μM, respectively. Time–course experiments showed that oleic acid (50 μM)-induced upregulation of IL36G mRNA expression began at 6 h and reached the maximum level at 24 h after stimulation (Figure ). Similar results were obtained for IL-36γ protein content in the conditioned media of oleic acid-treated keratinocytes (Figure ). In contrast, the upregulation of IL37 mRNA expression by oleic acid began at 24 h (Figure ), which resulted in a rapid and transient increase in the IL36G/IL37 ratio 6 h after of treatment (Figure ). Unexpectedly, IL-37 protein levels were negligible in the conditioned media of oleic acid-treated keratinocytes (data not shown). These results suggest that IL36G and IL37 mRNA expression are regulated in the keratinocytes by the same concentration range of oleic acid, but the time course of their expression patterns was different—oleic acid treatment led to the early increase in IL36G mRNA expression, followed by upregulation of IL37 mRNA.

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Involvement of NMDA receptors in oleic acid-induced 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 Ca²⁺concentration in cultured human keratinocytes. 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 , 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 dose-dependent manner by MK801 (Figure ). These results indicate that oleic acid regulates IL36G/IL37 ratio by utilizing the NMDA-type glutamate receptor for both IL36G and IL37 mRNA expression in keratinocytes.

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DISCUSSION

In this study, we demonstrated that the total lipid content and the proportion of FFAs (C16:1 and C18:1) in sebum were positively correlated with cheek redness in healthy Japanese subjects. IL-36γ/IL-37 ratio in the SC also positively correlated with cheek redness. We further provided evidence that oleic acid induces IL-36γ expression in keratinocytes in an NMDA-type glutamate receptor-dependent manner, which may in turn contribute to increased skin redness.

Sebum, being located on the outermost surface of the human body, is a target of various oxidative stressors including UV. SQOOH, the primary photooxidative product of SQ, induces inflammatory cytokines, and activates signaling pathways related to the development of acne vulgaris and inflammatory processes of UV-associated skin pathologies. However, in this study, the proportion of SQOOH in sebum did not positively correlate with the skin redness parameters, probably because we collected newly secreted sebum only 1.5 h after washing the face, and thus sufficient peroxidation of SQ was unlikely to occur. Instead, we clearly demonstrated that the proportions of FFAs (C16:1 and C18:1), SQ, and WE in sebum positively correlated with skin redness parameters. Conversely, the proportion of TAGs in sebum was negatively correlated with skin redness parameters. Since FFAs are generated from TAGs by the action of lipases secreted from bacteria, bacterial flora of the skin may play a role in skin redness in healthy subjects by hydrolyzing TAGs into FFAs, although further studies are required to confirm this hypothesis. In addition, the mechanism underlying the effect of SQ and WE on cheek redness in healthy subjects is still unclear. Further experimental studies are necessary to address this issue to obtain a better understanding of the relationship between skin redness and sebum.

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. Previous studies reported that the IL-1ra/IL-1α ratio in the SC temporarily decreased after a single application of surfactant or UVB radiation, whereas an elevated IL-1ra/IL-1α ratio was observed in the SC under chronic inflammatory conditions, including psoriasis, AD, and regular sun exposure. 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. In psoriatic skin, IL-36γ regulates keratinocyte-mediated inflammatory responses and contributes to the recruitment of neutrophils to the skin via the release of chemokines such as chemokine (C-X-C motif) ligand (CXCL) 8, chemokine (C-C motif) ligand (CCL) 20, and CXCL1. IL-36γ also induces angiogenesis and the branching of endothelial cells in a vascular endothelial growth factor (VEGF)-A-dependent manner. Importantly, infiltration of neutrophils contributes to the development of rosacea and mediates inflammation by releasing proteases, which compromise the integrity of capillary walls and reactive oxygen species, which directly induce VEGF and cause oxidative tissue damages. Therefore, although further studies are required, a plausible speculation is that IL-36γ-induced inflammation and angiogenesis mediated by oleic acid may contribute, at least in part, to facial skin redness in healthy subjects.

This study provides evidence that after the induction of IL36G in cultured keratinocytes, oleic acid induces the mRNA expression of IL-37, an anti-inflammatory cytokine belonging to the IL-1 family. A previous study reported that after treatment of AD skin condition with narrow-band UVB, IL-36γ was downregulated, whereas IL-37 was upregulated in the epidermis along with an improvement of AD conditions. Therefore, tight regulation of IL-36γ and IL-37 by oleic acid in sebum may be involved in maintaining healthy skin conditions, and an imbalance between them could trigger skin redness. As previously observed with oleic acid-induced IL1A mRNA expression, oleic acid-induced IL36G and IL37 mRNA expression was suppressed by MK801, suggesting that oleic acid-induced IL1A, IL36G, and IL37, all of which belong to the IL-1 family, share the NMDA-type glutamate receptor in keratinocytes. To date, several intracellular signaling pathways downstream of NMDA-type glutamate receptors have been identified, such as the calcium/calmodulin-dependent protein kinase II-dependent and calcineurin/cofilin-dependent pathways. Therefore, a likely speculation is that distinct pathways are utilized for the induction of the expression of each gene by oleic acid, as evidenced by their varied responses to oleic acid. Notably, the effects of palmitoleic acid and oleic acid on IL36G and IL37 mRNA expression in keratinocytes differed distinctly; palmitoleic acid predominantly increased IL37 expression, whereas oleic acid preferentially induced IL36G. This notion is supported by previous studies that demonstrated their different roles in the wound healing process of rat skin: the anti-inflammatory effect of palmitoleic acid accelerated wound healing and inhibited lipopolysaccharide-stimulated neutrophil migration by suppressing the release of cytokines such as IL-1β, whereas pro-inflammatory oleic acid increased the number of neutrophils in the wound healing area and stimulated the neutrophils to release IL-1β and VEGF-α. 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, on the IL-36γ/IL-37 and IL-1ra/IL-1α ratios in cultured keratinocytes.

In conclusion, our study provides evidence that oleic acid-induced 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.

AUTHOR CONTRIBUTIONS

A.K., T.K., E.W., T.I., Y.K., H.Y., and J.I. designed research; A.K., E.W., T.I., and K.K. performed research; A.K., T.K., E.W., T.I., Y.K., K.K., S.N., T.M., H.Y., and J.I. analyzed data; A.K., T.I., S.N., and H.Y. wrote the article; and T.K., Y.K., S.N., T.M., H.Y., and J.I. reviewed and revised the article. The authors approved the final article.

CONFLICT OF INTEREST STATEMENT

The authors have no conflict of interest to declare.

DATA AVAILABILITY STATEMENT

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.

ETHICS STATEMENT

The study was approved by the Ethical Committee of Kao Corporation (approval number: D123-200512) and was conducted under the Declaration of Helsinki.

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