Animals

The Institute of Cancer Research (ICR) and C57BL/6 IL-6 knockout mice (8–9 weeks old, female) were euthanized using 10% CO₂ in a sealed chamber (Boivin 2017; Russell et al. 2021). Mice were housed under controlled environmental conditions at an ambient temperature of 23 ± 2°C in standard cages with wood-chip bedding and ad libitum access to food and water (Russell et al. 2021). Animals were allowed to acclimate to the housing conditions for 3–7 days before data collection. Each experimental group comprised three randomly selected mice (n = 3 per group), and blinding was applied.

Bacterial culture

A single colony of C. acnes bacteria was inoculated into Reinforced Clostridium Medium (RCM) (Oxford, Hampshire, England) and cultured at 37°C under anaerobic conditions until the logarithmic growth phase. Bacterial pellets were collected by centrifugation at 5,000 × g for 10 min, washed, and resuspended in phosphate-buffered saline (PBS).

Cell culture

Sebocytes were cultured in Sebomed Basal Medium (Biochrom, Berlin, Germany) supplemented with 5 ng/ml human recombinant epidermal growth factor (Sigma, St. Louis, MO, USA) and 10% volume/volume (v/v) heatinactivated fetal bovine serum, and then incubated at 37°C in 5% (v/v) CO₂. Cells were cultured to 90% confluence before subculturing. Finally, the cells were seeded at a density of 8 × 10⁵ cells/ml in 10-cm Petri dishes containing 10 ml of media, which was renewed once every 2 days.

Expression of pro-inflammatory IL-6 in sebocytes treated with fatty acid

Sebocytes were cultured in 96-well culture plates and treated with 10 μM palmitoleic acid and 10 μM palmitic acid for 24 h. Samples treated with PBS served as controls. The expression of pro-inflammatory IL-6 in the cell culture medium was measured using enzymelinked immunosorbent assay (ELISA).

Preparation of lipidome metabolome extract using chloroform and methanol

Sebocytes (1 × 10⁷) were treated with 50 μg of recombinant green fluorescent protein (GFP) as a control, or 50 μg of lipase dissolved in 100 μl PBS. Cells were harvested, combined with 1 ml of water and 3.75 ml of a chloroform/methanol mixture (1 : 1, v/v), and then vortexed for 10–15 min. Subsequently, 1.25 ml of chloroform was added, followed by vortexing for 1 min, and then 1.25 ml of water was added and the mixture was vortexed again for 1 min. The sample was centrifuged at 13,000 × rpm for 10 min. The protein disc was carefully pierced with a pipette tip, and the lower organic phase was collected and transferred to a new tube. The organic solvent was evaporated under vacuum at room temperature (RT), and the dried sample was stored at –80°C. Before analysis, the lipid extract was reconstituted in 50 μl of a chloroform/methanol mixture (1 : 1, v/v).

Gas chromatography–mass spectrometry analysis of sebocyte metabolism

Lipid samples were analyzed using an HP 5890 gas chromatograph coupled with an HP 5971A mass spectrometer, an HP 7673A autosampler, and an Agilent Rtx-5MS column (30 m × 0.25 mm × 0.25 μm). A non-polar lipid standard mixture was used for compound identification and calibration. Relative lipid quantities were determined by comparing the single-ion response ratios of each analyte with the nearest eluting internal standard using HP ChemStation software (Agilent Technologies, Santa Clara, CA, United States, USA) for peak normalization (Kao et al. 2021; Keshari et al. 2020). Internal standards compensate for variability during extraction, sample preparation, and instrument response and are widely recommended for gas chromatography–mass spectrometry (GC-MS) analysis (Capoun and Krykorkova 2020). Quality control was maintained using calibration curves generated from multiple concentration levels of the Free Fatty Acids Test Standard (Restek Corporation) and by monitoring chromatographic peak areas or selected ion responses to verify consistent analytical performance (Capoun and Krykorkova 2020; Kao et al. 2021; Keshari et al. 2020).

Molecular cloning and expression of recombinant C. acnes lipase

Plasmids carrying the gene encoding either C. acnes lipase (accession number: YP_056770.1) or GFP (accession number: JS0692; control) were transformed into Escherichia coli (E. coli) BL21 (DE3) competent cells (Invitrogen, Carlsbad, CA, USA). Then, E. coli BL21 (DE3) clones expressing lipase or GFP were selected on Luria-Bertani (LB) (Biokar Diagnostics, Beauvais, France) medium containing 100 μg/ml ampicillin (Sigma-Aldrich, St. Louis, MO, USA). An aliquot of the overnight culture from each group was diluted with LB medium (1 : 10) and incubated at 37°C until it reached an OD₆₀₀ of about 0.6–0.8. Isopropyl-β-D-thiogalactoside (final concentration of 1 mM; Sigma, St. Louis, MO, USA) was added to the culture and incubated for 4 h at 30°C to induce protein expression. The recombinant lipase or GFP with 6 × His tag was purified by a ProBond™ Purification System (Invitrogen Carlsbad, CA, USA), followed by centrifugation with 10,000 molecular weight cut-offs Spin-X^® UF 6 concentrator (Cityva, Little Chalfont, Buckinghamshire, UK). Subsequently, the purified recombinant lipase or GFP was run on a 20% sodium dodecyl sulfate–polyacrylamide gel and stained with Coomassie Brilliant Blue R-250 (Biorad, Hercules, CA, USA) to detect protein expression.

Vaccination of mice and antibody detection

A 50 μg of recombinant GFP or lipase was dissolved in 100 μl PBS and mixed with an equal volume (100 μl) of 2% Alhydrogel (InvivoGen, San Diego, CA, USA). ICR mice were subcutaneously vaccinated on the dorsal skin three times at 2-week intervals. Mice were euthanized 2 weeks after the final vaccination to collect serum samples containing IgG, which were detected by a microplate reader at 450 nm subtracted from 570 nm (OD_570-450). For quantification, serum IgG antibody response was measured by ELISA, by coating 0.1 μg/well of recombinant GFP or lipase in 100 μl PBS at RT overnight. Next, the plate was blocked with 2% skimmed milk in PBS at RT for 1 h, and then incubated with diluted serum from mice vaccinated with GFP or lipase for 1 h. Subsequently, horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG (1 : 10,000, Promega, WI, USA) was added and incubated for 1 h. HRP activity was determined with NeA-Blue tetramethylbenzidine substrate (Clinical Science Product, Mansfield, MA, USA) for 1 h. Finally, the reaction was quenched by adding sulfuric acid as a stop solution (2 N; Merck, Billerica, MA, USA), and the optical density of each well was measured at 450 nm subtracted from 570 nm (OD_570-450). For test strip fabrication, an immunochromatographic strip consisting of a sample pad, conjugate release pad, and absorbent pad was assembled with a nitrocellulose (NC) membrane (Lovászi et al. 2017), with gold nanoparticles incorporated into the assembly. The NC membrane was spotted with Bovine Serum Albumin, anti-lipase or C. acnes serum lysates, and rabbit anti-mouse IgG secondary antibody at the designated (test–lower, control–upper) lines. The sample pad was loaded by soaking in 2.5% (v/v) serum (200 μl).

Immunogenicity of C. acnes lipase

ICR mice (n = 3 per group) were anesthetized using isoflurane (Panion and BF Biotech Inc., Taiwan). The ears of the ICR mice were intradermally injected with either recombinant lipase or GFP (5 μg in 10 μl PBS). Then, ear tissue homogenates were quantified by ELISA to evaluate the expression of pro-inflammatory IL-6.

Immune protection of anti-lipase/C. acnes in mice

Live C. acnes (10⁷ colony-forming units [CFU] in 10 μl of media) incubated with anti-lipase or GFP was subcutaneously injected into the central region of the right ear of the mice, while PBS as a control was injected in the left ear. After 72 h, ear tissue homogenates were collected and evaluated for the expression of proinflammatory IL-6 by ELISA.

Neutralization assays

C. acnes (10⁷ CFU) was pre-incubated with 5% (v/v) of anti-lipase or anti-GFP serum in culture medium at 37°C for 1 h. Complement in the serum was heat-inactivated at 58°C for 30 min. For in vivo neutralization assays, C. acnes (10⁷ CFU) pre-treated with anti-lipase or anti-GFP serum was intradermally injected into the ears of ICR mice and incubated for 72 h. For in vitro neutralization assays, pre-treated C. acnes (10⁷ CFU) was added to sebocyte cultures (8 × 10⁵) and incubated for 12 h (Nakatsuji et al. 2008).

Bacterial loads in the mouse ears

Following the intradermal injection of C. acnes along with either anti-lipase or GFP serum, the thickness of mouse ears was measured at 24, 48, and 72 h post-injection using an electronic digital caliper (Mitutoyo, Kanagawa, Japan). Redness or erythema of the ears was photographed using a USB digital microscope (GM019-#1, Global Camera Manufacturer Corp, Taipei, Taiwan). Ear tissues were then homogenized in 200 μl of sterile PBS using a tissue grinder. Serial dilutions (1 : 10⁰–1 : 10⁵) of the homogenates were plated on RCM agar to determine the CFU of C. acnes. Plates were incubated at 37°C for 3 days under anaerobic conditions using a Gas-Pak system. Colony formation was observed only in the ears injected with C. acnes, while no bacterial growth was detected in PBS-injected control ears.

ELISA

Ear samples from ICR or IL-6 knockout mice were collected 72 h after the injection of GFP/lipase or anti-GFP/lipase serum. Then, ear tissues were excised and homogenized with T-PER™ Tissue Protein Extraction Reagent (Thermo Fisher, Waltham, MA, USA), supplemented with ethylenediaminetetraacetic acid-free protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). The ear homogenates were centrifuged at 15,000 rpm for 30 min at 4°C, and the supernatants were collected. To measure the concentrations of pro-inflammatory IL-6 and MIP-2 in the supernatants of ear homogenates and sebocytes, a sandwich ELISA was performed using a Quantikine mouse IL-6 and MIP-2 kit (R&D Systems, Minneapolis, MN, USA).

Statistical analysis

Data are presented as the mean ± standard deviation from a minimum of three independent experiments. Statistical significance was evaluated using an unpaired t-test or a one-way analysis of variance followed by Tukey’s post hoc analysis, performed with SigmaStat software (Jandel Scientific, Palo Alto, CA, USA). P-values of < 0.05 (*), < 0.01 (**), and < 0.001 (***) were considered statistically significant. Different letters denote significant differences between groups (p < 0.05) (Negari et al. 2021).

GC–MS analysis of fatty acids in sebocytes and cytokine release

To identify fatty acids in sebocytes, cells (1 × 10⁷) were treated with 50 μg of recombinant GFP or lipase dissolved in 100 μl PBS. For targeted metabolite analysis, all detected metabolites had been previously characterized using GC-MS standards, based on accurate mass and retention time. For each compound, the peak area was determined from the extracted ion chromatogram corresponding to its specific retention time. Twelve compounds were detected in both lipase and GFP groups (Supplementary Table 1), demonstrating that lipase hydrolyzes lipids in sebocytes into a variety of fatty acids. Notably, palmitoleic and palmitic acid concentrations were significantly increased in the lipase group (Figures 1A and 1B). Several studies have reported that lipase can break down lipids into palmitoleic and palmitic acids, which may be associated with acne severity (Zhou et al. 2013; Jung et al. 2021; de Souza Pereira 2023; Al-Saedi et al. 2022). Furthermore, we investigated the potential role of palmitoleic and palmitic acids in inducing inflammatory responses in sebocytes by measuring the production of pro-inflammatory IL-6. In the presence of palmitic acid, pro-inflammatory IL-6 expression was significantly upregulated compared to the PBS-treated control (Figure 1C). A 10 μM concentration was selected for both palmitic and palmitoleic acids, as previous studies have shown that this concentration is physiologically relevant and can induce measurable biological responses, including inflammatory signaling and lipid metabolism regulation, without causing significant cytotoxicity (Ishikawa et al. 2026; Murakami et al. 2020). Additionally, a 24-h incubation period was selected, as it is widely applied in in vitro studies to allow for sufficient cellular response and gene expression changes following fatty acid treatment (Chlebicz and Śliżewska 2020; Figueiredo et al. 2017; Hipp et al. 2017). Conversely, sebocytes treated with palmitoleic acid showed a less pronounced difference in pro-inflammatory IL-6 levels. This finding demonstrates that sebocytes respond specifically to palmitic acid stimulation by upregulating inflammatory enzyme expression and increasing the production and secretion of pro-inflammatory cytokines. This aligns with previous research indicating that palmitic acid elevates intracellular lipid accumulation and stimulates pro-inflammatory IL-6 secretion in sebocytes (Choi et al. 2019; Törőcsik et al. 2021).

Figure 1

Gas chromatography–mass spectrometry analysis of fatty acids in sebocytes incubated with lipase or green fluorescent protein (GFP) for 24 h. (A) Mass spectra and retention times indicated palmitoleic acid, palmitic acid, and cholesterol. (B) The ion chromatogram and mass spectrum of palmitoleic and palmitic acids. (C) Sebocytes were treated with phosphate-buffered saline (PBS) as a control, 10 μM palmitoleic acid, or 10 μM palmitic acid for 24 h. The production of proinflammatory interleukin-6 (IL-6) was quantified by enzymelinked immunosorbent assay (ELISA). Different letters denote significant differences between groups (p < 0.05; n = 3)

Antibody production in mice vaccinated with C. acnes lipase

To examine antibody titers to lipase and validate the test strip fabrication, we vaccinated ICR mice with E. coli overexpressing lipase or GFP. Two weeks postvaccination, serum from lipase-immunized mice exhibited significantly higher IgG titers against lipase than serum from GFP-immunized controls, indicating that lipase exhibits immunogenic properties and can elicit a robust immune response. This result is supported by previous studies demonstrating that lipase influences immune activity in acne vulgaris (Ahle et al. 2023; Kovács et al. 2023). Serial dilution of the antibody titer to lipase (1 : 51,200) was performed to detect and quantify antibodies (Figure 2A). Moreover, immunochromatographic test strips were developed by coating with GFP, lipase, or lysates of C. acnes. Serum collected from mice vaccinated with lipase (Figure 2B) or lysates of C. acnes (Figure 3) was applied to the test strips. As shown in Figures 2B, and 3, lipase or lysates of C. acnes, but not GFP, were recognized by antibodies in both anti-lipase serum and C. acnes, suggesting that vaccination with lipase provoked antibodies that can cross-react with C. acnes.

Figure 2

The production of antibodies to lipase. The Institute of Cancer Research mice were subcutaneously vaccinated with lipase or green fluorescent protein (GFP) and exhibited detectable antibodies immunoglobulin G (IgG) to lipase. (A) The titer of antibodies to lipase. (B) The production of antibodies to lipase or GFP and their cross-reactivity with mouse serum to lipase were detected in test strips with NC membranes spotted with lipase or GFP (lower position) and rabbit anti-mouse IgG secondary antibody (upper position). Data are expressed as mean ± standard deviation (*p < 0.05; **p < 0.01; ***p < 0.001 vs. control, n = 3)

Figure 3

The production of antibodies to Cutibacteriumacnes (C. acnes). The Institute of Cancer Research mice were subcutaneously vaccinated with lipase, green fluorescent protein (GFP), or C. acnes and exhibited detectable antibodies immunoglobulin G (IgG) to lipase and C. acnes. The production of antibodies to lipase, GFP, or C. acnes and their cross-reactivity with mouse serum to C. acnes were detected in test strips with NC membranes spotted with lipase, GFP, or C. acnes (lower position) and rabbit anti-mouse IgG secondary antibody (upper position)

Immunogenicity of C. acnes lipase in mice

To investigate lipase as an immunogenic antigen and confirm its virulence, mice were intradermally injected with 50 μg of lipase or GFP into the right or left ears, respectively, and ear thickness was measured after 24, 48, and 72 h post-injection (Figure 4A). The ears of mice injected with lipase exhibited a relatively high level of inflammation, characterized by visible swelling and increased ear thickness, compared to those of mice injected only with GFP. These findings suggest that lipase plays a key role in virulence activity, as demonstrated by the localized inflammation in mice following lipase injection. Mice injected with lipase also exhibited significantly elevated levels of pro-inflammatory IL-6 (Figure 4B) and MIP-2 (Figure 5), suggesting that lipase possesses high virulence potential and induces excessive infection. Previous studies have shown that pro-inflammatory IL-6 and MIP-2 expression mediates acute-phase responses and drives innate immune activation (Josse et al. 2020; Flori et al. 2023; Negari et al. 2025). These findings indicate that C. acnes lipase exhibits immunogenic properties in mice vaccinated with the recombinant C. acnes lipase. This aligns with other research reporting that C. acnes lipase is immunogenic and contributes to the inflammatory processes involved in acne vulgaris (Mayslich et al. 2021).

Figure 4

The immunogenicity of Cutibacterium acnes lipase in mice. The ears of mice were intradermally injected with 50 μg lipase or green fluorescent protein (GFP) and observed for 72 h. (A) Ear morphology and changes in ear thickness (mm) are shown. Scale bar = 1 mm. (B) The production of pro-inflammatory interleukin-6 (IL-6) was quantified by enzyme-linked immunosorbent assay (ELISA). Data are expressed as mean ± standard deviation (*p < 0. 05; **p < 0.01; ***p < 0.001 vs. control, n = 3)

Figure 5

The production of macrophage inflammatory protein-2 (MIP-2) and the immunogenicity of Cutibacterium acnes lipase in mice. The ears of mice were intradermally injected with 50 μg lipase or green fluorescent protein (GFP) and observed for 72 h. The production of MIP-2 was quantified by enzyme-linked immunosorbent assay (ELISA). Data are expressed as mean ± standard deviation (*p < 0.05; **p < 0.01; ***p < 0.001 vs. control, n = 3)

Protective immunity by anti-lipase/C. acnes in mice

To evaluate the protective immune efficacy and assess the effectiveness of lipase vaccination in reducing C. acnes-induced inflammation, 10 μl of C. acnes (10⁷ CFU) was administered intradermally into the right ears of mice vaccinated with anti-lipase or GFP, while the left ears were injected with 10 μl of PBS to serve as controls. Inflammatory responses were monitored at 24, 48, and 72 h post-injection. As shown in Figure 6A, mice injected with anti-lipase/C. acnes showed significantly reduced ear redness and thickness, compared to mice injected with anti-GFP/C. acnes. Furthermore, the bacterial load, determined by CFU/ml counts from serial dilutions (1 : 10⁰–1 : 10⁵) of ear homogenates, was notably lower in the anti-lipase/C. acnes group (Figure 6B). Given that acne vulgaris is characterized as an inflammatory skin condition (Ahle et al. 2023; Dréno et al. 2018; Kim et al. 2020), we further investigated whether anti-lipase/C. acnes treatment influenced the production of pro-inflammatory IL-6 in mice (Figure 6C). As expected, anti-lipase/C. acnes treatment markedly decreased the production of pro-inflammatory IL-6, suggesting that vaccination with C. acnes lipase elicited protective immunity against C. acnes by neutralizing its secretory virulence activity.

Figure 6

Immune protection by anti-lipase/Cutibacterium acnes (C. acnes). C. acnes (10⁷ CFU) or phosphate-buffered saline (PBS) was injected into the right or left ears, respectively, of mice vaccinated with lipase or green fluorescent protein (GFP) and incubated for 72 h. (A) Ear morphology and changes in ear thickness (mm) are shown. Scale bar = 1 mm. (B) The CFUs (log₁₀CFU/ml) were enumerated by plating serially diluted (1:10⁰–1:10⁵) ear homogenates on agar plates. (C) The production of pro-inflammatory interleukin-6 (IL-6) was quantified by enzyme-linked immunosorbent assay (ELISA). Data are expressed as mean ± standard deviation (*p < 0.05; **p < 0.01; ***p < 0.001 vs. control, n = 3)

Passive immunization with neutralization antibodies to C. acnes lipase

We analyzed whether passive immunization with neutralization antibodies to C. acnes lipase can decrease C. acnes-induced inflammation. To achieve this, anti-lipase or anti-GFP serum was heated at 58^oC for 30 min to inactivate other protein complements in the serum. The complements were pre-treated with 5% (v/v) inactivated anti-lipase serum or GFP in the PBS solution at 37°C for 1 h. Subsequently, the serum was added to C. acnes (10⁷ CFU) in media and incubated at 37^oC for 1 h. The resulting mixture was intradermally injected into the right or left ears of mice, and inflammation was evaluated at 24, 48, and 72 h post-injection. Results showed that the ears injected with anti-lipase serum neutralized C. acnes and exhibited less inflammation in terms of both redness and thickness, compared with ears treated with anti-GFP serum (Figure 7A). Moreover, the anti-lipase serum significantly reduced bacterial colony counts (CFU/ml), as determined from serially diluted samples (1 : 10⁰–1 : 10⁵) of ear tissue homogenates, compared to the anti-GFP serum (Figure 7B). We further analyzed the levels of pro-inflammatory cytokines in the lipase-injected ears and observed decreased levels of IL-6 (Figure 7C) and MIP-2 (Figure 8). In addition, we evaluated the reduction of MIP-2 in the IL-6 knockout mouse (Figure 9) to determine whether this pro-inflammatory cytokine is influenced by C. acnes and lipase induction, thereby indicating whether MIP-2 may also serve as a marker of C. acnes-induced inflammation. This hypothesis is supported by the results shown in Figure 9, where IL-6 knockout mice injected with C. acnes or lipase exhibited visible ear redness and increased ear thickness (Figure 9A). According to previous studies, the levels of MIP-2 contributes significantly to the activation of innate immunity, and the elevated levels of MIP-2 cytokines induced by lipase in acne vulgaris may result in severe inflammation and the production of other proinflammatory cytokines (Wang et al. 2018; Parvin et al. 2020; Marito et al. 2020; Kao et al. 2022). Therefore, we analyzed the expression of MIP-2 to confirm its key role in C. acnes-induced inflammation. As shown in Figure 9B, the inflammatory response in mice injected with C. acnes was higher than that in mice injected with lipase alone, as indicated by elevated MIP-2 production. These findings suggest that passive immunization with neutralizing antibodies against lipase efficiently inhibits the secretion of the pro-inflammatory cytokines IL-6 and MIP-2, while simultaneously reducing bacterial colonization. Additionally, a marked increase in MIP-2 production was observed in IL-6 knockout mice, highlighting a compensatory inflammatory response in the absence of IL-6.

Figure 7

Neutralization with anti-lipase serum decreases Cutibacterium acnes (C. acnes)-induced bacterial growth and the production of pro-inflammatory interleukin-6 (IL-6) in mice. C. acnes was pre-treated with 5% (v/v) anti-lipase or green fluorescent protein (GFP) serum at 37°C for 1 h. Pre-treated C. acnes (10⁷ CFU) was then applied onto the ears of the Institute of Cancer Research mice and incubated for 72 h. (A) Ear morphology and changes in ear thickness (mm) are shown. Scale bar = 1 mm. (B) The CFUs (log₁₀CFU/ml) were enumerated by plating serially diluted (1:10⁰–1:10⁵) ear homogenates on agar plates. (C) The production of pro-inflammatory IL-6 was quantified by enzyme-linked immunosorbent assay (ELISA). Data are expressed as mean ± standard deviation (*p < 0.05; **p < 0.01; ***p < 0.001 vs. control, n = 3)

Figure 8

Neutralization with anti-lipase serum decreases Cuti bacterium acnes (C. acnes)-induced the production of macrophage inflammatory protein-2 (MIP-2) in mice. C. acnes was pre-treated with 5% (v/v) anti-lipase or green fluorescent protein (GFP) serum at 37°C for 1 h. Pre-treated C. acnes (10⁷ CFU) was then applied onto the ears of the Institute of Cancer Research mice and incubated for 72 h. The production of MIP-2 was quantified by enzyme-linked immunosorbent assay (ELISA). Data are expressed as mean ± standard deviation (*p < 0.05; **p < 0.01; ***p < 0.001 vs. control, n = 3)

Figure 9

The reduction of macrophage inflammatory protein-2 (MIP-2) in the pro-inflammatory interleukin-6 (IL-6) knockout mouse. The ears of the pro-inflammatory IL-6 knockout mice were intradermally injected with lipase as a control or Cutibacterium acnes (10⁷ CFU) and observed for 24 h. (A) Ear morphology and changes in ear thickness (mm) are shown. Scale bar = 1 mm. (B) The production of MIP-2 was quantified by enzyme-linked immunosorbent assay (ELISA). Data are expressed as mean ± standard deviation (*p < 0.05; **p < 0.01; ***p < 0.001 vs. control, n = 3)

Thus, we also evaluated the ability of the anti-lipase serum to inhibit C. acnes-induced IL-6 expression in sebocytes. Cells exposed to C. acnes pre-treated with either anti-lipase or anti-GFP serum secreted IL-6, with notable differences observed between the two conditions (Figure 10). These results demonstrated that passive immunization with neutralizing anti-lipase serum effectively decreases C. acnes-induced IL-6 production in sebocytes. This finding aligns with previous studies indicating that sebocytes located in the sebaceous glands serve as the primary targets for C. acnes in individuals affected by acne vulgaris (Schneider and Zouboulis 2018).

Figure 10

Neutralization with anti-lipase serum decreases Cutibacterium acnes (C. acnes)-induced the production of pro-inflammatory interleukin-6 (IL-6) in sebocytes. C. acnes was pre-treated with 5% (v/v) anti-lipase or green fluorescent protein (GFP) serum at 37°C for 1 h. Pre-treated C. acnes (10⁷ CFU) was then co-cultured with sebocytes (8 × 10⁵) for 12 h. The production of pro-inflammatory IL-6 was quantified by enzyme-linked immunosorbent assay (ELISA). Data are expressed as mean ± standard deviation (*p < 0.05; **p < 0.01; ***p < 0.001 vs. control, n = 3)