The regulation of cutaneous inflammatory processes is essential for the human skin to maintain homeostasis in the presence of the dense communities of resident microbes that normally populate this organ. Forming the hair follicle–associated sebaceous gland, sebocytes are specialized lipid-producing cells that can release inflammatory mediators. Cytokine and chemokine expression by pilosebaceous epithelial cells (i.e., sebocytes and follicular keratinocytes) has been proposed to contribute to the common human skin disease acne vulgaris. The underlying mechanisms that drive inflammatory gene expression in acne-involved pilosebaceous epithelial cells are still unknown because almost all sebaceous follicles contain dense concentrations of bacteria yet only some show an inflammatory reaction. In this study, we hypothesized that metabolites from the abundant skin-resident microbe Propionibacterium acnes can influence cytokine expression from human sebocytes. We show that short-chain fatty acids produced by P. acnes under environmental conditions that favor fermentation will drive inflammatory gene expression from sebocytes. These molecules are shown to influence sebocyte behavior through two distinct mechanisms: the inhibition of histone deacetylase (HDAC) activity and the activation of fatty acid receptors. Depletion of HDAC8 and HDAC9 in human sebocytes resulted in an enhanced cytokine response to TLR-2 activation that resembled the transcriptional profile of an acne lesion. These data provide a new insight into the regulation of inflammatory gene expression in the skin, further characterize the contribution of sebocytes to epidermal immunity, and demonstrate how changes in the metabolic state of the skin microbiome can promote inflammatory acne.

Acne vulgaris is an inflammatory skin disorder involving hair follicles and associated sebaceous glands. Ranking third in global disease burden of chronic skin conditions, acne is a significant health care problem and a leading source of medical expense (1). It is widely thought that the skin-resident bacterium Cutibacterium acnes [formerly known as Propionibacterium acnes (2) and referred to by that name throughout this article] is a driver of skin inflammation in this disease (3). TLR2 ligands present on P. acnes have been shown to initiate inflammatory gene expression from cells such as keratinocytes and monocytes (47). However, P. acnes is an abundant member of the microbiome in healthy skin in which inflammation is not activated by the presence of these TLR ligands (8, 9). Furthermore, despite the existence of this microbe in the majority of sebaceous follicles, only a select few at any time demonstrate the inflammation characteristic of acne. Thus, critical underlying events that regulate the inflammatory response to P. acnes are not completely understood.

Taking these observations into account, we hypothesized that a change in the local microenvironment of individual sebaceous follicles may lead to a change in the metabolic activity of P. acnes and that this local shift in metabolites may be then responsible for promoting the inflammatory responses observed in acne. Supporting this hypothesis, our previous work demonstrated that short-chain fatty acids (SCFAs) produced by P. acnes in hypoxic, lipid-rich conditions (conditions that mimic an occluded, sebaceous follicle) have a robust proinflammatory effect on epidermal keratinocytes (10). Keratinocytes treated with SCFAs showed an enhanced cytokine response to TLR activation, a distinct and striking phenotype of the keratinocyte inflammatory response that is opposite to the well-documented anti-inflammatory effects of SCFAs on cells of myeloid origin (1113). This proinflammatory response by keratinocytes could be linked to the inhibition of histone deacetylase (HDAC) activity by SCFAs, and the target of SCFAs was associated with unique HDACs (HDAC8 and HDAC9) because specific depletion of HDAC8 or HDAC9 led to an enhanced response to TLR activation in a manner similar to SCFA treatment. These observations suggested that these HDAC enzymes in particular are responsible for repressing excessive levels of proinflammatory gene expression in keratinocytes and that when SCFAs that inhibit HDAC activity are produced by P. acnes, then the epithelial surface breaks tolerance to these commensal microbes.

In addition to keratinocytes, sebocytes that make up the sebaceous glands associated with hair follicles have also been identified as a major contributor of proinflammatory cytokine and antimicrobial peptide expression in response to P. acnes (1417). In fact, recent studies have identified immunomodulatory roles of human sebocytes through their production of cytokines, chemokines, and lipid mediators (1820). Historically viewed primarily as lipid producers, sebocytes are now recognized as another immunocompetent cell type that may participate in, and have regulatory effects on, inflammatory and immune processes in the skin (2123). However, whether HDAC activity is involved in regulating the expression of inflammatory genes in sebocytes, and whether P. acnes may modulate this regulation through the production of SCFAs, is unknown.

In this study, we set out to explore the effects of P. acnes metabolites and HDAC inhibition on the inflammatory response of sebocytes. Through this work, we demonstrate that human sebocytes, like keratinocytes, exhibit an increased response to TLR activation and P. acnes stimulation when HDAC activity is depleted. Furthermore, the same HDAC enzymes that were demonstrated to be important in keratinocytes, HDAC8 and HDAC9, play a role in regulating proinflammatory gene expression in sebocytes because depletion of either of these proteins largely replicated the effects of SCFA treatment. Finally, we show that unlike keratinocytes, a portion of the proinflammatory effects of SCFAs in sebocytes is due to the activation of fatty acid receptors. Together, these observations show a role for lipids, hypoxia, and P. acnes in promoting the development of acne by both epigenetic and canonical inflammatory mechanisms and define the sebocyte as a unique cell type that may participate in the inflammatory response of the skin.

See Table I.

The sebocyte cell lines SEB-1 (24) and SZ95 (25) were maintained in Sebomed Basal Medium supplemented with 10% FBS, 1× antibiotic-antimycotic, and recombinant human epidermal growth factor (rhEGF; 5 ng/ml) in incubators maintained at 37°C in 5% CO2. Cells were passaged before reaching 100% confluence in tissue culture flasks. For experiments, cells were switched to medium containing 1% FBS overnight before stimulation.

BD OptEIA ELISA Kits were used to determine cytokine concentrations in cell supernatants (BD Biosciences). Kits were used according to the manufacturer’s instructions, and results were determined using a 96-well plate reader (Beckman Coulter Diagnostics).

RNA from cultured sebocytes was isolated using the PureLink RNA Mini Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. RNA concentration was determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific), and up to 1 μg of RNA was converted into cDNA using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories). Five nanograms of cDNA per sample was used in each subsequent quantitative RT-PCR (RT-qPCR), which was performed in 20 μl reactions using TaqMan Universal PCR Master Mix (Thermo Fisher Scientific) and TaqMan Gene Expression Assays (Thermo Fisher Scientific, assay numbers described in Supplemental Table I). All RT-qPCR were performed on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories).

Purified isolated RNA was submitted to the University of California, San Diego (UCSD) Institute for Genomic Medicine core facility for quality analysis, library preparation, and high-throughput sequencing. RNA quality was assessed using the Agilent TapeStation instrument (Agilent). Libraries were constructed using TruSeq Stranded mRNA Library Prep Kits (Illumina) and analyzed on a HiSeq 4000 instrument (Illumina). Raw data were obtained from the UCSD Institute for Genomic Medicine core and analyzed using Partek Flow software (Partek) to determine transcript abundance and identify differentially expressed genes. Data are deposited in the National Center for Biotechnology Information Sequence Read Archive (https://trace.ncbi.nlm.nih.gov/Traces/sra/?study=SRP151073) with the accession number SRP151073. Gene ontology analysis was performed using the Metascape Web site (www.metascape.org) (26).

Total protein was isolated from cultured sebocytes by cell lysis on ice in 1× RIPA buffer (150 mM NaCl, 25 mM Tris-HCl [pH 7.6], 0.1% SDS, 1% sodium deoxycholate, and 1% NP-40) supplemented with 1× protease inhibitor mixture (Roche) followed by incubation in a sonic water bath at 4°C for 30 min. Cellular debris was pelleted by centrifugation at 12,000 × g for 15 min at 4°C. Protein concentration in supernatants was then determined by bicinchoninic acid assay (Pierce Biotechnology/Thermo Fisher Scientific), and protein concentrations were normalized among samples. Equal amounts of protein were then denatured by heating at 100°C for 10 min in 1× Lamelli sample buffer (Bio-Rad Laboratories), separated on 4–20% Mini-Protean TGX gels (Bio-Rad Laboratories), and transferred to PVDF membranes using the Trans-Blot Turbo system (Bio-Rad Laboratories). Membranes were blocked with Odyssey Blocking Buffer (LI-COR Biosciences) for 1 h at room temperature, followed by overnight incubation at 4°C with primary Abs. Membranes were washed three times for 5 min in PBS plus 0.05% Tween-20 and then incubated for 1 h, protected from light, at room temperature with IRDye-labeled species-specific secondary Abs (LI-COR Biosciences). Membranes were subsequently washed with PBS plus 0.05% Tween-20 and PBS, after which membranes were visualized with the Odyssey Infrared Imager (LI-COR Biosciences). Odyssey program software was used to perform densitometry analysis.

Select genes were depleted from cultured sebocytes using Silencer Select small interfering RNA (siRNA) oligonucleotides (Thermo Fisher Scientific, catalog numbers listed in Supplemental Table I). Briefly, sebocytes at 60% confluence in 6-cm tissue culture dishes were treated with siRNA oligonucleotides diluted to a final concentration of 10 nM in 5 ml of a 9:1 Sebomed Basal Medium:Opti-MEM Medium mixture containing 0.25% RNAimax Lipofectamine reagent (Thermo Fisher Scientific). Cells were treated for 18 h with siRNA, after which media was changed and cells were allowed to recover for 24 h. Cells were then collected and plated into appropriate 12-well, 24-well, or 96-well plates for subsequent analysis. Cell treatments occurred within 120 h of original siRNA application, and RT-qPCR measurement of target transcript abundance was measured simultaneously with additional experiments.

P. acnes strain ATCC6919 was maintained in Difco Reinforced Clostridial Medium (BD Biosciences). For stimulation of sebocytes, P. acnes was inoculated into Sebomed Basal Medium (with 10% FBS and 5 ng/ml rhEGF, without antibiotics) with or without the addition of 2% glycerol. Bacteria were cultured in anaerobic conditions using BD GasPak EZ pouches (BD Biosciences) at 37°C for 7 d. Culture supernatants were then sterile filtered and added at 20% vol to Sebomed Basal Medium and used to stimulate sebocytes.

Measurements of HDAC activity in sebocytes in the presence of SCFAs were performed using the Epigenase HDAC Activity/Inhibition Direct Assay Kit (EpiGentek) according to the manufacturer’s instructions. Briefly, nuclear extracts from SEB-1 and SZ95 sebocyte cells were incubated in 8-well strips with an acetylated histone HDAC substrate in the presence or absence of ranging concentrations of propionate, butyrate, or trichostatin A (TSA) at 37°C for 90 min. Test wells were subsequently incubated with capture Ab (room temperature, 60 min), detection Ab (room temperature, 30 min), and detection solution (room temperature, 10 min) with washing of wells between incubation steps. Stop solution was added to test wells after 10 min of incubation with detection solution, and the absorbance of wells in the test plate at 450 nm was measured in a plate reader (Beckman Coulter Diagnostics). Inhibition of HDAC activity by propionate, butyrate, or TSA was calculated using the following formula as directed by the manufacturer:

Inhibition%=[1(Inhibitor sample ODBlank OD)/(No inhibitor sample ODBlank OD)]×100

Sebaceous glands in the skin are exposed to resident microbes and their products and have been previously shown to produce inflammatory cytokines (14). To test the hypothesis that metabolic products of C. acnes (P. acnes) could promote cytokine release from sebocytes, we first evaluated the release of IL-6 and IL-8 from a cultured sebocyte cell line (SEB-1) after exposure to supernatants of P. acnes (ATCC6919) that were grown under hypoxic conditions for 7 d in the presence or absence of glycerol. It has previously been demonstrated that glycerol, a naturally occurring metabolite on human skin that arises from the catalysis of sebaceous gland–derived triglycerides (27), is used by P. acnes under anaerobic conditions and results in the generation of SCFAs (28). The conditioned supernatant from these bacteria was then sterile filtered and added to sebocytes. Exposure of sebocytes to supernatant from P. acnes grown without glycerol led to a modest release of IL-6 and IL-8, but when exposed to bacterial supernatants from P. acnes grown with glycerol, a significant increase in these cytokines was seen (Fig. 1A). Similarly, measurement of mRNA by quantitative PCR for the inflammatory cytokines IL-8, TNF-α, CXCL10, and thymic stromal lymphopoietin (TSLP) demonstrated that sebocytes exposed to bacterial culture supernatant with glycerol had higher levels of gene expression than with exposure to supernatant from cultures grown without glycerol (Fig. 1B). These results did not appear to be a result of increased bacterial load in the glycerol-containing cultures as P. acnes growth and total abundance was similar in both culture conditions (Fig. 1C).

FIGURE 1.

C. acnes fermentation products increase cytokine response and histone acetylation in human sebocytes. (A) Conditioned media from cultures of C. acnes (P. acnes; strain ATCC6919) cultured with and without 2% glycerol was used to stimulate the SEB-1 human sebocyte cell line (20% vol of bacterial conditioned media in cell culture media). Levels of IL-6 and IL-8 were measured in cell culture supernatant by ELISA following 24 h of stimulation. (B) Proinflammatory cytokine induction as measured by RT-qPCR in SEB-1 sebocytes treated for 4 h with P. acnes conditioned media as described in (A); transcript abundances are normalized against the housekeeping gene GAPDH. (C) Growth of P. acnes cultured with and without glycerol determined by measuring OD at 600 nm on a spectrophotometer. (D) Levels of global histone H3 acetylation in SEB-1 sebocytes treated with MALP-2 (200 ng/ml), propionate (2 mM), or bacterial conditioned media (20% vol) for 24 h and measured using an AlphaLISA Assay kit. For graphs, data shown are mean ± SEM of one experiment representative of at least three independent experiments with n = 3 for each condition. *p < 0.05, **p < 0.01, ***p < 0.001 as determined by Student t test.

FIGURE 1.

C. acnes fermentation products increase cytokine response and histone acetylation in human sebocytes. (A) Conditioned media from cultures of C. acnes (P. acnes; strain ATCC6919) cultured with and without 2% glycerol was used to stimulate the SEB-1 human sebocyte cell line (20% vol of bacterial conditioned media in cell culture media). Levels of IL-6 and IL-8 were measured in cell culture supernatant by ELISA following 24 h of stimulation. (B) Proinflammatory cytokine induction as measured by RT-qPCR in SEB-1 sebocytes treated for 4 h with P. acnes conditioned media as described in (A); transcript abundances are normalized against the housekeeping gene GAPDH. (C) Growth of P. acnes cultured with and without glycerol determined by measuring OD at 600 nm on a spectrophotometer. (D) Levels of global histone H3 acetylation in SEB-1 sebocytes treated with MALP-2 (200 ng/ml), propionate (2 mM), or bacterial conditioned media (20% vol) for 24 h and measured using an AlphaLISA Assay kit. For graphs, data shown are mean ± SEM of one experiment representative of at least three independent experiments with n = 3 for each condition. *p < 0.05, **p < 0.01, ***p < 0.001 as determined by Student t test.

Close modal

P. acnes cultured with glycerol results in the generation of SCFAs, and the SCFAs propionate, butyrate, and valerate have been shown to inhibit HDAC activity and increase histone acetylation (2931). Increased histone acetylation can result in variable effects on cytokine expression depending on cell type (10, 32). We therefore next assessed if the crude bacterial supernatant resulted in a change in histone acetylation. Global histone H3 acetylation in sebocytes was increased by propionate but was not affected by stimulation with the TLR2/6 ligand macrophage-activating lipopeptide-2 (MALP-2) (Fig. 1D). Whereas the addition of supernatant from P. acnes grown without glycerol led to a modest increase in global H3 acetylation, supernatant from the glycerol-containing cultures led to a significantly higher increase in H3 acetylation to levels comparable to treatment with propionate (Fig. 1D).

To determine if SCFAs alone could recapitulate the responses observed by sebocytes to the crude P. acnes supernatant, sebocytes were next exposed to a TLR2/6 ligand (MALP-2) in the presence or absence of three purified SCFAs: propionate, butyrate, and valerate. Consistent with our previous findings in human keratinocytes, we saw that SCFAs significantly enhanced the TLR-mediated transcriptional induction of various proinflammatory cytokines, such as IL-1β, IL-8, TNF-α, and CXCL10 from SEB-1 sebocytes (Fig. 2A–D). This enhanced cytokine response in the presence of SCFAs was also seen with an independently derived sebocyte cell line (SZ95) (Fig. 2E–H) (25). Taken together with our previous findings that P. acnes can generate SCFAs under anaerobic, lipid-rich conditions, these results suggest that production of SCFAs by P. acnes increases inflammatory cytokine expression induced by its TLR ligands from sebocytes.

FIGURE 2.

SCFAs increase proinflammatory gene expression from human sebocytes in response to TLR activation. (AD) SEB-1 sebocytes or (EH) SZ95 sebocytes were stimulated with MALP-2 (200 ng/ml) for 4 h with or without the addition of propionate, butyrate, or valerate (2 mM). RT-qPCR was used to measure the induction of the proinflammatory cytokines IL-1β (A and E), IL-8 (B and F), TNF-α (C and G) and CXCL10 (D and H); transcript abundances are normalized against the housekeeping gene GAPDH. For graphs, data shown are mean ± SEM of one experiment representative of at least three independent experiments with n = 3 for each condition. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 as determined by two-way ANOVA.

FIGURE 2.

SCFAs increase proinflammatory gene expression from human sebocytes in response to TLR activation. (AD) SEB-1 sebocytes or (EH) SZ95 sebocytes were stimulated with MALP-2 (200 ng/ml) for 4 h with or without the addition of propionate, butyrate, or valerate (2 mM). RT-qPCR was used to measure the induction of the proinflammatory cytokines IL-1β (A and E), IL-8 (B and F), TNF-α (C and G) and CXCL10 (D and H); transcript abundances are normalized against the housekeeping gene GAPDH. For graphs, data shown are mean ± SEM of one experiment representative of at least three independent experiments with n = 3 for each condition. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 as determined by two-way ANOVA.

Close modal

To further assess if SCFAs lead to increased inflammatory response by inhibition of HDACs, we compared responsiveness to propionate to two well-established chemical HDAC inhibitors (TSA and suberoylanilide hydroxamic acid [SAHA]). We observed that both SCFAs and non-SCFA HDAC inhibitors all increased TLR-mediated production of IL-6 and IL-8 from SEB-1 sebocytes (Fig. 3A, 3B). Consistent with their known ability to inhibit HDAC activity, these molecules led to increased global levels of histone acetylation in sebocytes as demonstrated by Western blot analysis of multiple lysine residues of histone H3 (H3K9ac, H3K27ac) (Fig. 3C). Furthermore, we observed an increase in H3K9 acetylation throughout the promoter region of the IL-6 gene when sebocytes were treated with propionate (Fig. 3D), suggesting that the increased TLR-mediated induction of this and other inflammatory genes by HDAC inhibitors may be due to changes in chromatin accessibility. To verify that the observed increase in histone acetylation in sebocytes treated with SCFAs was a direct result of inhibition of HDAC activity, HDAC activity was measured from nuclear extracts of SEB-1 and SZ95 cells in the presence of SCFAs. Both propionate and butyrate were seen to directly inhibit HDAC activity to a comparable degree as the well-established HDAC inhibitor TSA (Supplemental Fig. 1).

FIGURE 3.

HDAC inhibitors increase TLR-mediated cytokine production in sebocytes. (A and B) Levels of IL-6 and IL-8 secretion from SEB-1 sebocytes stimulated with MALP-2 (200 ng/ml) with or without the addition of the HDAC inhibitors propionate (2 mM), SAHA (100 nM), or TSA (100 nM) for 18 h. (C) Levels of acetylated histone residues (H3K9ac and H3K27ac) measured by Western blot in SEB-1 sebocytes treated with the indicated HDAC inhibitors for 8 h. (D) Levels of H3K9 acetylation in the IL-6 promoter region in SEB-1 sebocytes treated with or without propionate (2 mM) for 8 h as measured by chromatin immunoprecipitation–quantitative PCR. (E and F) Levels of IL-6 and IL-8 secretion from SEB-1 sebocytes treated with MALP-2 (200 ng/ml) with or without the addition of the HDAC inhibitor butyrate (HDACi; 2 mM) or the HAT inhibitor anacardic acid (HATi; 4 μM) for 18 h. For Western blots, one representative experiment out of three is shown. For graphs, data shown are mean ± SEM of one experiment representative of at least three independent experiments with n = 3 for each condition. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 as determined by two-way ANOVA or Student t test.

FIGURE 3.

HDAC inhibitors increase TLR-mediated cytokine production in sebocytes. (A and B) Levels of IL-6 and IL-8 secretion from SEB-1 sebocytes stimulated with MALP-2 (200 ng/ml) with or without the addition of the HDAC inhibitors propionate (2 mM), SAHA (100 nM), or TSA (100 nM) for 18 h. (C) Levels of acetylated histone residues (H3K9ac and H3K27ac) measured by Western blot in SEB-1 sebocytes treated with the indicated HDAC inhibitors for 8 h. (D) Levels of H3K9 acetylation in the IL-6 promoter region in SEB-1 sebocytes treated with or without propionate (2 mM) for 8 h as measured by chromatin immunoprecipitation–quantitative PCR. (E and F) Levels of IL-6 and IL-8 secretion from SEB-1 sebocytes treated with MALP-2 (200 ng/ml) with or without the addition of the HDAC inhibitor butyrate (HDACi; 2 mM) or the HAT inhibitor anacardic acid (HATi; 4 μM) for 18 h. For Western blots, one representative experiment out of three is shown. For graphs, data shown are mean ± SEM of one experiment representative of at least three independent experiments with n = 3 for each condition. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 as determined by two-way ANOVA or Student t test.

Close modal

Although HDACs are responsible for the removal of acetylation marks in the genome, another class of enzymes, histone acetyltransferases (HATs), are responsible for the addition of these marks. Interestingly, when we stimulated sebocytes with anacardic acid, a chemical known to inhibit the action of HAT enzymes, we observed a decrease in TLR-mediated expression of IL-6 and IL-8 and a complete elimination of the proinflammatory effects of treatment with butyrate (Fig. 3E, 3F). Together, these data demonstrate that sebocytes exhibit a proinflammatory phenotype in response to HDAC inhibition, a finding that contrasts to the anti-inflammatory effects of HDAC inhibitors on bone marrow–derived immune cells like macrophages and dendritic cells.

As SCFAs and other chemical HDAC inhibitors used in these experiments are broad acting and inhibit the activity of numerous HDAC enzymes, we next sought to determine which individual HDACs may be important in the regulation of inflammatory gene expression in sebocytes. First, we characterized the expression of the 11 classical HDAC family members in sebocytes and compared them with expression levels from epidermal keratinocytes (Fig. 4A). HDAC8 and HDAC9 were partially depleted using siRNA-mediated gene silencing (Fig. 4B, Supplemental Fig. 2A–D). This resulted in a significant amplification of MALP-2–mediated induction of IL-1β, IL-6, IL-8, and CXCL10 (Fig. 4C–F). Expression of these cytokines was not increased in HDAC-depleted sebocytes without TLR activation. Furthermore, protein levels of IL-6 and IL-8 as measured by ELISA were also increased in sebocytes with partial silencing of HDAC8 and HDAC9 and treated with MALP-2 (Fig. 4G, 4H). To validate that these findings were indeed specific to depletion of HDAC8 and HDAC9 and not an off-target effect of the silencing oligonucleotides used, we tested additional siRNA oligonucleotides specific for HDAC8 and HDAC9. Consistent with our initial results, partial depletion of HDAC8 and HDAC9 using alternate oligonucleotides significantly enhanced the TLR-mediated induction of IL-6 and IL-8 from SEB-1 sebocytes (Supplemental Fig. 3A–C). These data indicate that HDAC8 and HDAC9 influence inflammatory cytokine expression in sebocytes and suggest that the activity of SCFAs on sebocytes is also mediated by the inhibition of HDACs.

FIGURE 4.

HDAC8 and HDAC9 control inflammatory gene expression in human sebocytes. (A) Expression pattern of the 11 classical HDAC enzymes in SEB-1 sebocytes and SZ95 sebocytes compared with normal human epidermal keratinocytes. (B) Levels of HDAC8 and HDAC9 expression in SEB-1 sebocytes following siRNA-mediated gene knockdown; transcript abundances are normalized against the housekeeping gene GAPDH. (CF) Expression levels of the proinflammatory cytokines IL-1β, IL-6, IL-8, and CXCL10 (normalized against GAPDH) in SEB-1 sebocytes stimulated with MALP-2 (200 ng/ml) following siRNA-mediated depletion of HDAC8 or HDAC9. (G and H) Levels of secreted IL-6 and IL-8 from SEB-1 sebocytes stimulated with MALP-2 (200 ng/ml) following siRNA-mediated depletion of HDAC8 and HDAC9. For graphs, data shown are mean ± SEM of one experiment representative of at least three independent experiments with n = 3 for each condition. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 as determined by two-way ANOVA or Student t test.

FIGURE 4.

HDAC8 and HDAC9 control inflammatory gene expression in human sebocytes. (A) Expression pattern of the 11 classical HDAC enzymes in SEB-1 sebocytes and SZ95 sebocytes compared with normal human epidermal keratinocytes. (B) Levels of HDAC8 and HDAC9 expression in SEB-1 sebocytes following siRNA-mediated gene knockdown; transcript abundances are normalized against the housekeeping gene GAPDH. (CF) Expression levels of the proinflammatory cytokines IL-1β, IL-6, IL-8, and CXCL10 (normalized against GAPDH) in SEB-1 sebocytes stimulated with MALP-2 (200 ng/ml) following siRNA-mediated depletion of HDAC8 or HDAC9. (G and H) Levels of secreted IL-6 and IL-8 from SEB-1 sebocytes stimulated with MALP-2 (200 ng/ml) following siRNA-mediated depletion of HDAC8 and HDAC9. For graphs, data shown are mean ± SEM of one experiment representative of at least three independent experiments with n = 3 for each condition. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 as determined by two-way ANOVA or Student t test.

Close modal

Having identified HDAC8 and HDAC9 as being important in restraining the sebocyte inflammatory response, we sought to use RNA sequencing to evaluate the global transcriptomic effects of depletion of these enzymes. In sebocytes treated with scrambled control siRNA, stimulation with MALP-2 led to an upregulation of 379 genes and a downregulation of 321 genes (Fig. 5A). Gene ontology analysis of the set of genes upregulated by TLR2 activation in these cells identified enrichment of immune- and inflammation-related pathways, such as inflammatory response, cytokine-mediated signaling pathway, and the TNF-signaling pathway, among others (Fig. 5B). Consistent with our hypothesis that HDAC8 and HDAC9 work to restrain inflammatory gene expression in sebocytes, cells depleted of HDAC8 and HDAC9 and stimulated by MALP-2 resulted in the induction of over twice as many genes (790 versus 379, Fig. 5C). Furthermore, a more significant enrichment of pathways involved in inflammatory responses was observed, such as cytokine-mediated signaling pathway, regulation of cell adhesion, and leukocyte migration (Fig. 5D).

FIGURE 5.

Depletion of HDAC8 and HDAC9 increases global inflammatory response from human sebocytes. SEB-1 sebocytes were treated with scrambled control siRNA or siRNA specific for HDAC8 and HDAC9, after which cells were treated with MALP-2 (200 ng/ml) for 4 h. RNA was isolated and analyzed by RNA sequencing. (AD) Numbers of genes upregulated and downregulated by 1.5-fold or greater by MALP-2 treatment in cells treated with control siRNA (A) or siRNA specific for HDAC8 and HDAC9 (C). Gene ontology analysis of the sets of genes upregulated by MALP-2 treatment in cells treated with control siRNA (B) or siRNA specific for HDAC8 and HDAC9 (D). (E and F) Genes induced by MALP-2 in SEB-1 sebocytes treated with control siRNA (E) or siRNA specific for HDAC8 and HDAC9 (F) were compared with the transcriptome of genes upregulated by 2-fold or greater identified from an inflamed clinical acne lesion. RNA sequencing was performed on n = 1 representative sample for each condition.

FIGURE 5.

Depletion of HDAC8 and HDAC9 increases global inflammatory response from human sebocytes. SEB-1 sebocytes were treated with scrambled control siRNA or siRNA specific for HDAC8 and HDAC9, after which cells were treated with MALP-2 (200 ng/ml) for 4 h. RNA was isolated and analyzed by RNA sequencing. (AD) Numbers of genes upregulated and downregulated by 1.5-fold or greater by MALP-2 treatment in cells treated with control siRNA (A) or siRNA specific for HDAC8 and HDAC9 (C). Gene ontology analysis of the sets of genes upregulated by MALP-2 treatment in cells treated with control siRNA (B) or siRNA specific for HDAC8 and HDAC9 (D). (E and F) Genes induced by MALP-2 in SEB-1 sebocytes treated with control siRNA (E) or siRNA specific for HDAC8 and HDAC9 (F) were compared with the transcriptome of genes upregulated by 2-fold or greater identified from an inflamed clinical acne lesion. RNA sequencing was performed on n = 1 representative sample for each condition.

Close modal

Next, we compared the global inflammatory response of sebocytes with or without functional HDAC8 and HDAC9 to the inflammatory signature of an acne lesion. A total of 2382 genes were upregulated in an inflamed acne lesion compared with healthy skin from the same donor. Comparison of this dataset with the list of genes induced by MALP-2 alone in sebocytes (treated with control siRNA) showed a relatively minor overlap of 157 genes (Fig. 5E). However, when an acne lesion was compared with sebocytes lacking HDAC8 and HDAC9 and stimulated by MALP-2, almost twice as many genes overlapped, with 277 genes common between the samples (Fig. 5F). These data demonstrate that when the activity of HDAC8 and HDAC9 are impaired in sebocytes, activation of TLR 2 leads to the induction of many more inflammatory genes that are detected in clinical acne samples.

Finally, in addition to the capacity to inhibit HDAC activity, SCFAs also have the potential to interact with cell surface G protein–coupled receptors (GPCRs). In particular, SCFAs are known ligands of GPR41 (also known as free fatty acid receptor [FFAR] 3), GPR43 (also known as FFAR2), and GPR109a (also known as hydroxycarboxylic acid receptor 2 [HCAR2]) (33, 34). We next assessed if there was a direct response to SCFAs by sebocytes and sought to distinguish this response from the amplification of TLR signaling by the epigenetic mechanism of histone acetylation. Prior observations in keratinocytes showed no detectable response to SCFAs alone, which may be explained by the extremely low expression levels of the three relevant GPCRs by this cell type (10). In contrast, sebocytes express FFAR2, FFAR3, and HCAR2 at relatively high levels, several hundred-fold higher than keratinocytes (Fig. 6A). Furthermore, SZ95 sebocytes treated with SCFAs in the absence of TLR2 ligands resulted in increased expression of the proinflammatory mediator TSLP, an effect that was lost when FFARs were depleted via siRNA-mediated knockdown (Fig. 6B). When FFAR2, FFAR3, or HCAR2 were depleted individually from sebocytes, the SCFA-mediated increase in MALP-2–induced cytokine secretion was partially decreased (Fig. 6C). Meanwhile, when cells were depleted of all three receptors in combination, the proinflammatory effect of SCFAs was almost completely abolished (Fig. 6C). Importantly, this did not occur to the same extent when cells were activated by the non-SCFA HDAC inhibitor TSA, demonstrating that these cells still respond appropriately to inhibition of HDAC activity (Fig. 6D). These data demonstrated that sebocytes, compared with other cell types in the skin, exhibit a unique response to SCFAs, which promote cytokine expression by both activation of GPCRs and inhibition of HDAC activity.

FIGURE 6.

FFAR activation by SCFAs induces cytokine expression from sebocytes. (A) Expression patterns of the major FFARs in SZ95 sebocytes compared with normal human epidermal keratinocytes; transcript abundances are normalized against GAPDH. (B) Expression levels of the inflammatory mediator TSLP expressed in SZ95 sebocytes following stimulation with butyrate (2 mM) for 4 h (transcript abundance normalized against GAPDH). (C and D) IL-8 secretion from SZ95 sebocytes stimulated with MALP-2 (200 ng/ml) with or without butyrate (2 mM) or TSA (100 nM) for 18 h following siRNA-mediated depletion of the indicated FFARs. For graphs, data shown are mean ± SEM of one experiment representative of at least two independent experiments with n = 3 for each condition. **p < 0.01, ***p < 0.001, ****p < 0.0001 as determined by two-way ANOVA or Student t test.

FIGURE 6.

FFAR activation by SCFAs induces cytokine expression from sebocytes. (A) Expression patterns of the major FFARs in SZ95 sebocytes compared with normal human epidermal keratinocytes; transcript abundances are normalized against GAPDH. (B) Expression levels of the inflammatory mediator TSLP expressed in SZ95 sebocytes following stimulation with butyrate (2 mM) for 4 h (transcript abundance normalized against GAPDH). (C and D) IL-8 secretion from SZ95 sebocytes stimulated with MALP-2 (200 ng/ml) with or without butyrate (2 mM) or TSA (100 nM) for 18 h following siRNA-mediated depletion of the indicated FFARs. For graphs, data shown are mean ± SEM of one experiment representative of at least two independent experiments with n = 3 for each condition. **p < 0.01, ***p < 0.001, ****p < 0.0001 as determined by two-way ANOVA or Student t test.

Close modal
Table I.
Reagents
ReagentSupplierCatalog No.
Sebomed Basal Medium Cedarlane/Thermo Fisher Scientific F8205 
Antibiotic-antimycotic Life Technologies 15240-062 
rhEGF Sigma-Aldrich E9644 
FBS Gemini Bio-Products 900-108 
Sodium propionate Sigma-Aldrich P1880 
Sodium butyrate Sigma-Aldrich B5887 
Isovaleric acid Sigma-Aldrich 78651 
TSA Sigma-Aldrich T8552 
SAHA Sigma-Aldrich SML0061 
MALP-2 Enzo Life Sciences/VWR 162-034-C500 
TaqMan Universal PCR Master Mix Life Technologies 4318157 
Complete protease inhibitor cocktail Roche 11697498001 
RNAiMAX Lipofectamine Thermo Fisher Scientific 56532 
HDAC activity/inhibition direct assay EpiGentek P-4034-96 
Silencer select siRNA oligonucleotides See Supplemental Table I 
TaqMan Gene Expression Assays See Supplemental Table I 
ELISA kits See Supplemental Table I 
ReagentSupplierCatalog No.
Sebomed Basal Medium Cedarlane/Thermo Fisher Scientific F8205 
Antibiotic-antimycotic Life Technologies 15240-062 
rhEGF Sigma-Aldrich E9644 
FBS Gemini Bio-Products 900-108 
Sodium propionate Sigma-Aldrich P1880 
Sodium butyrate Sigma-Aldrich B5887 
Isovaleric acid Sigma-Aldrich 78651 
TSA Sigma-Aldrich T8552 
SAHA Sigma-Aldrich SML0061 
MALP-2 Enzo Life Sciences/VWR 162-034-C500 
TaqMan Universal PCR Master Mix Life Technologies 4318157 
Complete protease inhibitor cocktail Roche 11697498001 
RNAiMAX Lipofectamine Thermo Fisher Scientific 56532 
HDAC activity/inhibition direct assay EpiGentek P-4034-96 
Silencer select siRNA oligonucleotides See Supplemental Table I 
TaqMan Gene Expression Assays See Supplemental Table I 
ELISA kits See Supplemental Table I 

These data show for the first time, to our knowledge, how metabolic products of skin-resident bacteria can induce inflammatory gene expression from sebocytes. Although it has been known that sebocytes can respond to P. acnes through activation of TLR 2, the question of why these bacteria only drive an inflammatory response in the skin in certain settings has remained unanswered. We have previously shown that when cultured in hypoxic conditions and supplied a lipid substrate (an environment reminiscent of a plugged, sebaceous follicle) P. acnes can generate high levels of SCFAs through anaerobic fermentation. Interestingly, these molecules were shown to amplify TLR-driven cytokine responses from epidermal keratinocytes through their inhibition of HDAC function. In this study, we now demonstrate that cytokine expression in sebocytes is also regulated through this epigenetic mechanism and also show that sebocytes have a unique capacity to enhance cytokine expression by direct activation of surface-bound FFARs. Thus, sebocytes in the skin are uniquely sensitive to a change in metabolites by P. acnes and respond to SCFAs by two independent mechanisms.

The proinflammatory effects of HDAC inhibition in both sebocytes and keratinocytes, combined with the similar pattern of expression of the 11 classical HDAC family members observed in the two cell types, suggested that the same HDAC enzymes might control inflammatory gene expression in both keratinocytes and sebocytes. Indeed, specific depletion of HDAC8 or HDAC9, which we had previously demonstrated to control cytokine expression in keratinocytes, resulted in an amplified response to TLR activation in sebocytes. When examined at a global level by RNA sequencing, the number of genes upregulated in response to TLR2/6 activation was nearly doubled when HDAC8 and HDAC9 activity was inhibited. Furthermore, the set of genes induced in sebocytes depleted of HDAC8 and HDAC9 more closely resembled the transcriptome of whole tissue samples from acne, suggesting that inhibition of HDAC8 and HDAC9 in the sebaceous gland by P. acnes–derived SCFAs is modeling aspects of the pathophysiology of the disease. Although the overlap with the sebocyte transcriptome still represents a relatively small portion of the genes identified from a clinical acne lesion, one must consider that the acne biopsy represents a heterogeneous mixture of various cell types from a physiological setting, whereas the sebocyte data represents a single cell type grown in culture. More precise measurements of the sebocyte contribution to an acne lesion (for example, through single cell sequencing of sebocytes from clinical specimens) could further elucidate the contribution of this cell type, and the importance of sebocyte HDAC activity, to acne inflammation in vivo.

Despite the commonalities in regard to HDAC8 and HDAC9, the impact of SCFAs on the inflammatory response of sebocytes and keratinocytes is not completely identical. As this work shows, SCFAs have a unique ability to induce cytokine expression from sebocytes through the activation of FFARs, a finding that was not observed in prior work with keratinocytes. Some proinflammatory mediators, such as TSLP, were induced from sebocytes by treatment with SCFAs alone, even in the absence of TLR ligands. These effects may be due to the high expression of these FFARs in sebocytes, which were detected at several hundred-fold higher levels than in keratinocytes. These findings are interesting when taken together with previous studies demonstrating that free fatty acids present in sebum, such as lauric, palmitic, and oleic acids, can induce antimicrobial peptide expression from sebocytes (35). Together, these findings illustrate how the local follicular microenvironment, consisting of fatty acids from the host, resident microbes, and their metabolic products, can influence inflammatory and antimicrobial processes in the skin. Future studies to characterize the fatty acid profiles of healthy and diseased skin and investigate how different mixtures of fatty acids may further inform the relative role of these metabolites in skin inflammation. Additionally, evaluating the generation of SCFAs and other bioactive metabolites by P. acnes cultured with lipid sources that more closely resemble human sebum, as opposed to simply glycerol, may shed interesting light onto the impacts of microbial metabolism on skin inflammation.

The effects of P. acnes–derived SCFAs on inflammatory gene expression from sebocytes through both epigenetic and receptor-dependent mechanisms described in this study invite the question of whether distinct strains of P. acnes have a differential capacity to generate these molecules. Whereas P. acnes proliferation and overgrowth in the follicle was once considered a driving factor behind the inflammation seen in acne, recent work has demonstrated that the overall abundance of microbes in healthy and affected follicles is actually quite comparable (8). Thus, the current view has shifted toward the idea that distinct strains of P. acnes are unique in their abilities to induce cytokine expression from sebocytes, keratinocytes, and cells of the innate immune system. Our results support speculation that SCFAs from P. acnes will drive cytokine expression prior to rupture of the follicle and may influence both the local pilosebaceous unit as well as surrounding skin. Indeed, characterizations of the P. acnes populations isolated from the skin of acne patients and healthy controls have identified “acne-associated” and “healthy-associated” strains of P. acnes, which exert distinct effects on the immune and inflammatory responses of the skin (9, 36, 37). However, whether the ability to produce SCFAs, or the ratios of SCFAs produced by these different bacteria, contribute to the host response has yet to be investigated.

Overall, this study expands our knowledge into the regulation of inflammatory gene expression in the skin, an organ that must strictly control its capacity to respond to microbes. Although this work was conducted using immortalized cell lines, the observation of the same trends in response to HDAC inhibition, specific depletion of HDAC8 and HDAC9, and depletion of FFARs in two independently derived cell lines supports that these responses are not restricted to a cell line but more likely reflect the sebocyte itself. Replicating these experiments in primary human sebocytes isolated from skin samples would help support the notion that these phenomena may occur in vivo, but even these culture-based studies are not a completely accurate representation of the physiological setting of the pilosebaceous unit. In the future, genetic mouse models can be used to generate tissue-specific deletions of HDAC8 and HDAC9 (from sebocytes, epidermal keratinocytes, or both) to gain a more in-depth understanding of the role of these enzymes in regulating skin inflammation and immunity. However, the lack of appropriate animal models for inflammatory acne vulgaris makes it difficult to specifically link our findings to acne rather than skin inflammation in a broader sense.

In conclusion, this work helps to promote a model of skin inflammation in which changes in the local microenvironment of the pilosebaceous unit drives metabolic shifts in resident skin microbes, resulting in the production of bioactive metabolites that promote the shift from the cutaneous immune system tolerating P. acnes colonization to one that is inflammatory. This shift may be the fundamental event in the pathophysiology of acne vulgaris.

We thank Dr. Diane Thiboutot from Pennsylvania State University Dermatology for providing the SEB-1 sebocyte cell line for use in these experiments. We also thank Dr. Yun “Larry” Tong from the UCSD Department of Dermatology for the acquisition of human samples used in immunohistochemistry and RNA sequencing.

This work was supported by National Institutes of Health Grants R01AR074302, R01AR069653, and R01AI052453 (to R.L.G.).

The sequences presented in this article have been submitted to the Sequence Read Archive under accession number SRP151073 (https://trace.ncbi.nlm.nih.gov/Traces/sra/?study=SRP151073).

The online version of this article contains supplemental material.

Abbreviations used in this article:

FFAR

free fatty acid receptor

GPCR

G protein–coupled receptor

HAT

histone acetyltransferase

HCAR2

hydroxycarboxylic acid receptor 2

HDAC

histone deacetylase

MALP-2

macrophage-activating lipopeptide-2

rhEGF

recombinant human epidermal growth factor

RT-qPCR

quantitative RT-PCR

SAHA

suberoylanilide hydroxamic acid

SCFA

short-chain fatty acid

siRNA

small interfering RNA

TSA

trichostatin A

TSLP

thymic stromal lymphopoietin

UCSD

University of California, San Diego.

1
Moradi Tuchayi
,
S.
,
E.
Makrantonaki
,
R.
Ganceviciene
,
C.
Dessinioti
,
S. R.
Feldman
,
C. C.
Zouboulis
.
2015
.
Acne vulgaris.
Nat. Rev. Dis. Primers
1
:
15029
.
2
Scholz
,
C. F.
,
M.
Kilian
.
2016
.
The natural history of cutaneous propionibacteria, and reclassification of selected species within the genus Propionibacterium to the proposed novel genera Acidipropionibacterium gen. nov., Cutibacterium gen. nov. and Pseudopropionibacterium gen. nov.
Int. J. Syst. Evol. Microbiol.
66
:
4422
4432
.
3
Bojar
,
R. A.
,
K. T.
Holland
.
2004
.
Acne and Propionibacterium acnes.
Clin. Dermatol.
22
:
375
379
.
4
Kim
,
J.
,
M. T.
Ochoa
,
S. R.
Krutzik
,
O.
Takeuchi
,
S.
Uematsu
,
A. J.
Legaspi
,
H. D.
Brightbill
,
D.
Holland
,
W. J.
Cunliffe
,
S.
Akira
, et al
.
2002
.
Activation of toll-like receptor 2 in acne triggers inflammatory cytokine responses.
J. Immunol.
169
:
1535
1541
.
5
Jugeau
,
S.
,
I.
Tenaud
,
A. C.
Knol
,
V.
Jarrousse
,
G.
Quereux
,
A.
Khammari
,
B.
Dreno
.
2005
.
Induction of toll-like receptors by Propionibacterium acnes.
Br. J. Dermatol.
153
:
1105
1113
.
6
Qin
,
M.
,
A.
Pirouz
,
M. H.
Kim
,
S. R.
Krutzik
,
H. J.
Garbán
,
J.
Kim
.
2014
.
Propionibacterium acnes induces IL-1β secretion via the NLRP3 inflammasome in human monocytes. [Published erratum appears in 2014 J. Invest. Dermatol. 134: 1779.]
J. Invest. Dermatol.
134
:
381
388
.
7
Vowels
,
B. R.
,
S.
Yang
,
J. J.
Leyden
.
1995
.
Induction of proinflammatory cytokines by a soluble factor of Propionibacterium acnes: implications for chronic inflammatory acne.
Infect. Immun.
63
:
3158
3165
.
8
Barnard
,
E.
,
B.
Shi
,
D.
Kang
,
N.
Craft
,
H.
Li
.
2016
.
The balance of metagenomic elements shapes the skin microbiome in acne and health.
Sci. Rep.
6
:
39491
.
9
Fitz-Gibbon
,
S.
,
S.
Tomida
,
B. H.
Chiu
,
L.
Nguyen
,
C.
Du
,
M.
Liu
,
D.
Elashoff
,
M. C.
Erfe
,
A.
Loncaric
,
J.
Kim
, et al
.
2013
.
Propionibacterium acnes strain populations in the human skin microbiome associated with acne.
J. Invest. Dermatol.
133
:
2152
2160
.
10
Sanford
,
J. A.
,
L. J.
Zhang
,
M. R.
Williams
,
J. A.
Gangoiti
,
C. M.
Huang
,
R. L.
Gallo
.
2016
.
Inhibition of HDAC8 and HDAC9 by microbial short-chain fatty acids breaks immune tolerance of the epidermis to TLR ligands.
Sci. Immunol.
1
:
eaah4609
.
11
Säemann
,
M. D.
,
G. A.
Böhmig
,
C. H.
Osterreicher
,
H.
Burtscher
,
O.
Parolini
,
C.
Diakos
,
J.
Stöckl
,
W. H.
Hörl
,
G. J.
Zlabinger
.
2000
.
Anti-inflammatory effects of sodium butyrate on human monocytes: potent inhibition of IL-12 and up-regulation of IL-10 production.
FASEB J.
14
:
2380
2382
.
12
Nastasi
,
C.
,
M.
Candela
,
C. M.
Bonefeld
,
C.
Geisler
,
M.
Hansen
,
T.
Krejsgaard
,
E.
Biagi
,
M. H.
Andersen
,
P.
Brigidi
,
N.
Ødum
, et al
.
2015
.
The effect of short-chain fatty acids on human monocyte-derived dendritic cells.
Sci. Rep.
5
:
16148
.
13
Chang
,
P. V.
,
L.
Hao
,
S.
Offermanns
,
R.
Medzhitov
.
2014
.
The microbial metabolite butyrate regulates intestinal macrophage function via histone deacetylase inhibition.
Proc. Natl. Acad. Sci. USA
111
:
2247
2252
.
14
Nagy
,
I.
,
A.
Pivarcsi
,
K.
Kis
,
A.
Koreck
,
L.
Bodai
,
A.
McDowell
,
H.
Seltmann
,
S.
Patrick
,
C. C.
Zouboulis
,
L.
Kemény
.
2006
.
Propionibacterium acnes and lipopolysaccharide induce the expression of antimicrobial peptides and proinflammatory cytokines/chemokines in human sebocytes.
Microbes Infect.
8
:
2195
2205
.
15
Nakatsuji
,
T.
,
Y. T.
Liu
,
C. P.
Huang
,
C. C.
Zoubouis
,
R. L.
Gallo
,
C. M.
Huang
.
2008
.
Antibodies elicited by inactivated propionibacterium acnes-based vaccines exert protective immunity and attenuate the IL-8 production in human sebocytes: relevance to therapy for acne vulgaris. [Published erratum appears in 2009 J. Invest. Dermatol. 129: 1590.]
J. Invest. Dermatol.
128
:
2451
2457
.
16
Selway
,
J. L.
,
T.
Kurczab
,
T.
Kealey
,
K.
Langlands
.
2013
.
Toll-like receptor 2 activation and comedogenesis: implications for the pathogenesis of acne.
BMC Dermatol.
13
:
10
.
17
Li
,
Z. J.
,
D. K.
Choi
,
K. C.
Sohn
,
M. S.
Seo
,
H. E.
Lee
,
Y.
Lee
,
Y. J.
Seo
,
Y. H.
Lee
,
G.
Shi
,
C. C.
Zouboulis
, et al
.
2014
.
Propionibacterium acnes activates the NLRP3 inflammasome in human sebocytes.
J. Invest. Dermatol.
134
:
2747
2756
.
18
Lovászi
,
M.
,
M.
Mattii
,
K.
Eyerich
,
A.
Gácsi
,
E.
Csányi
,
D.
Kovács
,
R.
Rühl
,
A.
Szegedi
,
L.
Kemény
,
M.
Ståhle
, et al
.
2017
.
Sebum lipids influence macrophage polarization and activation.
Br. J. Dermatol.
177
:
1671
1682
.
19
Mattii
,
M.
,
M.
Lovászi
,
N.
Garzorz
,
A.
Atenhan
,
M.
Quaranta
,
F.
Lauffer
,
A.
Konstantinow
,
M.
Küpper
,
C. C.
Zouboulis
,
L.
Kemeny
, et al
.
2018
.
Sebocytes contribute to skin inflammation by promoting the differentiation of T helper 17 cells.
Br. J. Dermatol.
178
:
722
730
.
20
Alestas
,
T.
,
R.
Ganceviciene
,
S.
Fimmel
,
K.
Müller-Decker
,
C. C.
Zouboulis
.
2006
.
Enzymes involved in the biosynthesis of leukotriene B4 and prostaglandin E2 are active in sebaceous glands.
J. Mol. Med. (Berl.)
84
:
75
87
.
21
Zouboulis
,
C. C.
2001
.
Is acne vulgaris a genuine inflammatory disease?
Dermatology (Basel)
203
:
277
279
.
22
Zouboulis
,
C. C.
,
E.
Jourdan
,
M.
Picardo
.
2014
.
Acne is an inflammatory disease and alterations of sebum composition initiate acne lesions.
J. Eur. Acad. Dermatol. Venereol.
28
:
527
532
.
23
Lovászi
,
M.
,
A.
Szegedi
,
C. C.
Zouboulis
,
D.
Törőcsik
.
2017
.
Sebaceous-immunobiology is orchestrated by sebum lipids.
Dermatoendocrinol
9
:
e1375636
.
24
Thiboutot
,
D.
,
S.
Jabara
,
J. M.
McAllister
,
A.
Sivarajah
,
K.
Gilliland
,
Z.
Cong
,
G.
Clawson
.
2003
.
Human skin is a steroidogenic tissue: steroidogenic enzymes and cofactors are expressed in epidermis, normal sebocytes, and an immortalized sebocyte cell line (SEB-1).
J. Invest. Dermatol.
120
:
905
914
.
25
Zouboulis
,
C. C.
,
H.
Seltmann
,
H.
Neitzel
,
C. E.
Orfanos
.
1999
.
Establishment and characterization of an immortalized human sebaceous gland cell line (SZ95).
J. Invest. Dermatol.
113
:
1011
1020
.
26
Tripathi
,
S.
,
M. O.
Pohl
,
Y.
Zhou
,
A.
Rodriguez-Frandsen
,
G.
Wang
,
D. A.
Stein
,
H. M.
Moulton
,
P.
DeJesus
,
J.
Che
,
L. C.
Mulder
, et al
.
2015
.
Meta- and orthogonal integration of influenza “OMICs” data defines a role for UBR4 in virus budding.
Cell Host Microbe
18
:
723
735
.
27
Fluhr
,
J. W.
,
R.
Darlenski
,
C.
Surber
.
2008
.
Glycerol and the skin: holistic approach to its origin and functions.
Br. J. Dermatol.
159
:
23
34
.
28
Shu
,
M.
,
Y.
Wang
,
J.
Yu
,
S.
Kuo
,
A.
Coda
,
Y.
Jiang
,
R. L.
Gallo
,
C. M.
Huang
.
2013
.
Fermentation of Propionibacterium acnes, a commensal bacterium in the human skin microbiome, as skin probiotics against methicillin-resistant Staphylococcus aureus.
PLoS One
8
:
e55380
.
29
Sealy
,
L.
,
R.
Chalkley
.
1978
.
The effect of sodium butyrate on histone modification.
Cell
14
:
115
121
.
30
Hinnebusch
,
B. F.
,
S.
Meng
,
J. T.
Wu
,
S. Y.
Archer
,
R. A.
Hodin
.
2002
.
The effects of short-chain fatty acids on human colon cancer cell phenotype are associated with histone hyperacetylation.
J. Nutr.
132
:
1012
1017
.
31
Waldecker
,
M.
,
T.
Kautenburger
,
H.
Daumann
,
C.
Busch
,
D.
Schrenk
.
2008
.
Inhibition of histone-deacetylase activity by short-chain fatty acids and some polyphenol metabolites formed in the colon.
J. Nutr. Biochem.
19
:
587
593
.
32
Roger
,
T.
,
J.
Lugrin
,
D.
Le Roy
,
G.
Goy
,
M.
Mombelli
,
T.
Koessler
,
X. C.
Ding
,
A. L.
Chanson
,
M. K.
Reymond
,
I.
Miconnet
, et al
.
2011
.
Histone deacetylase inhibitors impair innate immune responses to toll-like receptor agonists and to infection.
Blood
117
:
1205
1217
.
33
Brown
,
A. J.
,
S. M.
Goldsworthy
,
A. A.
Barnes
,
M. M.
Eilert
,
L.
Tcheang
,
D.
Daniels
,
A. I.
Muir
,
M. J.
Wigglesworth
,
I.
Kinghorn
,
N. J.
Fraser
, et al
.
2003
.
The orphan G protein-coupled receptors GPR41 and GPR43 are activated by propionate and other short chain carboxylic acids.
J. Biol. Chem.
278
:
11312
11319
.
34
Singh
,
N.
,
A.
Gurav
,
S.
Sivaprakasam
,
E.
Brady
,
R.
Padia
,
H.
Shi
,
M.
Thangaraju
,
P. D.
Prasad
,
S.
Manicassamy
,
D. H.
Munn
, et al
.
2014
.
Activation of Gpr109a, receptor for niacin and the commensal metabolite butyrate, suppresses colonic inflammation and carcinogenesis.
Immunity
40
:
128
139
.
35
Nakatsuji
,
T.
,
M. C.
Kao
,
L.
Zhang
,
C. C.
Zouboulis
,
R. L.
Gallo
,
C. M.
Huang
.
2010
.
Sebum free fatty acids enhance the innate immune defense of human sebocytes by upregulating beta-defensin-2 expression.
J. Invest. Dermatol.
130
:
985
994
.
36
Agak
,
G. W.
,
S.
Kao
,
K.
Ouyang
,
M.
Qin
,
D.
Moon
,
A.
Butt
,
J.
Kim
.
2018
.
Phenotype and antimicrobial activity of Th17 cells induced by propionibacterium acnes strains associated with healthy and Acne skin.
J. Invest. Dermatol.
138
:
316
324
.
37
Yu
,
Y.
,
J.
Champer
,
G. W.
Agak
,
S.
Kao
,
R. L.
Modlin
,
J.
Kim
.
2016
.
Different propionibacterium acnes phylotypes induce distinct immune responses and express unique surface and secreted proteomes.
J. Invest. Dermatol.
136
:
2221
2228
.

R.L.G. is a consultant and has equity interest in MatriSys Bioscience and Sente Inc. C.C.Z. owns an international patent on the SZ95 sebaceous gland cell line (WO2000046353). The other authors have no financial conflicts of interest.

Supplementary data