Juvenile atopic dermatitis models

Abstract

The invention relates to biomarkers in children's skin, in particular in the skin of infants, the expression of which changes when the skin is affected by atopic dermatitis. Such markers are particularly advantageous in that they allow the skin's response to atopic dermatitis to be monitored. The inventors have developed methods for evaluating the in vitro efficacy of formulations in preventing the effects of atopic dermatitis on a child's skin, using a skin model specifically capable of reproducing the characteristics of children's skin.

Claims

1. A method for evaluating the in vitro efficacy of a cosmetic active agent, an emollient or a formulation in reducing or treating the effects of atopic dermatitis affecting children's skin, said method comprising determining the efficacy of said cosmetic active agent, emollient or formulation in each of the four tests A, B, C and D, wherein: test A comprises the following steps: a) obtaining a reconstructed skin model from a skin sample from a child; b) contacting said cosmetic active agent, emollient or formulation with the reconstructed skin model; c) contacting the reconstructed skin model with a solution comprising poly(deoxyinosinic-deoxycytidylic) acid and interleukin 1 alpha (IL1α); d) measuring the expression level of at least one biological marker in the skin; and e) evaluating the efficacy of said cosmetic active agent, emollient or formulation based on the expression level of the at least one biological marker measured in step d); test B comprises the following steps: a) obtaining a reconstructed skin model from a skin sample from a child; b) contacting said cosmetic active agent, emollient or formulation with the reconstructed skin model; c) contacting the reconstructed skin model with a solution comprising at least two Th2 cytokines; d) measuring the expression level of at least one biological marker in the skin model; and e) evaluating the efficacy of said cosmetic active agent, emollient or formulation based on the expression level of the at least one biological marker measured in step d); test C comprises the following steps: a) obtaining a reconstructed skin model from a skin sample from a child; b) growing the reconstructed skin model in the presence of THP-1 monocytes; c) contacting said cosmetic active agent, emollient or formulation with the reconstructed skin model; d) inducing an impairment of the barrier function in the reconstructed skin model; e) contacting the reconstructed skin model with at least one pathogenic bacterium; f) measuring the expression and/or activation level of at least one biological marker in the skin model; and g) evaluating the efficacy of said cosmetic active agent, emollient or formulation based on the expression and/or activation level of the at least one biological marker measured in step f); and test D comprises the following steps: a) obtaining a reconstructed skin model from a skin sample from a child; b) contacting said cosmetic active agent, emollient or formulation with the reconstructed skin model; c) inducing an impairment of the barrier function in the reconstructed skin model; d) contacting the reconstructed skin model with at least one pathogenic bacterium; e) measuring the expression and/or activation level of at least one biological marker in the skin model; and f) evaluating the efficacy of said cosmetic active agent, emollient or formulation based on the expression and/or activation level of the least one biological marker measured in step e).

2. A method for evaluating the tolerance of a cosmetic active agent, an emollient or a formulation by children's atopic skin, said method comprising determining the tolerance of said cosmetic active agent, emollient or formulation by said children's atopic skin in each of the four tests A, B, C and D, wherein: test A comprises the following steps: a) obtaining a reconstructed skin model from a skin sample from a child; b) contacting said cosmetic active agent, emollient or formulation with the reconstructed skin model; c) contacting the reconstructed skin model with a solution comprising poly(deoxyinosinic-deoxycytidylic) acid and interleukin 1 alpha (IL1α); d) measuring the expression level of at least one biological marker in the skin model; and e) determining whether said cosmetic active agent, emollient or formulation is well tolerated by children's atopic skin based on the expression level of the least one biological marker measured step d); test B comprises the following steps: a) obtaining a reconstructed skin model from a skin sample from a child; b) bringing said cosmetic active agent, emollient or formulation into contact with the reconstructed skin model; c) bringing the reconstructed skin model into contact with a solution comprising at least two Th2 cytokines; d) measuring the expression level of at least one biological marker in the skin model; and e) determining whether said cosmetic active agent, emollient or formulation is well tolerated by children's atopic skin based on the expression level of the least one biological marker measured step d); test C comprises the following steps: a) obtaining a reconstructed skin model from a skin sample from a child; b) growing the reconstructed skin model in the presence of THP-1 monocytes; c) contacting said cosmetic active agent, emollient or formulation with the reconstructed skin model; d) inducing an impairment of the barrier function in the reconstructed skin model; e) contacting the reconstructed skin model with at least one pathogenic bacterium; f) measuring the expression and/or activation level of at least one biological marker in the skin model; and g) determining whether said cosmetic active agent, emollient or formulation is well tolerated by children's atopic skin based on the expression and/or activation level of the least one biological marker measured step f); and test D comprises the following steps: a) obtaining a reconstructed skin model from a skin sample from a child; b) contacting said cosmetic active agent, emollient or formulation with the reconstructed skin model; c) inducing an impairment of the barrier function in the reconstructed skin model; d) contacting the reconstructed skin model with at least one pathogenic bacterium; e) measuring the expression and/or activation level of at least one biological marker in the skin model; and f) determining whether said cosmetic active agent, emollient or formulation is well tolerated by children's atopic skin based on the expression and/or activation level of the least one biological marker measured step e).

3. A method for identifying a cosmetic active agent, an emollient or a formulation for reducing the effects of atopic dermatitis affecting children's skin, said method comprising determining the efficacy of said cosmetic active agent, emollient or formulation in reducing the effects of atopic dermatitis of children's skin in each of the four tests A, B, C and D, wherein: test A comprises the following steps: a) obtaining a reconstructed skin model from a skin sample from a child; b) contacting the reconstructed skin model with a solution comprising poly(deoxyinosinic-deoxycytidylic) acid and interleukin 1 alpha (IL1α); c) contacting said cosmetic active agent, emollient or formulation with the reconstructed skin model; d) measuring the expression level of at least one biological marker in the skin model; and e) determining whether said candidate cosmetic active agent, emollient or formulation is a cosmetic active agent, emollient or formulation for reducing the effects of atopic dermatitis on children's skin based on the expression level of the least one biological marker measured step d); test B comprises the following steps: a) obtaining a reconstructed skin model from a skin sample from a child; b) contacting the reconstructed skin model with a solution comprising at least two Th2 cytokines; c) contacting said cosmetic active agent, emollient or formulation with the reconstructed skin model; d) measuring the expression level of at least one biological marker in the skin model; and e) determining whether said candidate cosmetic active agent, emollient or formulation is a cosmetic active agent, emollient or formulation for reducing the effects of atopic dermatitis on children's skin based on the expression level of the least one biological marker measured step d); test C comprises the following steps: a) obtaining a reconstructed skin model from a skin sample from a child; b) growing the reconstructed skin model in the presence of THP-1 monocytes; c) inducing an impairment of the barrier function in the reconstructed skin model; d) contacting the reconstructed skin model with at least one pathogenic bacterium; e) contacting said cosmetic active agent, emollient or formulation with the reconstructed skin model; f) measuring the expression and/or activation level of at least one biological marker in the skin model; and g) determining whether said candidate cosmetic active agent, emollient or formulation is a cosmetic active agent, emollient or formulation for reducing the effects of atopic dermatitis on children's skin based on the expression and/or activation level of the least one biological marker measured step f); and test D comprises the following steps: a) obtaining a reconstructed skin model from a skin sample from a child; b) inducing an impairment of the barrier function in the reconstructed skin model; c) contacting the reconstructed skin model with at least one pathogenic bacterium; d) contacting said cosmetic active agent, emollient or formulation with the reconstructed skin model; e) measuring the expression and/or activation level of at least one biological marker in the skin model; and f) determining whether said candidate cosmetic active agent, emollient or formulation is a cosmetic active agent, emollient or formulation for reducing the effects of atopic dermatitis on children's skin based on the expression and/or activation level of the least one biological marker measured step e).

4. The method according to claim 1, wherein the at least two Th2 cytokines are selected from the group consisting of IL-4, IL-5, IL-10, IL-13, IL-22, IL-31, TSLP1 and TNFα.

5. The method according to claim 1, wherein the pathogenic bacterium is Staphylococcus aureus.

6. The method according to claim 1, wherein said marker is selected from markers of activities that inhibit bacterial physiology, markers of immunity, markers of inflammation, markers of the barrier function and markers preferentially expressed in stem cells.

7. The method according to claim 1, wherein step d) of tests A and B, step f) of test C, and step e) of test D comprise measuring a combination of biological markers, said combination comprising at least two markers, said markers being selected from at least two different categories of markers selected from markers of activities that inhibit bacterial physiology, markers of immunity, markers of inflammation, markers of the barrier function and markers preferentially expressed in stem cells.

8. The method according to claim 6, wherein the bacterial physiology-inhibiting activity is selected from inhibition of bacterial proliferation and inhibition of biofilm formation.

9. The method according to claim 6, wherein: the marker of immunity is HBD2 or TLR2, the marker of inflammation is CCL2, CXCL1, CCL7, IL6, IL18, CCL3, CCL5, CCL7, KLK5 or TSLP, the marker of barrier function is FLG, KRT1, KRT10, SCEL, BARX2, LOR, IVL, TGM1, DSG1, CDSN, CLDN1, CASP14, SMPD1, GBA, LASS6, NMF or the ceramides, or the marker preferentially expressed in stem cells is KRT15, KRT19, NOTCH1, BIRC5, ITGA6, ITGB1 or ITGB4.

10. The method according to claim 1, wherein the skin sample comes from a donor selected from the group consisting of newborns aged between 0 and 1 month, infants aged between 1 month and 2 years, and children aged between 2 years and 16 years.

11. The method according to claim 1, wherein the skin sample comes from skin with a phototype I, II, III, IV, V or VI.

12. The method according to claim 1, wherein the reconstructed skin model is selected from suspended skin cell cultures, monolayer skin cell cultures, bilayer skin cell cultures, reconstructed skin cultures and reconstructed mucosal cultures.

13. The method according to claim 12, wherein the cells of said model come from a skin tissue explant or from stem cells differentiated into skin cells.

14. The method according to that claim 1, wherein said model comprises at least fibroblasts or keratinocytes.

15. The method according to claim 2, wherein the at least two Th2 cytokines are selected from the group consisting of IL-4, IL-5, IL-10, IL-13, IL-22, IL-31, TSLP1 and TNFα.

16. The method according to claim 2, wherein the pathogenic bacterium is Staphylococcus aureus.

17. The method according to claim 3, wherein the at least two Th2 cytokines are selected from the group consisting of IL-4, IL-5, IL-10, IL-13, IL-22, IL-31, TSLP1 and TNFα.

18. The method according to claim 3, wherein the pathogenic bacterium is Staphylococcus aureus.

19. The method according to claim 2, wherein the biological marker is selected from markers of immunity, markers of inflammation, markers of barrier function, and markers preferentially expressed in stem cells.

20. The method according to claim 19, wherein: the marker of immunity is HBD2 or TLR2, the marker of inflammation is CCL2, CXCL1, CCL7, IL6, IL18, CCL3, CCL5, CCL7, KLK5 or TSLP, the marker of barrier function is FLG, KRT1, KRT10, SCEL, BARX2, LOR, IVL, TGM1, DSG1, CDSN, CLDN1, CASP14, SMPD1, GBA, LASS6, NMF or the ceramides, or the marker preferentially expressed in stem cells is KRT15, KRT19, NOTCH1, BIRC5, ITGA6, ITGB1 or ITGB4.

21. The method according to claim 3, wherein the biological marker is selected from markers of immunity, markers of inflammation, markers of barrier function, and markers preferentially expressed in stem cells.

22. The method according to claim 21, wherein: the marker of immunity is HBD2 or TLR2, the marker of inflammation is CCL2, CXCL1, CCL7, IL6, IL18, CCL3, CCL5, CCL7, KLK5 or TSLP, the marker of barrier function is FLG, KRT1, KRT10, SCEL, BARX2, LOR, IVL, TGM1, DSG1, CDSN, CLDN1, CASP14, SMPD1, GBA, LASS6, NMF or the ceramides, or the marker preferentially expressed in stem cells is KRT15, KRT19, NOTCH1, BIRC5, ITGA6, ITGB1 or ITGB4.

Description

FIGURE LEGENDS

(1) FIG. 1: Effect of product P8 on S. aureus biofilm formation on the surface of epidermises, observed after 6 h by scanning electron microscopy. A: Untreated control; Magnification ×750. B: Untreated control; Magnification ×10000. C: Product P8; Magnification ×750.

(2) FIG. 2: Effect of product P8 on S. aureus biofilm formation on the surface of epidermises, observed after 24 h by scanning electron microscopy. A: Untreated control; Magnification ×750. B: Untreated control; Magnification ×10000. C: Product P8; Magnification ×750. D: Product P8; Magnification ×10000.

(3) FIG. 3: Effect of products P1 and P5 and of Xylitol on S. aureus biofilm formation on the surface of epidermises, observed after 24 h by scanning electron microscopy at magnification ×10000 (A) or ×20000 (B).

EXAMPLES

(4) I. Model of TH2 Inflammation

(5) The biological efficacy of 6 products (P1, P2, P3, P4, P5 and P8: see Products 1 to 5 and 8 above) was evaluated in a model of atopic dermatitis induced by a mix of Th2 cytokines on 6-month-old epidermises.

A. Materials and Methods

(6) Reconstructed epidermises were made with keratinocytes from a 6-month-old donor.

(7) The epidermises were reconstructed according to the model derived from the method of Poumay et al. (Arch Dermatol Res 2004; 296: 203-11). The epidermises were treated or not (control) topically with the test products at a rate of 5 mg/cm.sup.2. After a 24-hour pre-incubation, the epidermises were again treated with the products at a rate of 2 mg/cm.sup.2 and optionally stimulated with the mixture of Th2 cytokines IL4/IL13/IL22/TNFα at 3 ng/ml.

(8) After 24 hours of incubation, gene expression of the barrier function, stem cell and inflammation markers listed in Table 1 was evaluated by qRT-PCR (quantitative real-time PCR).

(9) TABLE-US-00009 TABLE 1 Classification and name of the genes studied Cluster name Abbreviation Gene name Housekeeping RPS28 Ribosomal protein 28S GADPH Glyceraldehyde-3-phosphate dehydrogenase Stem cells ITGB1 Integrin beta 1 KRT15 Keratin15 KRT19 Keratin 19 BIRC5 Baculoviral IAP repeat-containing 5 (survivin) NOTCH1 Notch homolog 1 Epidermal FLG Filaggrine differentiation, KRT1 Keratin 1 Barrier function, KRT10 Keratin 10 Moisturizing SCEL Sciellin BARX2 Barx homeobox 2 LOR Loricrin IVL Involucrin TGM1 Transglutaminase 1 DSG1 Desmoglein 1 CDSN Corneodesmosin CLDN1 Claudin 1 CASP14 Caspase 14 SMPD1 Sphingomyelinase GBA Glucocerebrosidase LASS6 Ceramide synthase Inflammation CCL2 Chemokine (C-C motif) ligand 2 or monocyte chemoattractant protein 1 (MCP1) CXCL1 C—X—C motif chemokine ligand 1 CCL7 Chemokine (C-C motif) ligand 7 or monocyte-specific chemokine 3 (MCP3)

(10) After 48 hours of incubation, the amounts of natural moisturizing factors (NMFs) and ceramides produced by the epidermises were evaluated:

(11) Analysis of Ceramides:

(12) Epidermal lipids were extracted by stirring the epidermises from a mixture of organic solvents for 2 hours at room temperature. A solid/liquid extraction treatment was then carried out to isolate ceramides from the other lipids making up the epidermises.

(13) The presence of ceramides with a sphingosine [S], dihydrosphingosine [DS] and phytosphingosine [P] sphingoid base with an even chain length ranging from 16 to 22 carbon atoms was investigated by an LC/MS method.

(14) The ceramide content was normalized to the amount of total protein (BCA assay).

(15) Analysis of NMF Elements:

(16) The reconstructed epidermises were extracted under stirring for 2 h at room temperature from an aqueous mixture in the presence of a non-ionic surfactant to promote extraction of the markers of interest.

(17) Filaggrin catabolites were measured by an LC/UV method to screen urocanic acid (UCA) under its two isomers (cis and trans) and L-pyrrolidone carboxylic acid (PCA).

(18) The NMF content was normalized to the amount of total protein (BCA assay).

B. Results

(19) 1. Analysis of Barrier Marker Gene Expression

(20) The deficit of barrier function is one of the key parameters of atopic dermatitis pathophysiology. We evaluated the expression level of different markers of keratinocyte differentiation and of epidermal barrier function in the model of infant epidermises subjected to Th2 stress.

(21) Incubation of 6-month-old reconstructed epidermises in the presence of the Th2 cytokine mixture led to a strong inhibition of all the barrier markers studied (on average, 61% inhibition; Table 2).

(22) This is in correlation with the data in the literature and validates the model as mimicking the barrier function impairment induced by the Th2 inflammatory environment in the context of atopic dermatitis pathophysiology.

(23) TABLE-US-00010 TABLE 2 Gene expression level of barrier markers in 6-month-old epidermises treated with the Th2 cytokine mix Relative expression in % in relation to the Control 6-month-old control 6-month-old Th2 epidermises epidermises FLG 100 11 KRT1 100 25 KRT10 100 26 SCEL 100 29 BARX2 100 36 LOR 100 25 IVL 100 31 DSG1 100 30 CDSN 100 79 CLDN1 100 63 CASP14 100 26 SMPD1 100 40 GBA 100 81 LASS6 100 40 Mean expression of barrier 100 39 markers (%) Filaggrin (FLG), keratins 1 and 10 (KRT1, KRT10) and BARX2 are markers of epidermal differentiation. On the other hand, filaggrin, a precursor of NMFs, is a key marker involved in atopic dermatitis pathophysiology. Loricrin (LOR) and involucrin (IVL) are constitutive proteins of the corneal envelope. Sciellin (SCL) is a precursor of the corneal envelope. Desmoglein 1 (DSG1) and corneodesmosin (CDSN) are constitutive proteins of corneodesmosomes which ensure corneocyte cohesiveness within the corneal layer. Claudin 1 (CLDN1) is a constitutive protein of the tight junctions that play a role in the barrier function of the epidermis and are described as a second barrier against water loss after the stratum corneum. Caspase 14 (CASP14) is an enzyme involved in processing filaggrin to produce NMF. Sphingomyelinase (SMPD1), glucocerebrosidase (GBA) and ceramide synthase (LASS6) are enzymes involved in the synthesis of epidermal lipids, notably ceramides, which are key to ensuring barrier watertightness.

(24) The tested products induced an increase in gene expression of key barrier markers in 6-month-old epidermises incubated under Th2 stress conditions (Table 3).

(25) The efficacy of the products on barrier marker expression can be classified as follows: P1>P5>P3>P2>P4.

(26) TABLE-US-00011 TABLE 3 Gene expression level of barrier markers in 6-month-old epidermises treated with the Th2 cytokine mix - Product effect Relative expression in % compared with the Th2 Control Claudin 1 SMPD1 Involucrin TGM Average Th2 100 100 100 100 100 P1 639 346 1484 916 846 P2 180 105 210 142 159 P3 141 171 107 233 163 P4 115 76 144 96 108 P5 555 221 1767 635 795

(27) 2. Analysis of Inflammation Marker Gene Expression

(28) Incubation of 6-month-old epidermises in the presence of the Th2 cytokine mixture induced a significant overexpression of chemokines CXCL1 (Chemokine ligand 1), CCL2 (Chemokine ligand 2) and CCL7 (Chemokine ligand 7) (Table 4).

(29) These chemokines, responsible for recruiting inflammation cells in the skin, play an important role in amplifying the inflammatory response in the context of atopic dermatitis. In particular, chemokines CXCL1 and CCL2 have been described as being overexpressed in atopic skin. Thus, this model represents the amplification of the inflammatory response related to the Th2 environment in atopic dermatitis.

(30) Topical application of the test products modulated the overexpression of chemokines induced by Th2 stress, and the efficacy of the products can be classified as follows: P5≥1>P2>P3=P4.

(31) TABLE-US-00012 TABLE 4 Gene expression level of inflammation markers in 6-month-old epidermises treated with the Th2 cytokine mix - Product effect Relative expression in % in relation to the Control CCL2 CXCL1 CCL7 Expression % Expression % Expression % level Inhibition level Inhibition level Inhibition 6-month-old 100 100 100 control epidermises 6-month-old 9863 3688 2583 Th2 epidermises P1 99 −99% 2250 −39% 103 −96% P2 4636 −53% 1549 −58% 1653 −36% P3 3255 −67% 3688 0 1343 −48% P4 3057 −69% 3835  +4% 1188 −54% P5 0 −100%  996 −73% 129 −95%

(32) CXCL1 (Chemokine ligand 1): Chemoattractant for neutrophils, overexpressed in atopic dermatitis.

(33) CCL2 or MCP1 (Chemokine ligand 2): Chemoattractant for monocytes and basophils; overexpressed in atopic dermatitis, where it is responsible for recruiting dendritic cell precursors.

(34) CCL7 or MCP3 (Chemokine ligand 7): Chemoattractant for monocytes/macrophages.

(35) 3. Analysis of Stem Cell Marker Gene Expression

(36) Tissue stem cells in permanent renewal are traditionally defined as rare and relatively quiescent cells. They have a unique capacity for self-renewal and tissue regeneration that allows them to ensure the homeostasis and integrity of the tissue in which they reside.

(37) Among epidermal stem cells, interfollicular stem cells located in the basal layer constitute the main epidermal reservoir of stem cells. These cells reside in an anatomical and functional microenvironment, the niche, which helps to maintain their characteristics, especially when physiological conditions change. Interfollicular stem cells and their niches are involved in maintaining the integrity and regeneration of the epidermis. Stem cells can be identified only by following several markers. We have thus evaluated the expression level of different gene markers characteristic of stem cells in the model of Th2 inflammation.

(38) Incubation of 6-month-old epidermises in the presence of the Th2 cytokine mixture induced a significant decrease (−39%) in the mean expression level of the stem cell marker pool studied (Table 5).

(39) This is, to our knowledge, the first time that an effect of Th2 inflammation has been observed on stem cell markers. These observations tend to confirm the high vulnerability of the cell stock in infant epidermis to external insult modelled here by Th2 stress typical of atopic dermatitis pathogenesis.

(40) TABLE-US-00013 TABLE 5 Gene expression level of stem cell markers in 6-month-old epidermises treated with the Th2 cytokine mix Relative expression in % in relation to the Control 6-month-old 6-month-old Th2 control epidermises epidermises ITGB1 100 49 KRT15 100 86 KRT19 100 89 BIRC5 (SURVIVIN) 100 47 NOTCH1 100 35 Mean expression of the 100 61 stem cell marker pool (%)

(41) The application of product P1, and more moderately of product P5, restored the expression level of the stem cell marker pool to a level identical to that of the untreated control (Table 6). Products P2, P3 and P4 did not show any protective efficacy of the stem cell marker pool.

(42) TABLE-US-00014 TABLE 6 Gene expression level of the stem cell marker pool in 6-month-old epidermises treated with the Th2 cytokine mix - Product effect Relative expression in % in relation to the Control Marker pool ITGB1 KRT15 KRT19 BIRC5 TP63 NOTCH1 average Con- 100 100 100 100 100 100 100 trol Th2 49 86 89 47 91 35 66 P1 141 34 185 43 48 157 101 P2 61 29 105 22 109 54 63 P3 53 55 56 10 56 47 46 P4 57 15 91 39 65 37 51 P5 86 23 230 58 51 109 93

(43) 4. Analysis of NMF and Ceramide Amounts

(44) Incubation of 6-month-old epidermises in the presence of the Th2 cytokine mixture resulted in a significant decrease in the amount of NMFs (−52%, p<0.001) and ceramides (−41%, p<0.001; Tables 7 and 8, respectively).

(45) This is in correlation with the data in the literature and confirms the validity of the model to mimic the impairment of barrier function and of mechanisms of hydration regulation induced by the Th2 inflammatory environment in the context of atopic dermatitis pathophysiology.

(46) Under these Th2 stress conditions, products P1 and P8 induce a significant increase in the production of NMFs and ceramides by the reconstructed epidermises, with a greater effect of product P1 (Tables 7 and 8).

(47) TABLE-US-00015 TABLE 7 Quantification of NMF content in 6-month-old reconstructed epidermises under Th2 stress conditions - Statistics: One-way analysis of variance followed by Tukey's test NMF (Σ UCA and PCA in μg/mg protein) (mean ± standard deviation) Change (%) 6-month-old control epidermises 22.65 ± 1.878 6-month-old Th2 epidermises 10.84 ± 1.005 −52% p < 0.001 P1  16.5 ± 1.718 +52% p < 0.001 P8 12.88 ± 0.582 +19% p < 0.05

(48) TABLE-US-00016 TABLE 8 Quantification of ceramide content in 6-month-old reconstructed epidermises under Th2 stress conditions - Statistics: One-way analysis of variance followed by Tukey's test Ceramides (AU/mg protein) (mean ± standard deviation) Change (%) 6-month-old control epidermises 148.4 ± 8.045 6-month-old Th2 epidermises 88.27 ± 7.125 −41% p < 0.001 P1 110.7 ± 7.322 +25% p < 0.05 P8 100.6 ± 8.985 +14% p < 0.1

C. Conclusion

(49) Treatment of reconstructed epidermises with a mixture of Th2 cytokines reproduces the pathophysiological characteristics of atopic dermatitis related to the Th2 inflammatory environment: impairment of the skin barrier, amplification of local inflammation.

(50) Applied to epidermises obtained from keratinocytes from a 6-month-old donor, this model is, to our knowledge, the first baby-specific model mimicking the inflammatory environment of atopic dermatitis.

(51) Furthermore, this model demonstrated, for the first time, the possible impairment of the stem cell stock by the Th2 inflammatory environment.

(52) This model also allows a comparative study of the biological activity of topical products on the protection of barrier and stem cell markers as well as on modulation of the inflammatory response.

(53) II. Model of Initiation of Atopic Dermatitis

(54) The biological efficacy of 4 products (P1, P4, P5, P6) was evaluated in a model reproducing the initiation phase of atopic dermatitis on reconstructed epidermises, from a 6-month-old donor, stimulated by a Poly (I:C)+IL1α mixture.

(55) Poly I:C is a TLR3 agonist, IL1α is a pro-inflammatory cytokine. The stress induced by this mixture of molecules mimics the cascade of reactions induced by bacterial attack and leading to the induction of a Th2 inflammatory response. It is therefore a model that reproduces the initiation of an inflammatory response characteristic of atopic dermatitis.

A. Materials and Methods

(56) Reconstructed epidermises were made, as described above, with keratinocytes from a 6-month-old donor. The epidermises were optionally treated topically (at a rate of 5 mg/cm.sup.2) with the test products and pre-incubated for 24 h. The epidermises were again optionally treated topically (at a rate of 2 mg/cm.sup.2) with the products and optionally stimulated with the mixture of inducers: 10 μg/ml Poly(I:C)+10 ng/ml IL1α.

(57) After 4 h of incubation, gene expression of the inflammatory markers was evaluated by qRT-PCR (quantitative real-time PCR) with n=2.

(58) Table 9 lists the genes that were studied.

(59) TABLE-US-00017 TABLE 9 Classification and name of the genes studied Cluster name Abbreviation Gene name Housekeeping RPS28 Ribosomal protein 28S GADPH Glyceraldehyde-3-phosphate dehydrogenase Inflammation/ IL6 Interleukin 6 Pruritus IL18 Interleukin 18 CCL3 Chemokine (C-C motif) ligand 3 or macrophage inflammatory protein 1alpha (MIP1α) CCL5 Chemokine (C-C motif) ligand 5 or regulated on activation, normal T cell expressed and secreted (RANTES) CCL7 Chemokine (C-C motif) ligand 7 or monocyte-specific chemokine 3 (MCP3) KLK5 Kallikrein 5 or stratum corneum trypsin- like enzyme (SCTE)

B. Results: Analysis of Inflammation and Pruritus Marker Gene Expression

(60) Stimulation of 6-month-old epidermises by Poly(I:C)-FIL1α stress induced a very high overexpression of chemokines CCL3, CCL5 and CCL7, involved in the recruitment and activation of inflammatory cells (Table 10). In particular, chemokines CCL3 (or MIP1α) and CCL5 (or RANTES) are described as being overexpressed in atopic skin and responsible for the activation and recruitment of Th2 cells. Thus, this model effectively mimics the initiation phase of the inflammatory cascade leading to establishment of the Th2 inflammatory environment specific to atopic dermatitis.

(61) Furthermore, Poly(I:C)-FIL1α stress also induced overexpression of kallikrein 5. This protease, whose activity is increased in atopic dermatitis, plays a role in controlling desquamation, inflammation and pruritus (via PAR2 activation), thus contributing to atopic dermatitis pathogenesis related to barrier impairment and induction of inflammation and pruritus.

(62) The various products tested resulted in a more or less marked decrease in the gene expression of markers of inflammation and pruritus; the activity potential of the products can be classified as follows: P1>P5>P4P6. Only product P1 showed inhibitory efficacy on all markers studied.

(63) TABLE-US-00018 TABLE 10 Gene expression level of inflammation and pruritus markers Relative expression in %. IL6 IL18 CCL3 CCL5 CCL7 KLK5 Control nd nd 100 100 100 100 PolyI:C + IL1 100 100 5532 9280 8462 136 P1 12 54 664 3248 508 95 P4 246 95 2213 9373 6770 140 P5 188 65 2600 7610 4400 167 P6 114 138 3872 12806 6600 155 IL6 (Interleukin 6): pro-inflammatory cytokine IL18 (Interleukin 18): cytokine overexpressed in atopic skin, involved in induction of the Th2 response CCL3 or MIP1α (Chemokine ligand 3): Chemoattractant for inflammatory cells; overexpressed in atopic dermatitis. CCL5 or RANTES (Chemokine ligand 5): Chemoattractant for inflammatory cells; overexpressed in atopic dermatitis. CCL7 or MCP3 (Chemokine ligand 7): Chemoattractant for monocytes/macrophages. KLK5 or SCTE (Kallikrein 5): protease overexpressed in atopic dermatitis, involved in desquamation, inflammation and pruritus.

C. Conclusion

(64) Treatment of 6-month-old epidermises with the poly(I:C)/IL1α mixture effectively models the induction phase of the Th2 response in atopic dermatitis. Indeed, this stress led to an overexpression of chemokines and molecular messengers involved in the recruitment and activation of Th2 cells.

(65) This model of initiation of the inflammatory phase of atopic dermatitis made it possible to evaluate and classify the biological efficacy of different topical products on the markers of inflammation and pruritus.

(66) III. Model of Response to Staphylococcus aureus on Immunocompetent Epidermises

(67) The biological efficacy of products P1 and P5 was evaluated in a model mimicking S. aureus-induced atopic dermatitis in reconstructed 1-year-old epidermises made immunocompetent by co-culture with immune cells (THP1 monocyte cell line). Co-culture with the immune cells made it possible to establish an adaptive and protective response to S. aureus immune-mediated by induction of a Th2-type response.

A. Materials and Methods

(68) Reconstructed epidermises obtained with keratinocytes from a one-year-old donor were infected with a methicillin-resistant S. aureus strain (MRSA; ATCC 33591) at a rate of 2.Math.10.sup.6 cfu/epidermis for 4 hours, after undergoing slight surface abrasion. Non-adherent bacteria were then removed by rinsing and the epidermises were placed in co-culture with THP1 cells (10.sup.5 cells/ml). At the same time, the test products were applied to the surface of the epidermises (20 μl/epidermis). After 16 h of co-culture (T1), the gene expression of different markers was evaluated by qRT-PCR (quantitative real-time PCR) with n=3.

(69) On a second series of epidermises, the products were reapplied and the epidermises incubated for 24 h in the absence of THP1 cells. At the end of this incubation period, the gene expression of different markers was evaluated by qRT-PCR (quantitative real-time PCR) with n=3 (T2).

(70) Table 11 lists the genes that were studied.

(71) TABLE-US-00019 TABLE 11 Classification and name of the genes studied Cluster name Abbreviation Gene name Housekeeping GADPH Glyceraldehyde-3-phosphate dehydrogenase Innate immunity HBD2 Human beta-defensin 2 TLR2 Toll-like receptor 2 TH2 TSLP Thymic stromal lymphopoietin inflammation Barrier/Pruritus KLK5 Kallikrein 5 or stratum corneum trypsin- like enzyme (SCTE) Barrier CLDN1 Claudin-1 Stem cells K19 Keratin 19 ITGA6 Integrin alpha 6 ITGB1 Integrin beta 1

B. Results

(72) 1. Evaluation of the Epidermal Response

(73) T1: Atopic Dermatitis Induction Phase

(74) After 16 hours of co-culture (T1) with immune cells, analysis of gene expression in the 1-year-old epidermises showed overexpression of kallikrein-5, β-defensin 2 and TSLP, as well as a decrease in TLR2 (Table 12).

(75) Stimulation of kallikrein-5 would indicate an early epidermal response in favour of induction of the Th2 inflammatory phase (via TSLP), impairment of the barrier, and pruritus.

(76) TSLP itself is moderately increased, indicating the beginning of induction of Th2 inflammation.

(77) Stimulation of β-defensin 2, an anti-microbial peptide impaired in atopic dermatitis, shows that in this early phase, the epidermis sets up defence mechanisms to prevent colonization by S. aureus.

(78) TLR2, a receptor involved in pathogen recognition and induction of the immune response, whose expression is decreased in atopic dermatitis, is decreased in the model, which tends to show the inability of the skin to defend itself, with a deficit of this important element of innate immunity.

(79) Thus, in this early induction phase of the model, molecular signals related to the immune response appear to be in place to induce the Th2 shift characteristic of atopic dermatitis.

(80) TABLE-US-00020 TABLE 12 Gene expression level of markers in the model of “immunocompetent” epidermises colonized by S. aureus (T1, Early phase, 16 h post-colonization) KLK5 hBD2 TLR2 TSLP Control 1 1 1 1 S. aureus + THP1 1.425 1.469 0.308 1.299

(81) T2: Phase Modelling Established Atopic Dermatitis

(82) After 16 hours of co-culture then 24 hours of incubation, the response of the epidermises to colonization by S. aureus reproduces the main characteristics of atopic dermatitis pathophysiology (Table 13):

(83) Barrier impairment, represented by inhibition of claudin-1, the constitutive protein of tight junctions;

(84) Inhibition of defence mechanisms related to innate immunity: hBD2 and TLR2;

(85) Overexpression of TSLP, key cytokine in atopic dermatitis pathophysiology, involved in induction of a Th2 inflammatory response.

(86) In addition, a marked decrease in stem cell markers (Keratin 19, Integrins α6 and β1) was observed, comparable to the results obtained in the Th2 model described above.

(87) TABLE-US-00021 TABLE 13 Gene expression level of markers in the model of “immunocompetent” epidermises colonized by S. aureus (T2, Late phase, 16 + 24 h post-colonization) CLDN1 KRT19 ITGA6 ITGB1 hBD2 TLR2 TSLP Control 1 1 1 1 1 1 1 S. aureus + 0.576 0.412 0.05 0.129 0.047 0.201 2.446 THP1

(88) 2. Evaluation of Product Biological Efficacy

(89) Protection of the Stem Cell Stock

(90) Topical application of product P1 protected, in a manner clearly superior to product P5, the stem cell markers against the inhibition induced in the model (Table 14).

(91) TABLE-US-00022 TABLE 14 Gene expression level of stem cell markers in the model of “immunocompetent” epidermises colonized by S. aureus - Product effect (T2, Late phase, 16 + 24 h post-colonization) Control S. aureus + THP1 P1 P5 KRT19 1 0.412 3.14356 0.615528 ITGA6 1 0.05 0.18585 0.43795 ITGB1 1 0.129 0.57663 0.651063 Marker pool average 1.00 0.20 1.30 0.57

(92) Modulation of a Pruritus Marker

(93) The two products tested (P1 and P5) comparably inhibited the expression level of kallikrein 5 to restore a level comparable to the control (Table 15).

(94) Both products modulate the induction of this early marker, involved in pruritus, and which contributes to the development of atopic dermatitis.

(95) TABLE-US-00023 TABLE 15 Gene expression level of kallikrein 5 in the model of “immunocompetent” epidermises colonized by S. aureus - Product effect (T1, Early phase, 16 h post-colonization) Control S. aureus + THP1 P1 P5 KLK5 1.00 1.43 1.05 0.92

(96) Restoration of an Immune Defence Marker

(97) The two products tested, P1 and P5, induced a stimulation of TLR2, a receptor involved in the immune response and whose expression is impaired in atopic dermatitis (Table 16).

(98) Product P1 induced a greater increase in TLR2, compared with product P5, in favour of a greater efficacy in protecting mechanisms related to innate immune defences.

(99) TABLE-US-00024 TABLE 16 Gene expression level of TLR2 in the model of “immunocompetent” epidermises colonized by S. aureus - Product effect Control S. aureus + THP1 P1 P5 T1 (16 h) 1.00 0.31 0.64 0.44 T2 (16 h + 24 h) 1.00 0.20 3.04 0.71

(100) Modulation of TH2 inflammation

(101) In the early phase of the model (T1), both products P1 and P5 similarly inhibited the TSLP expression induced by the model (Table 17).

(102) In the late phase, modelling established atopic dermatitis (T2), only product P1 inhibited TSLP gene expression.

(103) Thus, via its TSLP inhibitory action, product P1 could modulate the induction of TH2 inflammation.

(104) TABLE-US-00025 TABLE 17 Gene expression level of TSLP in the model of “immunocompetent” epidermises colonized by S. aureus - Product effect Control S. aureus + THP1 P1 P2 T1 (16 h) 1.00 1.30 0.92 1.04 T2 (16 h + 24 h) 1.00 2.45 1.35 2.89

C. Conclusion

(105) This unique model, using reconstructed epidermises from a 1-year-old infant in co-culture with immunocompetent cells, reproduces the role of S. aureus in atopic dermatitis pathophysiology.

(106) The use of this model makes it possible to comparatively evaluate the biological activity of topical products on the protection of stem cells, the barrier and immune defences, as well as on the modulation of pruritus and TH2 inflammation.

(107) IV. Model for Studying S. aureus Biofilm

(108) The biological efficacy of products P1, P5 and P8 against S. aureus biofilm was evaluated in a model of reconstructed epidermises colonized by S. aureus and on which biofilm formation was visualized by electron microscopy.

(109) The biological efficacy of products P1, P5 or P8 against S. aureus biofilm was evaluated in a model of reconstructed epidermises colonized by S. aureus and on which biofilm formation was visualized by electron microscopy.

A. Materials and Methods

(110) The test products (P1, P5 or P8) or 5% xylitol (positive control for inhibition of biofilm formation) were applied topically to reconstructed epidermises at a rate of 30 μL/epidermis, after undergoing slight surface abrasion.

(111) After incubation overnight, the products were removed by rinsing and a 2.Math.10.sup.6 cfu inoculum of Staphylococcus aureus (methicillin-resistant S. aureus, MRSA; ATCC 33591) was applied to the surface of the reconstructed epidermises.

(112) Biofilm formation and the effect of the product thereon were observed visually by scanning electron microscopy (SEM) 6 and 24 hours after colonization.

B. Results

(113) As of 6 hours after colonization by S. aureus, planktonic bacteria cover the surface of the epidermises (FIG. 1A) and produce a polysaccharide matrix characteristic of the initiation of biofilm formation (FIG. 1B).

(114) The application of product P8 exerts an S. aureus anti-adhesion effect, visualized by the reduced number of bacteria present on the surface of the epidermises (FIG. 1C).

(115) After 24 hours of colonization (FIGS. 2 and 3), the bacteria are observed in numbers on the surface of the epidermises.

(116) Under the control conditions (untreated epidermises), a dense biofilm is seen (arrows; FIGS. 2A and 2B).

(117) In the control epidermises treated with xylitol, the bacteria appear well individualized. Under these conditions, the bacteria did not produce biofilm; this result validates the test (FIG. 3).

(118) In the presence of products P1 (FIG. 3) and P8 (FIGS. 2C and 2D), well individualized bacteria (cocci) are observed on the surface of the epidermises and are not enveloped in an exopolymeric matrix as in the untreated control conditions. These observations suggest that the topical application of products P1 and P8 inhibited the formation of S. aureus biofilm. And, particularly for product P8, an S. aureus anti-adhesion effect is observed (FIGS. 1C, 2C, 2D).

(119) P5, in turn, appears to induce partial inhibition of biofilm formation. Indeed, several areas where the bacteria appear well individualized (absence of biofilm) are observed on the surface of the epidermises, while most of the epidermal surface has bacteria covered with biofilm.

C. Conclusion

(120) This model allows electron microscopy monitoring of the kinetics of S. aureus biofilm formation on the surface of epidermises and comparative evaluation of the efficacy of topical products in limiting this biofilm, a factor of bacterial virulence and pathogenesis.

(121) Via this model it is possible to classify products according to their efficacy in inhibiting S. aureus biofilm: total inhibition, with anti-adhesion effect (e.g. products P1, P8), or partial inhibition (e.g. product P5).