Cutibacterium acnes regulates the epidermal barrier properties of HPV-KER human immortalized keratinocyte cultures

Our skin provides a physical barrier to separate the internal part of our body from the environment. Maintenance of complex barrier functions is achieved through anatomical structures in the skin, the stratified squamous epithelium specialized junctional organelles, called tight junctions (TJs). Several members of our microbial communities are known to affect the differentiation state and function of the colonized organ. Whether and how interactions between skin cells and cutaneous microbes, including Cutibacterium acnes (C. acnes), modify the structure and/or function of our skin is currently only partly understood. Thus, in our studies, we investigated whether C. acnes may affect the epidermal barrier using in vitro model systems. Real-time cellular analysis showed that depending on the keratinocyte differentiation state, the applied C. acnes strains and their dose, the measured impedance values change, together with the expression of selected TJ proteins. These may reflect barrier alterations, which can be partially restored upon antibiotic–antimycotic treatment. Our findings suggest that C. acnes can actively modify the barrier properties of cultured keratinocytes, possibly through alteration of tight cell-to-cell contacts. Similar events may play important roles in our skin, in the maintenance of cutaneous homeostasis.


Effect of C. acnes on the integrity of in vitro keratinocyte cultures.
Ohmic resistance and impedance measurements across a wide spectrum of frequencies are considered as good indicators of the integrity of cellular barriers [26][27][28][29] . Thus, we investigated how different culturing conditions affect the measurable impedance (Z), and the calculated Cell index (Ci) values of confluent NHEK (Fig. 1a) and HPV-KER (Fig. 1b) cultures using real-time cellular analysis, and interpreted the changes as alterations in barrier properties of the in vitro monolayer cultures [27][28][29] . We allowed the cells to form confluent monolayers using basal KSFM and then raised the concentration of extracellular Ca 2+ , which is known to induce keratinocyte differentiation 30 . We found that, after an initial growth phase, Ci values reached a plateau as the cultures became confluent and contact-inhibited. Replacing the media with fresh media containing high Ca 2+ concentrations (Ca-high) induced a marked and immediate increase in Ci compared to samples that were only contact inhibited and maintained in low Ca 2+ media (Ca-low). NHEK and HPV-KER cultures behaved similarly, suggesting that the latter cells may be used as a model to analyse keratinocyte barrier functions in monolayer cultures (Fig. 1a,b). These findings also agree with the available literature data and suggest that high extracellular Ca 2+ concentration leads to the stabilization of keratinocyte barriers. In addition, the capability of the xCELLigence RTCA system for real-time monitoring of these properties was confirmed 31,32 .
We used the same experimental setup to analyse how the addition of C. acnes to the Ca-low and Ca-high NHEK and HPV-KER cultures affected keratinocyte barriers. For this, C. acnes 889 strain (MOI = 100, 300) was added and nCi values were recorded in every 4 h for 95 h (Fig. 2). In Ca-low NHEK (Fig. 2a) and HPV-KER (Fig. 2c) cultures, we first observed a rapid and transient increase of nCi values, suggesting improved barrier properties of the cultures. Detailed analysis of these changes showed that the extent and exact shape of the curves depended on the MOI of C. acnes applied ( Supplementary Fig. 1).
The peak in nCi was followed by a drop in the Ca-low cultures, suggesting that, after reaching a threshold, continuously growing bacterial cells may have deleterious effects on the barrier properties of keratinocyte monolayers. We also repeated the experiments on differentiated, Ca-high NHEK and HPV-KER monolayers. In these cell cultures only the dose-dependent decrease of nCi values were detected, independent of the used cell type and C. acnes MOI (Fig. 2b,d).
For determining strain-specific effects, we repeated the experiment using different C. acnes strains and HPV-KER monolayers. Similar results were detected, suggesting that the observed nCi changes may be independent of the applied strains ( Supplementary Fig. 2).
When experiments were repeated using heat-killed C. acnes, no significant changes in nCi were detected, suggesting that the impedance changes observed in previous experiments depended on the presence of live bacteria ( Supplementary Fig. 3a-c). This prompted us to test whether PA, an important molecule produced during C. acnes metabolism, produced similar effects. Indeed, treatments with high concentrations of PA resulted in a transient increase in nCi, followed by marked decreases (Supplementary Fig. 4).

Figure 2.
Co-culturing HPV-KER and NHEK keratinocyte monolayers with C. acnes 889 strain leads to dose-dependent nCi changes. Ca-low and Ca-high NHEK (a, b, respectively) and HPV-KER (c, d, respectively) keratinocyte monolayers were established and co-cultured with C. acnes 889 strain (0 h time point, marked with an arrow) in different doses (MOI = 100, 300 www.nature.com/scientificreports/ Apart from the barrier properties, Ci changes may reflect differences in the number or in the specific dimensions of cells attached to the electrodes. To determine the exact nature of the C. acnes-induced cellular events, we monitored the effect of the bacterium on the number of cells both in the Ca-low and Ca-high HPV-KER cell cultures. No significant change in cell number was detected using a trypan blue exclusion assay ( Supplementary  Fig. 5a,b).
We also performed TEER measurement, a widely accepted quantitative technique to analyse tight junction dynamics 26 . Selected time-points (24 and 72 h) were chosen, based on our real-time investigation, and TEER measurement was performed using untreated samples as a control and samples treated with C. acnes (MOI = 300) in both Ca-low and Ca-high HPV-KER monolayers. Our results indicate that the TEER resistance values first increased in the Ca-low cultures, followed by a decrease in both models in the presence of the bacterium, similarly to what we found in the xCELLigence analysis (Fig. 3).
Altogether, these results strongly suggest that C. acnes is able to influence the state of the in vitro cultured keratinocyte barrier. This effect requires the presence of metabolically active, live bacteria, and the extent of the changes are dependent on the bacterial dose.
The effect of C. acnes 889 strain on the expression of selected TJ proteins in HPV-KER cultures. To study the cellular changes leading to C. acnes-induced nCi alterations, we treated Ca-low and Ca-high HPV-KER cultures with C. acnes 889 strain (MOI = 100, 300) and analysed the expression of selected genes encoding important TJ components: claudin 1 and 4 (CLDN1, CLDN4), occludin (OCLN) and zonula occludens 1 (ZO-1). mRNA expression studies were detected with real-time RT-PCR, and protein expression levels were detected with western analysis. ZO-1 mRNA levels did not markedly change, and OCLN expression increased upon bacterial treatment ( Fig. 4a,b,i,j). The expression of the two claudins exhibited opposing changes: CLDN1 levels decreased in both models, while CLDN4 increased in the Ca-low cultures (Fig. 4c,d,k,l).
We found that, upon the addition of the bacterium, ZO-1 levels increased markedly in the Ca-high cultures (Fig. 4m), and a small, but consistent elevation was detected in the Ca-low HPV-KER cultures (Fig. 4e). OCLN expression increased slightly (Fig. 4f,n), whereas CLDN1 protein levels decreased in both cultures (Fig. 4g,o). CLDN4 levels also increased but only in the Ca-low cultures 72 h after treatment (Fig. 4h,p).
Our findings suggest that C. acnes induces the expression changes of selected TJ structural proteins.
The effect of C. acnes 889 strain on the expression of selected TJ proteins in OS models. To determine whether the C. acnes-induced cellular changes were specific to monolayer cultures, we also investigated the effect of C. acnes on the expression of selected TJ structural components in ex vivo, three-dimensional OS cultures. In these experiments, we treated the upper, epidermal side of the skin samples with live C. acnes 889 strain. We collected samples at 24, 48 and 72 h after treatment, and IHC staining was performed to monitor the mRNA and protein expression changes. We found that ZO-1 and OCLN protein levels increased in all epidermal layers in the presence of C. acnes 889 strain. CLDN1 expression, which was strongly expressed throughout the epidermis in the untreated control samples, decreased everywhere except the basal layer. In contrast, CLDN4, which was restricted to the stratum granulosum in control samples, also appeared in the lower layers of the epidermis in the presence of the bacterium (Fig. 5). These findings suggest that the abundance as well as the localization of selected TJ proteins change in the presence of the bacterium. www.nature.com/scientificreports/ LY penetration assay of HPV-KER monolayer cultures and OS models. To analyse the functional consequences of the observed nCi changes, LY penetration assay was performed on the Ca-high HPV-KER monolayers and OS models. Ca-high cultures were first treated with C. acnes 889 strain (MOI = 300) for 24 or 72 h in a transwell system; controls were treated with PBS alone. We chose these time points based on the results of our previous TEER and xCELLigence experiments. LY dye was added to the upper chamber, and the penetration assay was carried out for 30 min. Samples from the cell supernatant collected from the lower chambers were subjected to fluorimetry.
We found that fluorescence intensities increased in treated samples compared to controls, indicating LY dye penetration by 72 h after treatment. No differences were detected in the 24-h samples (Fig. 6a).
We also repeated the dye penetration experiments in the OS models to confirm that the observed barrier changes were not specific to monolayer cultures. C. acnes 889 strain or PBS was pipetted to the upper, epidermal www.nature.com/scientificreports/ side of the OS samples and samples were incubated for 72 h before the addition of LY, and dye penetration was visualized by fluorescent microscopy. As observed for the monolayer cultures, LY dye penetration increased in the presence of high-MOI C. acnes 889 treatment, as indicated by the appearance of increased, diffuse fluorescent signal in the sub epidermal skin regions (Fig. 6b).
Antibiotic treatment of C. acnes co-cultured HpV-KeR cells leads to the partial reversal of decreased nci values. As many of the acne treatments currently available exhibit bacteriostatic and/or  www.nature.com/scientificreports/ antibacterial properties, we were interested to see if decreasing the C. acnes activity and/or viability in co-cultures with the addition of AB/AM treatment reverses the bacterium induced nCi decreases we noted in previous experiments. To this end, Ca-low HPV-KER cultures were co-cultured with C. acnes 889 strain (MOI = 100), and the effects of the bacterium were continuously monitored in real time using the xCELLigence system. When the nCi values started to drop, we changed the culturing media to KSFM supplemented with 1% or 5% AB/AM solution. Decreasing the load of live bacteria in the co-cultures by the addition of AB/AM solution partially reversed the deleterious effect of C. acnes 889 strain and led to a marked increase of nCi values, characteristic of improved barrier functions of in vitro keratinocyte cultures (Fig. 7a-e).

.Discussion
Although the cutaneous microbiota plays very important roles in the maintenance of the skin's homeostasis, the interactions between human cells and the various microbes also generate many challenges for the body. Skin cells, especially keratinocytes, must tolerate the presence of these microbes, even though they are equipped with all the conserved molecular structures that stimulate PRRs 19,20 . Thus, the skin must maintain a proper barrier against potentially harmful pathogens while simultaneously allowing continuous crosstalk between beneficial microbes and cutaneous immune cells [33][34][35] . Such complex interactions require fast, dynamic epidermal-barrier functions. In our study we aimed to investigate whether and how the cutaneous microbiota, particularly the C. acnes bacterium affects the properties of the skin barrier.
Our interest led us to perform in vitro studies using two keratinocyte monolayer cultures (NHEK, HPV-KER) as a model system for skin with real-time impedance measurement-based studies. These methods are widely used for the detection of cellular integrity and barrier changes of in vitro monolayer cultures 26,36 . Using the gathered data, together with the results we obtained by analysing full thickness OS models, we propose a possible model, how C. acnes may affect the cutaneous barrier in the healthy skin and during acne pathogenesis.
We found that C. acnes, a prominent member of the skin microbiota, had a complex effect on these cells. In less differentiated, Ca-low cultures, co-culturing with live bacterium induced rapid, transient nCi increases, which are characteristic of improved barrier properties. These changes were strain-specific and depended on the applied bacterial MOI. A fast growing pathogenic strain, C. acnes 889; IA, induced rapid and marked nCi increases compared to the C. acnes 6609 strain, which exhibits slower growth 37 . The effect of strain 6609, which was originally isolated from the facial region of a healthy subject, exhibited a less pronounced, but sustained effect.
C. acnes is not the only microbe that may strengthen the skin barrier. Ohnemus and colleagues reported similar results with another member of the skin microbiota, Staphylococcus epidermidis 38 . The observed phenomena are also very similar to those observed for the intestinal microbiota, which also aids the differentiation and the development of the anatomically mature, fully functioning gastrointestinal tract 24,25 .
When left long enough in contact with the cells, continuously growing C. acnes strains gradually exhibited deleterious effects on the integrity and barrier properties of keratinocyte monolayer cultures, as indicated by the marked decrease in nCi values. This effect was dependent on the dose of live bacteria applied. The decrease in nCi was independent of cell differentiation. In addition, the change in nCi was not caused by keratinocyte death, as no major changes were detected in the number of live cells in the HPV-KER monolayer cultures.
nCi changes were only observed when we co-cultured the keratinocytes with live C. acnes strains. It is possible that these changes are not solely caused by conserved microbial structural components, but these changes might also involve a variety of special compounds that are generated by bacterial metabolism. C. acnes produces short chain fatty acids as a result of anaerobic fermentation, enzymes (e.g., lipases) and other biologically active molecules, that impact important regulatory functions of a number of human cell types 39 . Identifying which of the many molecules are necessary for the regulation of cutaneous barrier functions requires further investigation. As an initial effort, we analysed the effect of PA, a special metabolic product of this bacterium that is linked to bacterial growth and metabolism 37 . We found that PA may be an important factor: Ca-low cultures treated with high doses of PA exhibited clear, transient Ci changes that were comparable to what we observed with live C. acnes strains.
Next, we aimed to see whether the detected resistance changes were correlated with changes in in vitro barrier integrity. Treatment with LY, a large molecular weight, fluorescently labelled compound that cannot freely permeate lipophilic barriers, clearly indicated that the nCi decrease could be the consequence of bacterium-induced deterioration of the integrity of the HPV-KER keratinocyte culture. This effect was not specific to monolayer cultures: similar results were found when the experiments were repeated in OS models, suggesting that C. acnes may also regulate cutaneous barrier properties, tightness and paracellular transport properties of the epidermis, even in a tissue environment.
Multiple different claudins, which are also essential proteins in TJ complexes, are present simultaneously in a given cell type, and the expression of various family members are highly context-dependent, controlled both spatially and temporarily. Many other factors, including tissue type, age, differentiation state, external and intracellular signals and stimuli, are involved in their regulation 40,41 . The claudin content of a given cell is not stable and can change significantly in a matter of hours. Such continuous molecular remodelling, called switching, is an important process that allows the epidermal barrier to adapt to the ever-changing external and internal environments 42 . TEER and the associated paracellular permeability are thought to depend on the profile of different claudins expressed in a given cell type at a given point in time 12 . In the skin, keratinocytes express several different claudin family members, but the most abundantly present proteins are CLDN1, CLDN 4 and CLDN 7 43 . It is interesting that apart from keratinocytes, sebocytes also express the same claudins. They play important roles in the regulation of a permeability barrier, and possibly also during the holocrine secretion process in these glands 44 . Whether and how C.acnes affects the expression of these claudins, and through that sebaceous gland functions requires further investigations. www.nature.com/scientificreports/ Immunostaining of characteristic TJ proteins has been used to gain insight into the integrity of different barriers 26 . In full-thickness skin organ cultures, ZO-1 and OCLN protein levels increased in all epidermal layers following treatment with C. acnes 889 strain, and we observed similar changes in monolayer cultures. CLDN1 and CLDN4 expressions exhibited opposite changes: while CLDN1 levels mostly decreased in the presence of the bacterium, CLDN4, which was restricted to the stratum granulosum in control OS samples, simultaneously appeared in the lower layers of the epidermis. These protein expression changes correlated well with changes in mRNA expression, especially in the case of OCLN, CLDN1 and CLDN4. These findings suggest that TJ protein levels and their localization change in the presence of the bacterium, and their regulation possibly involves transcriptional and post-transcriptional effects, as well 45 .
CLDN1 is a key determinant of epidermal barrier functions in human and mouse epidermis 46,47 . A strong positive correlation between epidermal integrity and CLDN1 expression levels was found in rodents. CLDN1deficient mice exhibit signs of hyperkeratosis and acanthosis with aging, suggesting that abnormal regulation of this protein affects multiple cellular mechanisms 48 . Reduced CLDN1 levels appears to be a common theme in different chronic inflammatory skin diseases, including psoriasis, atopic dermatitis and rosacea [47][48][49] .
CLDN4 levels increased when keratinocytes and cells of the OS model were co-cultured with C. acnes 889 strain. CLDN4 is also an important molecule of the epidermal barrier, and the decrease its expression in in vitro cultured keratinocytes has been associated with barrier defects. CLDN4 is considered a tightening claudin, and our results are compatible with the idea that its upregulation presents a compensatory mechanism to restore barrier functions caused by decreased CLDN1 expression levels 7,50 .
Opposing regulation of CLDN1 and CLDN4 has been reported before, and CLDN1/CLDN4 switching may be an important mechanism to regulate the epidermal barrier during inflammatory skin conditions 47,51 .
Overall, through the induction of mostly TLR-dependent innate immune and inflammatory changes and the production of bioactive molecules, the C. acnes bacterium may induce innate immune and inflammation activation and autophagy, as well as altering the differentiation state of skin cells and epidermal barrier functions [19][20][21]52 .
In our experiments, the extent of barrier changes in keratinocyte cultures appeared to be strain-and dosedependent: faster growing C. acnes strains (889 and ATCC 11828) exhibited more pronounced changes. These results confirm our earlier findings, suggesting that, in parallel with bacterial growth, the pathogenic potential of C. acnes increases 37,52 . This conclusion contradicts findings that the amount of the bacterium does not differ in control and acne skin 53,54 . The reason for these discrepancies may be complex. Only a limited number of follicles are inflamed at any given time, and C. acnes may not reach the skin surface as it becomes trapped inside the follicles, especially when present in a biofilm form. Once inside the pilosebaceous unit, its density may rapidly increase during lesion development.
Topical and systemic drugs are widely used for acne treatment 55 . We tested whether reducing the presence of viable C. acnes using antibiotics in the HPV-KER co-cultures restores the barrier properties and found clear increases in the measured nCi values. Our results suggest that acne pathogenesis may be an even more complex event than previously suspected, and that, apart from the role of immune and inflammatory events, changes in cutaneous barrier properties can play important roles. Further experimental and clinical studies are necessary to determine whether therapeutic modalities restoring supplementary barrier together with conventional antibacterial treatments would enhance the healing of lesions.

Materials and methods
C. acnes strains and culture conditions. C. acnes 889 (type IA) and 6609 (type IB) strains were cultured and stored as previously described in detail 20,37 . Keratinocyte cell cultures, organotypic skin models and C. acnes treatment. Normal human keratinocytes (NHEK) and ex vivo skin biopsies were obtained from skin specimens collected from the Plastic Surgery Unit of our department. Written, informed consent was obtained from the investigated individuals.   www.nature.com/scientificreports/ For generating the organotypic skin (OS) models, 1 cm 2 whole-thickness skin samples were placed onto the nitrocellulose membrane of a 6-well insert, with the dermal surface placed on the membrane. Samples were cultured in an air-liquid interphase using Dulbecco's Modified Eagle's/F12 medium supplemented with 10% fetal bovine serum. C. acnes culture (15 μl, 10 9 CFU/sample) was transferred to skin samples (epidermal surface) and further cultured for an additional 24, 48 or 72 h. Samples for immunofluorescent staining were fixed in 4% paraformaldehyde and embedded in paraffin. For PCR analysis, 6 mm punch biopsies were taken from the samples and the epidermis was separated from the dermis by incubating in dispase solution for 3 h at 37 °C. Epidermis samples were homogenized using the Ultra Turrax T8 homogenizer (IKA-WERKE). Total RNA was isolated using TRI-Reagent (Molecular Research Center; Cincinnati, USA).
Real-time, label-free cellular analysis of keratinocyte cultures. The integrity and the barrier properties of HPV-KER and NHEK monolayer cultures were detected in real-time using an impedance-measurement based technology (xCELLigence, Real Time Cell Analyser system, ACEA Biosciences, San Diego, USA) 31,56 . For contact inhibited cultures, NHEK and HPV-KER cultures were grown to confluency on fibronectin-coated 96-well E-plates (Ca-low cultures). After 24 h of growth, cells were co-cultured with live C. acnes strains belonging to different phylogenetic groups within the species (889, ATCC 11828, 6609)  For the differentiated NHEK and HPV-KER (Ca-high) cultures, KC-SFM culturing media was supplemented with 1.7 mM CaCl 2 after the cultures reached confluency. Forty-eight hours after treatment, the differentiated cultures were treated with C. acnes strains and nCi changes were monitored as described above.
The effect of propionic acid (PA) was determined using HPV-KER (Ca-low) cultures. After a confluent state was reached, PA was added to the cells at different concentrations, and nCi changes were monitored as described above.
Each treatment was performed in five technical replicates and the reported data points represent the mean ± SEM, unless otherwise noted.
Western blot analysis of TJ proteins. Cells were scraped with a cell scraper and harvested by centrifugation. The pellets were resuspended in protein lysis buffer (20 mM HEPES, 150 mM KCl 2 , 1 mM MgCl 2 , 1 mM dithiothreitol, 5% TritonX-100, 10% glycerol, 0.1% NP-40) containing 1% protein inhibitor cocktail, 1% phenylmethylsulfonyl fluoride, 0.5% sodium dodecyl sulfate (all components from Sigma). Protein concentrations were measured with the BCA detection kit (Thermo Scientific, Waltham, MA, USA). SDS-PAGE was carried out using 50 µg protein sample, and proteins were transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA). Membranes were blocked with Tris-buffered saline solution (150 mM NaCl, 25 mM Tris, pH 7.4) containing 3% and 5% non-fat dry milk powder (Bio-Rad). Rabbit anti-human actin (Sigma-Aldrich) and anti-ZO-1 (Thermo Fisher Scientific) antibodies were diluted at 1:1,000, mouse anti-claudin 1 (Abnova), anti-occludin (Abnova) and anti-Claudin-4 (Thermo Fisher Scientific) were diluted at 1:500, and membranes were incubated with antibodies overnight at 4 °C. Anti-rabbit and anti-mouse IgG horseradish peroxides-conjugated (HRP-conjugated) secondary antibodies (SouthernBiotech) were applied, and the resulting bands were visualized using the C-DiGit Blot Scanner (LI-COR Biosciences, Nebraska, USA) or the Omega Lum G Chemidoc Imageing System (Aplegen, Inc, Pleasanton, CA, USA). Original western blots are provided as Supplementary Figs. 6 and 7. total RnA isolation and real-time Rt-pcR. Total RNA was isolated from the monolayer cultures using TRI-Reagent (Molecular Research Center; Cincinnati, USA). cDNA synthesis was performed using EvoScript cDNA Synthesis Kit (Roche), and changes in the expression of TJ genes (CLDN1, CLDN4, OCLN and ZO-1) were analysed by real-time RT-PCR using the Universal Probe Library system (Roche, Indiana, USA). Supplementary Table 1 lists the PCR protocols and primer sequences used. TaqMan Gene Expression Assays (Thermo Fischer Scientific, Massachusetts, USA) were used to detect tumor necrosis factor α (TNFα; HS00174128_m1) and CXC motif chemokine ligand 8 (CXC8; HS00174103_m1). All data were normalized to the 18S rRNA using the ΔΔC t method before comparing to the time-matched untreated control samples. Supplementary table 1 lists the used primers and probes. immunohistochemical staining. Paraffin-embedded OS samples were used to analyse changes in TJ protein levels after C. acnes 889 treatment. A Bond-Max automated IHC/ISH stainer (Leica Biosystems, Wetzlar, Germany) was used for immunohistochemical (IHC) staining. The staining protocol is described here briefly. Six µm sections were cut, placed on glass slides, deparaffinized (dewax, 72 °C) and rehydrated. Subsequently, antigen retrieval was performed ( www.nature.com/scientificreports/ were performed for 8 min at room temperature (RT), subsequently, the polymer step was performed (15 min, RT). Peroxidase blocking was carried out (3 min, RT trypan blue exclusion assay. HPV-KER cells were seeded on 12-well plates at a starting density of 300,000 cells/well. After 48 h, the cultures were co-cultured with various doses of C. acnes 889 strain (MOI = 100, 300). Samples were trypsinized and collected at 0, 24, 48 and 72 h after treatment, washed with phosphate-buffered saline (PBS) and stained with trypan blue dye (Sigma-Aldrich, St. Louis, Missouri, United States). Viable cells were counted using a hemocytometer.
Lucifer yellow penetration assay. HPV-KER cells were seeded on porous 12-well plates (pore size 0.4 µm, Corning, New York, USA) at a density of 1.5 × 10 5 cells/well using KSFM (Life Technologies, Carlsbad, USA). Ca-high cultures were established and treated with C. acnes 889 strain (MOI = 300) for 24 and 72 h. Lucifer yellow (LY) penetration experiments were carried out in HBSS containing 100 µM LY (Sigma-Aldrich, Saint Louis, Missouri, USA). HBSS-LY media was added to the upper chamber and samples were collected from the bottom chamber after 30 min of incubation using standard culturing conditions (5% v/v CO 2 , 37 °C). Fluorescence intensities were measured with a BMG FLUOstar OPTIMA Fluorescence plate reader (Gemini BV Laboratory, Netherland) and relative fluorescence intensities were calculated. Fluorescence intensities were normalized to the time-matched untreated control values.
In the case of the OS models, C. acnes 889 strain (15 μl, 10 9 CFU/sample) was pipetted onto the upper, epidermal part of the skin samples, which were then incubated for 72 h. For the transport experiments, LY diluted to 1 mM LY with PBS (15 μl/sample) was applied to the top. After 3 h, skin samples were collected using 6 mm punch biopsies and embedded in Shandon Cryomatrix (Thermo Fischer Scientific, Massachusetts, USA) for frozen sectioning. Sections (6 µm) were cut from the samples, and LY dye penetration was analysed using a Zeiss Axio Imager Z1 fluorescence microscope (Carl Zeiss Microscopy GmbH, Munich, Germany) equipped with an AxioCam MRm camera.
Antibiotic treatment of c. acnes co-cultured HpV-KeR monolayers. The effect of antibiotic treatment of C. acnes co-cultured HPV-KER monolayers was observed in real time using the RTCA system. HPV-KER cells were plated at a density of 30,000 cells/well in fibronectin coated 96-well E-plates. After the cultures reached confluency, they were treated with C. acnes 889 strain (MOI: 100) and, 44 h later, 1% or 5% AB/AM was added to the cultures. Each treatment was performed in five technical replicates. Impedance values were measured every 60 min for 120 h, and a dimension-free cell index (Ci) was calculated from the data. Ci (average of the technical replicates) tracings were normalized to values recorded at the addition of the bacterium to the cultures, and the resulting nCi values were plotted. Statistical analysis. Unless otherwise noted, all the data are presented as mean ± SEM for three independent experiments. For xCELLigence analysis, treatment was performed in at least five times. For real-time RT-PCR and trypan blue exclusion experiments, each treatment was performed at least three times. For western blot analysis and IHC staining, each condition was repeated once in each independent experiment. Data were compared using paired Student's t-test and one-way ANOVA with post-hoc Tukey-test. A probability value of less than 0.05 was considered significant.