36 TESTING
3D skin model to study bacteria-host interactions
Valerie Borrel, Antoine Florin, Celine Lancelot, Michel Salmon - Straticell
While it was long thought that acne was due to the presence of a bacterium named at this time Propionibacterium acnes, it is now admitted that the development of acne spots is related to an imbalance in the phylotypes (phylogenetically related individuals) of this bacterium now renamed Cutibacterium acnes. The pathophysiology of acne is indeed the
result of an increase in the IA1 phylotype of C. acnes, in sebum-rich areas.1
The
hyperseborrhea combined to the hyperkeratinization form a lipid-rich anaerobic environment that favours the growth of C. acnes.2 Additionally, acne-prone skins also display
an overexpression of genes encoding pro- inflammatory molecules such as interleukin 8 (IL-8, encoded by the CXCL-8 gene) and interleukin 1 beta (IL-1β, encoded by the IL1B gene), as well as antimicrobial peptides like the human β-defensin 2 (hBD2, encoded by the DEFB4 gene).2,3 All these factors lead to the appearance of
uncomfortable acne breakouts in susceptible individuals. The population most affected by acne is the prepubertal population at 90%, even though acne is also found in 30% of adults.4 The prolonged wearing of masks during the pandemic further increases these percentages with the emergence of the new phenomenon of ‘maskne’, a contraction of ‘mask’ and ‘acne’. Assuming that the cause was bacterial,
several antibiotic treatments exist on the market to cure acne. However, in addition to the problem of bacterial resistance to antibiotics, it is now known that antibiotics will also impact the growth of commensal bacteria, thus slowing down the restoration of a balanced microflora. Retinoid derivatives such as isotretinoin or
benzoyl peroxide have also been prescribed to reduce acne spots, but their adverse side effects reduce their use and possible treatments.5,4 Therefore, it appears important to find new topical acne treatment solutions that can restore a balanced skin flora without undesired effects. Few in vitro models are available to
facilitate the preclinical phase of research and objectification of new anti-acne molecules. Microbiological tests and 2D cellular systems exist, but with poor similarity to the real micro- environment.
Animal models also exist, but their access is
limited, or even forbidden in the context of the development of active ingredients for cosmetic purposes. That is why access to 3D in vitro skin models replicating the cutaneous biology is
PERSONAL CARE June 2022
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0 0 hour Without inhibitor 72 hours
Figure 1: Cutibacterium acnes growth on reconstructed human epidermis (RHE). RHE were colonised for 72 hours with a strain of C. acnes, in the absence or presence of a bacterial growth inhibitor
becoming a priority to accelerate the preclinical phases of development of new solutions. In order to fill this gap, we decided to develop
a new model to study the interactions between the skin and its flora, by colonizing reconstructed human epidermis (RHE) with microorganisms of interest. Here, we present a model of RHE colonized
with a IA1 phylotype strain of C. acnes. After confirming the effective growth of the bacterium on the stratum corneum of the RHE, we studied the tissue response to this colonization by monitoring key biomarkers.
Colonization of reconstructed epidermis For the first step in the development of this new model, we reconstructed epidermis from primary normal human keratinocytes (NHEK). At the end of their reconstruction, we laid
on the stratum corneum a quantity of 100,000 colony forming units (CFU) per square centimetre of an acneic phylotype IA1 C. acnes reference strain in a lipid mixture representative of the ecological niche of this lipophilic bacterium. As a growth control, bacteria were applied in
the absence or presence of a molecule known for its inhibitory effect on this bacterium. After 72
hours of culture, all bacteria were harvested and the bacterial growth was evaluated by counting CFU on agar media under anaerobic culture conditions. This count allowed us to observe a bacterial CFU/cm2
growth up to 107 in the absence of
inhibitor, against a total absence of bacteria in the presence of the inhibitor molecule (Figure 1). These results confirm the survival and growth of the C. acnes strain on the RHE, and the possibility of inhibiting this growth with a specific inhibitor. From a histological point of view, we did not
notice any profound change in the morphology of the epidermis colonized by the bacteria. Indeed, after Hemalun/Eosin staining of paraffin-embedded tissues, we observed that the presence of bacteria is limited to the stratum corneum, without impact on the morphology of the epidermal layers (Figure 2). In contrast, the monitoring of three
biomarkers CXCL-8, IL-1β and DEFB4 revealed a significant increase in the expression of these genes in the presence of C. acnes (Figure 3). This analysis demonstrates that the presence
of bacterial elements on reconstructed epidermis induces a response similar to that described in the literature.2,3
www.personalcaremagazine.com With inhibitor
C. acnes growth on RHE (Log CFU/cm2
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