New insights into the role of Cutibacterium acnes-derived extracellular vesicles in inflammatory skin disorders

Abstract

Cutibacterium acnes (C. acnes) is one of the most prevalent bacteria that forms the human skin microbiota. Specific phylotypes of C. acnes have been associated with the development of acne vulgaris, while other phylotypes have been linked to healthy skin. In this scenario, bacterial extracellular vesicles (EVs) play a role in the interkingdom communication role with the human host. The purpose of this study was to examine the impact of EVs generated by various phylotypes of C. acnes on inflammation and sebum production using different in vitro skin cell types. The main findings of this study reveal that the proteomic profile of the cargo embodied in the EVs reflects distinct characteristics of the different C. acnes phylotypes in terms of life cycle, survival, and virulence. The in vitro skin cell types showed an extended pro-inflammatory modulation of SLST A1 EVs consistently triggering the activation of the inflammation-related factors IL-8, IL-6, TNFα and GM-CSF, in comparison to SLST H1 and SLST H2. Additionally, an acne-prone skin model utilizing PCi-SEB and arachidonic acid as a sebum inducer, was employed to investigate the impact of C. acnes EVs on sebum regulation. Our findings indicated that all three types of EVs significantly inhibited sebum production after a 24-h treatment period, with SLST H1 EVs exhibiting the most pronounced inhibitory effect when compared to the positive control. The results of this study highlight the protective nature of C. acnes SLST H1 EVs and their potential use as a natural treatment option for alleviating symptoms associated with inflammation and oily skin.

Figure 1

EVs isolation protocol and in vitro experimental design. (A) Process to isolate C. acnes-derived extracellular vesicles (EVs). (B) Schematic presentation of the different skin in vitro cell types: HaCaT cell line, SZ95 cell line, Jurkat cell line and the acne-prone skin model with PCi-SEB cells using AA to induce sebum production.

Figure 2

Visualization of C. acnes EVs by different microscopy techniques. (A) Transmission Electron Microscopy of C. acnes SLST H1 EVs and A1 EVs (bar 500 nm). (B) Nanoparticle Tracking Analysis of C. acnes SLST A1, H1 and H2 EVs. Prior to the measurement, the samples were diluted in PBS buffer at 1:5000, 1:1000 and 1:1000 respectively. EVs have a mean diameter of 96.8 ± 0.5 nm, 100.7 ± 0.7 nm and 133.0 ± 2.3 nm respectively. EV-concentration is expressed as a number of particles per mL on the y axis. All data represent the mean of three independent experiments ± standard error.

Figure 3

Proteomic characterization of C. acnes SLST A1, H1 and H2 EVs. (A) Proteomic fingerprint of C. acnes EVs and the corresponding protein whole cell content of each bacterium represented by an SDS-PAGE. (B) Venn diagram of the proteins contained in C. acnes SLST A1, H1 and H2 EVs. The number of overlapping proteins between the different phylotypes is indicated. All data is obtained from three independent experiments. (C) Gene Ontology analysis of C. acnes SLST A1, H1 and H2 EVs preparations. Biological process, Molecular function, and Cellular components of the identified vesicular proteins in C. acnes EVs are presented here. For each C. acnes phylotype, three independent batches were analysed in the proteomic analysis.

Figure 4

Uptake of C. acnes SLST A1 EVs in (A) HaCaT cell line and (B) SZ95 cell line. Visualization of internalized EVs by confocal fluorescence microscopy. Both cell lines were incubated for 24 h at 37 °C with rhodamine B-R18-labelled SLST A1 EVs (2 μg/ well).

Figure 5

Cytotoxicity assay of (A) HaCaT cell line and (B) SZ95 sebocyte cell line treated with different doses of C. acnes SLST A1, H1 and H2 EVs. All data are presented as mean ± standard deviation (SD) of triplicate measurements (*p ≤ 0.05 vs. non-stimulated controls).

Figure 6

In vitro analysis with direct incubation of C. acnes EVs. Human cells were treated for 24 h with different concentrations (12.5, 25 and 50 μg/mL) of C. acnes SLST A1, H1 and H2 EVs. Total RNA was extracted, and different biomarkers were assessed by RT-qPCR. (A) HaCaT (B) SZ95 and (C) Jurkat immortalized cell lines. All data are presented as mean ± standard deviation (SD) of triplicate measurements (*p ≤ 0.05, **p ≤ 0.001 vs. non-stimulated controls).

Figure 7

Total protein concentration secreted into the SN after direct incubation with C. acnes EVs. Human cells were treated for 24 h with different concentrations (12.5, 25 and 50 μg/mL of C. acnes SLST A1, H1 and H2 EVs. Filtered SN was evaluated by Multiplex analysis to measure protein secreted in it. Ranges for the standard curve for each of the biomarkers were: GM-CSF: 0.31–4978.62 pg/mL, TNFα: 0.11–1699.58 pg/mL and IL-6: 0.05–768.83 pg/mL. (A) HaCaT (B) SZ95 and (C) Jurkat immortalized cell lines were used. All data are presented as mean ± standard deviation (SD) of triplicate measurements (*p ≤ 0.05, **p ≤ 0.001 vs. non-stimulated controls).

Figure 8

Reduction of lipid production was assessed after treating PCi-SEB with AA5 for 24 h. PCi-SEB were treated during 48 h with AA at 5 µM (AA5) to induce lipid production. Afterwards PCi-SEB were treated for 24 h with 12.5 μg/mL of C. acnes SLST A1, H1 and H2 EVs (A) Metadata analysis on inter-EVs batches and (B) Fluorescence microscopy image of PCi-SEB stained with BODIPY 493/503; negative control (Vehicle), positive control (AA5). All data are presented as mean ± standard deviation (SD) of triplicate measurements (*p ≤ 0.05, **p ≤ 0.001 vs. non-stimulated controls).