Method for diagnosing skin exposed to ozone

Abstract

Method for diagnosing skin exposed to ozone. The present invention relates to a method for diagnosing skin exposure to ozone, as well as a method for identifying an active compound and a method for evaluating the efficacy of a compound, in order to prevent and / or treat the deleterious effects related to the oxidation of certain skin constituents and the modulation of biomarker expression in response to ozone exposure. The present invention also relates to a kit and its use. Figure for abstract: none

Description

Title of the invention: Method for diagnosing skin exposed to ozone

[0001] The present invention relates to a method for diagnosing skin exposure to ozone as well as a method for identifying an active compound and a method for evaluating the efficacy of a compound, in order to prevent and / or treat the deleterious effects linked to the oxidation of certain constituents of the skin and to the modulation of the expression of biomarkers in response to exposure to ozone. The present invention also relates to a kit and the use thereof.

[0002] Ozone or trioxygen has the chemical formula O3, it is an allotropic variety of oxygen, much less stable than dioxygen O2, into which it naturally tends to decompose. Ozone is naturally present in the Earth's atmosphere, forming an ozone layer in the stratosphere which intercepts the sun's ultraviolet rays. However, in the lower layers of the atmosphere (the troposphere) it is one of the most dangerous air pollutants for health. This very powerful oxidant can be produced by forest fires, high temperatures, it is also produced in industrial areas or urban centers with strong sunshine. Thus certain environments are regularly subject to ozone peaks. The individual in his daily environment and particularly in urban areas, can be subjected to multiple attacks on keratinous materials, and in particular the skin, by this air pollutant.Indeed, ozone has deleterious effects linked to the oxidation of certain constituents of the skin and to the modulation of the expression of the biomarkers that it induces.

[0003] From the above, it is understood the importance of finding biomarkers that can detect ozone exposure of the skin, in order to be able to treat the skin in a targeted manner. The present invention aims to satisfy this need.

[0004] The applicant surprisingly discovered several biomarkers significantly modulated in skin exposed to ozone: elafin (PI3), corneodesmosine (CSDN), and SPRR3.

[0005] The methods according to the invention are therefore based on the measurement of significantly modulated biomarkers in reconstructed tissues exposed to ozone and / or on skin samples exposed to ozone. The biomarkers of the present invention are genes or proteins encoded by them, involved in epidermal differentiation. The modulation of these biomarkers has in particular an impact on the barrier function of the skin. The biomarkers discovered by the applicant are in particular markers of depth and / or accumulation of exposure to ozone.

[0006] Thus, according to a first of its aspects, the invention relates to a method of diagnosis, preferably in vitro or ex vivo, of exposure to ozone of the skin, in a subject, said method comprising the following step: a. measuring the expression level of PI3, and / or CDSN, and / or SPRR3 in a skin sample of said subject.

[0007] Optionally, this diagnostic method according to the invention further comprises the following step:

[0008] b) based on the expression level measured in step a., determining whether the skin of said subject has been exposed to ozone.

[0009] The invention also relates to a method for identifying a compound for preventing and / or treating the harmful effects of ozone on the skin, said method comprising the following steps: a. measuring the expression level of PI3, and / or CDSN, and / or SPRR3 in a skin sample of a subject, said sample being treated with said compound; b. comparing said expression level measured in step a) to the corresponding expression level in a skin sample not treated with said compound; c. identify said candidate compound as a compound for preventing and / or treating the deleterious effects of ozone on the skin when the level of expression detected in the skin sample that has been treated with said compound is lower than the level of expression in the skin sample that has not been treated with said compound.

[0010] The invention also relates to a method for identifying a compound for preventing and / or treating the harmful effects of ozone on the skin, said method comprising the following steps: a. carrying out a treatment of a skin sample chosen from a reconstructed epidermis and a reconstructed skin, by bringing the skin sample into contact with said compound; b. carry out an ozone exposure of said skin sample; c. measuring the level of expression of PI3, and / or CDSN, and / or SPRR3 in the skin sample treated with said compound; d. comparing said expression level measured in step c) to the corresponding expression level in a skin sample exposed to ozone and not treated with said compound; e. identify said candidate compound as a compound for preventing and / or treating the deleterious effects of ozone on the skin when the level of expression detected in the skin sample that has been treated with said compound is lower than the level of expression in the skin sample that has not been treated with said compound;

[0011] it being understood that step b can be carried out after step a and vice versa, and that steps a and b can be repeated at least twice.

[0012] The invention also relates to a method for evaluating the effectiveness of a compound for preventing and / or treating the harmful effects of ozone on the skin of a subject, said method comprising the following steps: a. measuring the expression level of PI3, and / or CDSN, and / or SPRR3 in a skin sample of said subject prior to treatment with said compound. b. performing a treatment of the skin of said subject with said compound, c. measuring the level of expression of PI3, and / or CDSN, and / or SPRR3, after step b, in a second skin sample from said subject; d. compare said expression levels measured in step a. and in step c.; e. evaluate the efficacy of said candidate compound with regard to the prevention and / or treatment of the harmful effects of ozone on the skin.

[0013] In another aspect, the invention relates to a kit comprising: a. a means for measuring, in a skin sample, the level of expression of PI3, and / or CDSN, and / or SPRR3, and b. an instruction leaflet.

[0014] The invention also relates to the use of said kit for the identification of a compound making it possible to prevent and / or treat the harmful effects of ozone on the skin.

[0015] The invention will appear more clearly on reading the description which follows, given solely by way of non-limiting example.

[0016] General definitions

[0017] By “subject” we mean a human being, preferably aged from 1 month to 90 years.

[0018] Advantageously, the subject is likely to have been exposed to ozone, in particular to chronic or acute exposure.

[0019] The stratum corneum is the outermost layer of the epidermis, and comprises the surface of the skin. It is composed mainly of dead cells. By "stratum corneum sample" is meant here a sample taken from this layer. For example, such a sample can be taken by rubbing the surface of the skin or by using an adhesive surface such as a D-squame® disc applied to the skin.

[0020] By "exposure to ozone" is meant herein chronic or acute exposure to ozone.

[0021] By "chronic exposure" is meant here regular exposure, in particular daily, for a given period, preferably a period of more than one year, in particular a period of twelve months possibly interrupted on one or more occasions for short periods, typically periods of less than one month, preferably less than a week.

[0022] By "acute exposure" is meant here a momentary exposure, in particular from 12 hours to 5 days, to at least one ozone level greater than 0.05 ppm, preferably greater than 0.07 ppm, more preferably greater than 0.09 ppm, even better greater than 0.1 ppm.

[0023] Elafin (peptidase inhibitor 3, PI3) is a 117 amino acid protein encoded by the P13 gene. Elafin is expressed by many tissues and more particularly in epithelia (digestive, pulmonary, skin) and also by macrophages. Elafin has important anti-inflammatory properties, microbicidal and fungicidal properties. Human elafin is available in uniprot under number P19957.

[0024] Corneodesmosin (CDSN) encoded by the CDSN gene is a protein specific to comeodesmosomes, junctional structures of the stratum corneum of the epidermis. It is synthesized within the upper spiny keratinocytes. It has adhesive properties and reinforces the cohesion of the stratum corneum. Human corneodesmosin is available in uniprot under number Q15517.

[0025] SPRR3 (Small proline-rich protein 3) is a 169 amino acid protein encoded by the SPRR3 gene. The human SPRR3 protein is accessible in uniprot under the number Q9UBC9. SPRR genes encode proteins that are strongly induced during the differentiation of epidermal keratinocytes in vitro and in vivo. The SPRR family is characterized in the epidermis as being key for the formation of the protective barrier function of the skin, the quality of its mechanical properties and its surface protection against a large family of endogenous and external stimuli.

[0026] By "expression level of PI3, and / or CDSN, and / or SPRR3" is meant the level of expression in messenger RNA (mRNA) of the PI3 gene, and / or CDSN, and / or SPRR3 respectively, and / or the level of expression of the protein encoded by this gene. The expression level can be expressed as an amount, a concentration or a ratio.

[0027] Preferably, the level of expression in mRNA of the coding gene is measured using a nucleotide sequence complementary to the mRNA of the coding gene and specifically hybridizing to the mRNA of the coding gene or a fragment thereof specifically hybridizing to the mRNA of the coding gene, this sequence or this fragment comprising 5 to 50 nucleotides, preferably 10 to 20 nucleotides, or using a pair of primers or a probe of 10 to 60 nucleotides, preferably 15 to 30 nucleotides comprising said sequence or said fragment. The level of expression can then be measured by any method known to those skilled in the art, for example by quantitative PCR.

[0028] Preferably, the level of expression of the protein (here PI3, and / or CDSN and / or SPRR3) is measured using a ligand specific to the protein, such as for example an antibody, preferably monoclonal, a Fab fragment, a scFv or a nanobody, specific for this protein. The level of expression can then be measured by any method known to those skilled in the art, such as for example by a Western Blot or an ELISA test. In particular, the measurement of the level of expression of the protein can be carried out using a miniaturized system such as a microfluidic chip, such as that described in international application WO2011 / 126249, this chip containing ligands specific for the protein. The chip can then be analyzed using a suitable reader to determine whether said protein has bound to said ligand and to conclude as to the presence and quantity of the protein in the sample.

[0029] In the context of the invention, the terms "hybridize" or "hybridization", as well known to those skilled in the art, refer to the binding of a nucleic acid molecule to a particular nucleotide sequence under appropriate conditions, in particular under stringent conditions.

[0030] The term "stringent conditions" as used herein refers to conditions that are suitable for producing binding pairs between nucleic acids having a determined level of complementarity, while being unsuitable for forming pairs between binding nucleic acids having complementarity below said determined level. Stringent conditions are dependent on hybridization and washing conditions. These conditions may be modified according to methods known to those skilled in the art. Generally, high stringency conditions are a hybridization temperature about 5°C below the melting temperature (Tm), preferably close to the Tm of perfectly base-paired strands. Hybridization procedures are well known in the art.

[0031] High stringency conditions generally involve hybridization at a temperature of about 50°C to about 68°C in 5x SSC / 5x Denhardt's solution / 1.0% SDS, and washing in 0.2x SSC / 0.1% SDS at a temperature of about 60°C to about 68°C.

[0032] By "corresponding expression level" is meant the expression level of the same gene or protein as that whose level was measured in a previous step.

[0033] By "deleterious effects of ozone" is meant irritation or discomfort reactions at the level of the skin (also including the skin of the scalp) which can result in particular in redness, itching, sensations of heat, tingling and tightness.

[0034] Method for diagnosing ozone exposure of the skin

[0035] The subject of the present invention is a method for diagnosing exposure to ozone of the skin in a subject, said method comprising the following step: a. measuring the level of expression of PI3, and / or CDSN, and / or SPRR3 in a skin sample of said subject.

[0036] Preferably, this method of the invention is a method for diagnosing chronic exposure to ozone.

[0037] The method of the invention is preferably carried out in vitro or ex vivo.

[0038] The skin sample of the subject used in the diagnostic method according to the invention is a sample taken, preferably non-invasively, from the skin of the subject, preferably from the face of the subject, in particular from the cheek of the subject. Preferably, the skin sample is a biopsy, a microbiopsy in particular carried out using a needle, or a sample of stratum corneum.

[0039] Thus, according to one embodiment, this method according to the invention comprises a step of taking the skin sample from the subject. This step is preferably carried out in a non-invasive manner, and in particular does not require local anesthesia. According to a preferred embodiment, in particular in the case where the level of expression measured in step a. is the level of expression of CDSN and / or SPRR3, the step of taking the sample is carried out by rubbing the surface of the skin or by using an adhesive surface such as a D-squame® disc.

[0040] Optionally, the diagnostic method according to the invention further comprises the following step:

[0041] b. based on the expression level measured in step a., determining whether the skin of said subject has been exposed to ozone.

[0042] In a particular embodiment, step b. is carried out after comparing the expression level measured in step a. with a reference value. In one embodiment, the reference value is determined by the average value of the expression level measured in a determined population which has not been subjected to exposure to ozone, in particular acute or chronic exposure to ozone.

[0043] In a particular embodiment, the reference value is determined by the average value of the expression level in human beings aged from 1 month to 90 years. According to the invention, a reference value can be determined by a plurality of samples, preferably more than 20, 30, 50, 100, 200 or 500 samples.

[0044] The term "comparison" herein refers to determining whether the gene expression level is substantially the same as or differs from a reference value. Preferably, the gene expression level is considered different from a reference value if the observed difference is statistically significant. If the difference is not statistically significant, the gene expression level and the reference value are substantially the same.

[0045] Based on this comparison, it can be determined whether the skin has been exposed to ozone. Typically, the skin is considered to have been exposed to ozone when the gene expression level is significantly higher than the value of reference and the skin is not considered to have been exposed to ozone when the gene expression level is essentially the same as or significantly lower than the reference value.

[0046] Preferably, the method according to the invention comprises a step of treating the sample in order to prepare the measurement of the expression level. For example, if the skin sample was obtained using an adhesive surface such as a D-squame® disc, the soluble protein extract can be obtained by a protein extraction system, such as that described in international application WO2015 / 009085 which makes it possible to bring the D-squame samples into contact with a minimal volume of extraction buffer and thus to obtain a soluble protein extract. Suitable buffers for the extraction of skin proteins are well known and include, for example, RIPA buffer or a urea buffer, or a buffer comprising 0.5% sodium deoxycholate, 50 mM ammonium bicarbonate, 50 mM DTT, 1 qM Pepstatin as well as protease inhibitors.

[0047] Optionally, the diagnostic method according to the invention further comprises the following step:

[0048] c. if the skin of said subject is determined to have been exposed to ozone in step b., treating the skin of said subject with a cosmetic composition for preventing and / or treating the deleterious effects of ozone.

[0049] By "cosmetic composition for preventing and / or treating the harmful effects of ozone" is meant any cosmetic composition known to those skilled in the art for reducing and / or slowing down the appearance of the harmful effects of ozone such as irritations or reactions of discomfort in the skin, redness, itching, sensations of heat, tingling and tightness caused by ozone. In one embodiment of the invention, the cosmetic composition comprises a compound for preventing and / or treating the harmful effects of ozone on the skin identified by the method of the invention.

[0050] Methods for identifying a compound for preventing and / or treating the deleterious effects of ozone on the skin

[0051] The invention also relates to a method for identifying a compound for preventing and / or treating the harmful effects of ozone on the skin, said method comprising the following steps: a. measuring the expression level of PI3, and / or CDSN, and / or SPRR3 in a skin sample of a subject, said sample being treated with said compound; b. comparing said expression level measured in step a. to the corresponding expression level in a skin sample not treated with said compound; c. identify said candidate compound as a compound capable of preventing and / or to treat the deleterious effects of ozone on the skin when the level of expression detected in the skin sample which has been treated with said compound is lower than the level of expression in the skin sample which has not been treated with said compound.

[0052] According to a preferred embodiment, the skin samples of a subject used in the method for identifying a compound for preventing and / or treating the deleterious effects of ozone on the skin above, are chosen from an explant of human skin, a skin biopsy, a microbiopsy, in particular carried out using a skin needle, and a sample of stratum corneum.

[0053] According to one embodiment, this method according to the invention comprises at least one step of taking the skin sample from a subject. This step is preferably carried out in a non-invasive manner, and in particular does not require local anesthesia. According to a preferred embodiment, the step of taking the skin sample from a subject is carried out by rubbing the surface of the skin or by using an adhesive surface such as a D-squame® disc.

[0054] Preferably, this method according to the invention comprises a step of processing the samples in order to prepare the measurement of the expression level. For example, if the skin sample was obtained using an adhesive surface such as a D-squame® disc, the soluble protein extract can be obtained by a protein extraction system such as that described in international application WO2015 / 009085 which makes it possible to bring the D-squame samples into contact with a minimal volume of extraction buffer and thus to obtain a soluble protein extract. Suitable buffers for the extraction of skin proteins are well known and include, for example, RIPA buffer or a urea buffer, or a buffer comprising 0.5% sodium deoxycholate, 50 mM ammonium bicarbonate, 50 mM DTT, 1 qM Pepstatin as well as protease inhibitors.

[0055] In a particular embodiment, the method according to the invention above comprises an additional step of exposing the samples to ozone. This step is preferably carried out on both samples under the same conditions. It can be carried out before or after treatment of the skin sample with said compound.

[0056] The invention further relates to a method for identifying a compound for preventing and / or treating the deleterious effects of ozone on the skin, said method comprising the following steps: a. carrying out a treatment of a skin sample chosen from a reconstructed epidermis and a reconstructed skin, by bringing the skin sample into contact with a compound to be tested; b. carry out an ozone exposure of said skin sample; c. measuring the level of expression of PI3, and / or CDSN, and / or SPRR3 in the skin sample treated with said compound; d. comparing said expression level measured in step c) to the corresponding expression level in the skin sample exposed to ozone and not treated with said compound; e. identify said candidate compound as a compound for preventing and / or treating the deleterious effects of ozone on the skin when the level of expression detected in the skin sample that has been treated with said compound is lower than the level of expression in the skin sample that has not been treated with said compound;

[0057] it being understood that step b can be carried out after step a and vice versa; steps a. and b. can be repeated at least twice.

[0058] Preferably, this method according to the invention comprises a step of processing the samples in order to prepare the measurement of the level of expression.

[0059] In a preferred embodiment, the invention also relates to a method for identifying a compound for preventing and / or treating the deleterious effects of ozone on the skin, said method comprising the following steps: a. carrying out a first treatment of a skin sample chosen from a reconstructed epidermis and a reconstructed skin, by bringing the skin sample into contact with said compound; b. carry out a first exposure to ozone of said skin sample; c. carry out a second treatment of the skin sample by contacting of the skin sample with said compound; d. carry out a second exposure to ozone of said skin sample; e. carry out a third treatment of the skin sample by putting in contact of the skin sample with said compound; f. carry out a third exposure to ozone of said skin sample; g. measure the expression level of PI3, and / or CDSN, and / or SPRR3 in the skin sample treated with said compound; h. comparing said expression level measured in step g) to the corresponding expression level in a skin sample exposed to ozone and not treated with said compound; i. identify said candidate compound as a compound for preventing and / or treating the deleterious effects of ozone on the skin when the level of expression detected in the skin sample that has been treated with said compound is lower than the level of expression in the skin sample that has not been treated with said compound.

[0060] Preferably, this method according to the invention comprises a treatment step samples in order to prepare for the measurement of the expression level.

[0061] The methods for identifying a compound of the invention are preferably carried out ex vivo or in vitro.

[0062] Preferably, in the methods for identifying a compound of the invention, the only difference between the skin sample treated with said compound and the skin sample not treated with said compound is said treatment(s) with said compound.

[0063] Typically, treating the sample with said compound comprises contacting the skin sample with said compound for at least 5 minutes, at least 10 minutes or at least 20 minutes.

[0064] Typically, the measurement of the expression level of PI3, and / or CDSN, and / or SPRR3 is carried out at least 5 minutes, at least 10 minutes or at least 20 minutes after the last treatment of the sample with said compound.

[0065] Steps b, d or h of comparing the above identification methods refer to determining whether the expression level measured in steps a, c or g is essentially identical to the expression level of the same gene or protein measured in a skin sample not treated with said compound. Preferably, the expression level is considered different if the observed difference is statistically significant. If the difference is not statistically significant, the two expression levels are essentially identical.

[0066] Based on this comparison, it can be determined whether the compound is capable of preventing and / or treating the deleterious effects of ozone on the skin. Typically, the compound is considered to be capable of preventing and / or treating the deleterious effects of ozone on the skin when the level of expression is significantly lower in the skin sample treated with said compound compared to the level of expression measured in the sample that has not been treated with said compound, alternatively the compound is not considered to be capable of preventing and / or treating the deleterious effects of ozone on the skin when the level of expression is essentially the same or significantly higher in the skin sample treated with said compound compared to the level of expression measured in the sample that has not been treated with said compound.

[0067] Preferably, the ozone exposure steps are carried out in identical manners on the sample treated with said compound and the sample which has not been treated with said compound.

[0068] The ozone exposure steps b, d or f last at least 5 hours, more particularly at least 10 hours and preferably at least 18 hours. These steps may also be interspersed with incubation periods in a normal, ambient atmosphere. Typically the samples are incubated in an at ozone-enriched atmosphere for 18 hours, then a normal atmosphere for the next 6 hours, and then again in an ozone-enriched atmosphere for 18 hours.

[0069] By "atmosphere" in which the skin sample is incubated is meant the gas or mixture of gases in which the sample is placed. Preferably the sample is incubated in an atmosphere similar to ambient air.

[0070] By "ozone-enriched atmosphere" is meant an atmosphere similar to ambient air comprising at least 0.05 ppm (parts per million) of ozone, preferably at least 0.07 ppm of ozone, more preferably at least 0.09 ppm of ozone or at least 0.1 ppm of ozone.

[0071] Method for evaluating the effectiveness of a compound for preventing and / or treating the harmful effects of ozone on the skin

[0072] The invention also relates to a method for evaluating the effectiveness of a compound for preventing and / or treating the deleterious effects of ozone on the skin of a subject, said method comprising the following steps:

[0073] a. measuring the expression level of PI3, and / or CDSN, and / or SPRR3 in a skin sample of said subject prior to treatment with said compound,

[0074] b. performing a treatment of the skin of said subject with said compound,

[0075] c. measuring the expression level of PI3, and / or CDSN, and / or SPRR3, after step b, in a second skin sample of said subject;

[0076] d. comparing said expression levels measured in step a. and in step c.;

[0077] e. evaluating the effectiveness of said candidate compound with regard to the prevention and / or treatment of the harmful effects of ozone on the skin.

[0078] The method of the invention is preferably carried out in vitro or ex vivo.

[0079] The skin samples of the subject used in the evaluation method according to the invention are preferably taken non-invasively, from the skin of the subject, preferably from the face of the subject, in particular from the cheek of the subject.

[0080] Preferably the skin sample is a biopsy, a microbiopsy in particular carried out using a needle, or a sample of stratum corneum.

[0081] Thus, according to one embodiment, this method according to the invention comprises several steps of taking a skin sample from the subject. This step is preferably carried out in a non-invasive manner, and in particular does not require local anesthesia. According to a preferred embodiment, in particular in the case where the level of expression measured in step a. is the level of expression of CDSN and / or SPRR3, the step of taking the sample is carried out by rubbing the surface of the skin or by using an adhesive surface such as a D-squame® disc.

[0082] Preferably, the only difference between the skin sample of step a. and the skin sample of step c. is said treatment with the compound.

[0083] The skin sample of step c. is taken after the skin of said subject has been treated with said compound. In one embodiment, the skin sample of step c. is taken after the skin of said subject has been treated with said compound for at least 14 days, preferably at least 28 days. Typically, this sample is taken at least 5 minutes, at least 10 minutes or at least 20 minutes after the last treatment of the sample with said compound.

[0084] Preferably, this method according to the invention comprises a step of processing the samples in order to prepare the measurement of the expression level. For example, if the skin sample was obtained using an adhesive surface such as a D-squame® disc, the soluble protein extract can be obtained by a protein extraction system such as that described in international application WO2015 / 009085 which makes it possible to bring the D-squame samples into contact with a minimal volume of extraction buffer and thus to obtain a soluble protein extract. Suitable buffers for the extraction of skin proteins are well known and include, for example, RIPA buffer or a urea buffer, or a buffer comprising 0.5% sodium deoxycholate, 50 mM ammonium bicarbonate, 50 mM DTT, 1 qM Pepstatin as well as protease inhibitors.

[0085] Comparison step d. herein refers to determining whether the expression level measured in step a. is substantially identical to the expression level of the same gene or protein measured in step c. Preferably, the expression level is considered different if the observed difference is statistically significant. If the difference is not statistically significant, the two expression levels are substantially identical.

[0086] Based on this comparison, the effectiveness of a compound in preventing and / or treating the deleterious effects of ozone on the skin of a subject can be evaluated. Typically, the lower the expression level in the sample treated with said compound compared to the expression level measured in the sample that was not treated with said compound, the more effective the compound is considered to be in preventing and / or treating the deleterious effects of ozone on the skin when. Alternatively, the compound is not considered effective in preventing and / or treating the deleterious effects of ozone on the skin when the expression level is essentially the same or significantly higher in the skin sample treated with said compound compared to the expression level measured in the sample that was not treated with said compound.

[0087] Kit

[0088] The present invention also relates to a kit comprising: a. a means for measuring, in a skin sample, the level of expression of PI3, and / or CDSN, and / or SPRR3, and b. an instruction leaflet.

[0089] By "means for measuring the level of expression" is meant a ligand specific to the protein, such as for example an antibody, preferably monoclonal, a Fab fragment, a scFv or a nanobody, specific to this protein or even a microfluidic chip, such as that described in international application WO2011 / 126249, this chip containing ligands specific to the protein. Alternatively, it is understood to mean a nucleotide sequence complementary to the mRNA of the gene and, preferably, specifically hybridizing to the mRNA of the gene or a fragment thereof, preferably, specifically hybridizing to the mRNA of the gene, this sequence or this fragment typically comprising from 5 to 50 nucleotides, preferably 10 to 20 nucleotides, or a pair of primers and / or a probe of the mRNA of said protein, for example from 10 to 60 nucleotides, preferably 15 to 30 nucleotides.

[0090] Thus in one embodiment, the means for measuring the level of expression, in a skin sample, of the kit according to the invention is a ligand specific to the protein or a nucleotide sequence complementary to the mRNA or a fragment thereof, a pair of primers or a probe specific to the mRNA of said protein.

[0091] According to one embodiment of the invention, the kit does not comprise other ligands specific for other proteins or other nucleotide sequences complementary to the mRNA of genes other than PI3, and / or CDSN, and / or SPRR3.

[0092] The kit according to the invention may further comprise a standard range of the level of expression of the gene. This range comprises at least one sample comprising a known quantity or concentration of the protein encoded by said gene or of the mRNA of said gene.

[0093] According to one embodiment, the kit according to the invention further comprises a means for taking the skin sample such as a D-squame® disc, in particular in the case where the measuring means in step a. makes it possible to measure the level of expression of CDSN and / or SPRR3.

[0094] According to one embodiment, the kit according to the invention further comprises a protein extraction buffer.

[0095] The present invention further relates to the use of a kit according to the invention for the identification of a compound making it possible to prevent and / or treat the harmful effects of ozone on the skin. Example: Materials and methods: 1-In vitro ozone exposure system

[0096] To optimize the control of ozone concentrations used during exhibitions, all of the equipment used to generate and distribute ozone-enriched air was made with materials known to be relatively insensitive to the oxidizing power of ozone, such as PTFE (e.g. Teflon), PMMA (e.g. Plexiglas) and PEEK. 1.1- Device external to the incubator:

[0097] To mimic ozone pollution, the laboratory uses a MEGATEC 49i ozone analyzer / generator coupled with an external pump, and also connected to a culture incubator regulated at 37°C, 5% CO2 and saturated with humidity (approximately 95%).

[0098] The external pump is used to take ambient air and supply the MEGATEC ozone analyzer / generator. The air will then pass in front of the device's UV lamp (set to a wavelength of 185nm) which will transform some of the oxygen molecules (O2) in the air into ozone molecules (O3). The air is then enriched with ozone.

[0099] This ozone-enriched air can then be transferred into the incubator containing the culture plates with the reconstructed T-Skin™ skin model.

[0100] The MEGATEC analyzer / generator allows you to adjust and control: - The flow of ozone-enriched air (supplying the incubator) by means of a pressure regulator placed upstream of the circuit in the MEGATEC analyzer / generator. - The ozone concentration generated by adjusting the power of the UV lamp of the MEGATEC analyzer / generator.

[0101] Furthermore, the MEGATEC analyzer / generator has a second pump and an internal analysis system which allows the actual ozone concentration to be measured: either at the outlet of the device (generated concentration, upstream of exposure in the incubator), or in the incubator (residual concentration, downstream of exposure). 1.2- Device internal to the incubator:

[0102] Within the incubator, a fluidic system developed in the laboratory allows the same quantity of ozone to be distributed to each reconstructed skin insert. This system is semi-open to allow both: a homogeneous distribution of ozone on the top of the plates, and good regulation of the culture conditions of the reconstructed skins.

[0103] Furthermore, the cover of each culture plate (generally made of polystyrene, a material sensitive to ozone) is replaced by a specific cover for exposure: this is a PTFE or PEEK cover pierced above each reconstructed skin insert, and thus allowing direct exposure of the surface of the reconstructed skins to ozone-enriched air. In addition, this cover protects the culture medium to prevent its oxidation by ozone, in order to only expose the surface of the skins.

[0104] 2-Exposure to ozone of in vitro reconstructed skin, with or without application of sebum

[0105] 2.1- Reception and culture of the TSKIN reconstructed skin model

[0106] The reconstructed skin model used is the T-Skin™ reconstructed skin model produced and marketed by the company EPISKIN SA (FR-69007, Lyon).

[0107] This is a reconstructed skin model composed of: - A reconstructed dermis made up of a collagen 1 lattice, containing human-derived fibroblasts extracted from breast plastic surgeries from healthy adult donors (surgical waste obtained with the consent of patients during cosmetic breast reduction surgery). - A reconstructed epidermis made from human-origin keratinocytes extracted from breast plastic surgeries from healthy adult donors (surgical waste obtained with the consent of patients during cosmetic breast reduction surgery).

[0108] This model is grown in a 4.5cm2 culture insert which allows it to be suspended above the culture medium and grown at the air-liquid interface.

[0109] In this insert the collagen lattice, or dermal equivalent, covers a surface which can vary from 2 to 2.5 cm2 in the center of the insert. The epidermis generally covers the entire 4.5 cm2 of the insert.

[0110] The T-Skin™ model is received at the laboratory after 12 days of dermal culture (D12). The epidermis is 7 days into culture and is relatively immature (poorly differentiated). During transport, these inserts are placed on a semi-solid nutrient culture medium suitable for transport.

[0111] Upon receipt, the inserts are quickly transferred into new 6-well culture plates called “deep well” allowing 8.5 ml of liquid culture medium to be placed under each insert (culture at the air-liquid interface). These plates can be Corning brand plates reference 355467. The culture medium used is that marketed by the company EPISKIN for TSKIN kits: medium called “Culture medium for T-Skin”

[0112] The reconstructed skins are then placed in a culture incubator regulated at 37°C, 5% CO2 and with saturated humidity (approximately 95% humidity).

[0113] The culture medium is then replaced in the same way on D13 and D15 (dermis).

[0114] Finally, on D18, the inserts are placed in standard 6-well culture plates in SBS format (COSTAR brand plate type reference 3506) with 2.5 ml of culture medium for T-Skin™ under each insert.

[0115] Subsequently, these plates are replaced with the culture medium at least every day.

[0116] 2.2- Preparation of a hydrolipidic solution of sebum for application on the reconstructed skin model

[0117] A dry lipid mixture corresponding to an artificial sebum was prepared according to L'Oréal patent FR3022783A1.

[0118] This sebum contains approximately: - 25% free fatty acids (for example: palmitic acid, sapienic acid, stearic acid, oleic acid, linoleic acid) - 15% squalene - 3% cholesterol - 2% cholesterol esters (e.g.: cholesteryl oleate, cholesteryl palmitate, cholesteryl stearate) - 25% waxes (for example: palmityl palmitate, stearyl stearate, oleyl oleate, stearyl palmitate) - 30% glycerides (for example: 1,3-dipalmityl glycerol, 1,3-stearyl glycerol, 1,3-dioleyl glycerol, 1,3-dilinoleyl glycerol, tripalmitin, tristearin, tri-sapienin, triolein, trilinolein).

[0119] From this dry sebum, a hydrolipidic mixture is reconstituted extemporaneously to mimic a sebum-sweat mixture present on oily skin. Thus, the final concentration applied represents 300qg of sebum / cm2.

[0120] 2.3- Preparation of the hydrolipidic mixture at 50mg / ml in phosphate buffer without calcium and without magnesium: - The PBS is preheated for a few minutes in a water bath at 45°C, - It is then added to our bottle with dry artificial sebum to obtain a solution of 50mg of sebum / ml of PBS - Carrying out successive passes of the mixture in a vortex mixer and in a water bath at 45°C in order to dissolve the sebum. The final result is a milky emulsion.

[0121] When spreading on T-Skin™, 27qL of this solution is applied per insert, and this solution is distributed over the entire 4.5cm2 of the insert to obtain 300qg of hydrolipidic solution / cm2 of insert.

[0122] 2.3- Application of the sebum solution topically on the TSKIN model:

[0123] The sebum solution is applied according to the following protocol: - A precise volume of 27pL of solution is placed in the center of the model, - We take a glass rod with a round end that we pre-wet with a small volume of solution to spread, - We come into contact with the drop of solution placed in the center of the skin model, and we draw the drop in a circular movement which gradually moves away from the center towards the edge of the insert (4.5cm2 of surface application). During this application, the contact between the glass rod and the skin model must be as low as possible so as not to cause stress on the model.

[0124] 2.4- Repeated exposures of the TSKIN model to ozone:

[0125] For this type of exposure the ozone generator is set to low values: - Ozone generated at the device outlet: approximately 0.130 ppm for a concentration measured inside the incubator less than 0.1 ppm - The output flow rate of ozone-enriched air above each reconstructed skin is approximately 20 ml / min (note that the ozone outlets are more than 10 cm from the reconstructed skins inside the incubator to avoid direct stress from the output flow).

[0126] The exposures are carried out three times successively: 18 hours between D18 and D19, 18 hours between D19 and D20, and 18 hours between D20 and D21. Between each exposure the model is in the incubator without ozone for a little less than 6 hours.

[0127] To mimic oily skin, certain samples undergo, only before the first exposure to ozone on D18, the application of a hydrolipidic mixture of sebum at a rate of 300pg / cm2 (27pL of a solution at 50mg / ml of sebum spread over 4.5cm2) prepared according to the protocol described above.

[0128] Once the skins are ready for display, the standard polystyrene covers are replaced with perforated PEEK or PTFE covers.

[0129] A part of the samples are treated under ozone, and another part, the controls, are prepared in exactly the same way, but incubated in a conventional incubator without ozone.

[0130] At D21, the reconstructed skin samples were collected, the epidermis was separated from the dermis, cut in half and frozen at -80°C for transcriptomic analysis. At D22, the reconstructed skin samples were collected, the epidermis was separated from the dermis, cut in half and frozen at -80°C for proteomic and immunohistochemical analyses.

[0131] 3- Transcriptomic analysis of reconstructed skin exposed to ozone in vitro

[0132] For transcriptomic analysis by high-throughput sequencing (RNA-sequencing), the reconstructed skins were exposed 3 times to 0.1 ppm of ozone for 18 h on D18, D19 and D20, with or without topical application of sebum on D18. The epidermis samples were taken on D21. Each condition was carried out in triplicate and two different experiments were conducted.

[0133] Total RNA was isolated from these samples with the RNeasy Micro Kit (Qiagen, Hilden, Germany). RNA purity and integrity were assessed on the Agilent 2100 Bioanalyzer with the RNA 6000 NanoLabChip Kit (Agilent, Palo Alto, CA, USA) and the Nanodrop spectrophotometer. All samples had RIN values ​​> 8.8.

[0134] For library preparation and RNA sequencing, 200 ng of total RNA was reverse transcribed into complementary DNA (cDNA) of the first strand of RNA using a mixture of random and poly-dT primers, and omitting the DNase treatment step. Synthesis of the second strand, using a nucleotide analog for retention, generated the double-stranded cDNA. The latter was fragmented into pieces of 200 to 400 bases. Then, end repair was performed to generate a blunt-ended double-stranded cDNA, followed by ligation of indexing adapters, strand selection via targeted degradation by a nucleotide analog, and reduction of ribosomal RNA content. Finally, cDNA libraries were created by 13-cycle PCR enrichment. Equimolar amounts of each library were sequenced on an Illumina NextSeq 2000, for paired-end sequencing at 2x107 base pairs.

[0135] 4-Proteomic analysis of reconstructed skin exposed to ozone in vitro

[0136] 4.1- Exposure of reconstructed skin to ozone:

[0137] For non-targeted label-free proteomic analysis, reconstructed skins were exposed 3 times to 0.1 ppm ozone for 18 h on D18, D19 and D20, with or without topical application of sebum on D18. Epidermis samples were collected on D22. Each condition was performed in triplicate and two different experiments were conducted. 4.2- Protein extraction and digestion:

[0138] Total proteins were extracted in extraction buffer (0.5% sodium deoxycholate / 50 mM ammonium bicarbonate / 50 mM DTT, 1 μM Pepstatin and protease inhibitor cocktail) followed by vibratory grinding at 30 KHz for 2 minutes. Residual DNA was removed by sonication and proteins were precipitated in acetone and then diluted in 50 μL of 50 mM ammonium bicarbonate / 1% sodium deoxycholate buffer. Protein concentrations were determined by the Bradford method. 20 μg of proteins were then denatured (95°C for 5 minutes), then reduced (30 min at 37°C in the presence of 0.2 mM DTT) and alkylated (30 min at 37°C in the presence of 0.8 mM lodoacetamide). Finally, 0.4 μg of trypsin was added and digestion was carried out at 37°C for 16 h. Digestion was stopped by acidification (pH2) and the peptides were purified on a Stage Tip (C18).The peptides were dried in a concentrator and then taken up in 11 μl of a 2% ACN (Acetonitrile) / 0.05% TFA (Trifluoroacetic Acid) solution. The peptides were quantified on a NanoDrop2000 (ThermoFisher Scientific) at 205 nm. 4.3- Analysis by Nano LC-MS / MS

[0139] The analysis was carried out by a nanoLC-MS / MS, consisting of a chroma chain liquid tography nanoRSLC U3000 (ThermoScientific, Dionex Softron GmbH, Germering, Germany) coupled to an Orbitrap Fusion Tribrid -ETD mass spectrometer (ThermoScientific, San Jose, CA, USA) controlled by the Orbitrap Fusion Tune Application 3.3.2782.34 software and equipped with a Nanospray Flex source.

[0140] 5pg of peptide was injected onto a pre-column (300 mm id x 5 mm, C18 PepMaplOO, 5 mm, 100 Å; (Thermo Fisher Scientific)) in 5 min at a rate of 20 pL / min in a solvent containing 2% ACN / 0.05% TFA. The flow was then redirected from the pre-column onto the separation column (PepMaplOO RSLC, C18 3 mm, 100 Å, 75 pm id x 50 cm long (Thermo Fisher Scientific)) and the peptides were eluted in a linear gradient composed of 5-40% solvent B (A: 0.1% FA (Formic acid), B: 80% ACN / 0.1% FA) in 270 min at a flow rate of 300 nl / rnl. Spectra were acquired using Thermo XCalibur software 4.3.73.11. An internal calibration on the siloxane ion (m / z 445.12003) was performed. Peptide acquisition was done by DDA (Data Dependent Acquisition) mode.

[0141] 4.4- Identification and quantification of proteins

[0142] The raw data from the DDA acquisition were searched against the Human database (UniProt Reference Proteome - Proteome ID UP000005640 - 97065 entries - 2020.09) using the MaxQuant software version 2.0.2.0.

[0143] 5-Analysis of Elafine and SPRR3 proteins by immunohistochemistry (IHC)

[0144] For immunohistochemistry analysis, reconstructed skins were exposed 3 times to 0.1 ppm ozone for 18 hours on D18, D19 and D20, with or without topical application of sebum on D18. Epidermis samples were taken on D22. Each condition was performed in triplicate and two different experiments were conducted.

[0145] Formalin-fixed reconstructed skin samples were embedded in paraffin and 4 µm sections prepared for IHC. All IHCs were performed on a Roche Ventana Discovery Ultra. After deparaffinization, a heat-activated antigen retrieval step was performed and endogenous peroxidases were blocked for 10 minutes using 3% H2O2 solution.

[0146] A polyclonal anti-SPRR3 antibody (Reference PA5-60721 from Thermo Fisher) and a monoclonal anti-ELAFIN antibody (Reference SC-398075 from Santa Cruz Bio-technology) were used to label the proteins "small proline rich protein 3" (SPRR3) and "peptidase inhibitor 3 or skin-derived antileukoprotease" (SKALP, ELAFIN), respectively. The antibodies were revealed using IgG polymers or HRP-linked (OmniMap-HRP, Ventana). Immunostaining was revealed using an amplification system using Tyramide (TSA) linked to a fluorochrome (FAM-Disc from Ventana). The slides were counterstained with DAPI and covered with a coverslip. To attest to the specificity of the IHC, isotype control antibodies were were used under the same conditions leading to the absence of signal detection on positive samples, confirming the specificity of the signal.

[0147] All slides were scanned using Excilone's PathScanCombi at the X20 Objective with parameters adapted to avoid signal saturation. The same parameters were used to allow consistent signal detection and quantification for all skin samples. Results Corneodesmosin (CDSN), SPRR3, Elafine

[0148] Tables 1 and 2 show that the expression of the biomarkers elafin (P13 gene), Corneodesmosin (CDSN) and SPRR3 increase following ozone exposure at the gene level (transcriptomic analysis, Table 1) and at the protein level (proteomic analysis, Table 2). The results are similar with or without topical application of sebum before the first exposure to ozone.

[0149] Table 3 shows that the expression of the biomarkers elafin and SPRR3 increases following exposure to ozone, without topical application of sebum, at the protein level by immunohistochemistry.

[0150] [Table 1] Table 1: Gene expression ratios quantified in arbitrary units in vitro on reconstructed skin after 3 exposures to ozone at 0.1 ppm, with or without topical application of sebum, between conditions with and without exposure to ozone. P13 is the symbol of the gene corresponding to the Elafine protein. Transcriptomic Analysis Gene Ratio q-value Ratio q-value PI3 Sebum + Ozone Vs Sebum 2.65 5.69E-05 Ozone Vs Unexposed 2.51 l,74E-04 CDSN 2.40 l,56E-05 2.19 9.99E-05 SPRR3 5.17 2.69E-05 5.85 4.53 E-06

[0151] [Table 2] Table 2: Ratio of protein expression quantified in arbitrary units in vitro on reconstructed skin after 3 exposures to ozone at 0.1 ppm, with or without topical application of sebum, between conditions with and without exposure to ozone. Proteomic Analysis Protein Ratio q-value Ratio q-value Elafine Sebum + Ozone Vs Sebum 1.96 0.02 Ozone Vs Unexposed 2.05 0.017 CDSN 1.85 0.15 2.32 0.017 SPRR3 3.99 0.02 NA NA

[0152] [Table 3] Table 3: Expression ratio of the expression by immunolabeling of proteins quantified in arbitrary units in vitro on reconstructed skin after 3 exposures to ozone at 0.1 ppm, without topical application of sebum, between conditions with and without exposure to ozone. Immunohistochemistry Analysis Protein Ratio p-value Elafine Ozone Vs Unexposed 1.97 0.01 SPRR3 6.4 0.0001

Claims

Claims

1. A method of diagnosing ozone exposure of the skin in a subject, said method comprising the following step: a) measuring the level of expression of PI3, and / or CDSN, and / or SPRR3 in a skin sample of said subject.

2. A method of identifying a compound for preventing and / or treating the deleterious effects of ozone on the skin, said method comprising the following steps: a. measuring the expression level of PI3, and / or CDSN, and / or SPRR3 in a skin sample of a subject, said sample being treated with said compound; b. comparing said expression level measured in step a) to the corresponding expression level in a skin sample not treated with said compound; c. identify said candidate compound as a compound for preventing and / or treating the deleterious effects of ozone on the skin when the level of expression detected in the skin sample that has been treated with said compound is lower than the level of expression in the skin sample that has not been treated with said compound.

3.

4. The method of claim 2, wherein said skin samples are selected from a human skin explant, a skin biopsy, a microbiopsy and a stratum corneum sample. Method for identifying a compound for preventing and / or treating the harmful effects of ozone on the skin, said method comprising the following steps: a. carrying out a treatment of a skin sample chosen from a reconstructed epidermis and a reconstructed skin, by bringing the skin sample into contact with said compound; b. carry out an ozone exposure of said skin sample; c. measuring the level of expression of PI3, and / or CDSN, and / or SPRR3 in a skin sample treated with said compound; d. comparing said expression level measured in step c) to the corresponding expression level in a sample of skin exposed to ozone and not treated with said compound; e. identify said candidate compound as a compound for preventing and / or treating the deleterious effects of ozone on the skin when the level of expression detected in the skin sample that has been treated with said compound is lower than the level of expression in the skin sample that has not been treated with said compound; it being understood that step b. may be carried out after step a. and vice versa, and that steps a. and b. may be repeated at least 2 times.

5. Method according to one of claims 1 to 3, in which the skin sample(s) are taken using a D-squame® disc.

6. A method according to any one of claims 1 to 5, wherein the expression level is determined by the mRNA expression level or by measuring the protein expression level.

7. The method of claim 6, wherein the expression level of the protein is measured using a ligand specific for said protein.

8. Kit for implementing a method according to any one of the preceding claims, comprising: a. a means for measuring, in a skin sample, the level of expression of PI3, and / or CDSN, and / or SPRR3, wherein the means for measuring the level of expression is a ligand specific for the protein or a nucleotide sequence complementary to the mRNA or a fragment thereof, a pair of primers or a probe specific for the mRNA of said protein, and b. an instruction leaflet.

9. Use of a kit according to claim 8 for the identification of a compound for preventing and / or treating the harmful effects of ozone on the skin.