Sulforaphene Attenuates Cutibacterium acnes-Induced Inflammation

Acne is a chronic inflammatory disease of the sebaceous gland attached to the hair follicles. Cutibacterium acnes is a major cause of inflammation caused by acne. It is well known that C. acnes secretes a lipolytic enzyme to break down lipids in sebum, and free fatty acids produced at this time accelerate the inflammatory reaction. There are several drugs used to treat acne; however, each one has various side effects. According to previous studies, sulforaphene (SFEN) has several functions associated with lipid metabolism, brain function, and antibacterial and anti-inflammatory activities. In this study, we examined the effects of SFEN on bacterial growth and inflammatory cytokine production induced by C. acnes. The results revealed that SFEN reduced the growth of C. acnes and inhibited proinflammatory cytokines in C. acnes-treated HaCaT keratinocytes through inhibiting NF-κB-related pathways. In addition, SFEN regulated the expression level of IL-1α, a representative pro-inflammatory cytokine expressed in co-cultured HaCaT keratinocytes and THP-1 monocytes induced by C. acnes. In conclusion, SFEN showed antibacterial activity against C. acnes and controlled the inflammatory response on keratinocytes and monocytes. This finding means that SFEN has potential as both a cosmetic material for acne prevention and a pharmaceutical material for acne treatment.

hyperkeratinization and inflammatory reaction. Topical treatments include antibiotics, retinoids, benzoyl peroxide, and azelaic acid [20]. These are often accompanied by side effects such as dryness, contact allergic reaction, erythema, epidermal deprivation, burning sensation, and skin irritation [12]. Systemic therapy includes antibiotics, isotretinoin, and hormone preparations, which can also cause diarrhea, nausea, dry skin, depression, vomiting, and headaches [21]. As such, existing acne treatments are limited due their various side effects [22]. Therefore, in recent years, attempts have been made to develop acne treatments that lessen these side effects. In this study, we reveal the efficacy of treating acne by SFEN using HaCaT cells, which are keratinocytes of the skin. The mechanism of action was also elucidated.

Cell Culture
HaCaT cells were cultured in DMEM with 10% FBS at 5% CO 2 and 37°C. RPMI-1640 medium is used for culture of Human THP-1 promonocytic cells. Each cell was seeded and, when it reached 80% confluence, was replaced with serum-free media for starvation. After 1 h of treatment with SFEN, heat-killed C. acnes (multiplicity of infection [MOI] = 100) was added to the medium. In co-culture experiments, 1.0 × 10 5 cells/well of HaCaT cells were seeded in the upper chamber of a 12-well Transwell (Corning Inc., USA). THP-1 cells were seeded in the lower chamber at 2.0 × 10 5 cells/well. Then, following treatment with SFEN (5, 10, and 20 mM), heat-killed C. acnes (MOI = 100) was treated 1 h later.

Bacterial Culture
Brain heart infusion broth is used for culturing C. acnes ATCC 6919 under anaerobic conditions. A GasPak system was used to produce anaerobic conditions. The pellets were treated at 65°C for 30 min before being dissolved in DMEM.

qPCR
RNAs from HaCaT cells were prepared by using RNAiso Plus (Takara Bio Inc., Japan). The concentration and purity of the RNAs were measured using a NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific, USA). A PrimeScriptTM 1st Strand cDNA Synthesis Kit (Takara Bio Inc.) was used for reverse transcription. IQ SYBR (Bio-Rad Laboratories Inc., USA) was used for RT-PCR. cDNA (2 μl) was used in triplicate with GAPDH as an internal control. cDNA was amplified using the following primers: IL-8 forward (5'-TCT TGG CAG CCT TCC  TGA TT -3  results showed that SFEN did not exhibit cytotoxicity until 20 μM concentration. All graphs represent the means ± SEM (n = 3). Asterisks indicate a significant inhibition by SFEN compared with non-treated group (**p < 0.01) using Student's t-test.

Western Blot Assay
Protein lysates (60 mg) were separated by SDS-PAGE and transferred onto a PVDF membrane (MilliporeSigma, Inc.). The membrane was blocked in skim milk over 2 h and then incubated with an indicated primary antibody over 6 h. After washing 3 times, hybridization was carried out with an HRP-conjugated secondary antibody. A chemiluminescence detection kit from MilliporeSigma, Inc. was used for protein bands.

Statistical Analysis
One-way analysis of variance (ANOVA) and post-hoc Tukey's test were used. p-values < 0.05 were considered statistically significant. IBM SPSS Statistics v.23.0 (IBM, UAS) was used for statistical analysis. The data were expressed as means ± standard error of the mean (SEM).

Effects of SFEN on C. acnes Growth
An MIC test was performed to determine the antibacterial effect of SFEN. The strains used for MIC were C. acnes, E. coli CCARM 9008, CCARM 3102, or S. aureus CCARM 3102 (Table 1). In particular, SFEN inhibited the growth of C. acnes more so than that of other bacteria. Therefore, SFEN exhibits a direct antibacterial effect against C. acnes.

Effects of SFEN on Proinflammatory Cytokines in C. acnes-Treated HaCaT Keratinocytes
To conduct the experiment at a concentration without cytotoxicity, the MTT assay was performed. As in Fig. 1B, SFEN shows decreased viability at concentrations above 40 μM. Therefore, another experiment was conducted  with an SFEN concentration of less than 20 μM, which does not affect cell death. The protein levels of IL-6 and IL-8, the major cytokines produced by C. acnes, were measured through ELISA. SFEN effectively inhibited these cytokines (Figs. 2A and 2B). As in Figs. 2C and 2D, the mRNA levels of IL-6 and IL-8 were also effectively inhibited by SFEN.

Effects of SFEN on NF-κB Signaling Pathway Inhibition in HaCaT Keratinocytes
The transcription factor that plays a crucial role in the expression of IL-6 and IL-8 is NF-κB in HaCaT keratinocytes [23,24]. We used luciferase reporter gene assays to investigate the activation of NF-κB transcription factors. In HaCaT cells transduced with NF-κB reporter plasmid, treatment with C. acnes increases the activity of luciferase, and treatment with SFEN decreases it (Fig. 3A). In addition, SFEN inhibited the phosphorylation of IKKα/β and IκBα (Fig. 3B), which are upstream regulators of NF-κB.

Effects of SFEN on C. acnes-Induced IL-1β in Cocultured HaCaT Keratinocytes and THP-1 Monocytes
An experimental method of coculturing two cells was used to simulate a phenomenon occurring in the human body. In the human body, keratinocytes cause skin inflammation through interaction with Langerhans cells. The THP-1 cell line was originally known as a monocytic leukemia cell, but has characteristics of dendritic cells [25]. To show the protein level of IL-1β in the coculture model (Fig. 4A), the cells were divided into three conditioned groups: HaCaT only, THP-1 only, and HaCaT and THP-1. Heat-killed C. acnes was added to each group. IL-1β was increased significantly when HaCaT and THP-1 cells were cocultured (Fig. 4B). In our coculture model, SFEN inhibited IL-1β induced by heat-killed C. acnes in a concentration-dependent manner (Fig. 4C).

Discussion
The sebaceous glands are abundant on the face, back, and chest areas where acne is common. These glands are connected to hair follicles and produce an oily substance called sebum. [14]. Under normal conditions, sebum rises along the hair follicle wall and is discharged through the skin, but when the hair follicle is blocked, sebum cannot be discharged and gets trapped around the hair follicle, and bacteria that cause inflammation grow, which  leads to acne [11]. Among the bacteria resident in hair follicles, C. acnes, in particular, secretes lipolytic enzymes to form free fatty acids and stimulate hair follicles. It is also known that an immune response to these bacteria contributes to acne inflammation [15]. Therefore, if C. acnes can be prevented from growing on the skin and the inflammation caused by it can be alleviated, the damage to the skin caused by acne can be reduced [14]. In this study, we found that SFEN exerts antibacterial activity against C. acnes and relieves related inflammation.
C. acnes promotes this inflammation through Toll-like receptor (TLR) activation. Among TLRs, TLR2 plays the most important role in inflammation caused by C. acnes. Activation of TLR2 by C. acnes induces the MAPK and NF-κB pathways. Activated NF-κB transcription factors promote the expression of proinflammatory cytokines [26,27]. Activation of TLR2 by C. acnes induces proinflammatory cytokines such as IL-8 and IL-6 in keratinocytes [28]. These cytokines play roles in regulating inflammatory responses in keratinocytes and monocytes [29,30]. In this study, SFEN inhibited the transcriptional activity of NF-κB by suppressing IkBα and IKKα/β phosphorylation, which regulates NF-κB activation.
There are many reports that C. acnes contributes to the production of cytokines that are pivotal in inflammatory acne via a TLR2 pathway [31]. C. acnes-induced cytokine production is associated with TLR2 activation. C. acnes induces the production of IL-1β, IL-8, TNF-α and IFN-γ in keratinocytes [28]. It has been shown that peritoneal macrophages from knockout of TLR6 and TLR1 in mice produce IL-6 in response to C. acnes infection, but not TLR2 knockout mice [32]. Therefore, C. acnes activates TLR2 and TLR2 activates NF-κB. Activated NF-κB induces various inflammatory responses. SFEN can block NF-κB activation, thereby preventing inflammation caused by C. acnes.
A coculture model of HaCaT and THP-1 cells was used to evaluate the effects of the material more similar to an in vivo situation. The cytokines produced by C. acnes-induced keratinocytes act on immune cells in the skin. These cytokines induced other proinflammatory cytokines, such as IL-1β [33]. IL-1β secretion was strongly increased when C. acnes was treated in a coculture model than when HaCaT cells and THP-1 cells were treated with C. acnes, respectively. When SFEN was treated in a coculture model, IL-1β secretion induced by C. acnes was inhibited. This suggests that SFEN could inhibit inflammation occurring at acne sites.
In conclusion, we identified effects of SFEN on the growth of C. acnes and C. acnes-induced inflammation. Our findings demonstrate that SFEN has potential both as a cosmetic material for acne prevention, and as a pharmaceutical material for acne treatment.