Intradermal injection of Cutibacterium acnes and staphylococcus: A pustular acne‐like murine model

Skin 16S microbiome diversity analysis indicates that the Staphylococcus genus, especially Staphylococcus aureus (S. aureus), plays a crucial role in the inflammatory lesions of acne. However, current animal models for acne do not fully replicate human diseases, especially pustular acne, which limits the development of anti‐acne medications.


| INTRODUTI ON
Acne vulgaris is a common chronic inflammatory skin disease of the pilosebaceous gland with a high incidence.More than 90% of adolescents have varying degrees of acne, which is more common in the head and face, chest, and back. 1,2The pathogenesis is still unclear, mainly including the colonization of Cutibacterium acnes and other bacteria, excessive sebaceous secretion, hyperkeratosis of hair follicle ducts, and inflammatory factors.Acne vulgaris can be manifested as a variety of different types of skin lesions, including comedones, papules, pustules, nodules, cysts, etc., 3 which can be divided into inflammatory and non-inflammatory lesions.Clinical manifestations of pustules are inflammatory lesions. 4,5As a serious type of acne, pustular acne has attracted wide attention for its harmness and refractory nature.Moreover, pustular acne is prone to recurrent attacks and atrophic acne scars will appear after its rupture, causing great psychological pressure and spiritual pain to young people. 6In clinical practice, inaccurate use of antibiotics has led to delayed treatment and the emergence of bacterial resistance, becoming a major contributing factor to the prolonged nonhealing of pustular acne. 7 previous studies, C.acnes has been identified as a facultative anaerobic Gram-positive short rod bacterium.It is the dominant microbial species found in the hair follicles and sebaceous glands of acne patients and is closely associated with the pathogenesis of acne.Acne primarily occurs in areas with increased sebum secretion, where sebaceous glands significantly produce more sebum, leading to pore blockage.This provides a favorable anaerobic environment for C.acnes.C.acnes activates inflammatory signaling pathways through toll-like receptors (TLRs), proteasomes, and other receptors, thereby stimulating the release of a large number of inflammatory factors and triggering a potent inflammatory response. 8,91][12] In recent years, the role of S. aureus in the pathogenesis of acne has been a subject of controversy.However, an increasing body of research suggests that S. aureus is closely associated with the development of acne, although the underlying mechanisms remain unclear.Del Giudice P 13 reported that S. aureus infection of the skin can lead to diseases such as pyoderma, folliculitis, and primary abscesses.Brigitte Dreno 11 reported that the severity of acne lesions is positively correlated with the presence of Staphylococcus, particularly in pustular lesions where the main pathogenic strain is S. aureus.S. aureus is a highly pathogenic bacterium, Gram-positive bacterium, which widely exists in nature and can lead to a variety of suppurative diseases, causing severe inflammatory reactions. 14The mechanism by which S. aureus triggers skin inflammatory responses is highly complex and involves various cell types, including keratinocytes, macrophages, neutrophils, T cells, mast cells, and others.Through the secretion of toxic peptides and activation of TH17 cells, as well as interactions with TLRs and other pathways, S. aureus induces the production of inflammatory factors such as IL-1β, TNFα, IL-17, and others. 15,16 current research, any new candidate drugs for acne treatment need to undergo several rounds of screening using biochemical assays, cell culture, and animal models before proceeding to clinical trials.
Approximately 90% of drugs that enter clinical trials fail, and this high failure rate is partly attributed to the lack of accurate models representing the complexity and tissue structure of human tissues and/or models that capture the interactions within the human microbiome.
8][19] In clinical practice, doctors may overly rely on antibiotics as a treatment option for acne, especially in cases of moderate to severe acne.Prolonged or excessive use of antibiotics can lead to bacterial resistance, rendering previously effective treatment strategies ineffective. 20

| Experimental bacterial strains and growth conditions
The clinical strain of C. acnes (No.5 H3) used in the study was obtained from acne patients at Dongzhimen Hospital, Beijing University of Chinese Medicine.The bacteria were cultured in brainheart infusion broth (BHI) under anaerobic conditions (5% H 2 , 5% CO 2 , and 90% N 2 ) until reaching the logarithmic growth phase.The supernatant was removed by centrifugation at 4000 rpm for 10 min at 4°C.The pellets were washed three times and resuspended in BHI medium to a concentration of 5 × 10 8 CFU/mL for experiments.
Single colonies of Staphylococcus aureus strain MSSA (ATCC 25923) were grown in Luria-Bertani (LB) medium at 200 r/min for 12 h on a shaker, and the bacterial solution was collected and centrifuged at 4000 rpm for 10 min at 4°C to remove the supernatant.The pellets were washed three times and resuspended in BHI medium to a concentration of 3 × 10 7 CFU/mL for experiments.

| MALDI-TOF mass spectrometry identification
A small amount of single bacterial colony was taken and spread in a thin film on the target plate.The matrix solution from the mass spectrometry sample preparation kit was added to cover the sample after adding the lysis solution from the same kit, and the plate was left to dry at room temperature.The sample target was then placed into the mass spectrometer for identification.

| Determination of minimum inhibitory concentrations (MICs) of clinical isolates of C.acnes to different antibiotics
After 72 h of cultivation, the bacterial suspensions were adjusted to approximately 1 × 10 6 CFU/mL.A microdilution method was used in a 96-well plate to perform 2-fold dilutions of different concentrations of antibiotics, including erythromycin, clindamycin, lincomycin, tetracycline, chloramphenicol, streptomycin, fusidic acid, minocycline, azithromycin, and metronidazole.The plates were incubated anaerobically at 37°C for 72 h, and the MIC values were observed.The breakpoints were determined according to the CLSI 2016-M100-S23 standard, and for fusidic acid, the criteria from the European Committee on Antimicrobial Susceptibility Testing (EUCAST) 2016 were applied.

| Animals
The BALB/c male mice, aged 6-8 weeks, with a body weight of  For the C.acnes + S. aureus group, mice were injected intradermally with 50 μL mixed bacterial which mixed in a 7:3 volume ratio.

| Animal grouping and modeling
Immediately following injection, 20 μL freshly made synthetic sebum was applied to the skin.The acne sites were observed daily using in vivo imaging technology, and the dynamic changes were monitored for seven consecutive days.The synthetic sebum was formulated using 17% oleic acid, 45% triolein glyceride, 25% jojoba oil, and 13% squalene.

| Dermatoscopic imaging
The skin imaging system (BN-WG-1001, Nanjing Beining Medical Equipment Co., Ltd.-ACHEN, China) was employed for the experiment.After modeling, the mice were anesthetized with isoflurane and placed on the imaging platform.Using a 160× objective lens, enlarged images of the pustular acne sites in mice were captured.As for the patients, they were in a sitting or standing position, and using a 160× objective lens, enlarged images of the skin lesions in the acne patients were acquired.

| Laser speckle contrast imaging (LSCI)
LSCI was used to observe changes in blood flow in the pustular acne sites of both mice and acne patients.For the mice, they were anesthetized with isoflurane and placed under a body posture microscope at a distance of 20-30 cm.As for the patients, they were in a supine position, and the microscope lens was positioned above the area of interest, adjusted for image clarity, and real-time blood flow was captured for 15 s.The LED light intensity was set at 4000 times, and the optical magnification was 12 times.The brightness level was set at 5.0 for mouse images and 3.0 for acne patient images.Image analysis was performed using the Laser Speckle Blood Flow Imaging System (SIM BFI-WF; SIM Optical Technology Co., Ltd., China), and the measurement unit was perfusion units (PU).

| Photoacoustic imaging
Photoacoustic imaging was conducted using the optical imaging instrument (Hadatomo Z WEL5200, ADVANTEST, Japan) to observe changes in deep blood flow in the pustular acne sites.For mice, they were anesthetized with isoflurane and placed on the imaging platform.
As for the patients, they were in a supine position with even breathing, and both areas of interest were coated with glycerin.The imaging was performed with a 30 μm image resolution, covering a 9 × 9 mm area for detection.The WEL5200 system software displayed two-dimensional images (tomographic images) to adjust the sensor's focal position and signal intensity.The three-dimensional image data activated the sensor, and measurements were taken while recording the images.Image analysis was conducted using the EUCLID (Interim version) software.

| VISIA-CR imaging
VISIA-CR skin imaging system (Canfield Scientific, Inc) was used for capturing images with white light and cross-polarized light.For mice, after anesthesia, they were positioned in front of the camera for image acquisition.As for clinical patients, they were in a seated position, and images were captured in the following sequence: frontal view, left profile, and right profile.

| qRT-PCR analysis
Total RNA was extracted from mouse skin tissues using TRIzol reagent.The extracted total RNA was then subjected to reverse transcription using a reverse transcription kit (ABclonal Biotechnology Co., Ltd., Wuhan, China).Real-time polymerase chain reaction (qPCR) was performed using 2× universal SYBR Green Fast qPCR Mix (ABclonal Biotechnology Co., Ltd., Wuhan, China).The qRT-PCR was carried out using the Archimed Fluorescence Quantitative PCR Instrument (Kunpeng Genomics Co., Ltd., Beijing, China).All primers were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd.GAPDH was used as the internal reference gene.The 2 −∆∆Ct method was employed to calculate the fold change of the target gene as indicated by the qRT-PCR results.

| Enzyme-linked immunosorbent assay (ELISA)
A commercial ELISA kit (Beijing Dakewe Biotech Co., Ltd., China) was used to measure the protein expression levels of IL-1β, TNFα, and IL-6 in the mouse skin tissues of each group.The ELISA was performed following the instructions provided in the kit manual.

| Histology
Skin tissues from the acne lesions were collected at different time points, including before modeling, 1, 3, and 7 days after modeling for the pustular acne model, and 3 days after modeling for the conventional acne model.The skin tissues were carefully prepared by removing the subcutaneous connective tissues.They were then fixed in 4% tissue fixative, dehydrated, embedded in paraffin, and sectioned into 6 μm slices.The tissue sections were deparaffinized and subjected to Hematoxylin and Eosin (HE) staining and Gram stain.

| Statistical analysis
The experimental data was averaged at least from 3 parallel experiments and statistical analysis was performed with GraphPad Prism 7.0.The experimental data was analyzed using one-way analysis of variance (ANOVA) and subsequent Bonferroni multiple comparisons.If normality or equal variance tests failed, then a Kruskal-Wallis test and subsequent Dunn's multiple comparisons were used.The data was presented as mean ± SEM, p < 0.05 or less was considered as a significant difference.

| Clinical isolate identification and drug resistance
The contents of patients' hair follicles were cultured under anaerobic conditions, and seven single colonies were selected for bacterial identification.Five bacterial species were identified: C.acnes (F5, F7), C.acnes subspecies (H3), S. epidermidis (hemolytic) (F6, F8), K. aerogenes (H1), and S. lentus (H3).Under aerobic conditions, two single colonies were identified, and one bacterial species was identified as S. epidermidis (D11, D12).Further subtyping of C.acnes was conducted using anaerobic cultures of F7 and H3 colonies, with the H3 strain belonging to IA2 type and the F7 strain being a newly discovered variant.Drug susceptibility testing on the IA2 subtype of H3 strain showed resistance to erythromycin, clindamycin, azithromycin, minocycline, lincomycin, and metronidazole (Table 1).

| A pustular acne-like murine model that combines C.acnes and S. aureus
The onset of acne is related to the different lineages of C.acnes colonizing the skin, with the IA-2 strain showing a strong correlation with acne development, while the II-type strain has the highest abundance in the skin of healthy individuals. 21 The occurrence of acne is associated with different lineages of

| The mixed bacterial acne animal model is consistent with clinical pustular acne
In patients with pustular acne, the pathological changes manifest as early-stage pustules with a central area showing white, or pale yellow pus.The pustules are larger, with thin and flaccid walls and a soft texture, containing turbid, and thick pus that accumulates at the base.The surrounding skin may appear slightly red and swollen, with some patients experiencing pain, or itching.Blood flow variations in the affected area reveal reduced blood flow in the central region and increased blood flow in the peripheral area.
During the mid-stage, pustules may rupture, exposing a denuded surface that later dries to form yellowish crusts.Blood flow in the central region increases at this stage.In the later phase, if the pus drains spontaneously and the wound remains clean, crusts form and eventually fall off, leaving red acne marks.These marks may fade away after a few days.However, inadequate management or compromised immunity can lead to delayed healing or recurrent outbreaks.In summary, pustular acne is a severe form of acne that can cause significant distress due to its rapid progression and potential scarring.
To better assess pustular acne, we conducted a comprehensive analysis using multiple methods and perspectives.We compared images of clinical pustular acne patients at the peak of pustule formation and the first day of pustular acne animal model completion.

| Mixed bacterial acne animal model simulates the evolution of clinical pustular acne in patients
We In orthogonal polarization images, the central area changed from white to red, and its color was darker than normal skin.In LSCI blood flow images, the central area showed an increase in blood flow, where in acne patients, the blood flow in the central area was higher than in the surrounding region, while in the animal model, the blood flow was either the same, or slightly reduced compared to the surrounding region (Figure 4A).We statistically analyzed the difference between blood flow in the lesion area and the blood flow in the central area and found that both the animal model and clinical pustular acne patients exhibited a decreasing trend (Figure 4B).
These findings demonstrate that the pustular acne animal model we established can simulate the dynamic evolution of pustular acne in clinical patients.
In clinical practice, inflammation is a unique characteristic of different types of acne, and its diagnosis, treatment, and evaluation of therapeutic outcomes are closely related to changes in the inflammatory response.Therefore, we collected samples from different stages of the pustular acne animal model and detected the expression of inflammatory cytokines using ELISA.Both IL-1β and TNFα exhibited an initial increase followed by a decrease, while IL-6 showed no significant change in its expression trend (Figure 5A).

| DISCUSS ION
Acne is a disease unique to humans, with only mild manifestations observed in dogs and cats, and it does not occur naturally in any other species. 23,24However, the development of acne drugs and clinical therapeutic guidelines rely heavily on the establishment of animal models.Currently, mouse models are widely used to study the mechanisms of acne inflammation.These models exhibit similarities with human sebaceous follicular acne and offer advantages such as low cost and ease of operation. 25,26However, the intra- Therefore, establishing an animal model that closely replicates the clinical pathogenesis of pustular acne is a prerequisite for nonantibiotic drug screening.Currently, basic research primarily relies on intradermal injection of C. acnes to establish animal models of acne.However, this method lacks specificity to replicate the diverse clinical manifestations observed in acne patients.It fails to precisely correlate with clinical presentation, and most lesions formed are papules and cysts.Currently, there is a lack of animal models specifically replicating pustular acne.Therefore, there is a need for a pustular acne animal model to conduct drug efficacy evaluation and guide clinical treatment.In this study, we utilized a method of combining C. acnes and S. aureus to establish a pustular acne animal model on the back skin of mice.This model closely replicates the clinical manifestations observed in pustular acne patients.Through this model, we discussed the pathophysiological characteristics of the animal model of pustular acne and provided a solid foundation for its new drug development.

( 19 ± 2 )
g, were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd., with the animal license number SCXK-2021-0006.The mice were housed in a 12-h light/dark cycle, with ad libitum access to food and water.The animal experiments were conducted under the supervision and approval of the Ethics Committee of the Chinese Academy of Medical Sciences (Approval number ERCCACMS21-2111-27).
Establishment of the mouse dorsal acne model.Male BALB/c mice (SCXK2021-0006, Beijing Vital River Laboratory Animal Technology Co., Ltd., China) were randomly divided into three groups: the control group (CTRL), the C.acnes group, and the C.acnes + S. aureus group.All mice was shaved before application of Depilatory cream (Veet, Reckitt Home Chemical [China] Co., Ltd).The next day, the C.acnes group mice were injected intradermally with approximately 5 × 108 CFU/mL C.acnes in 50 μL BHI media.

2. 6 |
Clinical patient image acquisition This experiment has been approved by the Ethics Committee of Dongzhimen Hospital, Beijing University of Chinese Medicine (Clinical Ethics Number: 2022DZMEC-356-02).All study participants provided informed consent.The study included patients diagnosed with pustular acne by dermatologists.After cleansing the skin, patients underwent skin imaging using a dermatoscope, laser speckle instrument, and VISIA-CR imaging system, half an hour later.

C
.acnes residing in the skin, forming a balanced microbiome with other microbial species.The interaction between acne-causing bacteria like C.acnes and other pathogenic bacteria, including S. aureus, is an area of ongoing research.It has been reported that the supernatant of C.acnes culture can increase the antibiotic resistance of S. aureus.17Currently, acne models do not typically employ mixed bacterial models, so we tested whether the combination of C.acnes and S. aureus would lead to enhanced pathology.We further adjusted the experimental protocol, using a 7:3 ratio of 5 × 10 8 CFU/mL C.acnes and 3 × 10 7 CFU/mL S. aureus for intradermal injection into the back skin of mice, along with local application of artificial sebum.As shown in the figures, compared to the C.acnes group, the C.acnes + S. aureus group exhibited a significant pustular acne-like morphology.Skin microscopy revealed that both groups showed acne manifestations on the first day after modeling, with C.acnes group displaying prominent swelling and mild redness, while the C.acnes + S. aureus group presented with evident yellow pustular acne-like morphology, accompanied by more severe redness in the lesion area.On the third day after modeling, the C.acnes group showed obvious redness and the formation of small crusts with desquamation, while the C.acnes + S. aureus group displayed a yellow crust and a significant reduction in pustules, but with a more pronounced increase in redness in the lesion area (Figure 1A).Photoacoustic imaging and LSCI were used to analyze the changes of vascular morphology and blood flow in the acne lesions.Compared with the C.acnes group, the superficial blood vessels in the abscess area of the C.acnes + S.aureus group were significantly dilated and congested on the first day after modeling, and the pus was visible in the dermis under the epidermis in the photoacoustic z-axis imaging.The blood flow in the center of the lesion was significantly reduced.On the third day after modeling, the C.acnes + S. aureus group continued to exhibit significant dilation and increased congestion of shallow blood vessels, with a worsened abscess and the formation of crusts on the surface.The photoacoustic image displayed high-intensity areas in the shallow layer, and the blood flow in the center of the lesion area significantly increased compared to the first day (Figure 1B-D).Histological analysis of the skin damage showed that both groups exhibited epidermal thickening, with the epithelial granular layer and spinous layer significantly increased, and inflammatory cell infiltration in the dermis.However, the C.acnes + S. aureus group showed more pronounced inflammatory cell infiltration in the abscess region compared to the C.acnes group (Figure 1E).Gram staining revealed an increased aggregation of Gram-positive bacteria in the C.acnes + S. aureus group (Figure 1F).Pustular acne is characterized by enhanced inflammatory reactions.Three days after modeling, we examined the changes in F I G U R E 1 (A) Dynamic microscopic characterization of acne inflammation using skin microscopy observation.(B) PAI with Z-axis and XYaxis images.The yellow curved circle represents the abscess area, and the white box indicates the scab area.(C) Tracking of blood perfusion with LSCI, white circles highlighting the acne lesion area.(D) Dynamic measurements of blood flow, n = 6.(E) HE staining of the 3rd-day acne modeling samples (scale bar = 100 μm).(F) Gram stain of the 3rd-day acne modeling samples (scale bar = 100 μm).mRNA and protein levels of inflammatory factors in the lesion tissues.Compared to the CTRL group, the C.acnes + S. aureus group showed a significant increase in the mRNA expression of IL-1α, IL-1β, TNFα, and IL-6.Compared to the C.acnes group, the C.acnes + S. aureus group displayed significantly elevated mRNA expression of IL-1α and IL-1β, with a tendency for increased expression of TNFα and IL-6 (Figure 2A).In terms of protein expression, the C.acnes + S. aureus group showed significantly increased levels of TNFα and IL-1β compared to the CTRL group and C.acnes group, with no significant difference in IL-6 expression (Figure 2B).

Firstly, the morphological
presentation of the pustular acne model closely resembled that of clinical patients.Both the VISIA white light images and orthogonal polarization images showed central areas of the skin lesions with white or pale yellow pustules, exhibiting a soft texture, and slight redness and swelling in the surrounding skin.Secondly, the blood flow patterns in the pustular acne animal model highly resembled those in clinical patients.In the early stage of pustule formation, both exhibited reduced blood flow in the central area of the lesion and higher blood flow in the peripheral area.Additionally, in the optical coherence tomography images, pustules were observed within the dermis below the epidermis, consistent with LSCI imaging data (Figure 3A,B).Based on the analysis of morphological features and blood flow patterns in the acne lesion area, it can be concluded that there is a high degree of concordance between clinical pustular acne patients and the pustular acne animal model.F I G U R E 2 Skin tissue on the 3rd day after completion of modeling.(A) qRT-PCR was performed to detect the expression of inflammatory factors IL-1α, IL-1β, TNFα, and IL-6 in mouse skin tissue (n = 6).(B) ELISA was used to observe the expression of inflammatory factors TNFα, IL-1β, and IL-6 in mouse skin tissue (n = 3).**** p<0.0001, ***p<0.001,**p<0.01,*p<0.05versus CTRL; ###p<0.001,##p<0.01versus C. acnes.All data are presented as mean ± SEM.
classified clinical pustular acne in patients into three stages based on the different manifestations of pustules: Stage 1, characterized by larger pustule volume, white or pale yellow color, and soft texture; Stage 2, with reduced pustule volume, decreased content, and the appearance of scabs on the pustule surface, lasting approximately 1-2 days; Stage 3, where the scabs mostly disappear, and the pustule lesion area visibly shrinks and presents as red marks, lasting approximately 1 day.For the pustular acne animal model, the first day after modeling corresponds to Stage 1, characterized by the largest pustule appearance, white or pale yellow color, and soft texture, similar to Stage 1 of clinical pustular acne as described above.The third day after modeling corresponds to Stage 2, featuring prominent scabs with a yellow color and a slight reduction in pustule content.The seventh day after modeling corresponds to Stage 3, with the disappearance of yellow scabs, and the presence of red marks or small scabs indicating the healing stage.In Stage 1, both acne patients and the animal model exhibited pustules with central white color and peripheral redness in the orthogonal polarization images.LSCI blood flow images showed low blood flow in the central area and higher blood flow in the surrounding region.In Stage 2, pustules in both groups were smaller compared to Stage 1, with yellow scabs on the surface.The animal model exhibited larger pustules than clinical patients.Orthogonal polarization images showed similar characteristics, with a lighter central area compared to the previous stage.LSCI blood flow images demonstrated an increase in blood flow in the central area compared to the previous stage but still lower than the surrounding region.Entering Stage 3, both groups exhibited similar morphological changes, with a significant reduction in pustule area, presenting as red marks or red scabs.
In the pustular acne animal model, Stage 1 exhibited evident inflammatory infiltration, and Gram-positive bacteria were mainly distributed at the top of the pustule.Compared with Stage 1, Stage 2 showed more severe inflammatory infiltration, increased epidermal thickness, and similar pustule area but affecting a broader region.Gram-positive bacteria were primarily distributed throughout the pustule area.In Stage 3, the pustule was mostly cleared, and inflammatory infiltration subsided, with the epidermal thickness tending to recover, and Grampositive bacteria were largely eliminated (Figure 5B,C).Overall, our study indicates that the established pustular acne animal model can F I G U R E 3 Optical imaging comparison between skin lesions in clinical pustular acne patients and mouse models.(A) Optical acquisition images of pustular acne lesions in clinical patients.(B) Optical acquisition images of pustular acne lesions in the mouse model: (a) VISIA white light image (b) Cross-polarized image (red areas), (c) LSCI images, the large white circles represent the peripheral area of the acne, and the small white circles represent the central area of the acne.(d) Photoacoustic imaging Z-axis acquisition image, and (e) Photoacoustic imaging XY-axis acquisition image.mimic the dynamic progression of pustular acne observed in clinical patients.The different stages of the model were assessed for their inflammatory response by measuring the expression of inflammatory cytokines through ELISA.This animal model provides valuable insights for understanding the unique features of acne types and their diagnosis, treatment, and therapeutic efficacy evaluation, all of which are closely linked to changes in the inflammatory response.

F I G U R E 4
Comparison of the dynamic evolution process of acne between clinical acne patients and the animal model.(A) Multidimensional dynamic imaging in vivo during the evolution and development of acne in mice and patients.From left to right: VISIA-CR with white light image and cross-polarized image (red areas), skin microscopy image, LSCI tracking blood flow changes.(B) Dynamic measurements of blood flow at Stage 1 to Stage3 in acne lesions for clinical patients (left), n = 3, and in the pustular acne animal model (right), n = 6.
injection of pure Propionibacterium acnes to establish the model cannot distinctly differentiate between different stages of inflammation.As a result, the current acne animal models have certain limitations and cannot fully cover all manifestations of clinical acne.In clinical presentation, inflammatory lesions include papules, pustules, and nodules among which pustular acne differs from other types of acne lesions.Pustular acne is a red, inflamed lesion filled with white, yellow, or milky pus, which oozes out if the pustule is pierced or ruptured.Pustules may become firm and F I G U R E 5 (A) TNFα, IL-6, and IL-1β protein levels of acne lesions at different stages in mice.Data are presented as mean ± SEM. **p<0.01,*p<0.05;n = 3. (B) Staining of acne lesions, the white rectangle indicates local amplification of the inflamed area.(C) Gram staining, the blue area represents Gram positive bacteria.Scale bar = 100 μm.painful, and their size may increase.These can progress to cystic acne, which is a more severe form of acne. 27-29Hence, the establishment of a pustular acne model is significant for studying the pathogenesis and pathological process of inflammatory lesions in acne, and it holds valuable potential for the development of new treatments targeting acne inflammation.C.acnes is one of the critical factors in the occurrence and development of acne.Acnerelated systemic types IA-2 p+, IB-1, and IC induce high levels of inflammatory IFNγ and IL-17, leading to an increased tendency of inflammatory acne. 18However, infection of mice with C.acnes alone does not produce clinically similar pustular acne.Studies have shown that C.acnes dominates the content of acne lesions, with a small amount of S.aureus also present. 30,31S.aureus can trigger severe inflammatory responses and cause various pyogenic diseases.Therefore, in this study, we chose to infect mice with a mixture of C.acnes and S.aureus, and through preliminary experiments, we determined the appropriate ratio of 7:3 (volume ratio) for subsequent experiments (Data S1).In the morphological observations, on the first day after modeling, both the C.acnes group and the C.acnes + S.aureus group showed manifestations of acne.The C.acnes group exhibited distinct swelling and slight redness, while the C.acnes + S.aureus group showed evident yellow pustule-like morphology with a softer texture and more severe redness and swelling in the lesion area compared to the C.acnes group.In this study, the mixedbacteria inflammatory model's appearance more closely resembled clinical pustular lesions.Utilizing skin microscopy allows for a simple, rapid, and noninvasive observation of the morphological characteristics and vascular dilation at the site of mouse skin lesions.The VISIA-CR skin test can quickly and easily collect images with high definition and contains a variety of polarized light, which can evaluate the changes of acne in many aspects.Among them, the RBX-red technology can detect the content of hemoglobin from cross-polarized images, 32,33 and reflect the changes related to blood vessels and the degree of inflammation.Dermoscopy and VISIA-CR skin examination were used to detect the images of the model and clinical pustular lesions.The morphological changes of acne in the two groups were compared, and it was found that the animal model of mixed acne was partially consistent with the clinical appearance of pustular lesions.Additionally, we divided the clinical pustular lesions into three stages and analyzed the changes of the pustular acne animal model corresponding to the changes of the pustular acne animal model by dermoscopy, photoacoustic imaging, laser speckle imaging and VISIA-CR images.The results showed that the established animal model of pustular acne could well simulate the dynamic evolution of clinical pustular acne.This study laid the foundation for the basic research of drugs for pustular acne.The primary clinical manifestation of pustular acne is the occurrence of pustules, accompanied by intense inflammatory responses.Different stages of inflammatory reactions are associated with variations in blood flow at the lesion site.Through noninvasive and rapid live monitoring using photoacoustic imaging and scatterometry, our findings revealed consistent image representations of the various stages of acne, demonstrating similarities in blood flow changes between animal models and clinical pustular acne.The successful establishment of our animal model effectively mimics the evolution process of clinical pustular acne, thus holding significant implications for exploring the pathological mechanisms underlying acne progression.In addition to employing the aforementioned live imaging techniques, we also utilized conventional techniques such as HE staining and Gram stain to observe the pustular acne lesions in the animal model.In comparison with the single-bacterium acne model, the mixed-bacteria acne model exhibited more pronounced infiltration of inflammatory factors and a higher accumulation of Gram-positive bacteria.To assess the levels of inflammatory factors, we conducted qRT-PCR and ELISA tests, which revealed a significant increase in the expression of inflammatory cytokines, such as IL-1β.Interestingly, the mRNA expression of IL-6 in the mixed-bacteria acne model showed a clear elevation, but ELISA testing did not indicate a substantial change in the secretion of this inflammatory cytokine.We hypothesize that in this model, the response of IL-6 might occur later and involve a lower level of secretion.We conducted comparisons at three different stages, selecting day 3 after modeling for assessment.The mRNA expression of IL-6 was significantly higher, but the secretion had not yet significantly increased.However, there was an ascending trend in IL-6 secretion in Stage 3.This research demonstrates the use of advanced live imaging techniques in conjunction with traditional methods such as HE staining and Gram stain for the observation of pustular acne lesions in the animal model.By combining the data from these diverse techniques, we gained valuable insights into the inflammatory response and cytokine expression, particularly concerning IL-1β and IL-6, providing further understanding of the mixed-bacteria acne model's pathophysiology at different stages.In this study, we constructed an animal model of pustular acne intradermal injection of C. acnes and S.aureus (7:3), and used optical imaging techniques to realize noninvasive dynamic living observation besides its crucial role in early diagnosis, optical imaging also offers the additional advantage of significantly reducing the number of experimental animals required.34,35Furthermore, the animal model we established exhibits similar characteristics to the three developmental stages of clinical pustular lesions.This provides an observation model for the precise selection of anti-acne drugs targeting different stages of inflammation, including both the early and late inflammatory phases.This model has surpassed the single evaluation approach of antibiotic in vitro screening.By utilizing an in vivo animal model to simulate the developmental stages of clinical acne inflammation, it ultimately achieves a comprehensive assessment of new formulations and therapies aimed at reducing antibiotic resistance, thus enriching the treatment methods for acne.Additionally, the study suggests the practical application of this model through noninvasive dynamic in vivo observation methods.Therefore, regarding the extension of acne severity assessment methods, the animal model developed in this research can be utilized to develop novel diagnostic and evaluation methods, contributing to the improvement of clinical diagnosis for acne.However, this study has its limitations.The authors primarily conducted noninvasive and safe comparisons of clinical manifestations between pustular acne-like mice and clinical acne patients using optical imaging technology.They detected the expression and secretion of inflammatory factors in pustular acne-like mice, thus establishing a pustular acne-like mice model.However, the study did not involve research on clinical sample cells, transcriptomes, or molecular biology of clinical samples, leading to a lack of depth in this model's research.Additionally, due to the invasive nature of skin biopsies and strict indications in clinical practice, the study did not involve histopathological observations of clinical pustular acne lesions.Nevertheless, the pustular acne-like mice model established in this study closely resembles clinical pustular acne in terms of optical imaging and inflammatory cytokine expression levels.Therefore, this model provides a valuable tool for exploring the pathophysiology of acne and the development of therapeutic drugs.
Results of antimicrobial susceptibility testing for clinical isolates.
In this study, we selected a clinical isolate of IA-2 type C.acnes (No.5 H3) and constructed a back acne animal model by topically applying artificial sebum, following the method reported by Stacey L. Kolar et al. 22 TA B L E 1