Positive and ecobiological contribution in skin photoprotection of ectoine and mannitol combined in vivo with UV filters

Chronic exposure to ultraviolet (UV) irradiation causes immunosuppression, photoaging, and carcinogenesis by induction of a cascade of skin damages. Sunscreens currently on the market are not absorbing UV rays uniformly throughout the full UV range, high sun protection factor (SPF) sunscreens absorb most of UVB rays but are less effective in absorbing the UVA part of the spectrum. In the context, one approach could consist of preserving the skin natural resources and mechanisms, which is the foundation of the ecobiological approach, by combing UV filters and antioxidants to enhance their photoprotective effect.


| INTRODUC TI ON
Chronic exposure to ultraviolet (UV) irradiation causes immunosuppression, photoaging, and carcinogenesis. 1A cascade of reactions occurs upon UV exposure of human skin, including the generation of reactive oxygen species (ROS), oxidation of lipids and proteins, DNA damages, p53 mutations, sunburn cell formation, release of cytokines and matrix metalloproteinases (MMP), isomerization of trans-urocanic acid (UCA), and reduction of the antigen presentation function of Langerhans cells, key cells in the cutaneous immune response. 1UVB rays (280-315 nm) are predominantly absorbed by the skin's epidermis, while longer wavelength UVA rays (315-400 nm) penetrate the dermis more deeply, which makes them the primary driver of photoaging. 2 Moreover, UVA rays have been shown to induce oxidative stress through ROS, including singlet oxygen inducing oxidative stress leading to oxidative DNA lesions, which cause mutations. 3So photoprotection against UVA rays, in addition to UVB rays, is essential.
No sunscreen on the market is currently capable of significantly reducing the total UV exposure effect.Common commercialized sunscreens can provide a higher degree of UVB protection, evaluating erythema measured by the sun protection factor (SPF), than UVA protection for two reasons.First, there are less UVA filters available on the market than UVB filters, and it is therefore more complicated to absorb and/or reduce UV rays uniformly throughout the full UV range.Second, the European commission recommends for sunscreens marketed in Europe a minimal UVA protection factor to SPF ratio of 1:3, so do not required a total UVA protection, which would have an impact on the sunscreen cost.Moreover, for many decades the SPF has assessed sun protection using UV-induced erythema as an endpoint, but it does not accurately reflect all photoprotection benefits.An ecobiological approach would consider, not only to counteract the immediate consequences of sun exposure, but to better understand the causes, to prevent long term consequences.This original approach of ecobiology considers the skin, in relation with its environment, as an ever-evolving ecosystem, whose natural resources and mechanisms must be preserved, 4,5 and can be applied to adapted skincare product. 6 increase the protection derived from filters against UVA, which strongly induces oxidative stress, many studies have investigated the effect of additional molecules with physical and/or biological actions when applied topically.These molecules can act as UV blockers, SPF enhancers, or through biological effects. 7,810] Indeed, the body may not be able to completely neutralize ROS generated by excessive sun exposure, which can lead to significant skin damage.Thus, topical application of photoprotective antioxidants could maintain or restore skin functions.Cosmetic formulations combining UV filters with vitamin derivatives and/or botanical extracts have been shown to enhance antioxidant activity and increase protection against UVA and UVB radiation, 9-12 but few were human clinical studies. 10,12 this context, two active ingredients are interesting to investigate.5][16][17] In addition, ectoine, a cyclic amino acid produced by extremophile microorganisms, can prevent many UV/light consequences (Langerhans cells migration, 18 pro-inflammatory cytokines release, 19 DNA damages, 17 sunburn cell formation 18 ).Moreover, ectoine was initially described as a cell membrane protector reducing transepidermal water loss, chemical and physical penetration, and subsequent inflammation, and effective in atopic skin and prevention of skin aging. 20The present study aimed to characterize the photoprotection properties of an association of ectoine and mannitol and to investigate its additional photoprotection against UVA and UVB effect in humans in combination with UV filters based on the assessment of specific UV-induced biomarkers (squalene, trans-UCA, and catalase activity).

| MATERIAL S AND ME THODS
Additional details regarding the materials and methods are available in the supplementary data.

| Chemicals
The active association is composed of ectoine and mannitol.The combined UV filters comprised diethylhexyl butamido triazone, bisethylhexyloxyphenol methoxyphenyl triazine, butyl methoxydibenzoylmethane, and ethylhexyl triazone.In the in vivo study, the active association and the UV filters were formulated in a vehicle cream (Table S1) presenting an in vivo SPF of 30 (ISO 24444 method) and an in vivo UVA index of 15 (ISO 24442 methods).

| Glutathione assay
HaCaT cells were left untreated or pretreated for 24 h with the active association (0.01% ectoine and 0.05% mannitol diluted in Dulbecco's Modified Eagle Medium, DMEM) before total spectrum irradiation at 200 J/cm 2 (750 W/m 2 ) with a SUNTEST XLS (Atlas) device equipped with a daylight filter (300-800 nm) (Atlas), adapted to the cellular viability.Immediately after irradiation, the cells were re-incubated with the active ingredients.Twenty-four hours after irradiation, glutathione levels were quantified by GSH-Glo Assay (ref. V6911, Promega).The luminescence was measured with a Victor3™ 1420 Multilabel Counter plate reader (PerkinElmer) and the data analyzed with Wallac Workstation software (PerkinElmer).

| γH2A.X quantification
Normal human epidermal keratinocytes (NHEK) were left untreated or stimulated for 24 h with the active association (ectoine 0.01% and mannitol 0.05% diluted in the medium).The cells were then irradiated according to the cellular viability with a UVA lamp (PL 36W/09 PUVA, Waldmann) at 15 J/cm 2 (3.55 mW/cm 2 ), fixed, stained with anti-phosphorylated H2A.X (phosphor-Ser139) antibody (Millipore), and revealed with a secondary antibody coupled to fluorescein isothiocyanate (FITC; Santa-Cruz).The stained cells were imaged with a DP72 camera (Olympus) coupled to a BX-60 epifluorescence microscope (Olympus) and analyzed with Cell F® software (Olympus).Increased γH2A.X staining induced by UVA corresponds to an increase in the cellular response to doublestranded DNA breaks following single-stranded DNA breaks induced by oxidative stress.

| Isolation, culture, and irradiation of Langerhans cells
Epidermal cells were isolated from ex vivo normal human skin by trypsinization.After separation from the dermis, a suspension of epidermal cells was obtained following repeated agitation and filtration through sterile gauze.The epidermal cells were enriched in Langerhans cells by two cycles of centrifugation using Lymphoprep™ (Flobio SA).The Langerhans cells were incubated for 18 h with the active association (0.01% ectoine and 0.05% mannitol diluted in Hanks saline solution) and irradiated according to the cellular viability 21 at 4-6 J/cm 2 with a Bio-Sun UV irradiation system (Wilber-Lourmart) equipped with a UVA lamp emitting light at 365 nm and a plexiglass filter.

| Purification of lymphocyte cells and lympho-Langerhans cells mixed culture
Mononuclear peripheral blood cells were obtained from healthy unrelated subjects by centrifugation using Lymphoprep™.After monocyte adhesion for 2 h, T lymphocytes were purified along with sheep red blood cells, which were eliminated by osmotic shock with NH 4 Cl.
Lymphocyte proliferation was measured from the fifth to the sixth day of culture by the addition of tritiated thymidine.The incorporated radioactivity was measured using a β counter (Matrix, Packard Instrument Co.).

| H 2 DCF-DA assay
NHEK were pre-incubated for 24 h with the active association (0.01% ectoine and 0.01% mannitol diluted in medium or phosphate buffered saline [PBS]).After incubation with the fluorescent probe 2,7-DCDHF-DA, a quartz plate was deposited on each culture plate where the SPF30 filters were applied (1 mg/cm 2 ) or not (condition without filters) on quartz plate.The cells were left untreated or irradiated according to their viability with UVB at 100 mJ/cm 2 and UVA at 0.7 J/cm 2 using a SOL500 Sun Simulator equipped with an H2 filter (280-800 nm) (Dr.Hönle, AG).After irradiation, the cells were incubated for 30 min in PBS with and without the active association (0.01% ectoine and 0.01% mannitol) before quantification of the ROS production by measurement of the fluorescence emitted by the 2,7-DCDHF-DA probe (λex = 485 nm, λem = 538 nm) using a microplate reader (Perkin Elmer).

| Subjects and study design
This open intra-individual study was approved by the Internal Ethics Committee in February 2021 (Study #20E0976).Ten men aged 20-44 years with phototype II to III according to the Fitzpatrick scale were included with oily skin on the back (according to the supplier, with a sebum rate ≥27 μg/cm 2 verified using a Sebumeter®).The subjects received the requisite written and verbal information and provided their written informed consent.Each subject's minimal erythema dose (MED) was determined beforehand.At D0, a skin surface sampling allowed determination of the basal value of three biomarkers (described below) for each subject.Non-invasive samplings were performed with a swab sampling kit supplied by QIMA Synelvia.A swab wetted into a cocktail solution was applied to the selected area for 45 s using a template, then cut and placed in an Eppendorf tube.The sampling was repeated twice per area.From D0 to D3, the vehicle cream was applied twice daily alone or containing the active association (0.1% ectoine and 0.1% mannitol) and/or UV filters (2 mg/cm 2 ) to four defined areas on the back of the subjects.The subjects did not apply any other product to the back and washed their hair with head forward.At D3, the four selected UV sub-areas were irradiated by 2 MED (UVA and UVB spectrum) with a xenon lamp Monoport 300W (Solar Light).At D4, skin surface samplings were performed on the four irradiated and four non-irradiated sub-areas of each subject and stored at −20°C before biochemical analyses.Trans-UCA quantification was measured by LC/MS following a protocol similar to that for squalene.After centrifugation of swab homogenates, samples were stabilized and extracted using a buffer solution (QIMA Synelvia propriety) and the supernatant diluted with Tris-buffered saline (Sigma-Aldrich).The selected ion monitoring mode was recorded at 139 m/z.The concentration of trans-UCA in each sample was normalized to the total protein content of each sample expressed in μg/mg.

| Statistical analysis
For the in vitro experiments, the p-value was calculated with the non-paired equal variance Student's test.For the in vivo study, if normality was proven by the Shapiro-Wilk test, Student's test was used, otherwise the non-parametric Wilcoxon's signed-rank test was used.For all statistical analyses, a significance threshold level of 5% was chosen.
The coefficient of drug interaction (CDI) was used to analyze the synergistic inhibitory effect of the active association and UV filters combination.The CDI was calculated according to the formula described in detail elsewhere. 22A CDI <1 indicates synergy, a CDI = 1 indicates additivity, and a CDI >1 indicates antagonism.

| In vitro efficacy of ectoine and mannitol association on UV-induced oxidative stress
The glutathione level in HaCaT cells was significantly reduced by 24.5% following full solar spectrum irradiation compared to nonirradiated conditions but significantly maintained when pretreated with the active association (Figure 1).UVA-irradiation of NHEK significantly induced positive γH2A.xnuclei (77.3%) compared to non-irradiated non-treated cells, which was significantly higher than in cells pre-treated with the active association in terms of number (66.8%) and intensity (Figure 2).Only 10.5% of the UVA-irradiated NHEK pre-treated with the active association exhibited UVA-DNA strand breaks.Comet assay after UVA irradiation also showed activation of DNA damage mechanisms with the active association and with ectoine or mannitol alone (data not shown).

| In vitro efficacy of the active association on lymphoproliferation induced by UVA-irradiated Langerhans cells
UVA-irradiated Langerhans cells exhibited reduced T lymphocyte proliferation compared to non-irradiated cells.This reduction was significantly decreased by 17.5% in cells pre-treated with the active association (Figure 3).Ectoine and mannitol alone did not significantly alter the allostimulation function of UVA-irradiated Langerhans cells compared to the non-irradiated controls (data not shown).

| In vitro efficacy of UV filters with and without the active association on UV-induced oxidative stress
UV irradiation significantly increased the fluorescence (2.5-fold; p < 0.001) in NHEK, corresponding to intracellular ROS production, compared to non-irradiated cells (Figure 4).ROS formation was significantly reduced in irradiated cells protected by UV filters compared to non-protected irradiated cells (p < 0.05) but was still significantly higher compared to non-irradiated non-treated cells (1.75-fold; p < 0.05).Interestingly, the formation of ROS was not significantly increased in NHEK protected with UV filters and F I G U R E 1 Glutathione levels in untreated and active association treated HaCaT cells before and after solar spectrum irradiation (300-800 nm, 750 W/m 2 ).The mean values of the relative luminescent units, with standard deviations, are presented.**p < 0.01; ns, not significant.Similarly, higher genomic protection was also observed by comet assay after UVA irradiation and treatment with the active association combined with UV filters compared to UV filters alone (data not shown).

| In vivo efficacy of the active association with the UV filters after UV-irradiation on oxidative stress and immunosuppression
This study was conducted on 10 men aged 20-44 years (mean age of 27.

| DISCUSS ION
Our work shows that the ectoine and mannitol association protected UV-irradiated skin cells from UV-induced alterations by maintaining glutathione levels, reducing both DNA lesions and ROS formation, and preserving Langerhans cells functionality.Interestingly, the production of ROS in irradiated NHEK was not significantly increased in cells pre-treated with the active association in combination with UV filters compared to non-irradiated cells, unlike UV filter protection alone.Furthermore, in vivo application of the active association combined with UV filters significantly protected three natural defense systems, namely squalene oxidation, catalase activity, and trans-UCA photo-isomerization; so more than the UV filter application alone.This suggests that the active association provides an ecobiological composition which provide significant additional skin photoprotection to UV filters with an SPF of 30.
To our knowledge, to date, only one study in humans has investigated skin UV protection by active ingredients alone (caffeine, vitamin E, vitamin C, Echinacea pallida extract, gorgonian extract, and chamomile essential oil) and in combination to UV filters, but this was with an invasive method. 12In this preliminary study on biopsies from five subjects, Matsui et al. showed no significant difference in the Langerhans cells number but significant collagen protection based on measurement of MMP-1 levels on the two MED-irradiated areas treated with an SPF25 sunscreen containing the antioxidants compared to the area treated with the sunscreen alone. 12In our current studies, the active association, comprising ectoine and mannitol, also exhibited antioxidative properties.5][16][17] Ectoine, on the contrary, is a cyclic amino acid produced by extremophile microorganisms that exhibits many interesting dermatological properties.It was initially described as a cell membrane protector with outstanding water-binding and stabilizing properties that could reduce transepidermal water loss, chemical and physical penetration, and subsequent inflammation. 20At concentrations of 2% and higher, ectoine improves dryness and impaired skin barrier induced by UVA irradiation than with UV filters.Interestingly, the active association also preserved both in vitro Langerhans cells functionality and in vivo trans-UCA transformation to cis-UCA (known to be immunosuppressive 25 ), suggesting that it could protect skin from UV-induced immunosuppression.Moreover, the active association synergically increased the photoprotection of UV filters against UCA transformation.Since UCA transformation is mainly due to UVB rays 25 and that cis-UCA was recently shown as a sensitive noninvasive biomarker to assess UVB photoprotection, 26 this suggests that the active association may also protect against UVB rays.
Following the principle of ecobiology, which is an original approach considering the skin as an ever-evolving ecosystem whose natural resources and mechanisms must be preserved, 4,5 the association of ectoine and mannitol combined the UV filters protects skin components (lipids, proteins, DNA) and preserves the skin antioxidant and immune defense systems.
The main limitation of this work is the small sample size of subjects in the in vivo study and the irradiation protocol, not totally matching with real conditions of use.The irradiation after each sunscreen application during 4 days is not ethical, however applying daily a sunscreen is realistic.A study including more subjects and investigating other sun protection biomarkers, such as sunburn cells formation, p53, caspase-3, and DNA repair enzymes, could be interesting but would require the use of invasive technique to obtain biopsies.Another interesting aspect that warrants investigation is how the active association prevents UCA photo-isomerization (direct UVB absorption, trans-UCA form stabilization, etc.).
In conclusion, our study performed in vitro and in vivo with noninvasive analysis of biomarkers showed that this active association

2. 6 . 2 |
Quantification of oxidized squalene, catalase activity, and trans-UCA Squalene oxidation was quantified as the ratio of the concentrations of monohydroperoxide squalene (oxidized squalene) and squalene (non-oxidized squalene) measured by LC/MS.After centrifugation of swab homogenates, samples were extracted by double liquid/liquid extraction method and evaporated under nitrogen at 60°C, and the residue was dissolved in ethanol.Squalene monohydroperoxide and squalene were detected with an UltiMate 3000 liquid chromatography system (Dionex) coupled to an ISQ Plus detector (Fisher Scientific).For mass spectroscopy detection, the ion source was atmospheric pressure chemical ionization.Positive ion spectra were recorded in the 50-450 m/z range.The ratios of the concentration of monohydroperoxide squalene and squalene were normalized to the total protein content.The catalase activity in the swabs was determined using a colorimetric method set up by QIMA Synelvia.This method consisted of measurement of the residual content of hydrogen peroxide (Sigma-Aldrich) following the catalase action, which induced resorufin fluorescence.Resorufin fluorescence intensity is inversely proportional to catalase activity.The activity of catalase in each sample was normalized to the total protein content of each sample expressed as IU/ mg.
with the active association compared to non-irradiated non-treated cells.Although the formation of ROS was decreased by 35% in cells protected by UV filters and pre-treated with the active association compared to UV filters alone, the difference was not significant (p = 0.08).The combination of UV filters and the active association promoted a level of antioxidant protection of 86% compared to the irradiated non-treated cells.

F I G U R E 2 F I G U R E 3
γH2A.X (phospho-Ser139) quantification in NHEK pre-treated with the active association before UVA irradiation (315-400 nm, 3.55 mW/cm 2 ).The mean percentage of cells positive for γH2A.X (phosphor-Ser139) divided by the total number of cell nuclei, with standard deviations, are presented.Corresponding epifluorescence microscopy images of the cells (×20) are shown below each bar.**p < 0.01, ***p < 0.001.In vitro measurement of proliferation of lymphocytes co-cultured with Langerhans cells pre-treated with the active association before UVA irradiation (365 nm, 4-6 J/cm 2 ).The results are means with standard deviations of the percentage inhibition compared to the non-irradiated controls.**p < 0.01.proliferation compared to non irradiated controls (%)

F I G U R E 4 5
In vitro assessment of intracellular ROS production quantified by H 2 DCFDA fluorescence in NHEK pre-treated with active association with and without UV filters before full solar spectrum irradiation (280-800 nm, 100 mJ/cm 2 /0.7 J/cm 2 ).The mean values of the relative fluorescence units, with standard deviations, are presented.*p < 0.05, ***p < 0.001; ns, not significant.In vivo quantification of the oxidized squalene/squalene ratio (A), catalase activity (B), and quantity of trans-UCA (C) induced by UV irradiation after application of the active association with and without UV filters.The results are presented as box plots, with whiskers representing the maximum values or 1.5 times the interquartile range of the data, whichever was smaller.Student's test for catalase and Wilcoxon's signed-rank test for squalene and trans-UCA.*p < 0.05, and **p < 0.01; ns, not significant.it has been described as being effective for treatment of atopic skin and prevention of skin aging.20 Interestingly, other studies have shown that ectoine prevents UV-induced migration of Langerhans cells 18 and pro-inflammatory cytokines release,19 protects DNA against visible light, 17 mtDNA against UV rays, 23 and prevents sunburn cell formation.18The antioxidant potential of ectoine remained controversial until the study by Brands et al., demonstrating that ectoine is a good hydroxyl radical scavenger, similar to mannitol,13 thus explaining its irradiation protective effect.24Our current studies confirmed the antioxidative properties of ectoine and mannitol association by H 2 DCF-DA assay, in addition to showing maintenance of glutathione levels and prevention of DNA strand breaks consecutive to oxidative stress.These antioxidant properties of the active association can explain the higher protection against squalene oxidation and catalase activity reduction provides significant additional skin photoprotection to UV filters in terms of oxidative stress reduction and skin immunosuppression prevention induced by UV rays.Sunscreens containing active ingredients, especially antioxidants, appear to provide better photoprotection at a cellular level than UV filters alone, and this ecobiological approach should be taken into consideration by dermatologists and users of such products.AUTH O R CO NTR I B UTI O N S Conception or design of the work: Arnaud Fontbonne, Baba Teme, and Elise Abric (in vivo study), Guillaume Lecerf (in vivo study), Alain Moga (in vivo study), Nathalie Ardiet (in vivo study), Aurélie Guyoux, and Sandra Trompezinski.Design of sampling protocol: Sylvie Callejon.Data collection: Baba Teme, Guillaume Lecerf, and Alain Moga (in vivo study).Data analysis and interpretation: Arnaud Fontbonne, Baba Teme, and Elise Abric (in vivo study), Guillaume Lecerf (in vivo study), Alain Moga (in vivo study), Félix Giraud (in vivo study), Marlène Chavagnac-Bonneville, Sandra Trompezinski, and Aurélie Guyoux.Drafting the article: Arnaud Fontbonne and Marlène Chavagnac-Bonneville. Critical revision of the article: Arnaud Fontbonne, Baba Teme, Elise Abric, Sylvie Callejon, Alain Moga, Benoît Cadars, Félix Giraud, Nathalie Ardiet, and Sandra Trompezinski.Final approval of the version to be published: Arnaud Fontbonne, Baba Teme, Elise Abric, Guillaume Lecerf, Sylvie Callejon, Alain Moga, Benoît Cadars, Félix Giraud, Marlène Chavagnac-Bonneville, Nathalie Ardiet, Aurélie Guyoux, and Sandra Trompezinski.