Pro-inflammatory activity of Cutibacterium acnes phylotype IA1 and extracellular vesicles: An in vitro study

Abstract

Acne is a chronic inflammatory skin condition that involves Cutibacterium acnes (C. acnes), which is classified into six main phylotypes (IA₁, IA₂, IB, IC, II and III). Acne development is associated with loss of C. acnes phylotype diversity, characterised by overgrowth of phylotype IA₁ relative to other phylotypes. It was also shown that purified extracellular vesicles (EVs) secreted by C. acnes can induce an acne-like inflammatory response in skin models. We aimed to determine if the inflammatory profile of EVs secreted by C. acnes phylotype IA₁ from an inflammatory acne lesion was different from C. acnes phylotype IA₁ from normal skin, thus playing a direct role in the severity of inflammation. EVs were produced in vitro after culture of two clinical strains of C. acnes phylotype IA₁, T5 from normal human skin and A47 from an inflammatory acne lesion, and then incubated with either human immortalised keratinocytes, HaCaT cells, or skin explants obtained from abdominoplasty. Subsequently, quantitative PCR (qPCR) was performed for human β-defensin 2 (hBD2), cathelicidin (LL-37), interleukin (IL)-1β, IL-6, IL-8, IL-17α and IL-36γ, and ELISA for IL-6, IL-8 and IL-17α. We found that EVs produced in vitro by C. acnes derived from inflammatory acne lesions significantly increased the pro-inflammatory cytokines and anti-microbial peptides at both transcriptional and protein levels compared with EVs derived from normal human skin. We show for the first time that C. acnes EVs from inflammatory acne play a crucial role in acne-associated inflammation in vitro and that C. acnes phylotype IA₁ collected from inflammatory acne lesion and normal skin produce different EVs and inflammatory profiles in vitro.

Keywords: Cutibacterium acnes; acne; extracellular vesicles; inflammation; skin.

FIGURE 1

Growth and morphological differences between the two different clinical isolates of Cutibacterium acnes (C. acnes): T5 (phylotype IA₁, normal skin) and A47 (phylotype IA₁, lesional acne). (A) Growth analysis of C. acnes strains T5 (phylotype IA₁, normal skin) and A47 (phylotype IA₁, acne lesion). Bacterial cultures of T5 and A47 were inoculated into fresh medium at an optical density (OD) of 0.1, and then OD measurements of the bacterial cultures were taken at 10 days post‐inoculation. (B) EVs derived from C. acnes T5 and A47 were subjected to scanning transmission electron microscopy (STEM), and EV size was measured from at least five images from each sample. (C) Images of EVs derived from C. acnes T5 and A47 by SEM with STEM detector. Experiments were performed at least three times in triplicate. Significance was considered when p < 0.05 as determined by Student's t‐test. ***p < 0.001, ****p < 0.0001.

FIGURE 2

Transcriptional analysis by qPCR for hBD2, IL‐1β, IL‐6, IL‐8 and IL‐36ɣ in extracellular vesicles (EVs)‐treated immortalised human keratinocytes (HaCaT cells). qPCR for innate immune markers (A) hBD2, (B) IL‐1β, (C) IL‐6, (D) IL‐8 and (E) IL‐36ɣ in HaCaT cells treated for 24 h with EVs isolated from Cutibacterium acnes strains T5 (phylotype IA₁, normal skin) and A47 (phylotype IA₁, acne skin). Vehicle control was the incubation of cells with the same volume of phosphate‐buffered saline (PBS). Experiments were performed at least three times in triplicate. Significance was considered when p < 0.05 as determined by multiple comparisons using ANOVA analysis. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 3

Transcriptional analysis by qPCR for hBD2, LL‐37, IL‐1β, IL‐6, IL‐8, IL‐17α and IL‐36ɣ in extracellular vesicles (EVs)‐treated human skin explants. qPCR for innate immune markers (A) hBD2, (B) LL‐37, (C) IL‐1β, (D) IL‐6, (E) IL‐8, (F) IL‐17α and (G) IL‐36ɣ in ex vivo skin explants treated for 24 h with EVs derived from Cutibacterium acnes (C. acnes) strains T5 (phylotype IA₁, normal skin) and A47 (phylotype IA₁, acne skin). Experiments were performed at least three times in triplicate. Significance was considered when p < 0.05 as determined by multiple comparisons using ANOVA analysis. *p < 0.05, **p < 0.01, ****p < 0.0001.

FIGURE 4

Protein analysis by ELISA for innate immune markers IL‐6, IL‐8 and IL‐17α in extracellular vesicles (EVs)‐treated immortalized human keratinocytes (HaCaT cells) and human skin explants. ELISA for innate immune markers (A) IL‐6 and (B) IL‐8 in HaCaT cells, and (C) IL‐6, (D) IL‐8, and (E) IL‐17α in human skin explants treated for 24 h with EVs derived from Cutibacterium acnes (C. acnes) strains T5 (phylotype IA₁, normal skin) and A47 (phylotype IA₁, acne skin). Experiments were performed at least three times in triplicate. Significance was considered when p < 0.05 as determined by multiple comparisons using ANOVA analysis. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.