The innate immune system activates the adaptive response (cytokine/chemokine release or co-stimulatory signals expressed by antigen-presenting cells [APCs]), by recognition of molecules on or produced by microorganisms. These pathogen-associated molecular patterns (PAMPs) are recognised by specific host receptors, especially toll-like receptors (TLRs) and nucleotide-binding oligomerisation domain (NOD) receptors (Medzhitov and Janeway, 2000). TLRs are expressed at the surface of monocytes, macrophages, and dendritic cells (DCs). DC maturation represents a bridge between the innate and adaptive immunity systems and depends on the binding of PAMPs to TLRs expressed at their surface.

TLRs play an essential role in immune development into conventional T helper (Th) for humoral immune response (Th2), cellular immune response (Th1), or regulatory T cells (Tr1, Th3, and CD4+CD25+) responses according to the type of stimulation (Amati et al., 2006).

TLR2 predominantly recognises Gram-positive bacterial components, especially from probiotic lactobacilli, such as peptidoglycans. TLR4 recognises bacterial endotoxin lipopolysaccharides (LPSs), which constitute the major component of the outer membrane of Gram-negative bacteria (Mukhopadhyay et al., 2004).

The epidermis also plays an important role in innate immune responses through epidermal keratinocyte TLR activation. It is now well established that the TLR2 subtype present in keratinocytes responds locally to initiate the immune pathway and thus may recognise commensal microorganisms and probiotics (Medzhitov and Janeway, 2000; Kawai et al., 2002; Pivarcsi et al., 2003). However, no chemical compounds or biotechnological biomass (or constituents) have yet been identified that induce the TLR2 pathway locally in the skin.

TLR2 activation by bacteria can result in either inflammatory or tolerogenic immune responses (Oliveira-Nascimento et al., 2012). It has been demonstrated for Neisseria meningitidis, which is a closely related Gram-negative bacteria within the Neisseriaceae family as V. filiformis, that the TLR2 activating ligand is a bacterial porin with anti-inflammatory properties (Strohl, 2005).

In contrast to Gram-negative pathogenic bacteria eliciting proinflammatory immune responses, innate immune sensing of non-pathogenic Gram-negative bacteria like V. filiformis was characterised by TLR2 signalling over TLR4 signalling (Volz et al., 2014). Similar to the way in which oral probiotics act on DC in the gut, Vfe regulates skin homeostasis by predominantly inducing the release of interleukin-10 (IL-10) through the TLR2 pathway in DC. The release of IL-10 leads to a very high IL-10/IL-12 ratio for an optimal Th1/Th2 balance and induction of T-regulatory cell function ( Figure 1 ). Most of the following properties are maintained in TLR4 knock-out cells, demonstrating that the observed efficacy is independent of the LPS of Vfe (Volz et al., 2014).

These pathways to induce tolerance may help explain the efficacy of Vfe on AD prone skin that was demonstrated in three proof-of-concept studies (Gueniche et al., 2006; Gueniche et al., 2008b; Gueniche et al., 2008c).

Interestingly, in vitro experiments also demonstrated that Vfe (0.033%–0.3%) significantly stimulated the chemotactic migration of polymorphonuclear neutrophils using a Boyden chamber assay in a dose-dependent manner and significantly increased the phagocytosis of monocytes and Langerhans cells using fluorescent beads. Monocytes freshly isolated from whole blood and Langerhans cells were seeded in a 96-well plate in culture medium with or without (control) Vfe. The phagocytosis of fluorescent beads was calculated by gating and analysing fluorescein isothiocyanate (FITC)-positive (fluorescent) cells. Vfe significantly increased the phagocytosis of Langerhans cells 1% (48.2% ± 8.0% compared with 14.2% ± 2.3% of FITC-positive cells; p < 0.01) and monocytes 0.3% (74.1% ± 1.3% compared with 45.4% ± 2.1% of FITC-positive cells; p < 0.01).

Vfe was also shown to stimulate the expression of TNFAIP3 (A20) in human epidermal keratinocytes. Since TNFAIP3 (A20) regulates proinflammatory genes link to NFkb, it indicates an anti-inflammatory activity (Urbano et al., 2018).

Furthermore, inflammation induced by Cutibacterium acnes ATCC^®6919, as measured by stimulation of IL-8 secretion on human sebocytes (2,206 vs. 45 pg/ml), was significantly inhibited in the presence of 2 μg/ml of Vfe (1,195 vs. 2,206 pg/ml).

In recent in vitro studies to evaluate the resolutive pathways, Vfe significantly stimulated lipoxins A4 and B4, protectins D1 and DX, resolvins D4 and D5, and resolvins E1 and E2 and decreased thromboxane B2, prostaglandin E2, and leukotriene B4 ( Table 1 ). This efficiency in stimulating pro-resolution is a fundamental property especially during chronic inflammatory processes (Serhan and Levy, 2018; Sugimoto et al., 2019; Fu et al., 2020).

In order to demonstrate the protective activity of the Vfe against irritation induced by methyl nicotinate, blood microcirculation was analysed using Doppler laser on ten subjects of mean age of 28 years. In the area treated with Vfe, there was a significantly smaller increase in blood flow following the application of methyl nicotinate compared with placebo.

Vfe has also been shown to reduce gene expression of thymic stromal lymphopoietin receptor (TSLPR), IL31R and cytokines TSLP, IL-31, which are stimulated by LPS, lipoteichoic acid, and zymosan, which are used to mimic pathogenic colonisation in keratinocytes, endothelial cells, or T cells (Kempkes et al., 2012). IL-31 is a T cell-derived cytokine linked to pruritus in skin inflammation, while TSLP is an excellent candidate for mediating the innate immune response triggered by viruses or bacteria. All these results may explain the efficacy of Vfe in reducing pruritus of seborrheic dermatitis or AD (Gueniche et al., 2008a).

Vfe stimulated in vitro sensory neurons cultured by a modified reported method with and without capsaicin decreased calcitonin gene-related peptide (CGRP) ( Table 2A ). In addition, the effect of Vfe on substance P-induced neurogenic skin inflammation was tested ex vivo in human abdominal plastic skin explant models. In the positive control, substance P stimulated a statistically significant increase in vasodilation, oedema, and mast cell degranulation. These substance P-induced vasodilation and oedema were abrogated by the presence of Vfe ( Table 2B ). Also, mast cell degranulation induced by substance P, as measured by CGRP liberation after capsaicin stimulation of sensitive neurons, was significantly lower in the presence of Vfe compared with the control ( Table 2C ).

The efficacy against neurogenic inflammation has been validated by results from stinging tests in a clinical trial in volunteers with sensitive skin. After 4 weeks of daily applications, the sensitive skin was significantly less reactive to lactic acid, showing a significant soothing effect over time for the Vfe formula (Li et al., 2006).

The human skin forms the interface between the body and outside environment and is endowed with innate immune defence mechanisms against infection. The epidermis (epithelial skin compartment), which covers the surface of the whole body, acts as a mechanical barrier and frontline of defence against pathogenic microbial invasion. It is an active immune organ playing a crucial role in innate immune responses that protects the body against microbial invasion by killing pathogenic microorganisms through the production of cationic antimicrobial peptides (AMP), such as human β-defensins and cathelicidin (Harder et al., 2004; Braff et al., 2005; Sumikawa et al., 2006). It also provides a home to the commensal microbiota and regulates the microbial balance of these complex microbial communities (Grice et al., 2009; Grice and Segre, 2011). In addition, the skin microbiome plays an integral role in the maturation, protection of the skin barrier function, and homeostatic regulation of keratinocytes and host immune networks; thus, it is fundamental to keep it well balanced (Suter et al., 2009; Chen et al., 2018).

β-Defensin 2 and S100A7 are AMPs that are able to decrease the growth of Malassezia restricta and Malassezia globosa fungi, as well as Gram-negative and Gram-positive bacteria. In keratinocytes (Vfe 0.3%) and reconstructed epidermis (topically applied Vfe 10%), Vfe significantly stimulated β-defensin 2 and S100A7 at the gene expression and protein levels (specific PCR, immunolabelling, and ELISA dosage) by 7,500 vs. 0 pg/ml and 50 vs. 25 ng/ml, respectively.

Although Vfe does not kill bacteria or fungi, Vfe was recently shown to specifically stimulate the growth of Staphylococcus epidermidis, one of the main beneficial bacteria ( Figure 2 ). This finding demonstrates that it might be of interest on skin health.

In addition to the direct immunomodulatory effect of Vfe on skin-associated immune responses, the stimulation of epidermal AMP and growth of S. epidermidis may help to rebalance the skin microbiota due to a reduction in S. aureus, which may partly explain the beneficial effects of Vfe on AD (Gueniche et al., 2008c).

In vitro studies using keratinocytes and fibroblasts have shown that Vfe stimulates endogenous anti-oxidant defences by stimulating mitochondrial manganese superoxide dismutase 2 (MnSOD2) activity at both the mRNA and protein levels (Mahé et al., 2006; Mahe et al., 2013). These results suggest that Vfe could induce skin cells to produce their own endogenous protective defences against both exogenous environmental stressors, such as UV radiation, as well as to combat endogenous sources of deleterious free radicals involved in skin ageing. Furthermore, Vfe was found to significantly inhibit the appearance of sunburn cells in UVB‐exposed areas, a signature of skin alteration that may be linked to a defect in MnSOD protective activity (Mahé et al., 2006; Mahe et al., 2013).

Microbial stress from PAMP or other damage-associated molecular patterns induces a decoupling of the mitochondria that results in the release of reactive oxygen species (ROS). These ROS are then capable of activating the inflammasome, causing the pro-cytokine precursors to mature into effective and inflammatory cytokines. Vfe showed a dose–response decrease in mitochondria uncoupling following microbial stress and thus protected the respiratory chain, significantly limiting the release of ROS. Moreover, this dose-dependent activity was maintained when the number of altered mitochondria was increased by a respiratory chain uncoupling agent, particularly on complex III (CoQ target) such as rotenone ( Figure 3 ). This unique activity, to reduce the number of altered mitochondria and thus the quantity of intracellular ROS, is important as the activation of the inflammasome NLRP3 can be induced by skin microbiota dysbiosis, for example, an imbalance of Malassezia spp. during seborrheic dermatitis leading to inflammation (Gueniche et al., 2008a; Kistowska et al., 2014).

CD59 protectin is a protective shield that protects the membranes of cutaneous cells against non-specific cell lysis. In keratinocytes incubated with Vfe 0.1%, CD59 protectin mRNA expression was stimulated 4.9 times (quantitative RT/PCR) and CD89 protectin levels increased by 1.8 times (p < 0.01) (fluorescence cell sorter analysis). When keratinocytes were incubated at 37°C for 24 h without thermal stress, Vfe did not induce the expression of HSP70. However, 30 min after being subjected to thermal stress, Vfe 0.2% stimulated the production of HSP70 by 500% to 700% compared with control keratinocytes, indicating that it stimulates cellular defences.

Several epidemiological studies have demonstrated that exposure to UV induces dramatic changes in immune functions. Among these changes, a decrease in the number and morphological modifications of Langerhans cells, as well as an alteration in their capacity to present antigens, has been demonstrated. Vfe was found to protect Langerhans cells in the reconstructed epidermis or skin explants exposed to UV, indicating preservation of the skin immune homeostasis even when exposed to solar radiation ( Table 3 ).

Skin barrier structure and function are essential to human health. The most important function of the skin is to act as a barrier between the “inside” and the “outside” of the organism to prevent invasion of pathogens and fend off chemical assaults, as well as the unregulated loss of water and solutes. The stratum corneum, which consists of protein-enriched cells and lipid-enriched intercellular domains, is the main physical barrier. Any modifications in epidermal differentiation and lipid composition result in altered barrier function, a central event in various skin alterations and diseases.

Keratinocyte growth was evaluated by measuring 3H-thymidine incorporation in DNA. When cells were treated with Vfe, an increase in 3H-thymidine incorporation was observed demonstrating that Vfe increases keratinocyte proliferation. The turnover time of the stratum corneum is an important parameter since it is linked to epidermal cell turnover, which decreases with age. The in vitro efficacy was corroborated by clinical studies evaluating the epidermal turnover. In vivo, the application of Vfe significantly increased the stratum corneum renewal rate, as measured by a faster reduction in dansyl chloride fluorescence, compared with the control skin. The mean cell turnover was faster for Vfe treated skin (17.3 ± 3.4 days) compared with the untreated skin (20.7 ± 2.2 days; p < 0.0001), and the mean area under the curve was lower at 57.2 vs. 66.8, respectively (p < 0.0001). The cell renewal rate was also significantly faster in the presence of Vfe when measured by the decrease in colour from staining with 10% dihydroxyacetone (DHA) ( Figure 4 ).

Vfe 1% induced faster in vitro keratinocyte (monolayers seeded on collagen 1) migration than the control with better efficacy in reepithelisation (297 ± 84 migrative cells compared with 201 ± 28 for the control). Additionally, using a new in vitro epidermal regeneration 3D model for keratinocyte migration, mimicking phase II of wound healing (Deshayes et al., 2018), Vfe significantly increased wound healing at day 16 (Vfe 0.3%) and day 17 (Vfe 0.1% and 0.3%).

The adherens and tight junctions in the epidermis, in addition to the abundance of corneodesmosome, play a fundamental role in skin physiology by mediating firm mechanical stability between the cells. Indeed, these structures are crucial components of the skin barrier function and link structural integrity to proliferation and inflammatory responses in the skin. Tight junctions in the stratum granulosum regulate “apical” protein and lipid vesicle targeting toward the stratum corneum. Vfe was shown to increase the quantity of zonula occludens-1 in normal keratinocytes, which is one of the main tight junction proteins (198% increase with Vfe 0.05% and 354% increase with Vfe 2% compared with 340% for the positive control CaCl₂, 1.5 mM).

Each maturation step contributing to the formation of an effective moisture barrier, especially corneocyte strengthening, lipid processing, and natural moisturising factor generation (a complex mixture of low-molecular-weight, water-soluble compounds formed within the corneocytes by degradation of filaggrin), is influenced by the level of stratum corneum hydration. Inefficient degradation of corneodesmosomes and accumulation of corneocytes on the skin’s surface cause dry, flaky, and dull-looking skin. In normal human keratinocytes, Vfe was shown to increase the expression of loricrin (a protein precursor of the stratum corneum) and three proteins fundamental for differentiation, including transglutaminase (epidermal TGK are involved in cornified envelope formation by cross-linking structural proteins), LEP16 (late envelope proteins are linked to cornified envelope proteins), and corneodesmosin (a protein found in corneodesmosomes required for desquamation) ( Table 4 ).

The efficacy of Vfe on reinforcing the skin barrier function was shown in vitro using reconstructed skin. The rate of diffusion of caffeine through reconstructed skin was slowed after the application of Vfe, indicating an improvement in skin barrier function ( Figure 5 ). Furthermore, Vfe improved barrier function recovery in vivo after tape stripping compared with the control area, as evaluated by Tewameter^® on healthy volunteers ( Figure 6 ). A significantly better improvement of skin barrier was found after 1 (p = 0.017) and 2 days (p < 0.001) of treatment with Vfe compared with the control skin that was left untreated after tape stripping.

Laminin 5 is found in the hemidesmosome adhesive junction and contributes to the cohesion of epithelial cells for a strong dermal–epidermal junction (DEJ), while perlecan is a proteoglycan found in the DEJ and is able to store and protect cellular growth factors. The application of Vfe 0.05% stimulated perlecan 180% with taking the control as 100%. Also, Vfe had a protective effect on laminin 5 and perlecan altered by corticoids in ex vivo skin.

Finally, type I and IV collagens are important constituents of the skin. High proportions of type I collagen are found in all dermal layers, while type IV collagen is localised in the basement membrane of the DEJ. Although they possess closely related amino acid compositions, they present important structural differences that confer specific biophysical properties. When the expression of proteins was measured in normal human dermal fibroblast monolayers, Vfe significantly stimulated the expression of collagen I and IV ( Table 4 and Figure 7 ).