Establishment of a Dynamic Ear Inflammation Model in Rats for Acne Vulgaris and Evaluation of Adjuvanted Inactivated Cutibacterium acnes-Based Vaccines Efficacy

TL;DR

Researchers created a rat model of acne inflammation and tested vaccines that target the bacteria responsible for acne, showing promising results in reducing inflammation and bacterial load.

Contribution

A novel rat ear inflammation model for acne and evaluation of adjuvanted inactivated C. acnes vaccines for acne treatment.

Findings

Inflammation in the rat ear model peaked within 1-3 days and resolved by day 7 with bacterial clearance.

Vaccinated rats showed higher IgG levels, reduced swelling, and lower bacterial burden upon challenge.

Vaccine-induced protection was linked to humoral immunity and suppression of inflammatory mediators.

Abstract

Background/Objectives: Acne vulgaris is a chronic inflammatory skin disorder characterized by sebaceous gland hyperactivity, follicular hyperkeratinization, proliferation of Cutibacterium acnes (C. acnes), and subsequent inflammation. The development of effective therapeutics necessitates reliable preclinical models that accurately replicate key pathological aspects of the human disease. Methods: In this study, we established an inflammatory acne model in Wistar rats via the intradermal injection of live C. acnes into the ear pinnae and thoroughly characterized its temporal dynamics of the induced inflammation. Utilizing this model, we evaluated the protective efficacy of a whole-cell inactivated C. acnes vaccine (HI-C. acnes) formulated with adjuvants WS03 or MA107b. Results: Inflammation peaked between days 1 and 3 post-infection, manifesting as pronounced erythema, ear swelling, increased ear thickness, elevated bacterial load, and significant upregulation of pro-inflammatory cytokines (IL-6, IL-1β, and MCP-1). Histopathological examination revealed extensive neutrophil infiltration and microabscess formation, while immunohistochemistry confirmed localized overexpression of TNF-α, IL-1β, and CXCL1 within the lesional tissue. Inflammatory manifestations gradually subsided by day 5 and were fully resolved by day 7, which coincided with complete bacteria clearance and normalization of pro-inflammatory cytokine levels. Vaccinated rats developed significantly higher C. acnes-specific IgG titers and, upon challenge, exhibited markedly reduced ear swelling, diminished bacterial burden, and suppressed expression of key inflammatory mediators compared to control groups, indicating that vaccine-induced protection is associated with humoral immunity. Conclusions: Collectively, our standardized and quantifiable rat ear inflammation model provides a robust platform for mechanistic investigations and preclinical assessment of novel anti-acne vaccines and therapeutic agents.

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Full text

1. Introduction

Acne vulgaris is the eighth most prevalent disease globally, affecting approximately 9.4% of the population [1,2]. Specifically, it is a chronic inflammatory dermatosis involving the pilosebaceous unit. Key contributing factors include excess sebum production, abnormal follicular keratinization, colonization by C. acnes, and dysregulated inflammation and immune responses [3,4]. Acne vulgaris is characterized by high incidence, prolonged duration, recurrence, and scarring, which can impact patients’ appearance, psychosocial well-being, and quality of life [5].

Animal models are indispensable in the preclinical stage of studying acne pathogenesis and evaluating novel therapies. Their necessity is threefold: first, human studies face ethical and technical limitations, preventing dynamic, invasive sampling to dissect the changes in the follicular microenvironment. The multifactorial nature of acne (genetics, hormones, environment) is also difficult to control for in clinical studies [6]. Second, animal models provide a controllable, reproducible system to precisely simulate key disease aspects, enabling systematic sampling at defined timepoints to elucidate the causal relationships among etiology, pathology, and phenotype and validate therapeutic targets. Finally, any new drug, biologic, or vaccine must undergo comprehensive preclinical efficacy and safety assessments in an animal model that approximates human disease complexity before human trials. This preclinical evaluation is crucial for ensuring predictive preclinical value, as well as mitigating the risks and costs of drug development.

Due to interspecies differences in the sebaceous glands, hair follicles, skin microbiota, and immune responses, different animals yield different modeling outcomes. Species commonly used for acne research include rabbits [7,8,9] and mice [10,11,12]. The rabbit ear model has significant limitations: its skin differs from human facial skin in sebaceous gland structure and follicular distribution, which often exhibits exaggerated reactions and leads to false positives. It also fails to adequately replicate key aspects of human acne like bacterial proliferation, sebum secretion, and chronic inflammation [13]. In contrast, commonly used mouse acne models also have several constraints. Most rely on a single intervention (e.g., bacterial infection, chemical stimulus), failing to recapitulate the complex multi-factorial pathological interactions in humans. Hair-bearing mice require frequent shaving, and hair growth interferes with observation, while hairless mice often have atrophic sebaceous glands, differing significantly from human skin. Their small size complicates experimental procedures and hinders continuous, objective observation and assessment of acne progression, thereby introducing subjectivity in evaluations [14].

This study aimed to establish a standardized and quantifiable animal model that recapitulates complex in vivo pathology, enabling multi-dimensional, dynamic, and systematic evaluation, thereby providing a platform for subsequent mechanistic and therapeutic research. Accordingly, this C. acnes-induced inflammatory model in rats simulates the inflammatory processes in acne patients and was utilized to assess novel prophylactic or therapeutic acne vaccine candidates.

2. Materials and Methods

2.1. Ethics Approval

All animal experiments were performed using specific pathogen-free (SPF) male Wistar rats (175–200 g) sourced from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The animals were housed under SPF conditions in the Experimental Animal Facility at the Wuhan Institute of Biological Products Co., Ltd. (WIBP) (Wuhan, China). All experimental procedures were conducted for research purposes at the WIBP Animal Experiment Center and were approved by the Institutional Animal Care and Use Committee (IACUC) (Approval No. WIBP-AII382025001). The study adhered to internationally recognized guidelines and regulations for the humane care and use of laboratory animals. Additionally, our study follows ARRIVE guidelines for animal research reporting.

2.2. Bacterial Strain

Cutibacterium acnes (Scholz and Kilian) ATCC 6919 was obtained from the American Type Culture Collection (ATCC) (Virginia, USA). The bacterium was cultured anaerobically in Reinforced Clostridial Medium (RCM) (Hope Bio-Technology, Qingdao, Shandong, China). Cultures were prepared in RCM broth contained in test tubes overlaid with sterile liquid paraffin to maintain anaerobic conditions and incubated at 37 °C in a 5% CO_2_ incubator for 3–5 days [15]. For experimental use, single colonies from freshly revived or subcultured RCM agar plates were inoculated into RCM broth and incubated under anaerobic conditions at 37 °C for 3–5 days to reach mid-logarithmic phase. Bacterial cells were harvested by centrifugation at 3000 rpm for 10 min. The supernatant was discarded, and the resulting pellet was washed twice with sterile phosphate-buffered saline (PBS). The final bacterial pellet was resuspended in an appropriate volume of sterile PBS to achieve the desired concentration. Bacterial concentration was quantified using the standard plate count method. Briefly, the suspension was subjected to 10-fold serial dilutions (e.g., 10^−1^, 10^−2^, 10^−3^). From each dilution, 100 µL was plated in triplicate onto RCM agar plates. Plates were sealed in anaerobic bags containing gas-generating sachets (Mitsubishi Gas Chemical Company, Inc., Tokyo, Japan) and incubated at 37 °C for 3–5 days. Colonies were counted on plates yielding 50–200 CFUs, and the original concentration was calculated as colony-forming units per milliliter (CFU/mL) based on the dilution factor and plated volume.

2.3. Animal Infection and Immunization

The animals were randomized using stratified block randomization, with body weight as the stratification factor to ensure baseline balance among groups. For outcome measurements (ear thickness and histopathology scores), blinded assessments were performed by investigators unaware of the experimental group allocations.

An ear infection model was established in Wistar rats. Each animal received a 50 µL intradermal injection of phosphate-buffered saline (PBS) into the left ear as an internal control, while the right ear was injected with 50 µL of a C. acnes suspension (8 × 10^9^ CFU/mL). Clinical and pathological assessments were performed at days 1, 3, 5, and 7 post-infection (n = 6 rats per time point). C. acnes whole cells were inactivated by heating at 56 °C for 30 min to generate the HI-C. acnes vaccine antigen. Wistar rats (n = 5 per group) were randomly assigned to one of six groups: PBS, WS03 adjuvant alone, MA107b adjuvant alone, HI-C. acnes alone, WS03 + HI-C. acnes, or MA107b + HI-C. acnes. WS03 was provided by WIBP, and MA107b was kindly gifted by Chengdu Huarenkang Biotechnology Co., Ltd. (Chengdu, China). Specifically, WS03, an AS03-like adjuvant, is an oil-in-water emulsion adjuvant composed of squalene, α-tocopherol, and polysorbate 80, with the capacity to activate innate immunity, enhance antigen presentation, and strongly promote humoral immunity. MA107b, an AS01-like adjuvant, is a liposomal adjuvant containing monophosphoryl lipid A (MPL) and Quillaja saponaria saponin QS-21, which synergistically induces robust and balanced humoral immunity (elevated IgG titers) and cellular immunity. All animals received three intramuscular immunizations (0.5 mL per dose) at 14-day intervals.

2.4. Ear Thickness Measurement

Ear thickness was measured using a digital caliper (SATA Tools (Shanghai) Co., Ltd., Shanghai, China). The caliper jaws were gently positioned at the midpoint of the most inflamed region of the rat ear (or a at the corresponding anatomical site in control animals), and the thickness was recorded in millimeters.

2.5. Bacterial Load

Ear tissues were immediately placed in 1.5 mL microcentrifuge tubes containing 1 mL of ice-cold PBS. Tissues were first minced with sterile scissors and then homogenized using a sterile tissue grinder. The resulting homogenate was subjected to 10-fold serial dilutions. From each dilution, 100 µL was plated in triplicate onto RCM agar plates. Plates were incubated anaerobically at 37 °C for 3–5 days. Colonies were counted on plates yielding 50–200 CFUs, and the bacterial load was expressed as colony-forming units per milliliter (CFU/mL) of tissue homogenate, calculated based on the dilution factor and plated volume.

2.6. Inflammatory Cytokine Levels

For tissue processing, rat ear tissues were isolated under aseptic conditions and immediately immersed in pre-chilled sterile phosphate-buffered saline (PBS) for rinsing to eliminate surface contaminants. Subsequently, the cleaned ear tissues were transferred to a grinding tube containing 1 mL of sterile PBS, followed by thorough homogenization on an ice bath using a tissue homogenizer. The homogenization was performed at a rotation speed of 4000 rpm for 30 s, and this procedure was repeated three times with ice bath cooling intervals to prevent tissue homogenate denaturation caused by heat generation. After homogenization, the tissue homogenate was centrifuged at 5000 rpm for 5 min at 4 °C. The resulting supernatant was carefully collected and stored appropriately until subsequent experimental assays.

Cytokine concentrations in ear tissue homogenates were quantified using the LEGENDplex™ Rat Inflammation Panel (13-plex) kit (BioLegend, San Diego, CA, USA) according to the manufacturer’s instructions. All reagents and samples were equilibrated to room temperature for at least 30 min prior to use. Lyophilized standards were reconstituted and serially diluted in a 4-fold manner to generate the standard curve. To each well of the assay plate, 25 µL of assay buffer, standards, or samples was added, followed by 25 µL of the premixed capture bead cocktail. The plate was sealed, wrapped in foil to protect from light, and incubated on an orbital shaker for 2 h at room temperature. After two washes with wash buffer, 25 µL of detection antibody mixture was added per well, and the plate was incubated for 1 h with shaking. Subsequently, 25 µL of streptavidin–phycoerythrin (SA-PE) was added to each well, followed by a 30 min incubation under shaking conditions. Following two final washes, the beads were resuspended in 120 µL of wash buffer and analyzed on a flow cytometer CytoFlex S (Beckman Coulter, Inc., Brea, CA, USA). Cytokine concentrations were determined by interpolating sample median fluorescence intensities against the standard curve using the LEGENDplex™ data analysis software version 8.0 (BioLegend, San Diego, CA, USA).

2.7. Histopathology and Immunohistochemistry (IHC)

At days 0, 1, 3, 5, and 7, rats were humanely euthanized, and both ears were harvested for histopathological analysis. Tissue samples were fixed in 4% paraformaldehyde (pH 7.4) for 24 h, then dehydrated through a graded series of ethanol solutions, cleared in xylene, infiltrated with paraffin, and embedded. Paraffin blocks were sectioned at 4–5 µm thickness, floated in a 40 °C water bath, and mounted onto adhesive-coated slides before being baked at 60 °C for 1 h. Hematoxylin and eosin (H&E) staining was performed using a Leica ST5010 Autostainer XL (Leica Biosystems Nussloch GmbH, Nussloch, Germany) according to standard protocols. Sections were examined under a light microscope, and pathological changes were systematically recorded and scored based on established pathological scoring criteria [16]. The scoring system focused on key indicators including neutrophil infiltration, microabscess formation, and follicular structural damage, ensuring objective evaluation of inflammatory responses.

For IHC, baked slides were loaded onto a Leica Bond RX automated stainer. The protocol comprised the following steps: automated deparaffinization and rehydration, followed by heat-induced epitope retrieval using either Bond Epitope Retrieval Solution 1 (ER1; 95 °C for 20 min) or ER2 (100 °C for 5 min), selected based on antibody validation requirements. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 5 min at room temperature. Primary antibodies—rabbit anti-TNF-α (1:300), rabbit anti-IL-1β (1:100), and rabbit anti-CXCL1 (1:100)—were applied and incubated for 30 min at room temperature. This was followed by sequential incubation with the secondary antibody and polymer reagents from the Bond Polymer Refine Detection Kit (8 min each, room temperature). Immunoreactivity was visualized using 3,3′-diaminobenzidine (DAB) chromogen for 5 min at room temperature, and nuclei were counterstained with hematoxylin. Finally, slides were dehydrated through a graded ethanol series, cleared in xylene, and mounted with permanent mounting medium. Stained sections were examined and imaged using a light microscope to assess the expression and spatial distribution of target inflammatory mediators. Stained sections were examined and imaged using a light microscope (consistent with H&E staining procedures) to assess the expression level and spatial distribution of target inflammatory mediators.

2.8. Antibody Detection

Serum C. acnes-specific IgG antibodies were quantified by enzyme-linked immunosorbent assay (ELISA). Briefly, C. acnes was cultured, harvested by centrifugation, washed, and resuspended in lysis buffer (50 mM Tris–HCl, 100 mM NaCl, 2% SDS, 0.5 mM PMSF) to a concentration of 6 × 10^6^ CFU/mL. The bacterial suspension was sonicated on ice using a probe sonicator (Ningbo Scientz Biotechnology Co., Ltd., Ningbo, China) (400 W; 5 s pulse, 5 s pause, for 3 cycles) to ensure complete lysis and antigen solubilization. ELISA plates were coated with an equivalent of 6 × 10^5^ CFU of lysed antigen per well in carbonate-bicarbonate coating buffer (pH 9.6) and incubated overnight at 4 °C. Following three washes with PBST (PBS containing 0.05% Tween-20), plates were blocked with 1% bovine serum albumin (BSA) in PBS for 1 h at 37 °C. After additional washes, serial two-fold dilutions of rat serum (starting at 1:100, for a total of 8 dilution steps) were added to the wells and incubated for 1 h at 37 °C. Plates were then washed and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rat IgG (1:10,000) for 1 h at 37 °C. After a final series of washes, the reaction was developed with 3,3′,5,5′-tetramethylbenzidine (TMB) substrate for 30 min at 37 °C in the dark. The enzymatic reaction was stopped by adding 2 M H_2_SO_4_, and absorbance was measured at 450 nm using a microplate reader. The endpoint IgG titer was defined as the highest serum dilution yielding an optical density (OD_450_) equal to or greater than the pre-established cut-off value of 0.15, which was determined based on background signal from naïve rat sera.

2.9. Statistical Analysis

Statistical analyses and graphical presentations were performed using GraphPad Prism version 9.0 (GraphPad Software, Inc., La Jolla, CA, USA). Comparisons between two independent groups were conducted using the Mann–Whitney U test. A p-value of less than 0.05 was considered statistically significant. Post hoc power analysis was conducted via G*Power software version 3.1.9.7 (Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany) for the datasets involved in the differential statistical comparisons. The results demonstrated that all calculated power values reached or exceeded the threshold of 0.8, indicating sufficient statistical power to detect potential group differences.

3. Results

3.1. Inflammatory Phenotype Evaluation in the Wistar Rat Acne Model

To establish an inflammation model, live C. acnes was intradermally inoculated into the ears of Wistar rats. Macroscopic evaluation revealed a characteristic time-dependent inflammatory response in the inoculated group compared to untreated (naïve) and PBS control groups (Figure 1A). Erythema at the injection site became evident as early as day 1 post-challenge. Between days 1 and 3, both the extent and intensity of erythema progressively increased, with visible pustule formation apparent by day 3. Thereafter, the inflammation entered a resolution phase: erythema and pustules gradually diminished, and by day 7, macroscopic signs of inflammation were nearly absent, indicating complete clinical resolution.

To objectively quantify the inflammatory response, we monitored dynamic changes in ear thickness following C. acnes challenge (Figure 1B). Compared with the naive and PBS control groups, ear thickness in the C. acnes-challenged group increased significantly (p < 0.01), peaking on day 3, which was consistent with the peak of macroscopic inflammation. Subsequently, ear swelling gradually subsided, with thickness measurements showing a progressive reduction on days 5 and 7. This objective measurement provides a quantitative temporal profile of C. acnes-induced inflammation in this model.

3.2. Bacterial Load and Inflammatory Cytokines Levels in Ear Tissue

To elucidate the local dynamics of C. acnes following inoculation, we quantified bacterial loads in ear tissue at multiple time points post-challenge (Figure 2A). In the model group, a high initial bacterial burden-approximately 10^8^ CFU/mL-was detected on day 1 after intradermal inoculation. The bacterial load declined steadily over time, reaching nearly undetectable levels by day 7. In contrast, no significant bacterial growth was observed in the PBS control group throughout the observation period. Meanwhile, we assessed the temporal dynamics of key pro-inflammatory cytokines in ear tissue following C. acnes challenge (Figure 2B–D). Compared with naïve and PBS control groups, the C. acnes-challenged group exhibited a consistent and rapid upregulation of interleukin-6 (IL-6), interleukin-1β (IL-1β), and monocyte chemoattractant protein-1 (MCP-1), with all three mediators peaking on day 1 post-challenge. Subsequently, their levels declined progressively and returned to near-baseline values by day 7, levels that were comparable to those in control groups. These findings reveal the kinetic profiles of bacterial burden and pro-inflammatory cytokine expression in rat ears following C. acnes infection, which closely parallel the observed clinical manifestations.

3.3. Histopathological Observations and Immunohistochemical Assay

Hematoxylin and eosin (H&E) staining of ear skin revealed normal tissue architecture with no significant pathological alterations in the naïve and PBS control groups (Figure 3A). In contrast, the C. acnes-challenged group displayed hallmark features of acute inflammation. As early as day 1 post-challenge, histological examination showed epidermal hyperplasia, dermal edema, and dense inflammatory cell infiltration—predominantly neutrophils—with microabscess formation evident in focal areas. Over time (days 3, 5, and 7), the intensity of inflammatory infiltration progressively decreased but remained detectable. Semi-quantitative histopathological scoring (Table 1) further corroborated these observations, demonstrating significantly elevated pathology scores in the challenged group during the early phase (days 1 and 3) compared to controls, followed by a gradual decline that aligned with the temporal resolution of macroscopic inflammation and reductions in ear thickness. Two independent pathologists, who were blinded to group assignments, scored all histopathological sections according to the established criteria. Cohen’s Kappa coefficient was calculated to assess the consistency between the two scorers, and the result (kappa = 0.862) indicates good inter-rater consistency, confirming the objectivity and reproducibility of the scoring system.

Immunohistochemical (IHC) analysis of key inflammatory mediators revealed their distinct expression patterns and spatial localization in ear skin (Figure 3B–D). Compared with naïve and PBS control groups, the C. acnes-challenged group exhibited significantly elevated protein levels of TNF-α, IL-1β, and CXCL1. These mediators displayed characteristic spatial distributions: TNF-α and IL-1β were predominantly localized within regions of dense inflammatory cell infiltration on day 1 post-challenge, whereas CXCL1 showed strong perifollicular expression, closely co-localizing with neutrophil aggregates observed in H&E-stained sections. These findings underscore the coordinated upregulation and anatomical compartmentalization of pro-inflammatory signals during C. acnes-induced inflammation.

3.4. Humoral Immune Response Induced by HI-C. acnes Vaccine

To evaluate vaccine-induced humoral immunity, we measured serum levels of C. acnes-specific IgG antibodies at multiple time points following a three-dose immunization regimen (Figure 4). On day 14 post-primary immunization, IgG titers in the combined vaccine groups (WS03 + HI-C. acnes and MA107b + HI-C. acnes) did not differ significantly from those in their respective single-component control groups (WS03 or MA107b alone; p > 0.05; Figure 4A). By day 28, the WS03 + HI-C. acnes group exhibited a significantly higher IgG titer compared to the WS03-only group (p < 0.05; Figure 4B), whereas no significant difference was observed between the MA107b + HI-C. acnes and MA107b-only groups at this time point. Following completion of the full immunization schedule on day 42, both combined vaccine groups demonstrated markedly elevated IgG levels relative to their corresponding single-component controls (p < 0.01; Figure 4C). These findings indicate that co-administration of whole-cell inactivated C. acnes (HI-C. acnes) with adjuvants WS03 or MA107b elicits a stronger and more sustained antigen-specific humoral immune response.

3.5. HI-C. acnes Vaccine Attenuates Ear Swelling and Thickness

To evaluate the immunoprotective efficacy of the whole-cell inactivated vaccine, we assessed the inflammatory phenotype across immunization groups following C. acnes challenge (Figure 5A). Prior to challenge, all control groups-receiving three doses of PBS, adjuvant alone (WS03 or MA107b), or antigen alone (HI-C. acnes)-showed no signs of baseline inflammation or tissue damage, comparable to the naïve group. However, after bacterial challenge, these control groups developed varying degrees of erythema and microabscess formation. In stark contrast, animals immunized with combined formulations (WS03 + HI-C. acnes or MA107b + HI-C. acnes) exhibited markedly attenuated inflammatory responses, characterized by minimal ear redness and swelling. Notably, the MA107b + HI-C. acnes group demonstrated the most robust protection, displaying a significantly milder inflammatory phenotype compared to the WS03 + HI-C. acnes group. Following C. acnes challenge, ear thickness was determined. Both combined immunization regimens (WS03 + HI-C. acnes and MA107b + HI-C. acnes) showed significantly attenuated ear swelling relative to their respective single-component controls (p < 0.01), demonstrating that co-administration of adjuvant with inactivated whole-cell antigen effectively suppressed challenge-induced inflammation (Figure 5B).

3.6. The Efficacy in C. acnes Clearance and Inflammation Reduction

In the vaccine evaluation, post-challenge tissue bacterial load was a key metric for assessing immunoprotection (Figure 6A). At the same time point following C. acnes challenge, rats immunized with combined formulations (WS03 + HI-C. acnes or MA107b + HI-C. acnes) exhibited significantly lower bacterial burdens compared to those receiving corresponding single-component regimens (p < 0.01). These results demonstrate that co-administration of whole-cell inactivated C. acnes antigen with adjuvant markedly enhances the host’s capacity to clear the pathogen, resulting in accelerated reduction in bacterial load post-challenge.

We further evaluated the impact of immunization on local cytokine responses following C. acnes challenge (Figure 6B–D). Compared to immunization with WS03 alone, the WS03 + HI-C. acnes combination significantly suppressed post-challenge levels of IL-6, IL-1β, and MCP-1 in ear tissue (p < 0.05). Similarly, MA107b + HI-C. acnes immunization markedly reduced IL-1β and MCP-1 expression relative to MA107b alone (p < 0.05). Collectively, these findings indicate that combined immunization with whole-cell inactivated C. acnes effectively attenuates excessive local inflammation after bacterial challenge, thereby mitigating inflammatory tissue damage.

4. Discussion

This study demonstrates that intradermal inoculation of live C. acnes into the ears of Wistar rats establishes a reproducible acute inflammatory model with well-defined temporal kinetics [17]. The model faithfully recapitulates key features of early acne inflammation, including rapid neutrophil infiltration, microabscess formation, and upregulated expression of critical pro-inflammatory mediators-such as IL-1β, TNF-α, and CXCL1-thereby mirroring the initiation, peak, and resolution phases observed in clinical inflammatory acne lesions [18,19].

The core innovation of this study lies in the development of an integrated, four-dimensional quantitative evaluation system that simultaneously assesses macroscopic phenotype, tissue thickness, microbial burden, multiplex cytokine profiles, and histopathological/immunohistochemical features. This comprehensive framework overcomes a critical limitation of traditional single-parameter models-such as the rabbit ear comedogenesis model or simple murine bacterial abscess models-which fail to recapitulate the complex “pathogen–host” interactions inherent to acne vulgaris and lack the capacity for multi-parametric correlation analysis [20,21]. By enabling synchronized, multi-scale readouts of inflammation dynamics, our approach offers a more physiologically relevant and refined platform for mechanistic investigations and preclinical evaluation of anti-acne interventions.

We further validated the utility of this model for therapeutic evaluation by testing whole-cell inactivated C. acnes vaccines formulated with distinct adjuvants (WS03 or MA107b). Combined immunization strategies significantly reduced post-challenge bacterial burden in ear tissue, attenuated inflammatory edema, and suppressed local pro-inflammatory cytokine levels. Notably, the magnitude and cytokine-specific profile of this anti-inflammatory effect differed modestly between the two adjuvant formulations, suggesting adjuvant-dependent modulation of the immune response. The model also detected markedly elevated levels of C. acnes-specific IgG antibodies in the combined immunization groups, with antibody kinetics closely paralleling the observed protective outcomes. Nevertheless, whether humoral immunity is the sole mechanism of protection—or whether cellular immune responses also contribute-and the precise causal relationship between IgG titers and clinical protection warrant further investigation [22]. This successful application underscores the model’s robustness and translational relevance for screening and optimizing immune-based interventions, such as next-generation anti-acne vaccines [23,24].

A key limitation of the current model is its focus on the C. acnes-triggered acute inflammatory phase, which does not recapitulate other hallmark features of human acne vulgaris-namely, chronicity, follicular hyperkeratosis (comedone formation), and sebaceous gland hyperplasia. To address this, future studies could integrate this dynamic inflammatory framework with keratolytic agents (e.g., oleic acid) or hormonal modulators to develop a composite model that simultaneously mimics aberrant keratinization and sustained inflammation. A further limitation of the present study is the absence of assessments of vaccine-induced cellular immunity. This gap in our analyses prevents a full characterization of the mechanistic basis underlying the protective efficacy of the vaccine. In subsequent investigations, we will incorporate a panel of assays targeting cellular immune responses, including the quantification of antigen-specific T cell proliferation, cytokine secretion profiles, and cytotoxic T lymphocyte activity. These additional measurements will enable a more holistic and integrated evaluation of the vaccine’s protective effects, thereby providing a more complete understanding of how the vaccine mediates immunity against C. acnes.

5. Conclusions

Our study successfully established a C. acnes–induced ear infection model in Wistar rats and characterized the dynamic progression of acute inflammation. Using this model, we preliminarily evaluated the protective efficacy of whole-cell inactivated vaccines formulated with different adjuvants. Our findings demonstrate that this standardized, quantifiable, and reproducible preclinical platform faithfully recapitulates key features of acne-associated inflammation and provides a robust foundation for elucidating inflammatory mechanisms as well as for the high-throughput screening of novel anti-inflammatory therapeutics and anti-acne vaccine candidates.