INTRODUCTION: Human skin is naturally covered with a population of microorganisms, specialized or opportunists, so called skin microbiota.
Among the multiple commensal microorganisms present in the healthy skin flora, Cutibacterium acnes (previously named Propionibacterium acnes) is a ubiquitous gram-positive aerotolerant anaerobic bacterium belonging to the Actinobacteria phylum, that predominantly resides deep within the sebaceous follicle in contact with keratinocytes. Specific metabolic features allow C. acnes to colonise the hostile lipid-rich sebaceous follicle environment. In particular, it can degrade triglycerides present in sebum to generate short-chain fatty acids that can irritate the follicular wall and induce inflammation which subsequently leads to cutaneous infections (Dréno et al., 2018). The most well-known skin ailment associated with C. acnes is acne vulgaris. Furthermore, the severity of acne might not only be due to a specific C. acnes strain but also to host and environmental factors. C acnes infection induces keratinocytes activation and stimulates production of proinflammatory cytokines such as interleukins (IL-8, IL-1β and IL-12) and tumour necrosis factor-α (TNF-α). The major factors contributing to acne are the hypercornification of the outer root sheath and the pilosebaceous duct, increased sebum production and, potentially, the overgrowth of C. acnes and biofilm formation (Brandwein et al., 2016; Dréno et al., 2018).
Like other bacterial species, C. acnes shows phenotypic and genotypic diversity. The species has been subdivided into three types, I–III and two subtypes IA and IB. In patients with severe acne, there is a loss of diversity of these phylotypes compared to healthy individuals, with a clear predominance of the IA1 phylotype.
Although C. acnes is predicted to play roles in acne pathogenesis, the exact mechanism of action at the molecular level has not been clarified.
Bacteria secrete diverse factors to communicate with or evoke cellular responses from target cells. Like mammalian cells, in addition to soluble factors, most gram-negative and -positive bacteria release extracellular vesicles (EVs). By harboring diverse proteins, lipids, nucleic acids, and metabolites originating from the parent cells, EVs transfer biologically active molecules to neighboring and distant cells for communication and influence (Brown et al., 2015). In particular, bacterial EVs are known to mediate pathophysiological functions in bacteria–bacteria and bacteria–host interactions.
In this context, we investigated the effects of C. acnes EVs isolated from phylotype IA1 (DSM1897 strain) on both human keratinocytes and sebocytes.

METHODS: C. acnes DSM1897 was grown in Brain Heart Infusion (BHI) and incubated at 37°C for 2 days in anaerobic conditions. Subcultures of C. acnes were inoculated in 3 L of BHI and were grown in anaerobic conditions for 2 days. Then, EVs were isolated by ultrafiltration. Some of EVs were labeled with the lipophilic fluorescent dye Vybrant DiI cell-labeling solution for 30 min at 37 °C. EVs were added in the culture medium of keratinocytes or sebocytes. EV internalisation was studied by fluorescent microscopy.
Production of IL-8 and TNF-α protein in culture supernatants as well as filaggrin, β-defensin 2 and S100A7, was assayed by ELISA and normalized with regard to the quantity of total proteins. Autophagy was evaluated by western blot using LC3b antibody.

RESULTS & DISCUSSION: EVs were internalized by these cutaneous cells. Then, we found that C. acnes-derived EVs induced acne-like phenotypes such as increased secretion of inflammatory cytokines and dysregulated epidermal differentiation in primary human keratinocytes. Indeed, EVs significantly induced inflammatory cytokine IL-8 production and dysregulated epidermal differentiation by increasing filaggrin protein expression. Moreover, extracellular vesicles seemed to stimulate the production of antimicrobial peptides (β-defensin 2 and S100A7). However, this study did not reveal any modulation of TNF-α in keratinocytes.
Moreover, extracellular vesicles isolated from the virulent C. acnes strain stimulate the production of IL-8 and TNF-α on human sebocytes derived from iPS. This inflammation induced by C. acnes EVs is a typical component of acne. C. acnes-derived EVs also induce autophagy (LC3b) on sebocytes.

CONCLUSION: Collectively, these results suggest that EVs induce acne-like phenotypes. Therefore, inhibiting the release of EVs from C. acnes or targeting EV-mediated signaling pathways could represent an alternative method for alleviating acne occurrence and phenotypes.