Method for the selection of an ecobiological compound capable of increasing the skin tolerance threshold

Abstract

The present invention relates to a method for selecting at least one preferably eco-biological compound for the prevention and / or care of sensitive and / or sensitized skin, involving identifying a modulation of the content of TAFA4 and / or of nucleic acid encoding same.

Description

[0001] DESCRIPTION

[0002] TITLE: METHOD FOR SELECTING AN ECOBIOLOGICAL COMPOUND CAPABLE OF INCREASING THE SKIN'S TOLERANCE THRESHOLD

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to a method for selecting at least one compound capable of increasing the tolerance threshold of the skin, in particular via the activity of the chemokine TAFA4. In particular, it is a method for selecting at least one compound, preferably ecobiological, intended for the prevention and / or care of sensitive and / or sensitized skin, comprising in particular a step of quantifying the content of TAFA4, and / or of nucleic acids encoding TAFA4, and a step of comparing this quantification with a reference value so as to identify the presence or absence of a variation in the content of TAFA4 and / or of nucleic acids encoding TAFA4.

[0005] STATE OF THE ART

[0006] The skin is the main barrier that separates the body from the external environment and is composed of the superposition of three main layers, respectively from the deepest to the most superficial: the hypodermis, the dermis and the epidermis, colonized by the microbiota which is often considered as the last cutaneous layer. Due to its constant interaction with the environment, the skin undergoes multiple attacks that alter its balance. In parallel with these exogenous attacks, each individual presents internal factors that have an impact on the health of the skin, namely, genetic heritage or hormones. These different exogenous and endogenous factors generate remarkable differences between individuals in the levels of skin tolerance and skin sensitivity, with a growing part of the population perceiving their skin as irritable or sensitive.

[0007] The results of epidemiological studies show that sensitive skin syndrome is a global problem that is constantly increasing, with a prevalence that varies from one country to another (Misery et al., 2018b). In France, 59% of the population reports having sensitive skin (Misery et al., 2018a). More generally, in Europe in recent years, the prevalence of sensitive skin has increased considerably, namely by +7% in 10 years (Misery et al., 2018a). Comparable prevalences have also been found in Asia and Latin America.

[0008] Sensitive skin is characterized by skin hyperreactivity: the skin reacts excessively to external and internal aggressions and stimuli that should normally be well tolerated (Stânder et al., 2009). In other words, sensitive skin reacts more quickly due to a lowering of its tolerance threshold.

[0009] Sensitive skin is now recognized as a real syndrome by the medical community with an official definition established by the International Forum for the Study of Itch (IFSI): “a syndrome defined by the occurrence of unpleasant sensations (tingling, tightness, warming, itching and irritation) in response to stimuli that should not normally cause such sensations. The skin may appear normal or be accompanied by erythema. Sensitive skin can affect all parts of the body, particularly the face” (Misery et al., 2017).

[0010] According to the current model accepted by the scientific community, sensitive skin syndrome is the result of a cumulative effect of a set of skin changes described below:

[0011] - an altered barrier function, which allows aggressive agents to penetrate the skin more easily, leading to skin inflammation;

[0012] - an alteration of type C cutaneous nerve endings, notably a neuropathy of small nerve fibers (Huet et al., 2018);

[0013] - non-specific inflammation of keratinocytes involving pro-inflammatory mediators such as cytokines (IL-1, IL-8, TNFa), inflammatory lipid mediators (prostaglandins E2, prostaglandins F2a and leukotrienes) and histamine; and

[0014] - neurogenic inflammation generated by the release of neurotransmitters, which maintain cutaneous inflammation and lead to vasodilation (Stânder et al., 2009).

[0015] Skin hypersensitivity has a considerable impact on overall quality of life and often leads people suffering from this condition to reduce or stop using cosmetic products in order not to exacerbate irritation. Indeed, cosmetic products generally contain compounds that can cause inflammatory phenomena, such as surfactants or preservatives. These intolerance reactions can be specific to an individual and limited to very specific conditions and / or combinations of compounds, and are therefore difficult to predict. However, in the case of specific products such as sunscreens and products targeting specific dermatological conditions, reducing the application frequency and / or the applied quantities of cosmetic products is not recommended as it risks leading to an aggravation of skin hypersensitivity.

[0016] Furthermore, it is important to be able to control sensitivity at the skin level, for example that generated by atmospheric agents (pollution, temperature, humidity, etc.), allergy phenomena, healing or even sunburn by using non-pharmaceutical cosmetic compounds, advantageously ecobiological, in particular presented in a form suitable for topical application.

[0017] For example, EP 1 457 780 describes a screening method in which the reduction in the production of stem cell factor (SCF), used as a specific biomarker, allows the selection of an ingredient of interest capable of treating pruritus and dry or sensitive skin syndromes. However, SCF is known at the cutaneous level to be mainly involved in the survival of melanocytes and in the induction of melanin synthesis (Grabbe et al., 1994; Atef et al., 2019). Therefore, the modulation of this factor could lead to significant adverse effects on skin pigmentation.

[0018] There therefore remains a clear need for a process enabling the identification of compounds, preferably non-pharmaceutical cosmetics, in particular capable of being presented in a galenic form suitable for topical and / or oral administration, advantageously ecobiological, which make it possible to increase the skin tolerance threshold and / or reduce skin discomfort also by acting on the endogenous mechanisms for controlling pain and inflammation specific to human skin.

[0019] To date, the protein TAFA4 (TAFA Chemokine Like Family Member 4), also called FAM19A4 (family with sequence similarity 19 (chemokine (CC motif)-like) member A4), a neurokine mainly produced in humans by certain nerve fibers (Wang et al., 2015; Yoo et al., 2021), is identified as an anti-inflammatory and soothing agent. In humans, the amino acid sequence of TAFA4 corresponds to the sequence SEQ ID NO: 1 and / or to the sequence SEQ ID NO: 2 and / or is encoded by the nucleotide sequence of mRNAs corresponding, partially or totally, to the sequence SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.

[0020] The TAFA4 protein is composed of two domains, including a signal peptide and a highly conserved chemokine-like sequence (Wang et al., 2015). In humans, TAFA4 is highly expressed in the brain, and more weakly in other organs such as the colon, spleen, pancreas, prostate, liver, and lungs (Leeman et al., 2020; Liu et al., 2017; Tom Tang et al., 2004; Wang et al., 2015).

[0021] At the cutaneous level, in vivo in mice, TAFA4 is expressed and secreted by certain neurons whose free endings are found in the skin, notably DRG (dorsal root ganglion) neurons, and C-LTMRs (C-low-threshold mechanoreceptors) sensory nerve fibers, the latter being involved in the transmission of sensory signals (Delfini et al., 2013; Hoeffel et al., 2021; Salio et al., 2021). Studies have shown that TAFA4 knockout mice are viable, without apparent abnormalities and respond to acute thermal and mechanical stimuli (Delfini et al., 2013). On the other hand, they present defects in skin regeneration after exposure to UV (Hoeffel et al., 2021) and hypersensitivity to pain (Delfini et al., 2013, Kambrun et al., 2018; Salio et al., 2021).In particular, TAFA4 knockout mice have a defective resolution of inflammation 35 days post-irradiation in an actinic erythema-like model via UVC irradiation. This condition can be compensated by intradermal injection of recombinant TAFA4 protein post-irradiation (Hoeffel et al., 2021).

[0022] Furthermore, TAFA4 KO mice develop significantly increased pain hypersensitivity in pathological conditions (via intradural injection of inflammatory pain agonist or via partial sciatic nerve injury) compared to control mice. Intradural injection of recombinant human TAFA4 compensates for the condition of TAFA4 KO mice. In mice, TAFA4 also appears to play a restorative role during healing, particularly following sunburn (Hoeffel et al., 2021). In addition, it could control allergic phenomena by inducing the synthesis of IL-10, which in turn acts on immune-mediated inflammation (Qiu et al., 2022).

[0023] In vivo in mice, an injection of the human TAFA4 protein, subcutaneously or at the level of neurons present in the bone marrow, reduces pain / hypersensitivity felt locally in the skin, up to 4 hours post-injection, particularly in a neuropathic pain model or a post-operative pain model (Kambrun et al., 2018; Yoo et al., 2021).

[0024] The use of the TAFA4 protein, its component peptide fragments, or a nucleic acid encoding it as an anti-inflammatory and soothing agent has already been proposed. For example, WO2014 / 180853A1 discloses the use of TAFA4 in pharmaceutical compositions for preventing, relieving, or treating pain. WO2020 / 064907A1 describes the use of TAFA4 or a nucleic acid encoding it for combating skin inflammation. WO2021 / 156310A1 discloses the use of TAFA4 or a nucleic acid molecule encoding it for treating inflammatory diseases.

[0025] Although TAFA4 is described as an anti-inflammatory and soothing agent, its formulation in cosmetic or dermatological compositions, particularly for topical use, is difficult due in particular to bioavailability issues, its molar mass of 15-20 kDa preventing its cutaneous penetration, but also to the maintenance of biological activity, metabolism by skin cells or bacteria of the microbiota, etc. There therefore remains a clear need to identify compounds, preferably non-pharmaceutical cosmetics, particularly capable of being presented in a galenic form suitable for topical application, advantageously ecobiological, which allow the prevention and / or care of sensitive and / or sensitized skin, in particular by increasing the skin tolerance threshold and / or reducing skin discomfort, also by acting on the endogenous mechanisms of pain control and inflammation specific to human skin.

[0026] DETAILED DESCRIPTION OF THE INVENTION

[0027] Surprisingly, the Applicant, pursuing its ecobiological approach in the search for action on the real causes of skin problems and to give the skin the means to strengthen itself, demonstrated that the modulation of the endogenous content of TAFA4 and / or the nucleic acid encoding it (i.e., modulation of the transcription and / or protein expression of TAFA4) was possible, in particular at the cutaneous level, more particularly by a topical intervention (excluding exogenous supply of TAFA4 and / or the nucleic acid encoding it).

[0028] Even more surprisingly, the Applicant has demonstrated that the main living cells of the skin, namely keratinocytes and fibroblasts, also express TAFA4 endogenously, and that they can modulate this expression.

[0029] These major discoveries open the way to a completely new approach to skin sensitivity, particularly through topical, particularly cosmetic, treatments that are more effective, more durable, and more respectful of the skin's ecosystem, acting with respect and via its own natural mechanisms, thus elucidated.

[0030] The Applicant has thus identified that the modulation of the endogenous content of TAFA4 and / or the nucleic acid encoding it is an indicator making it possible to classify candidate compounds according to their capacity or not to prevent and / or treat sensitive and / or sensitized skin, in particular by increasing the skin tolerance threshold, in particular via the use of relevant experimental models.

[0031] Based on this observation, the Applicant has developed a reliable means (i.e., having an acceptable sensitivity and / or specificity), easy to read / interpret and integrable into routine research and development, particularly in the cosmetic field, in order to decide on the selection or not of a compound, preferably ecobiological, intended for the prevention and / or care of sensitive and / or sensitized skin, this means being based on the modulation of the content of TAFA4 and / or of nucleic acid encoding it. Thus, a first subject of the present invention relates to a method for selecting at least one compound, preferably ecobiological, intended for the prevention and / or care of sensitive and / or sensitized skin, advantageously following stress, more advantageously an allergy, implementing the identification of a modulation, advantageously an increase, of the content of TAFA4 and / or of nucleic acid, advantageously of mRNA, encoding it (i.e., encoding TAFA4).In other words, it is a method comprising in particular a step of identifying a modulation, advantageously an increase, in the content of TAFA4 and / or nucleic acid, advantageously mRNA, coding TAFA4.

[0032] In particular, the method according to the invention comprises the following steps: a) Bringing said at least one compound into contact with at least one skin cell; b) Measuring the content of TAFA4 and / or nucleic acid, advantageously messenger ribonucleic acid (mRNA), encoding TAFA4; c) Comparing the value obtained in step b) with a reference value; d) Selecting said at least one compound inducing an increase in the content of TAFA4 and / or nucleic acid encoding TAFA4 in step c).

[0033] In the context of the invention, the term "compound" designates a substance or compound which has biological and / or therapeutic properties which underlie a physiological and / or galenic effect. It may be an active ingredient, an excipient or their mixtures.

[0034] In the context of the invention, the expressions “ecobiological composition”, “ecobiological compound”, “ecobiological ingredient”, “ecobiological active ingredient” or “ecobiological excipient” respectively designate a composition, a compound, an ingredient or an excipient that is respectful of the person, of their interactions with the world, and of the planet. In particular, they designate a composition, a compound, an ingredient, an active ingredient or an excipient that is respectful of the communities of living cells constituting the skin (i.e., skin microbiota, keratinocytes, fibroblasts, etc.) which constantly interact with each other and with their environment.

[0035] In the context of the invention, the term "ecobiological approach" refers to the particular approach initiated and developed by the inventor / Applicant which combines skin biology and ecology to help the skin live according to its natural mechanisms, over the long term.

[0036] In the context of the present invention, the expression "TAFA4" designates, together and individually, any protein, polypeptide or peptide derived from a mammalian cell, advantageously human or murine, more advantageously human, corresponding to the active peptide sequence of the protein, including the different existing or predicted isoforms generated by alternative mRNAs, as well as all the peptide fragments composing it and all its biologically active derivatives. In particular, the amino acid sequence of TAFA4 corresponds to the sequence SEQ ID NO: 1 and / or to the sequence SEQ ID NO: 2. More precisely, the term “derivative or fragment” means a protein sequence identical to the sequence SEQ ID NO: 1 and / or to the sequence SEQ ID NO: 2 to at least 60%, preferably to at least 70%, more preferably to at least 80%, more preferably to at least 85%, or even to at least 90%, which therefore includes TAFA4 of different origins (non-human mammals, etc.).

[0037] In the context of the invention, the expression "biologically active derivatives" designates isoforms, fragments or modified versions of isoforms or fragments, in particular covalently modified with grafts of organic groups, such as capryloyl, stearoyl, palmitoyl fatty chains or even by acetylation, methylation, etc. Said derivatives may have a biological activity similar to or even superior to the protein from which they are derived.

[0038] In the context of the present invention, the expression "nucleic acid coding for TAFA4" designates, together and individually, any nucleic acid, preferably any ribonucleic acid, more preferably any messenger ribonucleic acid (mRNA), originating from a mammalian cell coding for TAFA4, including its different isoforms. In particular, the nucleotide sequence of TAFA4 corresponds, partially or totally, to the sequence SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7 or to a sequence identical to one of the sequences SEQ ID NO: 3 to 7 at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, or even at least 90%, which therefore includes the nucleotide sequence coding for a TAFA4 protein of different origins (non-human mammals, etc.). These complementary deoxyribonucleic acids (cDNA) correspond to as many alternative mRNAs identified in humans.

[0039] In the context of the invention, the term “care” designates a topical care, in particular cosmetic and non-therapeutic.

[0040] In the context of the invention, the expression “sensitized skin” designates skin that is damaged and / or attacked and / or in an inflammatory state and / or made painful and / or disrupted, advantageously following stress, more advantageously an allergy.

[0041] In the context of the invention, the expression "bringing into contact with at least one skin cell" designates any direct exposure (e.g., cells in culture) or indirect exposure (e.g., by topical application) to said at least one candidate compound according to the invention. In particular, indirect exposure by topical application can be carried out on the skin of a whole living organism, advantageously of a human being, on a skin explant, on a reconstructed epidermis or on reconstructed skin.

[0042] In the context of the present invention, the expression “skin explant” designates a biopsy of human skin from surgical waste and which comprises the entire epidermis and a dermal and / or hypodermal compartment.

[0043] In the context of the present invention, the expression "skin substitute" designates a reconstructed skin or a reconstructed epidermis, preferably human, which contains differentiated cells distributed in several layers. In particular, within the meaning of the invention, the reconstructed skin contains at least two compartments, namely: a dermal compartment and an epidermal compartment. These models can be generated by 3D printing or by cell differentiation and can include several cell types in order to come as close as possible to the complete functionalities of the human or animal epidermis (innervation, immunization, pigmentation, vascularization, etc.). Examples of suitable models of this type are marketed by the companies LabSkin Creations or Straticell, Skin Ethic, Episkin or Phenion.

[0044] In the context of the present invention, the expression “reconstructed epidermis” designates an epidermis generated in vitro by conventional techniques well known to those skilled in the art (eg, Limât and Hunziker 2002; Poumay et al., 2004). The keratinocytes used in this epidermis are advantageously derived from sensitive human skin.

[0045] In the context of the present invention, the expression "reconstructed skin" means an epidermal component, containing keratinocytes, generated in vitro and a dermal component, containing fibroblasts, generated in vitro integrated into a dermal matrix tissue. The reconstructed skin is commercially available. The keratinocytes and fibroblasts used in this skin can advantageously be derived from sensitive human skin.

[0046] In the context of the present invention, the expression "skin cells" designates any cell type likely to be found, at least partially, in one of the three layers of the skin, i.e. epidermal cells (for example keratinocytes, melanocytes, sebocytes, Merkel cells, immune cells such as Langerhans cells, nerve endings of type C fibers), dermal cells (for example mast cells, vascular smooth muscle cells, specialized muscle cells, fibroblasts and immune cells, dermal dendritic cells, fibrocytes, endothelial cells), hypodermal cells (for example adipocytes), nerve cells and macrophages. It is clear that according to the invention, the expression "skin cell" includes any neuronal cell whose endings or fibers are found in the skin.In particular, the skin cells are fibroblasts or keratinocytes, advantageously human, in particular, the skin cells are macrophages, advantageously Ml-type macrophages, preferably human. The methods for obtaining Ml-type macrophage cells are known to those skilled in the art, for example by differentiating THP-1 monocytes (Horiba et al., 2022) or PMBC monocytes (human peripheral blood mononuclear cells; Wang et al., 2015). In particular, these are GINIP+ type C neurons (Salio et al., 2021; Jung et al., 2023).

[0047] In the context of the invention, the expressions “reference value”, “normal value”, “normal”, “standard”, “usual value” or “reference biological sample” are used interchangeably and designate a value or an average of the values ​​of a parameter (e.g., gene expression, in particular at the mRNA level, etc.), originating from one or more subjects and representing the basal state of the measurement envisaged; as opposed to a “tested value”, “test value” or “test biological sample”. The reference value and the test value are obtained by implementing the same method of detection, quantification, identification, etc.

[0048] According to a particular embodiment, the reference value according to the invention is the measurement obtained without bringing the at least one skin cell according to the invention into contact with the at least one compound according to the invention. In other words, the reference value according to the invention is the measurement obtained under the same conditions (in particular experimental, measurement, etc.) as in step b) but in the absence of step a) in the method according to the invention.

[0049] Preferably, the measurement of the reference value according to the invention is carried out before or at the same time as step b) according to the invention, more advantageously under the same conditions, even more advantageously using the same culture of the at least one skin cell.

[0050] In the context of the invention, the terms "content", "quantity" and "level" are used interchangeably.

[0051] In the context of the invention, the expression "content of TAFA4 and / or of the nucleic acid(s) encoding it" designates the levels present in the cells (i.e., intracellular medium) and / or in the extracellular medium after secretion by said cells. In the context of the invention, the expression "cutaneous content of TAFA4 and / or of the nucleic acid(s) encoding it" designates the levels present in the skin cells (i.e., intracellular medium) and / or in the extracellular medium after secretion by said skin cells.

[0052] In the context of the invention, the expression "endogenous cutaneous content of TAFA4 and / or of the nucleic acid(s) encoding it" designates the basal levels present in the cutaneous cells (Ie., intracellular medium) and / or in the extracellular medium after secretion by said cutaneous cells under normal conditions, in particular under normal culture conditions, and without induction or depletion of the expression of TAFA4.

[0053] According to a particular embodiment, the content of TAFA4 and / or nucleic acid encoding TAFA4 according to the invention is measured in the intra and / or extracellular medium of at least one skin cell according to the invention.

[0054] For the purposes of the invention, the term "intracellular medium" or "intracellular space" refers to the environment within a cell where intracellular biological processes take place. It includes the cytoplasm, the nucleus, and organelles. The term "extracellular medium" or "extracellular space" refers to the space outside a cell, composed of fluids such as blood, lymph, interstitial fluid, and in particular, in the case of skin cells, the extracellular matrix, or ECM, which is secreted by the skin cells. By definition, the secretion of intracellular constituents, such as extracellular matrix precursors or cytokines, takes place in the extracellular medium.

[0055] In the context of the invention, the expression "modulation of the content of TAFA4 and / or of the nucleic acid encoding TAFA4" designates any modification of the content of TAFA4 and / or of the nucleic acid encoding it, of the transcription and / or of the protein expression of TAFA4.

[0056] According to a particular embodiment, said modulation according to the invention corresponds to an increase in the content of TAFA4 and / or in the nucleic acid encoding it compared to the reference value according to the invention. Preferably, the increase in the content of TAFA4 is at least 120%, more preferably 150%, even more preferably 200% compared to the reference value according to the invention; and / or the increase in the content of nucleic acids encoding TAFA4 is at least 120%, more preferably 150%, even more preferably 200%, or even more than 1000% compared to the reference value according to the invention.

[0057] In the context of the invention, the expression "variation of expression" designates an overexpression or an underexpression of the gene. In particular, the term "overexpression" means that the expression of a gene is increased compared to a reference value; the term "underexpression" means that the expression of a gene is decreased compared to a reference value.

[0058] Unless otherwise indicated, for the purposes of the invention, a "nucleotide sequence coding for an amino acid sequence" means all nucleotide sequences which code for the amino acid sequence, including degenerate nucleotide sequences making it possible to obtain said amino acid sequence. The nucleotide sequence which codes for a protein or an RNA or a cDNA may optionally comprise introns.

[0059] The terms "coding" or "coding for," "code" or "codes for" are used interchangeably and refer to the inherent property of specific nucleotide sequences in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of polypeptides having a defined sequence of amino acids, and the biological properties that result therefrom. Thus, a gene codes for a protein if transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and which is generally described in sequence listings and databases, and the non-coding strand, used as a template for transcription of a gene or cDNA, may be designated as coding for the protein or other product of that gene or cDNA.

[0060] For the purposes of the invention, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. A protein by definition contains at least two amino acids, without limitation as to the maximum number of amino acids. Polypeptides include indiscriminately several peptides and / or proteins, which themselves comprise two or more amino acids linked to each other by peptide bonds. As used herein, the term refers both to short chains, which are also commonly referred to in the art as peptides, oligopeptides and oligomers for example, and to longer chains, which are generally referred to in the art as proteins, of which there are many types.“Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, among others. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0061] The terms "identical" and "similar" refer to sequence similarity, which is measured as the percentage of sequence identity between two polypeptides or two nucleic acid molecules. When a position in each of the two sequences being compared is occupied by the same amino acid base or monomeric subunit (for example, when a position in each of two DNA molecules is occupied by an adenine), then the molecules are identical at that position. The percentage identity, or similarity, between two sequences is a function of the number of matching positions shared by the two sequences, and is that number divided by the number of positions being compared and multiplied by 100. For example, if 6 out of 10 of the positions in two paired sequences are identical, then the two sequences are 60% identical or similar.Typically, the comparison is performed by aligning the two sequences to give maximum identity and, for protein sequences, without considering conservative substitutions as part of the sequence identity.

[0062] In the context of the invention, the expression "amplification primer" designates a nucleotide fragment which may comprise from 5 to 100 nucleotides, preferably from 15 to 30 nucleotides, and which has a specificity of hybridization with a target nucleotide sequence, under conditions determined for the initiation of an enzymatic polymerization, for example in an enzymatic amplification reaction of the target nucleotide sequence. Generally, "primer pairs" are used, consisting of two primers hybridizing to the two complementary strands. When it is desired to carry out the amplification of several different genes, several different primer pairs are preferably used, each preferably having a capacity to hybridize specifically with a different gene.

[0063] In the context of the invention, the expression "hybridization probe" designates a nucleotide fragment typically comprising from 5 to 100 nucleotides, preferably from 15 to 90 nucleotides, even more preferably from 15 to 35 nucleotides, having a hybridization specificity under determined conditions to form a hybridization complex with a target nucleotide sequence. The probe also comprises a reporter (such as a fluorophore, an enzyme or any other detection system), which will allow the detection of the target nucleotide sequence. In the present invention, the target nucleotide sequence may be a nucleotide sequence included in a messenger RNA (mRNA) or a nucleotide sequence included in a complementary DNA (cDNA) obtained by reverse transcription of said mRNA.When it is desired to target several different genes, several different probes are preferably used, each preferably having the ability to hybridize specifically with a different gene.

[0064] In the context of the invention, "hybridization" means the process during which, under appropriate conditions, two nucleotide fragments, such as for example a hybridization probe and a target nucleotide fragment, having sufficiently complementary sequences, are capable of forming a double strand with stable and specific hydrogen bonds. A nucleotide fragment "capable of hybridizing" with a polynucleotide is a fragment capable of hybridizing with said polynucleotide under hybridization conditions, which can be determined in each case in a known manner. The hybridization conditions are determined by the stringency of the operating conditions. The more stringent the hybridization, the more specific it is. This is defined in particular as a function of the base composition of a probe / target duplex, as well as by the degree of mismatch between two nucleic acids.All these data are well known and the appropriate conditions can be determined by those skilled in the art. In general, depending on the length of the hybridization probes used, the temperature for the hybridization reaction is between about 20 and 70°C, in particular between 35 and 65°C in a saline solution at a concentration of about 0.5 to 1 M. A step of detecting the hybridization reaction is then carried out.

[0065] In the context of the invention, the expression "enzymatic amplification reaction" designates a process generating multiple copies of a target nucleotide fragment, by the action of at least one enzyme. Such amplification reactions are well known to those skilled in the art and the following techniques may be cited in particular: Polymerase Chain Reaction (PCR), Ligase Chain Reaction (LCR), Repair Chain Reaction (RCR), Self Sustained Sequence Replication (3SR) with document WO-A-90 / 06995, Nucleic Acid Sequence-Based Amplification (NASBA), Transcription Mediated Amplification (TMA) with document US5399491 and Loop mediated isothermal amplification (LAMP) with document US6410278.When the enzymatic amplification reaction is a PCR, we will speak more specifically of RT-PCR (RT for "reverse transcription"), when the amplification step is preceded by a step of reverse transcription of messenger RNA (mRNA) into complementary DNA (cDNA), and of qPCR or RT-qPCR when the PCR is quantitative.

[0066] In the context of the invention, the term "inducer" and the expression "inducer of the synthesis of TAFA4 and / or nucleic acid encoding it" are used interchangeably and designate an agent, a treatment or a culture condition capable of triggering a determined reaction or a programmed sequence of reactions in the biological system, namely the production (i.e., transcription and / or translation) of TAFA4 and / or nucleic acid encoding it.

[0067] In the context of the invention, the term "inhibitor" and the expression "inhibitor of the synthesis of TAFA4 and / or of the nucleic acid encoding it" are used interchangeably and designate an agent, a treatment or a culture condition capable of triggering a determined reaction or a programmed sequence of reactions in the biological system, namely the partial or total inhibition of the production (i.e., transcription and / or translation) of TAFA4 and / or of the nucleic acid encoding it. In the context of the invention, the expression "depletion of the endogenous cutaneous content of TAFA4 and / or of the nucleic acid encoding it" designates the application of an agent, a treatment or a culture condition capable of inducing a reduction or even an elimination of the content of TAFA4 and / or of the nucleic acid encoding it, in the biological system.

[0068] In the context of the invention, the expressions "sensitizing agent" or "irritant agent" or "polluting agent" are used interchangeably and designate an agent, a treatment or a culture condition at the origin of a deregulation or sensitization of the skin, namely a skin presenting different physiological constants to degrees which deviate from normal, in particular presenting an inflammatory state such as that observed during an increase in the expression and / or the concentration of IL-8, IL-1, TNFa or other inflammatory cytokines.Advantageously, such an agent may be a surfactant, more advantageously sodium lauryl sulfate (SDS), or a preservative, more advantageously methylisothiazolinone, or another substance, not corresponding to a cosmetic raw material but which may be found as a contaminant of cosmetic raw materials, more advantageously a chromium salt, a nickel salt, cobalt, or a substance present in the environment, more advantageously microparticles, pollen.

[0069] In the context of the invention, the expression “non-sensitized cell” designates a cell derived from a donor which is not characterized by sensitive and / or sensitized skin, and which has not been subjected to stress likely to sensitize it.

[0070] Preferably, the subject of the present invention is a method for selecting at least one compound, as defined above and having the following technical characteristics, taken alone or in combinations:

[0071] - Said modulation of the content of TAFA4 and / or of the nucleic acid encoding TAFA4 is an increase;

[0072] - Said increase in the TAFA4 content corresponds to at least 120%, more preferably 150%, even more preferably 200% compared to the reference value according to the invention;

[0073] - Said increase in the content of nucleic acid coding TAFA4 corresponds to at least 120%, more preferably 150%, even more preferably 200%, or even more than 1000% compared to the reference value according to the invention;

[0074] - said at least one compound is not the TAFA4 protein nor / or one of its biologically active derivatives nor / or one of its isoforms;

[0075] - said method is an in vivo, in vitro or ex vivo method;

[0076] - said method is an in vivo or ex vivo method and is characterized in that the contacting of step a) is carried out by topical application of said at least one compound to the skin of a whole living organism, advantageously the skin of a whole living non-human organism, a skin explant, a reconstructed epidermis or a reconstructed skin;

[0077] - said method is an in vivo method in a human subject;

[0078] - said method is an in vivo method in a non-human subject, advantageously a rodent (eg, mouse, rat, guinea pig or rabbit), a cat, a dog, a primate (eg, a chimpanzee), a bird (eg, a hen), a reptile, an amphibian or even a fish (eg, zebrafish);

[0079] - said method is an in vitro method comprising the culture of skin cells, advantageously selected from the group consisting of keratinocytes, melanocytes, sebocytes, Merkel cells, vascular smooth muscle cells, specialized muscle cells, fibroblasts, mast cells, lymphocytes, monocytes, dendritic cells, advantageously dermal dendritic cells; Langerhans cells, adipocytes, nerve cells, macrophages, advantageously macrophages of type M1 or M2; endothelial cells; and mixtures thereof; preferentially chosen from keratinocytes, fibroblasts and mixtures thereof; and is characterized in that the contacting of step a) is carried out by addition to the culture medium;

[0080] - said at least one skin cell is at least one macrophage, advantageously at least one macrophage of type Ml, preferably at least one macrophage of human type Ml;

[0081] - said at least one skin cell is at least one keratinocyte, advantageously at least one human keratinocyte, more advantageously at least one normal human keratinocyte;

[0082] - said at least one skin cell is at least one fibroblast, advantageously at least one human fibroblast, more advantageously at least one normal human fibroblast;

[0083] - said method further comprises, before implementing step a), a step of adding to the culture medium an inducer of the synthesis of TAFA4 and / or of nucleic acid encoding it, by said at least one skin cell, advantageously chosen from: phorbol 12-myristate 13-acetate (PAM), preferably at a concentration of between 1 and 20 ng / ml; lipopolysaccharide (LPS), preferably at a concentration of between 0.1 pg / ml and 2 mg / ml; calcium, preferably at a concentration of between 0.1 mM and 10 mM; forskolin, preferably at a concentration of between 10 pM and 200 pM; the combination of calcium, preferably at a concentration of between 0.1 mM and 10 mM, and forskolin, preferably at a concentration of between 10 pM and 200 pM; epidermal growth factor (EGF), preferably at a concentration between 10 ng / ml and 200 ng / ml;fetal calf serum (FCS), advantageously in a concentration of between 0.1 and 5% by weight of the total culture medium; exposure to UV light, preferably exposure at a dose of between 2 and 10 J / cm; 2 UVA for at least 5 minutes, preferably at least 10 minutes; and mixtures thereof; - said addition step is carried out in an interval of between 30 minutes and 72 hours, preferably under the following culture conditions: 37°C, 5% CO2 and / or saturated humidity atmosphere;

[0084] - said method further comprises, before implementing step a), a step of adding to the culture medium an inhibitor of the synthesis of TAFA4 and / or of nucleic acid encoding it, by said at least one skin cell;

[0085] - said method further comprises, before carrying out step a), a step of depletion of the endogenous cutaneous content of TAFA4 and / or of nucleic acid encoding it produced by said at least one cutaneous cell;

[0086] - said step b) of measuring the content of TAFA4 and / or nucleic acid, advantageously mRNA, encoding it is carried out at a protein level by an ELISA test, immunohistochemistry (including colorimetry, fluorescence, luminescence), other colorimetric method, or western-blot, dot-blot, simplified western by capillary immunoelectrophoresis (WES) and / or at the nucleic level by in situ hybridization, northern blot, sequencing or RT-PCR, advantageously RT-qPCR;

[0087] - said content of TAFA4 and / or nucleic acid, advantageously mRNA, coding TAFA4 is measured in the intra and / or extracellular medium of said at least one skin cell;

[0088] - before implementing said step a), said at least one skin cell has been brought into contact, preferably by topical application or by addition to the culture medium, with a sensitizing agent, advantageously a cosmetic sensitizing agent, more advantageously a surfactant or a preservative;

[0089] - the amino acid sequence of TAFA4 corresponds, partially or totally, to the sequence SEQ ID NO: 1 and / or to SEQ ID NO: 2, or to a sequence identical to one of the sequences SEQ ID NO: 1 to 2 at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, or even at least 90%;

[0090] - the nucleotide sequence of TAFA4 corresponds, partially or totally, to the sequence SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7, or to a sequence identical to one of the sequences SEQ ID NO: 3 to 7 at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, or even at least 90%;

[0091] - said method comprises, in said step b), the additional measurement of the content of at least one indicator agent different from TAFA4 and / or the nucleic acid encoding it, for example IL-8, IL-1, TNF-a or other anti-inflammatory cytokines, said indicator agent content also being compared with a reference value specific to said step c);

[0092] - said reference value is the measurement obtained without bringing the at least one skin cell into contact with said at least one compound; - said reference value is the measurement obtained under the same conditions as in said step b) but in the absence of said step a);

[0093] - the measurement of said reference value is carried out before or at the same time as said step b) according to the invention, more advantageously under the same conditions, even more advantageously using the same culture of said at least one skin cell; - said selection step d) is associated with another selection step based on the induction of an increase or a decrease in the content of said indicator agent (modulation dependent on the indicator agent chosen) to decide on the selection or not of said at least one compound;

[0094] - said method further comprises, after the implementation of step d), a step e) of formulating said at least one selected compound in a non-therapeutic, advantageously ecobiological, cosmetic and / or food composition, more advantageously presented in a galenic form suitable for topical and / or oral administration;

[0095] - said step e) of formulating said at least one selected compound corresponds to the addition of at least one active ingredient usually used in the cosmetic, food and / or dermatological fields, advantageously selected from an anti-radical or more generally an antioxidant, a whitening agent, a pigmenting agent, an emollient, a moisturizer, an anti-seborrheic agent, an anti-inflammatory agent, an anti-acne agent, a keratolytic and / or desquamating agent, an anti-wrinkle and / or tightening agent, a mineral or organic sunscreen which is hydrophilic or lipophilic, a draining agent, an anti-irritant agent, a soothing agent, a vitamin and their mixtures, a mattifying agent, an anti-aging active ingredient such as retinol, a healing agent, an antiseptic and an essential oil; and

[0096] - said step e) of formulating said at least one selected compound corresponds to the addition of at least one excipient usually used in the cosmetic, food and / or dermatological fields, advantageously selected from a fatty substance, an emulsifier, a co-emulsifier, a hydrophilic or lipophilic gelling agent, a preservative, an antioxidant, a solvent, an exfoliating agent, a perfume, a filler, a neutralizer, a pro-penetrating agent, and a polymer.

[0097] Thus, and according to one aspect of the invention, the method according to the invention is an in vitro method, said contacting is carried out by addition to the culture medium, and the at least one skin cell is chosen from keratinocytes, melanocytes, sebocytes, Merkel cells, vascular smooth muscle cells, specialized muscle cells, fibroblasts, mast cells, lymphocytes, monocytes, dendritic cells, advantageously dermal dendritic cells; Langerhans cells, adipocytes, nerve cells, macrophages, advantageously of type M1 or M2; endothelial cells; and mixtures thereof. According to one aspect, the at least one skin cell is chosen from keratinocytes and fibroblasts, preferably human, more preferably normal; and mixtures thereof.

[0098] According to one aspect of the invention, the at least one skin cell is a murine cell, advantageously a cell derived from an immortalized murine line of B16 melanocytes.

[0099] The measurement of the content of TAFA4 and / or nucleic acid, advantageously mRNA, encoding it can be carried out with any method known to those skilled in the art. Examples include Northern blot, Southern blot, PCR, RT-PCR, quantitative RT-PCR, SAGE and its derivatives, nucleic acid chips, including cDNA chips, oligonucleotide chips and mRNA chips, tissue chips and RNA-Seq, immunohistology methods, immunoprecipitation, Western blot, simplified Western by capillary immunoelectrophoresis (WES), dot-blot, ELISA or ELISPOT, protein chips, antibody chips, or tissue chips coupled with immunohistochemistry, FRET or BRET techniques, microscopy or histochemistry methods, including confocal microscopy and electron microscopy methods, methods based on the use of one or more excitation wavelengths and a suitable optical method,such as electrochemical methods (voltammetry and amperometry techniques), atomic force microscopy, and radiofrequency methods, such as multipole, confocal and non-confocal resonance spectroscopy, detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, and birefringence or refractive index (e.g., by surface plasmon resonance, ellipsometry, resonant mirror method, etc.), flow cytometry, radioisotope or magnetic resonance imaging, analysis by polyacrylamide gel electrophoresis (SDS-PAGE); by HPLC spectrophotometry, by liquid chromatography / mass spectrophotometry / mass spectrometry (LC-MS / MS).,

[0100] Advantageously, quantification of TAFA4 content can be performed by immunohistochemistry or immunocytochemistry on sections of skin or cultured cells and other methods; by western blot; by dot blot; by simplified western by capillary immunoelectrophoresis (WES). In particular, protein dot blotting is based on the principle of hybridization between a probe, generally labeled antibodies, and the desired target. It is simple and rapid, requiring no complex purification steps. To perform a dot blot, the biological sample (cell supernatants or protein extracts) is deposited on a nitrocellulose or nylon membrane, thus forming small points or "dots". After drying, the membrane is incubated with the probe to detect the desired protein.The simplified Western blotting method using capillary immunoelectrophoresis (WES) combines the principles of Western blotting and capillary immunoelectrophoresis to detect and quantify specific proteins in a biological sample without the laborious process of membrane transfer. In this approach, proteins are first separated by capillary electrophoresis based on their size and electrical charge; detection of target proteins is then performed using specific antibodies labeled directly on the capillaries, followed by automated analysis in a dedicated instrument.Alternatively, the quantification of the TAFA4 content can be carried out by an ELISA test, for example on the supernatants of cell cultures or cell extracts, by interpolating the optical density of the sample on a calibration curve obtained with serial dilutions of the recombinant human TAFA4 protein (for example from 0.1561 ng / ml to 10 ng / ml). In practice, on the cell lawn, a viability test with ((3-4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) or MTT, known to those skilled in the art, can be carried out. The TAFA4 assays are used only if the cell viability is greater than 80%, and are reported at this cell viability value.

[0101] The measurement of the content of messenger ribonucleic acid (mRNA) encoding TAFA4 can be advantageously carried out by in situ hybridization on cell sections; by northern blot; or by RT-qPCR. Advantageously, total RNA is extracted from tissues, for example using commercially available kits (such as the Rneasy Plus Mini QIAGEN). The extraction can be followed by a total RNA assay which makes it possible to know the quantity and quality of RNA in the cell extract; this step can be carried out for example using the Bioanalyzer and the RNA 6000 Nano kit. cDNA is generated using the QuantiTect Reverse Transcription Kit (QIAGEN), followed by Quantitative Real-Time PCR at each treatment time point for the TAFA4 gene, using for example the QuantiFast SYBR Green PCR Kit (QIAGEN), and following the expression of a housekeeping gene (e.g., GAPDH) as a control and TAFA4 with specific primers based on SEQ ID NOs 3, 4, 5, 6 or 7.

[0102] Thus, and according to a particular embodiment, the measurement of the TAFA4 content is carried out by an ELISA test, by immunohistochemistry on cell sections; by western blot; the measurement of the content of messenger ribonucleic acids (mRNA) coding TAFA4 is carried out by immunohistochemistry, in situ hybridization on cell sections, by northern blot, sequencing or RT-PCR, advantageously RT-qPCR.

[0103] The quantification of the content of at least one marker different from TAFA4 as mentioned above, and / or of the nucleic acid encoding it can be carried out according to the methods specified above.

[0104] According to a particular embodiment, the method for selecting at least one compound, preferably ecobiological, intended for the prevention and / or care of sensitive and / or sensitized skin, advantageously following stress, more advantageously an allergy, according to the invention comprises the following steps: a) Bringing said at least one compound into contact in vitro with at least one skin cell by adding it to the culture medium; b) Measuring the content of TAFA4 and / or nucleic acid encoding TAFA4, preferably in the culture medium and / or in the intracellular medium of said at least one skin cell; c) Comparing the value obtained in step b) with a reference value, preferably obtained without bringing said at least one skin cell into contact with said at least one compound;d) Selecting said at least one compound inducing an increase in the content of TAFA4 and / or nucleic acid encoding it in step c), preferably of more than 120%, advantageously of more than 150%, compared to said reference value.;

[0105] According to an alternative embodiment, the method for selecting at least one compound, preferably ecobiological, intended for the prevention and / or care of sensitive and / or sensitized skin, advantageously following stress, more advantageously an allergy, according to the invention comprises the following steps: a) Bringing said at least one compound into ex vivo contact with at least one skin cell by topical application to a skin explant, a skin substitute, a reconstructed epidermis or a reconstructed skin; b) Measuring the content of TAFA4 and / or of the nucleic acid encoding it, preferably in the intra and / or extracellular medium of said at least one skin cell; c) Comparing the value obtained in step b) with a reference value, preferably obtained without bringing said at least one skin cell into contact with said at least one compound;d) Selecting said at least one compound inducing an increase in the content of TAFA4 and / or nucleic acid encoding it in step c), preferably of more than 120%, advantageously of more than 150%, compared to said reference value.;

[0106] According to yet another embodiment, the method for selecting at least one compound, preferably ecobiological, intended for the prevention and / or care of sensitive and / or sensitized skin, advantageously following stress, more advantageously an allergy, according to the invention comprises the following steps: a) Bringing said at least one compound into contact in vivo with at least one skin cell by topical application to the skin of a whole living organism, advantageously a human or non-human being; b) Measuring the content of TAFA4 and / or nucleic acid encoding it, preferably in the intra- and / or extracellular medium of said at least one skin cell; c) Comparing the value obtained in step b) with a reference value, preferably obtained without bringing said at least one skin cell into contact with said at least one compound;d) Selecting said at least one compound inducing an increase in the content of TAFA4 and / or nucleic acid encoding it in step c), preferably of more than 120%, advantageously of more than 150%, compared to said reference value.;

[0107] According to a particular embodiment, said at least one skin cell, to be used in the method according to the invention, is not sensitized.

[0108] According to an alternative embodiment, said at least one skin cell, to be used in the method according to the invention, is sensitized.

[0109] According to another embodiment, said at least one cell comes from a donor characterized by sensitive or sensitized skin, advantageously irritable, or reactive.

[0110] According to an alternative embodiment, said at least one cell has been previously brought into contact, by topical application or by addition to the culture medium, with a sensitizing agent, advantageously a cosmetic sensitizing agent, even more advantageously chosen from a surfactant and a preservative.

[0111] According to a particular aspect of the invention, the endogenous cutaneous contents of TAFA4 and / or of the nucleic acid encoding it of said cell according to the invention were depleted, before bringing said cell into contact with the at least one candidate compound.

[0112] According to a particular aspect, said compound according to the invention does not correspond to the TAFA4 protein nor / or to one of its isoforms nor / or to one of its biologically active derivatives nor / or to TAFA4 nor / or to a ribonucleic acid encoding it nor / or to a nucleic acid encoding it, nor / or to their formulation.

[0113] Another subject of the present invention relates to a kit for implementing the method for selecting at least one compound, preferably ecobiological, intended for the prevention and / or care of sensitive and / or sensitized skin, advantageously following stress, more advantageously an allergy, as defined above, comprising means for amplification and / or means for detecting the content of TAFA4 and / or of the nucleic acid encoding it, advantageously said means of amplification and / or detection are reagents specific for the expression products of TAFA4 target genes chosen from amplification primers, hybridization probes or antibodies specifically binding to TAFA4, advantageously the TAFA4 protein and / or to one of its biologically active derivatives. Another subject of the present invention also relates to a composition comprising at least one compound selected by the method described above.

[0114] Preferably, the subject of the present invention is a composition as described above and having the following technical characteristics, taken alone or in combination:

[0115] - the composition comprises at least one compound selected by the method described above;

[0116] - the composition consists of at least one compound selected by the method described above;

[0117] - the composition is presented in at least one of the galenic forms suitable for topical application to the skin and / or mucous membranes, and / or appendages, for example in anhydrous form, in the form of an oil-in-water emulsion, a water-in-oil emulsion, a multiple emulsion, a silicone emulsion, a microemulsion, a nanoemulsion, a gel, an aqueous solution or a hydroalcoholic solution;

[0118] - the composition is fluid or not and is presented in the form of a cream, an ointment, a milk, a lotion, a serum, or a gel;

[0119] - the composition is presented in at least one of the galenic forms suitable for non-therapeutic food application, advantageously by oral route; and

[0120] - the composition is colored or not, advantageously colored using a coloring agent.

[0121] Another object of the present invention also relates to the use of modulation of the content of TAFA4 and / or nucleic acid, advantageously mRNA, encoding it, as an indicator making it possible to classify candidate compounds according to their capacity or not to increase the skin tolerance threshold.

[0122] Another subject of the present invention also relates to a method for formulating a non-therapeutic, advantageously ecobiological, cosmetic and / or food composition, more advantageously in a galenic form suitable for topical and / or oral administration, comprising the incorporation into said composition of at least one compound selected by the selection method according to the invention.

[0123] The following examples, without being limiting, form an integral part of the invention and any characteristic which appears to be new compared to the state of the prior art is claimed as such and as a general means.

[0124] Figure 1 shows the effect of valproic acid, TGF-β and LPS tested at different concentrations and LPS at one concentration (1 pg / ml) on TAFA4 synthesis in normal human fibroblasts (NHF) expressed as percentage induction. NS: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001. Figure 2 shows the effect of EGF protein at one concentration (30 ng / ml) on TAFA4 synthesis in normal human keratinocytes (NHF) expressed as percentage induction. NS: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001.

[0125] EXAMPLES OF ACHIEVEMENT

[0126] Example I - Keratinocyte and fibroblast protocol

[0127] 1.1 Introduction

[0128] The aim of the study is to provide a simple and rapid protocol to study the modulation of TAFA4 expression or transcription in keratinocyte and fibroblast lines by cosmetic compounds and compositions.

[0129] It should be noted that, optionally, an inducer of the expression of TAFA4 can be used to potentiate the expression of the gene coding for this protein, in particular in the case where the quantity of gene coding for the TAFA4 protein or the quantity of TAFA4 protein is less than or equal to the detection limit imposed by the method implemented by the person skilled in the art and / or to provide a reference TAFA4 production value, Le., a positive control, this value having to be reached in order to classify the candidate compound in the category of compounds, preferably ecobiological, suitable for the prevention and / or care of sensitive and / or sensitized skin.

[0130] 1.2 Cell culture

[0131] Normal human keratinocytes (NHK) and normal human fibroblasts (NHF) were used in this study. They were obtained from Promocell (France), Lonza (France).

[0132] The conditions tested for the development of the model are:

[0133] • Cell type: KHN, FHN

[0134] • Culture medium: KBM + 1% P / S for KHN, DMEM + 1% P / S for FHN.

[0135] • Processing time: 48h and 72h

[0136] • Seeding density: 15,000 cells / well in 96-well plates at 48 hours

[0137] 10,000 cells / well in 96-well plates at 72 hours.

[0138] We chose to test different induction conditions for the 2 cell types:

[0139] These inducers were selected as positive controls based on literature data.

[0140] • 24h pretreatment with PMA at 10 ng / ml (P1585-1MG, SIGMA)

[0141] • 24h pretreatment with PMA at 50 ng / ml

[0142] • LPS at 1 mg / ml (L2880-10MG, SIGMA)

[0143] - LPS at 1 mg / ml + PMA at 10 ng / ml (24h pretreatment for PMA) - LPS at 1 mg / ml + PMA at 50 ng / ml (24h pretreatment for PMA)

[0144] • Calcium 1.2 mM (C7902-500G, SIGMA)

[0145] - Calcium 1.2 mM + Forskolin at 10 pM

[0146] • lOOpM Forskolin (93049-10MG, SIGMA)

[0147] • 10 pM Forskolin

[0148] Specifically for KHN:

[0149] • 10 ng / ml EGF (AF100-15, Peprotech)

[0150] • 30 ng / ml EGF

[0151] • 10 ng / ml EGF + 100 pM Forskolin

[0152] • 30 ng / ml EGF + 100 pM Forskolin

[0153] Specifically for FHN

[0154] • 0.5% SVF (S1900-500B, Dominique Dutscher)

[0155] • 2% SVF

[0156] • 0.5% SVF + lOOpM Forskolin

[0157] • 2% FCS + 100 pM Forskolin

[0158] 1.3 Screening of compounds or compositions

[0159] The cells (KHN, FHN) are seeded in 96-well plates in the corresponding complete culture medium. After 24 hours of culture at 37°C, 5% CO2, the compounds or compositions to be evaluated are added at the chosen concentrations in the culture medium and the cells are incubated for 48 hours or 72 hours at 37°C 5% CO2. A positive induction control is evaluated in parallel with the active ingredients.

[0160] After the different incubation times, the supernatants are harvested and stored at -80°C before performing the TAFA4 assay by ELISA. The assay is performed only if cell viability is greater than 80%. For the evaluation of TAFA4 expression by RT-qPCR, the cells are recovered after incubation of the active or positive controls on shorter kinetics (30 min to 24 h).

[0161] 1.4 Selection of compounds or compositions to be evaluated

[0162] Different pharmaceutical or cosmetic molecules can be pre-selected and tested. These molecules will be directly solubilized in the culture medium or solubilized in ethanol or DMSO depending on their nature.

[0163] 1.5 TAFA4 assay in culture supernatants by ELISA

[0164] Quantification of TAFA4 in supernatants or cell extracts is performed using an ELISA test, Enzyme Linked Immunosorbent Assay (Ref: abx522987, Cliniscience, France) with the reagents recommended by the manufacturer. The Optical Density (OD) is read with a spectrophotometer at 450 nm. The concentration of TAFA4 is calculated from the standard range (0.1561 ng / ml to 10 ng / ml) established with the recombinant human TAFA4 protein.

[0165] Results are expressed in ng / ml of TAFA4 per well. On the cell lawn, an MTT viability test is performed. TAFA4 assays are used only if cell viability is greater than 80%, and are reported at this cell viability value.

[0166] 1.6 Quantification of TAFA4 expression by RT-qPCR

[0167] After 30 min to 24 h of incubation with the active ingredients, an extraction of total RNA is carried out using the Rneasy Plus Mini kit according to the supplier's recommendations (Qiagen).

[0168] Total RNA assays determine the quantity and quality of RNA in cell extracts. The assay is performed using the Bioanalyzer and the RNA 6000 Nano kit.

[0169] Reverse transcription using the QuantiTect Reverse Transcription kit (Qiagen) produces cDNA.

[0170] Quantitative real-time PCR at each treatment time is performed for the TAFA4 gene. The Qiagen QuantiFast SYBR Green PCR kit is used.

[0171] • Housekeeping gene: GAPDH

[0172] • Gene of interest: TAFA4 -> see gene-specific primers

[0173] 1.7 MTT Cell Viability Test

[0174] After incubation and harvesting of culture supernatants, the wells are rinsed with D-PBS. After removing the D-PBS, 100 μl of 1 mg / ml MTT solution are added per well and incubated for 3 hours at 37°C. The MTT is then removed and 100 μl of DMSO are added to each well to dissolve the formazan crystals. After homogenization of the color, the optical density is read with a spectrophotometer at 540 nm (Victor 3, Perkin Elmer). The results are then expressed using the formula: % viability = (OD treated condition / OD (100% viability)) x 100. The cells that represent 100% viability correspond to the cells incubated at different times in the control culture medium.

[0175] NB: MTT ((3-4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) is a soluble yellow dye that is metabolized by mitochondrial enzymes (succinate dehydrogenase) into a dark blue compound: formazan. The formazan crystals are then solubilized in DMSO and the optical density is measured spectrophotometrically at 540 nm.

[0176] 1.8 Statistical analyses

[0177] Data were collected from independent experiments performed in triplicate. Quantitative analyses are expressed as mean ± standard deviation. Statistical significance was determined by Student's t-test. Differences were considered statistically significant at p < 0.05.

[0178] (NS: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001).

[0179] Example II - Macrophage Protocol

[0180] II.1 Introduction

[0181] The aim of the study is to provide a simple and rapid protocol to study the modulation of TAFA4 expression or transcription in an inflammatory macrophage model by cosmetic compounds and compositions.

[0182] It should be noted that a depletion of the TAFA4 content (depletion of the expression of the gene encoding the TAFA4 protein and / or of the quantity of TAFA4 protein) can be implemented to provide a model in which the endogenous constitutive production of TAFA4 must be restored in order to classify the candidate compound in the category of compounds, preferably ecobiological, intended for the prevention and / or care of sensitive and / or sensitized skin.

[0183] 11.2 Cell culture

[0184] Macrophages are immune cells capable of infiltrating tissues. Macrophages are differentiated from monocytes (blood cells), which in turn are differentiated from bone marrow stem cells. Both macrophages and monocytes are cells capable of phagocytosis. Their role is to phagocytose cellular debris and pathogens and they participate more broadly in the immune response by synthesizing different types of mediators.

[0185] There are 3 main types of macrophages:

[0186] • MO which are non-activated macrophages.

[0187] • Ml which have pro-inflammatory properties. At the cutaneous level, they are notably involved in the destruction of pathogenic agents, particularly during healing.

[0188] • M2s which have anti-inflammatory and pro-inflammatory properties. At the cutaneous level, they are notably involved in the repair phase of healing.

[0189] At the in vitro level, there are different cellular models that approximate a macrophage-like phenotype. These different models can be used in this context:

[0190] • Human monocyte lines: THP-1, U-937, SC, AML-193, HL-60 / S4

[0191] THP-1 are the most described and used cells in the literature.

[0192] For THP-1: Culture medium: RPMI 1640 (Roswell Park Memorial Institute) + 10% FBS (fetal bovine serum) (Wang et al., 2015) + / - 100 mg / L gentamicin, 4.5 g / L glucose, 1 mM pyruvate, 0.05 mM 2-mercaptoethanol and 2 mM L-glutamine (Shen et al., 2014) and + / - penicillin (100 units / mL) and streptomycin (100 pg / mL) in a humid environment at 5% CO2 in air and 37°C (Kawano et al., 2015). Tl

[0193] THP-1 can be differentiated into MO, Ml and M2 type macrophages according to the culture conditions described by Horiba et al., 2022.

[0194] • Monocyte-derived macrophages from human PBMC (human peripheral blood mononuclear cells) obtained from human peripheral blood samples.

[0195] PMBCs were isolated from blood by Polymorphprep. Monocytes (CD14+) were then recovered after 4 hours of PMBC adhesion. Culture medium: RPMI 1640 + 10% FBS. Monocytes could be differentiated into MO macrophages by M-CSF (macrophage colony-stimulating factor) stimulation in vitro followed by treatment with LPS (Escherichia coli 055:B5), combined with IFN-γ (interferon gamma) or IL-4 (interleukin-4), to differentiate them into M1 or M2 macrophages respectively, for 24 hours (Wang et al., 2015) in a humid environment at 5% CO2 in air and 37°C.

[0196] • Macrophages derived from CD34+ hematopoietic stem cells

[0197] These cells are recovered from samples of bone marrow, peripheral blood and human umbilical cord blood in vivo (Clanchy and Hamilton, 2013). Conventionally these cells are recovered from cord blood.

[0198] 11.3 Selection of compounds or compositions effective for inducing the expression or synthesis of TAFA4

[0199] 11.3.1. Protocol

[0200] The cells are seeded in 96-well plates in the corresponding complete culture medium. After 24 hours of culture at 37°C, 5% CO2, the active ingredients to be evaluated are added at the chosen concentrations in the culture medium and the cells are incubated for 48 hours or 72 hours at 37°C 5% CO2. A positive induction control is evaluated in parallel with the active ingredients.

[0201] After the different incubation times, the supernatants are harvested and stored at -80°C before performing the TAFA4 assay by ELISA. The assay is performed only if cell viability is greater than 80%. For the evaluation of TAFA4 expression by RT-qPCR, the cells are recovered after incubation of the active or positive controls on shorter kinetics (30 min to 24 h).

[0202] 11.3.2. Inducing factors (positive controls)

[0203] During bibliographic research, 2 inducers were identified as being able to induce the synthesis of TAFA4 at the cutaneous level: LPS and UV (Hoeffel et al., 2021; Wang et al., 2015).

[0204] 11.3.3. Case of LPS stimulation

[0205] Pre-treatment of monocytes / macrophages with PMA (Phorbol 12-myristate 13-acetate, 10 ng / ml) for 24 h and then stimulated with LPS (1 pg / ml) for 12 h or 24 h (Wang et al., 2015). 11.3.4. Case of UV stimulation

[0206] After removing the culture medium and rinsing with D-PBS, the cells are irradiated with UV.

[0207] Data on the described macrophage exposure protocols:

[0208] - at a dose of 6.7 J / cm 2UVA for 15 min in a Biosun System comprising an irradiation chamber equipped with a 365nm illumination lamp, a dosimeter / sensor, a calibrator and software (Shen et al., 2014); or

[0209] - UVA by a black lamp with a peak emission energy at 360 nm. The emitted dose was measured by a radiometer (UVX-36; UVP, Inc., San Gabriel, CA). No UVB was detected with a UVX-31 sensor. The irradiance at the samples is 2.5 mW / cm 2 (Kawano et al., 2015).

[0210] 11.4 Selection of compounds or compositions to be evaluated

[0211] Different molecules can be pre-selected and tested. These molecules will be directly solubilized in the culture medium or solubilized in ethanol or DMSO depending on their nature.

[0212] 11.5 MTT Cell Viability Test

[0213] See point 1.7.

[0214] 11.6 Quantification of TAFA4 expression by RT-qPCR

[0215] After 30 min to 24 h of incubation with the active ingredients, an extraction of total RNA is carried out using the Rneasy Plus Mini Kit according to the supplier's recommendations (Qiagen).

[0216] Total RNA assays determine the quantity and quality of RNA in cell extracts. The assay is performed using the Bioanalyzer and the RNA 6000 Nano kit.

[0217] Reverse transcription using the QuantiTect Reverse Transcription kit (Qiagen) produces cDNA.

[0218] Quantitative real-time PCR at each treatment time is performed for the TAFA4 gene. The Qiagen QuantiFast SYBR Green PCR Kit is used.

[0219] • Housekeeping gene: GAPDH

[0220] • Gene of interest: TAFA4 -> see gene-specific primers

[0221] 11.7 TAFA4 assay in culture supernatants by ELISA

[0222] The quantification of TAFA4 in the supernatants is carried out using an ELISA test, Enzyme Linked Immunosorbent Assay (Ref: abx522987, Cliniscience, France) with the reagents recommended by the manufacturer. The Optical Density (OD) is read with a spectrophotometer at 450 nm. The concentration of TAFA4 is calculated from the standard range (0.1561 ng / ml to 10 ng / ml) established with the recombinant human protein of

[0223] TAFA4. Results are expressed in ng / ml of TAFA4 per well. On the cell lawn, an MTT viability test is performed. TAFA4 assays are used only if cell viability is greater than 80%, and are reported at this cell viability value.

[0224] II.8 Statistical analyses

[0225] Data were collected from independent experiments performed in triplicate. Quantitative analyses are expressed as mean ± standard deviation. Statistical significance was determined by Student's t-test. Differences were considered statistically significant at p < 0.05.

[0226] (NS: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001).

[0227] Example III - Differentiation of SH-SY5Y cells into sensory neuronal cells and analysis of TAFA4 levels

[0228] III.l Introduction

[0229] The aim of the study is to test the modulation of TAFA4 expression in a neuronal cell type likely to produce it.

[0230] III.2 Materials and methods

[0231] III.2.1 SH-SY5Y cell culture and differentiation

[0232] The SH-SY5Y human neuroblastoma cells used in this study were obtained from Sigma Aldrich (94030304). The cells were thawed at P13 in a 37°C water bath and then immediately placed in DMEM. The cell suspension was centrifuged at 125 x g for 7 min at 4°C and the pellets were suspended in a 1:1 mixture of Eagle's Minimum Essential Medium and F12 Medium, supplemented with 10% fetal bovine serum, and PS solution.

[0233] Cells were seeded into the 96-well plate at a density of 7,000 cells per well and cultured at 37°C in a 95% air, 5% CO2 incubator. To avoid any edge effects, the first and last column as well as the first and last row of the plate were not used in the study. Empty wells were filled with water. Cells were allowed to grow to confluence (80%).

[0234] At confluence (1 day later), cells grow in a mixture of 1:1 DMEM and F12 Medium, supplemented with 1% fetal bovine serum, NGF (5 ng / mL), PS solution (1%), and 100M retinoic acid (RA), to allow their differentiation. The medium is changed every 2 days. For 96-well plates, only 60 wells are used. 111.2.2 TAFA4 expression / release at different time points, D8, D10, D12, after exposure to compounds After 8, 10, or 12 days of differentiation with RA, cells are stressed for 24 hours with two compounds individually diluted at different concentrations in the culture medium. In parallel, a non-stressed control condition is performed at each differentiation time point (8, 10, and 12 days; reference values).

[0235] Only 100 pL of culture medium is applied.

[0236] 111.2.3 qPCR: TAFA4 gene expression

[0237] Several hours (depending on stressors and steps) after stressor application, cells are lysed with the nucleo spin RNS XS kit (Macherey Nagel). Briefly, 100 pL of lysis buffer is added per well, according to the manufacturer's instructions (Macherey Nagel). The procedure includes DNase treatment. RNA extraction is performed in a laboratory room dedicated to biomolecular experiments, with sterile, DNase / RNase-free solutions and laboratory consumables (e.g., filtered tips, tubes). The total amount and purity of RNA present in each sample are evaluated spectrophotometrically using a Nanodrop device (NanoDrop technologies LLC). RNA samples are stored below -70°C.cDNAs were obtained by reverse transcription using prime script reverse transcriptase (SensiFAST cDNA synthesis kit, Bioline meridian) from 25 ng of total RNA according to the manufacturer's instructions. cDNA samples were stored at -15 / -25°C. The PCR reaction was performed using the CFX96™ real-time system (Biorad). An equivalent of 10 ng of initial RNA was subjected to PCR amplification using 3 pM of primers and the SYBR Premix (ONEGreen Fast qPCR Premix, OZYME). The PCR reaction was performed using the following cycling procedure: 95°C for 30s, then 95°C for 5s, 60°C for 30s, and a total of 40 cycles were performed.

[0238] Each sample is analyzed for the selected gene (GAPDH and TAFA4) using RT-qPCR to measure the relative expression levels of mRNAs encoding the selected genes. Gene expression analysis is assessed using the 2-AACt method (Livak and Schmittgen, 2001), where Ct is the cycle threshold value. A negative control (no cDNA) is performed for each primer pair. Expression is normalized to a housekeeping gene (e.g., GAPDH).

[0239] 111.2.4 Statistical analyses

[0240] All values ​​are expressed as mean ± SEM (standard error of the mean). Statistical analysis was performed by one-way ANOVA followed by Fisher's LSD test. p<0.05 was considered significant.

[0241] III.3 Results and conclusions

[0242] These tests made it possible to identify modulators, i.e. inhibitors and inducers of TAFA4 expression in SH-SY5Y cells. Example IV - Evaluation of the effect of compounds on TAFA4 synthesis in normal human fibroblast cultures

[0243] IV.l Introduction

[0244] The aim of the study is to develop a cellular model to quantify the synthesis of TAFA4 in skin cells.

[0245] IV.2 Materials and methods

[0246] IV.2.1 Cell culture

[0247] Normal human fibroblasts (NHF) were used in this study (Lonza - Switzerland). The cells were seeded at 10,000 cells in 24-well plates in DMEM + 1% Penicillin-Streptomycin (P / S). After 24 hours of incubation, the cells were incubated with the compounds presented in Table 1.

[0248] [Table 1]

[0249] After 72 hours of incubation at 37°C, 5% CO2, the supernatants and protein extracts (obtained from the cell lawn) are collected and stored at -80°C.

[0250] Prior to the TAFA4 protein assay, the toxicity of the tested compounds is assessed using the MTT assay. Only concentrations achieving viability greater than 80% are selected for quantification by ELISA and Dot blot.

[0251] Data were collected from independent experiments performed in triplicate.

[0252] IV.2.2 MTT cell viability test

[0253] See point 1.7.

[0254] IV.2.3 TAFA4 assay by dot-blot

[0255] Dot blotting is an immunoenzymatic detection method used to quantify a protein in a sample after depositing it on a nitrocellulose membrane in a well. Specific equipment (Biorad, France) is used with a nitrocellulose membrane. The equipment and consumables used in this experiment are shown in Table 2 below.

[0256] [Table 2]

[0257] The device is subjected to a vacuum pressure which will create the suction of the sample (supernatant or protein extract) and therefore the fixation of the proteins on the nitrocellulose membrane. The equipment is placed at a height in relation to the vacuum pump, to respect the principle of gravity.

[0258] The membrane is first hydrated with 100 μl of TBS buffer (Tris base + NaCl diluted in water adjusted to pH 7.5) per well. This operation is repeated twice to optimize the hydration of the membrane for optimal fixation of the proteins contained in the samples. The samples to be analyzed (supernatants and protein extracts) and the different solutions of TAFA4 recombinant proteins at different concentrations of the TAFA4 standard range (0.78 to 100 ng / ml) are placed in the plate (volume 100 μl / well used for protein extracts and 500 μl / well used for supernatants) then the aspiration flow is activated. When all the samples are aspirated, 100 μl of TBS is added per well to ensure that the entire sample has passed over the membrane. The proteins contained in the samples will thus be deposited on the nitrocellulose membrane and create a 1 cm spot. 2 .

[0259] For labeling and revelation, a saturation step of the aspecific sites is carried out by incubating the membrane in a bath containing 5% skimmed milk in TBS buffer (volume: 30 ml) with shaking at 4°C overnight. The next day, the membrane is washed with 20 ml of TBS-Tween buffer (TBS + Tween 20 at 0.05%) with shaking for 10 minutes. This wash is repeated 3 times, changing the washing buffer (volume: 30 ml). The membrane is incubated in a bath containing primary antibody diluted 1 / 200 in TTBS buffer + 1% skimmed milk (volume: 20 ml) for 2 hours at room temperature with shaking. Then, the membrane is washed in the same way as described above. The membrane is then incubated in a solution containing the secondary antibody (HRP-coupled antibody) diluted 1 / 500 in TTBS buffer + 1% skimmed milk (volume: 20 ml) for 2 hours at room temperature with stirring.A membrane wash is performed in the same manner as described above. The secondary antibody alone is used as a negative control.

[0260] The revelation of the proteins present in each of the deposits is carried out by incubating the membrane in a bath of pure ECL, a chemiluminescent substrate allowing the detection of the activity of peroxidase (HRP, horseradish peroxidase) conjugated to the secondary antibody. The presence of the TAFA4 protein on the membrane results in the formation of a gray to black spot depending on the intensity of the reaction and therefore the quantity of protein. The membrane is read with the Fusion FX device (Vilber Lourmat) coupled with the VisionCapt software (Vilber Lourmat). The quantification of the TAFA4 protein in the tested samples is carried out with the VisionCapt software which allows the intensity of the different spots to be measured. The quantity of the samples is then carried out using the TAFA4 standard curve established with the recombinant protein.

[0261] IV.2.4 Statistical analyses

[0262] Data were collected from independent experiments performed in triplicate. Quantitative analyses are expressed as mean ± standard deviation. Statistical significance was determined by Student's t-test. Differences were considered statistically significant at p < 0.05.

[0263] (NS: p>0.05; *: p=0.05; **: p=0.01; ***: p=0.001).

[0264] IV.3 Results and conclusions

[0265] Figure 1 reports the effect of valproic acid, TGF-β, and LPS on TAFA4 modulation in FHN supernatant after 72 h of incubation. Results are expressed as percentage of TAFA4 expression normalizing to 100% control (DMEM medium control). Data were obtained from 2 independent experiments performed in triplicate.

[0266] Valproic acid and TGF-β induce TAFA4 synthesis in a dose-dependent manner (statistically significant induction for 2 out of 2 doses for TGF-β; 1 out of 2 doses for valproic acid).

[0267] LPS inhibits TAFA4 synthesis (statistically significant inhibition) at the dose tested. Example V - Evaluation of the effect of compounds on TAFA4 synthesis in normal human keratinocyte cultures

[0268] Vl Introduction

[0269] The aim of the study is to develop a cellular model to quantify the synthesis of TAFA4 in skin cells.

[0270] V.2 Materials and methods

[0271] V.2.1 Cell culture

[0272] Normal human keratinocytes (NHK) were used in this study. They were obtained from Promocell (Germany). The cells were seeded at 100,000 cells / well in 24-well plates in KGM. After 24 hours, the cells were incubated with the compound shown in the table below in KBM + P / S medium at 37°C, 5% CO2.

[0273] After 72 hours of incubation, the supernatants and protein extracts (obtained from the cell lawn) are collected and stored at -80°C. The compound tested is indicated in Table 3 below.

[0274] [Table 3]

[0275] After 72 hours of incubation at 37°C, 5% CO2, the supernatants and protein extracts (obtained from the cell lawn) are collected and stored at -80°C.

[0276] Prior to the TAFA4 protein assay, the toxicity of the compounds is assessed using the MTT assay. Only concentrations achieving viability greater than 80% are selected for quantification by ELISA and Dot blot.

[0277] Data were collected from independent experiments performed in triplicate.

[0278] K2.2 MTT cell viability test

[0279] See point 1.7.

[0280] V.2.3 TAFA4 assay by dot-blot

[0281] See point IV.2.3.

[0282] V.2.4 Statistical analyses

[0283] See point IV.2.4. V.3 Results and conclusions

[0284] Figure 2 reports the effect of EGF on TAFA4 modulation in KHN supernatant after 72 h of incubation. The results are expressed as percentage of TAFA4 expression by normalizing the control to 100% (KGM medium control). Data were obtained from 1 independent experiment performed in triplicate.

[0285] EGF tested at 30 ng / ml inhibits TAFA4 synthesis (statistical significance).

[0286] BIBLIOGRAPHY

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[0297] Livak KJ, Schmittgen TD. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 25(4):402-8

[0298] Liu, D., Peng, H., Sun, Q„ Zhao, Z., Yu, X., Ge, S., Wang, H., Fang, H., Gao, Q„ Liu, J., et al. (2017). The Indirect Efficacy Comparison of DNA Methylation in Sputum for Early Screening and Auxiliary Detection of Lung Cancer: A Meta-Analysis. Int J Environ Res Public Health 14: E679. Misery L., Stander S., Szepietowski J.C., Reich A., Wallengren J, Evers A.W., Takamori K., Brenaut E., Le Gall- lanotto C., Fluhr J., Berardesca E., Weisshaar E. (2017) Definition of Sensitive Skin: An Expert Position Paper from the Special Interest Group on Sensitive Skin of the International Forum for the Study of Itch. Acta Derm Venereol. 97:4-6

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Claims

CLAIMS 1 / Method for selecting at least one compound, preferably ecobiological, intended for the prevention and / or care of sensitive and / or sensitized skin, implementing the identification of a modulation, advantageously an increase, in the content of TAFA4 and / or nucleic acid, advantageously messenger ribonucleic acid (mRNA), coding TAFA4. 2 / Method according to claim 1, characterized in that it comprises the following steps: a) Bringing said at least one compound into contact with at least one skin cell; b) Measuring the content of TAFA4 and / or nucleic acid encoding TAFA4; c) Comparing the value obtained in step b) with a reference value; d) Selecting said at least one compound inducing an increase in the content of TAFA4 and / or nucleic acid in step c). 3 / Method according to claim 2, characterized in that said reference value is the measurement obtained without bringing said at least one skin cell into contact with said at least one compound. 4 / Method according to one of claims 2 or 3, characterized in that said content of TAFA4 and / or of nucleic acid coding TAFA4 is measured in the intra and / or extracellular medium of said at least one skin cell. 5 / Method according to one of claims 2 to 4, characterized in that: Said increase in the TAFA4 content corresponds to at least 120%, more preferably 150%, even more preferably 200% compared to said reference value; and / or Said increase in the content of nucleic acid coding TAFA4 corresponds to at least 120%, more preferably 150%, even more preferably 200%, or even more than 1000% compared to said reference value. 6 / Method according to one of claims 1 to 5, characterized in that the at least one compound is not the TAFA4 protein or one of its biologically active derivatives. 7 / Method according to any one of the preceding claims, characterized in that it is an in vivo or ex vivo method and in that the contacting of step a) is carried out by topical application of said at least one compound to the skin of a whole living organism, advantageously the skin of a whole living non-human organism, a skin explant, a reconstructed epidermis or reconstructed skin. 8 / Method according to any one of claims 2 to Ç>, characterized in that: it is an in vitro method, said at least one skin cell is chosen from keratinocytes, melanocytes, sebocytes, Merkel cells, vascular smooth muscle cells, specialized muscle cells, fibroblasts, mast cells, lymphocytes, monocytes, dendritic cells, advantageously dermal dendritic cells; Langerhans cells, adipocytes, nerve cells, macrophages, advantageously macrophages of type M1 or M2; endothelial cells; and mixtures thereof; preferentially chosen from keratinocytes, fibroblasts and mixtures thereof; and said contacting of step a) is carried out by addition to the culture medium. 9 / Method according to claim 8, characterized in that the at least one skin cell is: - at least one macrophage, advantageously at least one macrophage of type Ml, preferably at least one macrophage of human type Ml; and / or - at least one keratinocyte, advantageously at least one human keratinocyte, preferably at least one normal human keratinocyte; and / or - at least one fibroblast, advantageously at least one human fibroblast, preferably at least one normal human fibroblast; and / or - at least one nerve cell. 10 / Method according to any one of claims 2 to 9, characterized in that step b) of measuring the content of TAFA4 and / or nucleic acid, advantageously mRNA, coding TAFA4 is carried out at a protein level by an ELISA test, immunohistochemistry, colorimetric method, western-blot, dot-blot or simplified western by capillary immunoelectrophoresis (WES) and / or at the nucleic level by in situ hybridization, northern blot, sequencing or RT-PCR, advantageously RT-qPCR. 11 / Method according to any one of claims 2 to 10, characterized in that, before carrying out step a), said at least one skin cell has been brought into contact, by topical application or by addition to the culture medium, with a sensitizing agent, advantageously a cosmetic sensitizing agent, more advantageously a surfactant or a preservative. 12 / Method according to any one of the preceding claims, characterized in that: the amino acid sequence of TAFA4 corresponds to the sequence SEQ ID NO: 1 and / or to SEQ ID NO: 2 and / or to a sequence identical to one of the sequences SEQ ID NO: 1 to 2 at least 60%, of preferably at least 70%, more preferably at least 80%, more preferably at least 85%, or even at least 90%; and / or the nucleotide sequence of TAFA4 corresponds to the sequence SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7 or to a sequence identical to one of the sequences SEQ ID NO: 3 to 7 at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, or even at least 90%. 13 / Process for formulating a non-therapeutic, advantageously ecobiological, cosmetic and / or food composition, more advantageously in a galenic form suitable for topical and / or oral administration, comprising the incorporation into said composition of at least one compound selected by the process according to any one of the preceding claims.

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