Method for screening ecological biological compounds capable of improving skin tolerance threshold

Abstract

The present invention relates to a method for selecting at least one compound for preventing and / or caring for sensitive and / or sensitizing skin, preferably an eco-biotype compound, by identifying the regulation of the content of TAFA4 and / or the nucleic acid encoding TAFA4.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for selecting at least one compound capable of increasing the tolerance threshold of the skin, in particular by the action of the chemokine TAFA4. In particular, it is a method for selecting at least one compound, preferably an eco-biotype compound, for the prevention and / or care of sensitive and / or sensitised skin, which method notably comprises a step of quantifying the content of TAFA4 and / or of a nucleic acid encoding TAFA4, and a step of comparing this quantification with a reference value in order to determine whether there is a change in the content of TAFA4 and / or of a nucleic acid encoding TAFA4. BACKGROUND

[0002] The skin constitutes the main barrier that isolates the organism from the external environment, and is composed of three main layers superimposed, from the deepest to the shallowest: the subcutaneous layer, the dermal layer and the epidermal layer, which is colonized by a microbiota and is generally considered as the outermost layer of the skin. Due to its constant interaction with the environment, the skin is subjected to multiple attacks that modify its balance. In parallel with this exogenous aggression, each individual has intrinsic factors that affect the health of the skin, i.e. the genetic makeup or the hormones. These different exogenous and endogenous factors lead to significant differences in the level of tolerance and sensitivity of the skin of different individuals, and an increasing number of people consider that their skin is stressed or sensitive.

[0003] Epidemiological studies have shown that the sensitive skin syndrome is a global problem, with a prevalence that varies from country to country and that continues to rise (Misery et al., 2018b). In France, 59% of people claim to have sensitive skin (Misery et al., 2018a). More generally, the prevalence of sensitive skin in Europe has increased significantly in recent years, i.e. by 7% in 10 years (Misery et al., 2018a). Similar prevalence rates have been found in Asia and Latin America.

[0004] Sensitive skin is characterized by an overreaction of the skin: the skin overreacts to external and internal attacks and to stimuli that it should normally be able to withstand well (Bergaud et al., 2009). In other words, sensitive skin reacts more quickly, because its tolerance threshold is lowered.

[0005] ​Sensitive skin is now recognized by the medical community as a true syndrome, with the official definition established by the International Forum for the Study of Itch (IFSI) as: “A syndrome defined by unpleasant sensations (stinging, tightness, warmth, itching, and irritation) in response to stimuli that should not normally cause such sensations. The skin may appear normal or may be accompanied by erythema. Sensitive skin can affect various areas of the body, especially the face” (Misery et al., 2017).

[0006] According to the currently accepted model, sensitive skin syndrome is the cumulative effect of the following skin changes:

[0007] - Changes in barrier function, making it easier for aggressive substances to penetrate the skin, leading to skin inflammation;

[0008] - Type C changes in cutaneous nerve endings, particularly neuropathy of small nerve fibers (Huet et al., 2018);

[0009] - Nonspecific inflammation of keratinocytes involving pro-inflammatory mediators such as cytokines (IL-1, IL-8, TNFα), inflammatory lipid mediators (E2 prostaglandins, F2α prostaglandins, and leukotrienes), and histamine; and

[0010] - Neurogenic inflammation resulting from the release of neurotransmitters, which maintain skin inflammation and cause vasodilation ( et al., 2009 ).

[0011] Allergic skin reactions have a significant impact on overall quality of life, often prompting patients to reduce or discontinue cosmetic use to avoid exacerbating irritation. Indeed, cosmetics often contain compounds, such as surfactants or preservatives, that can cause inflammatory reactions. These intolerances can be individualized and limited to very specific conditions and / or combinations of compounds, making them difficult to predict. However, for certain products, such as sunscreens and those targeting specific skin conditions, reducing the frequency and / or amount of cosmetic use is not recommended, as this may worsen skin allergies.

[0012] Furthermore, it is important to be able to control skin sensitivities, such as those caused by atmospheric factors (pollution, temperature, humidity, etc.), allergic phenomena, healing or sunburn, by using non-pharmaceutical cosmetic compounds, preferably of biocompatible type, in particular in a form that can be applied topically.

[0013] For example, document EP 1 457 780 describes a screening method in which stem cell factor (SCF) is used as a specific biomarker whose production is decreased in order to select ingredients of interest capable of treating pruritus and dry or sensitive skin syndromes. However, at the level of the skin, SCF is known to be mainly involved in the survival of melanocytes and the induction of melanin synthesis (Grabbe et al., 1994; Atef et al., 2019). Thus, the modulation of this factor can have a significant adverse effect on skin pigmentation.

[0014] Thus, it is clear that there is still a need for a method for determining a compound, preferably a non-pharmaceutical cosmetic compound, in particular capable of being presented in a pharmaceutical dosage form, suitable for topical and / or oral administration, preferably of an ecological and biological type, capable of increasing the tolerance threshold of the skin and / or reducing skin discomfort, also acting through endogenous mechanisms specific to human skin that control pain and inflammation.

[0015] To date, the TAFA4 (T cell activation G protein-coupled receptor 4) protein, also known as FAM19A4 (family with sequence similarity 19 (chemokine (C-C motif)-like) member A4), is a neurokine mainly produced by certain nerve fibres in the human body (Wang et al., 2015; Yoo et al., 2021), which has been 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 a nucleotide sequence corresponding in part or in whole to the mRNA of the sequences SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.

[0016] The TAFA4 protein consists of two domains, including a signal peptide and a highly conserved chemokine-like sequence (Wang et al., 2015). In humans, TAFA4 is strongly expressed in the brain and weakly expressed in other organs such as the colon, spleen, pancreas, prostate, liver or lungs (Leeman et al., 2020; Liu et al., 2017; Tom Tang et al., 2004; Wang et al., 2015).

[0017] At the level of the skin in mice, TAFA4 is expressed and secreted by certain neurons whose free endings are present in the skin, in particular DRG neurons (dorsal root ganglion) and sensory nerve fibres C-LTMRs (C- low threshold mechanoreceptors), the latter being involved in the transmission of sensory signals (Delfini et al., 2013; Hoeffel et al., 2021; Salio et al., 2021).

[0018] Studies have shown that TAFA4 gene knockout mice can survive, have no obvious abnormalities and respond to acute heat stimulation and mechanical stimulation (Delfini et al., 2013). However, they have a skin regeneration defect after exposure to ultraviolet light (Hoeffel et al., 2021) and are sensitive to pain (Delfini et al., 2013, Kambrun et al., 2018; Salio et al., 2021). Specifically, TAFA4 gene knockout mice have a 35-day post-irradiation inflammation resolution defect in the model of actinic erythema irradiated with UVC. This condition can be compensated by subcutaneous injection of recombinant TAFA4 protein after irradiation (Hoeffel et al., 2021).

[0019] In addition, compared with control mice, TAFA4 gene knockout mice have significantly increased sensitivity to pain under pathological conditions (by intrathecal injection of inflammatory pain agonists or by partial injury of the sciatic nerve). Intrathecal injection of recombinant human TAFA4 can compensate for the condition of TAFA4 gene knockout mice. In mice, TAFA4 also appears to play a repair role in the healing process, especially in the healing after sunburn (Hoeffel et al., 2021). In addition, it can also control allergic phenomena, especially by inducing the synthesis of IL-10, which in turn acts on immune-derived inflammation (Qiu et al., 2022).

[0020] In mice, especially in models of neuropathic pain or postoperative pain, subcutaneous injection or injection into the neuronal layer in the bone marrow of human TAFA4 protein reduces the pain / allergic reaction felt by the skin locally for up to 4 hours after injection (Kambrun et al., 2018; Yoo et al., 2021).

[0021] It has been proposed to use TAFA4 protein, peptide fragments constituting it or nucleic acids encoding it as anti-inflammatory and soothing agents. Thus, document WO2014 / 180853A1 discloses the use of TAFA4 in a pharmaceutical composition to prevent, alleviate or treat pain. Document WO2020 / 064907A1 describes the use of TAFA4 or a nucleic acid encoding it against skin inflammation. Document WO2021 / 156310A discloses the use of TAFA4 or a nucleic acid molecule encoding it for the treatment of inflammatory diseases.

[0022] Although TAFA4 is described as a substance with anti-inflammatory and soothing effects, it is difficult to formulate it into a cosmetic or dermatological composition, especially a composition for topical use, mainly due to problems of bioavailability, a molecular weight of 15-20 kDa hindering its skin penetration, as well as problems of maintaining biological activity, being metabolized by skin cells or microbiota bacteria, etc.

[0023] It is therefore apparent that there is a need to identify certain compounds, preferably non-pharmaceutical cosmetic compounds, and in particular compounds that can be presented in a dosage form suitable for topical application, preferably of the eco-biological type, that are capable of preventing and / or caring for sensitive and / or sensitized skin, in particular by increasing the skin tolerance threshold and / or reducing skin discomfort, also by acting on the endogenous control mechanisms specific to human skin pain and inflammation. SUMMARY

[0024] Surprisingly, the Applicant, who continues to use an eco-biological approach to find the real causes of skin problems and to give the skin means of self-strengthening, has demonstrated that modulating the endogenous content of TAFA4 and / or of the nucleic acid encoding it, i.e. modulating the transcription of TAFA4 and / or the protein expression of TAFA4, in particular at the level of the skin, more particularly by a topical intervention, not involving the exogenous supply of TAFA4 and / or of the nucleic acid encoding it, is possible.

[0025] More surprisingly, the Applicant has demonstrated that the main living cells of the skin, i.e. the keratinocytes and fibroblasts, also express TAFA4 in an endogenous manner and that they can modulate this expression.

[0026] These major findings lay the foundations for a completely new way of managing sensitive skin, which is more effective, more sustainable and more respectful of the skin ecosystem, by respecting and by explaining through its own natural mechanisms, in particular in the field of cosmetics, in terms of topical application.

[0027] The Applicant has thus identified modulation of the endogenous content of TAFA4 and / or of the nucleic acid encoding it as an indicator that makes it possible to classify candidate compounds according to their ability to prevent and / or treat sensitive and / or sensitized skin, in particular by increasing the skin tolerance threshold, in particular by using relevant experimental models.

[0028] From this observation, the Applicant has developed a reliable method, i.e. with acceptable sensitivity and / or specificity, which is easy to read / interpret and can be integrated into the daily research and development work, in particular in the field of cosmetics, to decide whether or not to select a compound, preferably of the eco-biological type, for preventing and / or caring for sensitive and / or sensitized skin, based on modulation of the content of TAFA4 and / or of the nucleic acid encoding it.

[0029] Thus, a first object of the application relates to a method for screening at least one compound, preferably an eco-biological compound, for preventing and / or caring for sensitive and / or sensitized skin, advantageously after stress, more advantageously after allergy, implementing the recognition of the modulation, advantageously the increase, of the content of TAFA4 and / or of a nucleic acid, advantageously of mRNA encoding it, i.e. encoding TAFA4. In other words, such a method notably comprises a step of determining the modulation, advantageously the increase, of the content of TAFA4 and / or of a nucleic acid, advantageously of mRNA, encoding TAFA4.

[0030] In particular, the method according to the application comprises the following steps:

[0031] a) contacting said at least one compound with at least one skin cell;

[0032] b) measuring the content of TAFA4 and / or of a nucleic acid, advantageously of a messenger ribonucleic acid (mRNA) encoding TAFA4;

[0033] c) comparing the value obtained in step b) with a reference value;

[0034] d) selecting at least one compound inducing an increase in the content of TAFA4 and / or of a nucleic acid encoding TAFA4 in step c).

[0035] Within the scope of the application, the term "compound" refers to a substance or a compound having biological and / or therapeutic properties, these properties being the basis of physiological and / or pharmacological effects. It can be an active principle, an excipient or a mixture thereof.

[0036] Within the scope of the application, the terms "eco-biological composition", "eco-biological compound", "eco-biological principle", "eco-biological active principle" or "eco-biological excipient" refer respectively to a composition, a compound, a principle or an excipient respecting the interactions of the human being, of the human being with the world and of the Earth. In particular, they refer to a composition, a compound, a principle, an active principle or an excipient respecting the populations of living cells constituting the skin, i.e. the skin microbiota, the keratinocytes, the fibroblasts, etc., which are constantly interacting and interacting with the environment.

[0037] Within the scope of the application, "eco-biological method" refers to a particular method initiated and developed by the inventors / applicants, which combines the biology and ecology of the skin, helping the skin to survive in the long term according to its natural mechanisms.

[0038] Within the scope of the present application, the term "TAFA4" means any protein, polypeptide or peptide, collectively or individually, from a mammalian cell, advantageously human or murine, more advantageously human, corresponding to an active peptide sequence of the protein, including different existing or predicted isoforms generated by alternative mRNAs, as well as all peptide fragments thereof and all biologically active derivatives thereof. 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 particularly, "derivative" or "fragment" means a protein sequence having at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, even at least 90% identity with the sequence SEQ ID NO: 1 and / or with the sequence SEQ ID NO: 2, thus including TAFA4 of different origin (non-human mammal, etc.).

[0039] Within the scope of the present application, the term "biologically active derivative" means an isoform, a fragment or a covalently modified isoform or fragment, in particular grafted with an organic radical, such as octanoyl, stearyl, palmitoyl fatty chain or by acetylation, methylation, etc. Said derivatives can have a biological activity similar to or even better than the protein from which they are derived.

[0040] Within the scope of the present application, the term "nucleic acid encoding TAFA4" means any nucleic acid, preferably any ribonucleic acid, more preferably any messenger ribonucleic acid (mRNA), collectively or individually, from a mammalian cell, encoding TAFA4, including different isoforms thereof. In particular, the nucleotide sequence of TAFA4 corresponds in part or in whole 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 has at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, even at least 90% identity with one of the sequences SEQ ID NO: 3 to 7, thus including nucleotide sequences encoding TAFA4 protein of different origin (non-human mammal, etc.). These complementary deoxyribonucleic acids (cDNAs) correspond to the various alternative mRNAs found in humans.

[0041] Within the scope of the present application, the term "care" means topical care, in particular cosmetic and non-therapeutic care.

[0042] Within the scope of the present application, the term "sensitive skin" means a skin that is injured and / or attacked and / or in an inflammatory state and / or painful and / or disturbed, advantageously a skin due to stress, more advantageously a skin due to allergies.

[0043] Within the scope of the present invention, the term "contact with at least one skin cell" means any direct (e.g. cultured cells) or indirect (e.g. topical administration) exposure of said at least one candidate compound according to the invention. In particular, indirect exposure by topical administration can be performed on the skin of the whole organism (advantageously a human being), on a skin explant, on a reconstructed epidermis or on a reconstructed skin.

[0044] Within the scope of the present invention, the term "skin explant" means a human skin biopsy from surgical waste, comprising the whole epidermis and dermis and / or subcutaneous layer.

[0045] Within the scope of the present invention, the term "skin substitute" means a reconstructed skin or a reconstructed epidermis, preferably of human being, containing differentiated cells distributed in several layers. In particular, in the sense of the present invention, the reconstructed skin comprises at least two layers, namely: a dermal layer and an epidermal layer. These models can be generated by three-dimensional printing or cell differentiation and can include various cell types to get as close as possible to the complete functionality of the human or animal epidermis (innervation, immunity, pigmentation, blood vessels, etc.). Companies such as LabSkin Creations or Straticell, SkinEthic, Episkin or Phenion have put such engineered models on the market.

[0046] Within the scope of the present invention, the term "reconstructed epidermis" means an epidermis generated in vitro by conventional techniques well known to the person skilled in the art (e.g. Limat and Hunziker 2002; Poumay et al., 2004). The keratinocytes used in the epidermis are advantageously from sensitive human skin.

[0047] Within the scope of the present invention, the term "reconstructed skin" means an epidermis component containing keratinocytes generated in vitro and a dermis component containing fibroblasts generated in vitro combined together with a dermal matrix tissue. Reconstructed skin is commercially available. The keratinocytes and fibroblasts used in this skin are advantageously from sensitive human skin.

[0048] Within the scope of the present application, the term "skin cell" refers to any cell type that can at least partially be found in one of the three layers of the skin, i.e. cells in the epidermis (such as keratinocytes, melanocytes, sebocytes, Merkel cells, immune cells such as Langerhans cells, C-type fibres nerve endings), cells of the dermis (such as mast cells, vascular smooth muscle cells, specialized muscle cells, fibroblasts and immune cells, dermal dendritic cells, fibrocytes, endothelial cells), cells of the subcutis (such as adipocytes), nerve cells and macrophages. Obviously, according to the present application, the term "skin cell" includes any nerve cell whose endings or fibres can be found in the skin. In particular, the skin cell is a fibroblast or a keratinocyte, advantageously human, in particular the skin cell is a macrophage, advantageously of the M1 type, advantageously human. Methods for obtaining M1 type macrophage-like cells are known to the person skilled in the art, for example by differentiating THP-1 monocytes (Horiba et al., 2022) or mononuclear cells of human peripheral blood (Wang et al., 2015). In particular, GINIP+ type, C-type neurons (Salio et al., 2021 ; Jung et al., 2023).

[0049] Within the scope of the present application, the terms "reference value", "normal value", "normal", "standard", "usual value" or "reference biological sample" are used interchangeably to refer to the numerical value or average of the numerical values of the parameter values (such as gene expression, in particular mRNA levels, etc.) from one or more subjects, representative of the basal state of the measurement considered; as opposed to the "test value", "test value" or "test biological sample". Reference and test values are obtained by carrying out the same detection, quantification, identification methods, etc.

[0050] According to one particular embodiment, the reference value according to the present application refers to the measurement obtained in the absence of contact of at least one skin cell according to the present application with at least one compound according to the present application. In other words, the reference value according to the present application is the measurement obtained in the method according to the present application, under the same conditions as in step b) (in particular experiments, measurements, etc.), without carrying out step a).

[0051] Preferably, the measurement of the reference value is carried out before or simultaneously with step b) according to the present application, more advantageously under the same conditions, even more advantageously using the same culture of at least one skin cell.

[0052] Within the scope of the present application, the terms "content", "quantity" and "level" are used interchangeably.

[0053] Within the scope of the present application, the "content of TAFA4 and / or of a nucleic acid encoding the same" refers to the level present within a cell (i.e. in the intracellular medium) and / or in the extracellular medium after secretion by said cell.

[0054] Within the scope of the present application, the term "cutaneous content of TAFA4 and / or of a nucleic acid encoding the same" refers to the level present within a skin cell (i.e. in the intracellular medium) and / or in the extracellular medium after secretion by said skin cell.

[0055] Within the scope of the present application, the term "endogenous cutaneous content of TAFA4 and / or of a nucleic acid encoding the same" refers to the basal level present within a skin cell (i.e. in the intracellular medium) and / or in the extracellular medium after secretion by said skin cell under normal conditions, in particular under normal culture conditions, and without conditions inducing or depleting the expression of TAFA4.

[0056] According to one particular embodiment, the content of TAFA4 and / or of a nucleic acid encoding TAFA4 according to the application is measured in the intracellular and / or extracellular medium of at least one skin cell according to the application.

[0057] Within the scope of the present application, the term "intracellular medium" or "intracellular space" refers to the intracellular environment where intracellular biological processes take place. It includes the cytoplasm, the nucleus and the organelles. The term "extracellular medium" or "extracellular space" refers to the space outside the cell, composed of fluids such as blood, lymph, interstitial fluid, etc., in the case of skin cells, of the extracellular matrix or ECM secreted by the skin cells. By definition, the secretion of intracellular components such as extracellular matrix precursors or cytokines takes place in the extracellular medium.

[0058] Within the scope of the present application, the term "modulation of the content of TAFA4 and / or of a nucleic acid encoding TAFA4" refers to any modulation of the content, of the transcription and / or of the expression of the TAFA4 protein of TAFA4 and / or of a nucleic acid encoding the same.

[0059] According to one particular embodiment, said modulation according to the application corresponds to an increase in the content of TAFA4 and / or of a nucleic acid encoding the same relative to the reference value according to the application. Preferably, the content of TAFA4 is increased by at least 120%, more preferentially 150%, even more preferentially 200% relative to the reference value according to the application; and / or the content of a nucleic acid encoding TAFA4 is increased by at least 120%, more preferentially 150%, even more preferentially 200%, or even more than 1000% relative to the reference value according to the application.

[0060] Within the scope of the present application, the term "expression change" refers to overexpression or underexpression of a gene. In particular, the term "overexpression" refers to an increase in the amount of expression of a gene relative to a reference value; the term "underexpression" refers to a decrease in the amount of expression of a gene relative to a reference value.

[0061] Unless otherwise indicated, in the meaning of the present application, "nucleotide sequence encoding an amino acid sequence" refers to all nucleotide sequences which encode the amino acid sequence, including degenerate nucleotide sequences which can result from the degeneracy of the genetic code. A nucleotide sequence which encodes a protein or an RNA or a cDNA can optionally contain introns.

[0062] The terms "encodes" or "is for encoding", "encoding" or "is for encoding" are used interchangeably and refer to the inherent property of a specific sequence of nucleotides in a polynucleotide, such as a gene, a cDNA or an mRNA, to serve as a template for synthesis of a polypeptide with a defined sequence of amino acids, and as a result of this synthesis, the biological property of the organism. Thus, if the transcription and translation of a gene corresponding to the mRNA results in a protein in a cell or other biological system, the gene encodes the protein. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence, usually described in sequence listings and databases) and the non-coding strand (which serves as the template for synthesis of mRNA) can be designated as encoding the protein or other product of the gene or cDNA.

[0063] In the present application, 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 contains at least two amino acids by definition, but is not limited in the maximum number of amino acids. A polypeptide likewise contains multiple peptides and / or proteins, which themselves contain two or more amino acids covalently linked by peptide bonds. As used in this case, the term refers to short and long chains, the former being also commonly referred to in the art as peptides, oligopeptides and oligomers, the latter being commonly referred to in the art as proteins, which have various types. For example, "polypeptide" includes biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0064] The terms "identity" and "similarity" refer to sequence similarity, measured as the percent of sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the compared sequences is occupied by the same amino acid monomer base or subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules have an identity at that position. The percent of identity or similarity between two sequences is a function of the number of corresponding positions in the two sequences that share the same amino acid monomer base or subunit, i.e., the number of positions occupied by the same nucleotide in the two molecules. For example, if six of ten paired sequences are occupied by the same nucleotide then the two sequences have 60% identity or similarity. Generally, the method of comparison is to align the two sequences to achieve the maximum identity, and for protein sequences, conservative substitutions are not considered part of sequence identity.

[0065] Within the scope of the present invention, the term "amplification primer" refers to a nucleotide fragment that can comprise from 5 to 100 nucleotides, preferably from 15 to 30 nucleotides, and that has the specificity of hybridizing to a nucleotide sequence of interest under defined conditions for the initiation of polymerization by an enzyme, for example, in an enzymatic amplification reaction of the nucleotide sequence of interest. Generally, a "primer pair" is used, which consists of two primers that hybridize to two complementary strands. When amplification of several different genes is desired, several different primer pairs are preferably used, each of which preferably hybridizes specifically to a different gene.

[0066] Within the scope of the present invention, the term "hybridization probe" refers to a nucleotide fragment that generally comprises from 5 to 100 nucleotides, preferably from 15 to 90 nucleotides, and even more preferably from 15 to 35 nucleotides, and that has the specificity of hybridizing to a nucleotide sequence of interest under conditions defined to form a hybridization complex. The probe also comprises a reporter (such as a fluorophore, an enzyme, or any other detection system) that can detect the nucleotide sequence of interest. In the present invention, the nucleotide sequence of interest can be a nucleotide sequence contained in a messenger ribonucleic acid (mRNA) or in a complementary DNA (cDNA) obtained by reverse transcription of said mRNA. When amplification of several different genes is desired, several different probes are preferably used, each of which preferably hybridizes specifically to a different gene.

[0067] Within the scope of the present application, "hybridization" refers to the process in which two nucleotide fragments, such as a hybridization probe and a target nucleotide fragment, having sufficient complementary sequences, are able to form a double strand with stable and specific hydrogen bonds under appropriate conditions. A nucleotide fragment "capable of hybridizing" to a polynucleotide is a fragment that is capable of hybridizing to said polynucleotide under hybridization conditions, which can be determined in each case in a known manner. The exactness of the hybridization conditions determines the specificity of the hybridization. The higher the exactness of the hybridization, the stronger the specificity of the hybridization. This is determined in particular according to the base composition of the probe / target double strand and the degree of mismatch between the two nucleic acids. All these data are well known and a person skilled in the art can determine the appropriate conditions. Generally, the temperature of the hybridization reaction is about 20 to 70°C, in particular 35 to 65°C, in a physiological saline solution having a concentration of about 0.5 to 1 M, depending on the length of the hybridization probe used, followed by a step of detecting the hybridization reaction.

[0068] Within the scope of the present application, the term "enzymatic amplification reaction" refers to a process in which a plurality of copies of a target nucleotide fragment is produced under the action of at least one enzyme. Such amplification reactions are well known to a person skilled in the art and mention can be made in particular of the following techniques: polymerase chain reaction (PCR), ligase chain reaction (LCR), repair chain reaction (RCR), self-sustained sequence replication (3SR) according to document WO-A-90 / 06995, nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA) according to document US 5399491 and loop-mediated isothermal amplification (LAMP) according to document US 6410278. When the enzymatic amplification reaction is a PCR, more particularly when the amplification step is preceded by a reverse transcription of messenger RNA (mRNA) into complementary DNA (cDNA), it is preferable to refer to RT-PCR (RT standing for "reverse transcription"); when the PCR is a quantitative PCR, it is preferable to use a qPCR or a RT-qPCR.

[0069] Within the scope of the present application, the terms "inducer" and "inducer of the synthesis of TAFA4 and / or of the nucleic acid encoding the same" are used interchangeably and refer to an agent, a treatment or a culture condition capable of triggering a determined reaction or a programmed sequence of reactions in a biological system, i.e. the production (i.e. the transcription and / or the translation) of TAFA4 and / or of the nucleic acid encoding the same.

[0070] Within the scope of the present application, the terms "inhibitor" and "inhibitor of the synthesis of TAFA4 and / or of the nucleic acid encoding the same" are used interchangeably and refer to an agent, a treatment or a culture condition capable of triggering a determined reaction or a programmed sequence of reactions in a biological system, i.e. the partial or total inhibition of the production (i.e. the transcription and / or the translation) of TAFA4 and / or of the nucleic acid encoding the same.

[0071] Within the scope of the application, the term "depletion of the endogenous cutaneous content of TAFA4 and / or of the nucleic acid encoding it" means the application in a biological system of an agent, of a treatment or of a culture condition capable of causing a decrease or even the elimination of the content of TAFA4 and / or of the nucleic acid encoding it.

[0072] Within the scope of the application, the terms "sensitizing agent" or "stimulating agent" or "polluting agent" are used interchangeably to mean an agent, a treatment or a culture condition which causes a skin disorder or an allergy, i.e. has different physiological constants of the skin which deviate from the norm, in particular has an inflammatory state, such as that observed when the expression and / or concentration of IL-8, IL-1, TNF alpha or other inflammatory cytokines is increased. Advantageously, such a substance can be a surfactant, more advantageously sodium dodecyl sulfate (SDS), or also a preservative, more advantageously methylisothiazolinone, or another substance which does not correspond to a cosmetic raw material but can be found as a contaminant of a cosmetic raw material, more advantageously a chromium salt, a nickel salt, cobalt, or a substance present in the environment, more advantageously a microparticle, a pollen.

[0073] Within the scope of the application, the term "non-sensitized cell" means a cell from a donor which is characterized by not being sensitive and / or allergic skin, nor having been subjected to a stress which can have sensitized it.

[0074] Preferably, the application aims to provide a method for selecting at least one compound, for example a compound as described above, having the following individual or combined technical features:

[0075] - said modulation of the content of TAFA4 and / or of the nucleic acid encoding TAFA4 is an increase;

[0076] - said content of TAFA4 is increased by at least 120%, more preferentially 150%, even more preferentially 200% compared to the reference according to the application;

[0077] - said content of the nucleic acid encoding TAFA4 is increased by at least 120%, more preferentially 150%, even more preferentially 200%, or even more than 1000% compared to the reference according to the application;

[0078] - said at least one compound is not one of TAFA4 protein and / or a biologically active derivative thereof and / or an isomer thereof;

[0079] - said method is an in vivo, in vitro or ex vivo method;

[0080] - the method is an in vivo or ex vivo method, characterized in that the contacting of step a) is performed by topical application of the at least one compound onto the skin of the whole organism, advantageously onto the skin of the whole non-human organism, onto a skin explant, onto a reconstructed epidermis or onto a reconstructed skin;

[0081] - the method is an in vivo method in a human subject;

[0082] - the method is an in vivo experimental method in a non-human subject, advantageously in a rodent (such as a mouse, a rat, a guinea pig or a rabbit), a cat, a dog, a primate (such as a chimpanzee), a bird (such as a chicken), a reptile, an amphibian or a fish (such as a zebrafish);

[0083] - the method is an in vitro method, comprising a culture of skin cells, advantageously 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 Ml or M2 type macrophages), endothelial cells; and mixtures thereof; preferably selected from keratinocytes, fibroblasts and mixtures thereof; and characterized in that the contacting operation of step a) is performed by addition to the culture medium;

[0084] - the at least one skin cell is at least one macrophage, advantageously at least one Ml type macrophage, preferably at least one human Ml type macrophage;

[0085] - the at least one skin cell is at least one keratinocyte, advantageously at least one human keratinocyte, more advantageously at least one normal human keratinocyte;

[0086] - the at least one skin cell is at least one fibroblast, advantageously at least one human fibroblast, more advantageously at least one normal human fibroblast;

[0087] - the method further comprises, before implementing step a), adding to the culture medium at least one inducer of synthesis of TAFA4 and / or of the nucleic acid encoding it by the at least one skin cell, the inducer being advantageously the following substances: phorbol 12-myristate 13-acetate (Pam) at a concentration preferably between 1 and 20 ng / ml; lipopolysaccharide (LPS) at a concentration preferably between 0.1 pg / ml and 2 mg / ml; calcium at a concentration preferably between 0.1 mM and 10 mM; forskolin at a concentration preferably between 10 mM and 200 mM; a combination of calcium and forskolin at a concentration preferably between 0.1 mM and 10 mM, the calcium concentration preferably being between 0.1 mM and 10 mM; forskolin at a concentration preferably between 10 mM and 200 mM; epidermal growth factor (EGF) at a concentration preferably between 10 ng / ml and 200 ng / ml; fetal bovine serum (FBS) at a concentration advantageously between 0.1 and 5% of the total culture medium by weight; ultraviolet irradiation, preferably exposure to 2 to 10 J / cm 2 of UVA for at least 5 minutes, preferably for at least 10 minutes; and mixtures thereof;

[0088] - the addition step is implemented between 30 minutes and 72 hours, preferably under the following culture conditions: 37°C, 5% CO2 and / or a humidity-saturated atmosphere;

[0089] - the method further comprises, before implementing step a), adding to the culture medium an inhibitor of synthesis of TAFA4 and / or of the nucleic acid encoding it by the at least one skin cell;

[0090] - the method further comprises, before implementing step a), depleting the endogenous skin content of TAFA4 and / or of the nucleic acid encoding it produced by the at least one skin cell;

[0091] - the step b) of measuring the content of TAFA4 and / or of the nucleic acid encoding it, advantageously mRNA, is implemented at the protein level by ELISA tests, immunohistochemistry (including colorimetry, fluorimetry, luminometry), other colorimetric methods, or Western blotting, dot blotting, Western blotting simplified by capillary immunoelectrophoresis (WES), and / or at the nucleic acid level by in situ hybridization, Northern blotting, sequencing or RT-PCR, advantageously RT-qPCR;

[0092] - the content of TAFA4 and / or of the nucleic acid thereof, preferably mRNA, is measured in the intracellular and / or extracellular medium of the at least one skin cell;

[0093] - before carrying out said step a), said at least one skin cell has been contacted with a sensitising agent, advantageously a cosmetic sensitising agent, more advantageously a surfactant or a preservative, preferably by topical application or addition in culture medium;

[0094] - the amino acid sequence of TAFA4 corresponds partially or totally to the sequence SEQ ID NO: 1 and / or SEQ ID NO: 2, or to a sequence having at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, even at least 90% identity with one of the sequences SEQ ID NO: 1-2;

[0095] - 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 having at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, even at least 90% identity with one of the sequences SEQ ID NO: 3-7;

[0096] - said method comprises, in said step b), the additional measurement of the content of at least one indicator different from TAFA4 and / or the nucleic acid encoding it, such as IL-8, IL-1, TNF-alpha or other anti-inflammatory cytokine, the content of said indicator also being compared to a specific reference value of said step c);

[0097] - said reference value is a measurement obtained in the absence of contact of at least one skin cell with said at least one compound;

[0098] - said reference value is a measurement obtained under the same conditions as said step b) but in the absence of said step a);

[0099] - the measurement of said reference value is carried out before or simultaneously with said step b) according to the application, 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 decrease in the content of said indicator (the modulation depending on the selected indicator) to decide whether or not to select said at least one compound;

[0100] - said method further comprises, after carrying out step d), a step e) of formulating said at least one compound selected from cosmetic and / or food compositions, said compound being non-therapeutic, advantageously of the eco-biological type, more advantageously presented in a dosage form suitable for topical and / or oral application;

[0101] - 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 field of cosmetics, food and / or dermatology, advantageously chosen from among anti-radical agents or more commonly antioxidants, whitening agents, pigmentation agents, emollients, moisturizers, anti-seborrheic agents, anti-inflammatory agents, anti-acne agents, keratolytic and / or keratolytic agents, anti-wrinkle and / or firming agents, hydrophilic or lipophilic mineral or organic sunscreens, draining agents, anti-irritant agents, soothing agents, vitamins and mixtures thereof, mattifying agents, anti-aging active agents such as retinol, healing agents, preservatives and essential oils; and

[0102] - said step e) of formulating said at least one selected compound corresponds to the addition of at least one excipient usually used in the field of cosmetics, food and / or dermatology, advantageously chosen from among fats, emulsifiers, co-emulsifiers, hydrophilic or lipophilic gelling agents, preservatives, antioxidants, solvents, exfoliating agents, fragrances, fillers, neutralizing agents, penetration enhancers and polymers.

[0103] Thus, according to one aspect of the application, the method according to the application is an in vitro method, said contacting being performed by addition to a culture medium, said at least one skin cell being chosen from among keratinocytes, melanocytes, sebocytes, Merkel cells, vascular smooth muscle cells, specialized muscle cells, fibroblasts, mast cells, lymphocytes, monocytes, dendritic cells, preferably dermal dendritic cells, Langerhans cells, adipocytes, nerve cells, macrophages, preferably Ml or M2 type macrophages, endothelial cells; and mixtures thereof.

[0104] According to one aspect, the at least one skin cell is chosen from among keratinocytes and fibroblasts, preferably human cells, more preferably normal cells; and mixtures thereof.

[0105] According to one aspect of the application, the at least one skin cell is a mouse cell, advantageously an immortal cell line from B16 melanocytes.

[0106] The determination of the content of TAFA4 and / or of a nucleic acid encoding it, advantageously mRNA, can be carried out using any method known to those skilled in the art. For example, mention can be made of Northern blotting, Southern blotting, PCR, RT-PCR, quantitative RT-PCR, SAGE and derived methods thereof, nucleic acid chips (including cDNA chips, oligonucleotide chips and mRNA chips), tissue chips, RNA-Seq, immunohistochemistry methods, immunoprecipitation, Western blotting, Western blotting simplified by capillary immunoelectrophoresis (WES), dot blotting, ELISA or ELISPOT, protein chips, antibody chips or tissue chips combined with immunohistochemistry, FRET or BRET techniques, microscopic or histochemical methods (including notably confocal microscopy and electron microscopy methods), methods based on the use of one or more excitation wavelengths and suitable optical methods, such as electrochemical methods (voltammetry and amperometry techniques), atomic force microscopy and radiofrequency methods, such as multipolar, confocal and non-confocal resonance spectroscopy, fluorescence detection, luminescence, chemiluminescence, absorbance, reflectance, transmittance and birefringence or refractive index (such as surface plasmon resonance, ellipsometry techniques, resonant mirror methods, etc.), flow cytometry, isotope or magnetic radiofrequency resonance imaging, polyacrylamide gel electrophoresis (SDS-PAGE) analysis; HPLC spectrophotometry, liquid chromatography / mass spectrometry spectrophotometry / mass spectrometry analysis (LC-MS / MS).

[0107] Advantageously, the quantification of the TAFA4 content can be done by immunohistochemistry or immunocytochemistry analysis on skin sections or cells in culture and other methods; by Western Blot; by Dot Blot; by Western Blot simplified by capillary immunoelectrophoresis (WES). Especially Western Blot, which is based on the principle of hybridization of a probe, usually a labeled antibody, to the sought target. It is simple and fast, and does not require any complex purification steps. When making a Dot Blot, the biological sample (cell supernatant or protein extract) is deposited on a nitrocellulose or nylon membrane to form small dots. After drying, the membrane is incubated with the probe to detect the sought protein. Western Blot simplified by capillary immunoelectrophoresis (WES) combines the principles of Western Blot technique and capillary immunoelectrophoresis, allowing the detection and quantification of a specific protein in a biological sample without the laborious procedure of membrane transfer. In this method, proteins are first separated by capillary electrophoresis according to their size and charge; the target protein is then detected using specific antibodies labeled directly on the capillary, and finally analyzed automatically in a dedicated instrument. In addition, the quantification of the TAFA4 content can also be done by ELISA detection, for example, on cell culture supernatant or cell extract, with a calibration curve obtained with serial dilutions of recombinant human TAFA4 protein (for example from 0.1561 ng / ml to 10 ng / ml) to interpolate the optical density of the sample. In practice, on cell layers, viability tests can be performed using (3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) or MTT known to the person skilled in the art. The TAFA4 detection method can only be used if the cell survival rate is greater than 80% and is correlated to the cell survival rate value.

[0108] The measurement of the content of messenger ribonucleic acid (mRNA) encoding TAFA4 can advantageously be performed by in situ hybridization of cell sections, Northern Blot or RT-qPCR. Total RNA is advantageously extracted from tissues, for example using a commercial kit such as Rneasy Plus Mini QIAGEN. After extraction, the total RNA can be tested to possibly know the quantity and quality of the RNA in the cell extract; this step can be performed using a bioanalyzer and a RNA 6000 Nano kit, etc. The cDNA is generated using the QuantiTect Reverse Transcription Kit (QIAGEN) and then the real-time quantitative PCR of the TAFA4 gene is performed at each treatment time using, for example, the QuantiFast SYBR Green PCR Kit (QIAGEN), followed by the expression of a housekeeping gene (for example GAPDH) as a control, and the expression of TAFA4 with specific primers based on SEQ ID NOs: 3, 4, 5, 6 or 7.

[0109] According to a particular embodiment, the determination of the content of TAFA4 can be carried out by enzyme-linked immunosorbent assay, immunohistochemistry on cell sections, Western blotting. The determination of the content of the messenger ribonucleic acid (mRNA) coding for TAFA4 can be carried out by immunohistochemistry, in situ hybridization on cell sections, Northern blotting, sequencing or RT-PCR, advantageously RT-qPCR.

[0110] The content of at least one marker different from TAFA4, such as mentioned above, and the nucleic acid coding therefor can be quantitatively determined according to the methods described above.

[0111] According to a particular embodiment, a method for selecting at least one compound, preferably an eco-biological compound, aimed at preventing and / or caring for sensitive and / or sensitized skin, advantageously after a stress, more advantageously after an allergy, comprises the following steps:

[0112] a) contacting at least one skin cell in vitro with at least one compound by adding it in a culture medium;

[0113] b) determining the content of TAFA4 and / or the content of the nucleic acid coding for TAFA4, preferably in the medium and / or in the intracellular medium of the at least one skin cell;

[0114] c) comparing the value obtained in step b) with a reference value, preferably obtained in the absence of contact of the at least one skin cell with the at least one compound;

[0115] d) selecting the at least one compound which induces an increase in the content of TAFA4 and / or of the nucleic acid coding therefor, preferably an increase of more than 120%, advantageously of more than 150%, compared with the reference value in step c).

[0116] According to another embodiment, a method for selecting at least one compound, preferably an eco-biological compound, aimed at preventing and / or caring for sensitive and / or sensitized skin, advantageously after a stress, more advantageously after an allergy, comprises the following steps:

[0117] a) contacting at least one skin cell ex vivo with the at least one compound by topical application on a skin explant, a skin substitute, a reconstructed epidermis or a reconstructed skin;

[0118] b) measuring the content of TAFA4 and / or of the nucleic acid coding for TAFA4, preferably in the intracellular and / or extracellular medium of the at least one skin cell;

[0119] c) comparing the value obtained in step b) with a reference value, the reference value being preferably obtained without contact of said at least one skin cell with said at least one compound;

[0120] d) selecting said at least one compound which induces an increase in the content of TAFA4 and / or of the nucleic acid encoding it in step c), preferably an increase of more than 120%, more preferably of more than 150% compared to said reference value.

[0121] According to another embodiment, a method for selecting at least one compound, preferably an eco-biological compound, aimed at preventing and / or caring for sensitive and / or sensitized skin, advantageously after a stress, more advantageously after an allergy, comprises the following steps:

[0122] a) contacting said at least one compound in vivo with at least one skin cell by topical application on the skin of an entire organism, preferably a human or non-human;

[0123] b) measuring the content of TAFA4 and / or of the nucleic acid encoding it, preferably in the intracellular and / or extracellular medium of said at least one skin cell;

[0124] c) comparing the value obtained in step b) with a reference value, the reference value being preferably obtained without contact of said at least one skin cell with said at least one compound;

[0125] d) selecting said at least one compound which induces an increase in the content of TAFA4 and / or of the nucleic acid encoding it in step c), preferably an increase of more than 120%, advantageously of more than 150% compared to said reference value.

[0126] According to a particular embodiment, said at least one skin cell used in the method according to the application has not been treated with an allergic reaction.

[0127] According to an alternative embodiment, said at least one skin cell used in the method according to the application has been treated with an allergic reaction.

[0128] According to another embodiment, said at least one cell is from a donor individual characterized by a sensitive or sensitized, advantageously an allergic or reactive, skin.

[0129] According to an alternative embodiment, said at least one cell has previously been contacted with a sensitizing agent, advantageously a cosmetic sensitizing agent, more advantageously selected from surfactants and preservatives, by topical application or by addition to the culture medium.

[0130] According to a particular aspect of the application, the endogenous content of TAFA4 and / or of the nucleic acid encoding it in the skin of the cells according to the application has been depleted before said cells are contacted with at least one candidate compound.

[0131] According to a particular aspect, the compound according to the application does not correspond to the TAFA4 protein nor to any of its isomers, or to any of its biologically active derivatives, nor to TAFA4 or to the ribonucleic acid encoding it or to the nucleic acid encoding it, or to a preparation thereof.

[0132] Another object of the application relates to a kit for implementing the method for selecting at least one compound, preferably of ecological and biological type, intended for the prevention and / or care of sensitive and / or sensitized skin, advantageously after stress, more advantageously after allergy, such as defined above, comprising means for amplifying and / or for detecting the content of TAFA4 and / or of the nucleic acid encoding it, said amplifying and / or detecting means being advantageously specific reagents of the TAFA4 target gene expression product, chosen from amplification primers, hybridization probes or antibodies specifically binding to TAFA4, advantageously to one of the TAFA4 protein and / or to a biologically active derivative thereof.

[0133] Another object of the application also relates to a composition comprising at least one compound selected by the method described above.

[0134] Preferably, the application aims to provide a composition as described above, alone or in combination, having the following technical features:

[0135] - the composition comprises at least one compound selected by the method described above;

[0136] - the composition comprises at least one compound selected by the method described above;

[0137] - the composition is presented in at least one pharmaceutical form suitable for topical application to the skin and / or mucous membranes and / or dandruff, such as anhydrous form, oil-in-water emulsion form, water-in-oil emulsion form, multiple emulsion form, silicone emulsion form, microemulsion form, nanoemulsion form, gel form, aqueous solution form or hydroalcoholic solution form;

[0138] - the composition is liquid or not, presented in the form of a cream, ointment, emulsion, lotion, serum or gel;

[0139] - the composition is presented in at least one pharmaceutical form suitable for non-therapeutic food applications, preferably in oral form; and

[0140] - the composition is colored or not, preferably by a colorant.

[0141] Another object of the application also relates to the use of modulating the content of TAFA4 and / or of a nucleic acid encoding it, preferably mRNA, as an indicator for classifying candidate compounds according to their ability to increase the threshold of tolerance of the skin.

[0142] Another object of the application also relates to a method for formulating a cosmetic and / or food composition, which is non-therapeutic, advantageously of the eco-biological type, more advantageously in a pharmaceutical dosage form suitable for external and / or oral application, said method comprising adding to said composition at least one compound selected according to the selection method of the application.

[0143] Without limitation, the following examples form an integral part of the application, any feature seemingly novel with respect to the prior art being claimed as a general means.

[0144] Figure 1 The effect of different concentrations of valproic acid, TGF-β and LPS and LPS at a concentration of 1 μg / ml on the synthesis of TAFA4 in normal human fibroblasts (NHF) is shown in percent induction. NS: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001.

[0145] Figure 2 The effect of EGF protein at a concentration of 30 ng / ml on the synthesis of TAFA4 in normal human keratinocytes (NHF) is shown in percent induction. NS: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001.

[0146] Examples of embodiments

[0147] Example I: Keratinocyte and fibroblast protocol I.1 Introduction

[0148] The aim of the present study is to provide a simple, rapid protocol to study the modulating effect of cosmetic compounds and compositions on the expression or transcription of TAFA4 in keratinocyte and fibroblast cell lines.

[0149] It must be noted that, optionally, a TAFA4 expression inducer can be implemented to enhance the expression of the gene encoding this protein, in particular in the case where the amount of gene encoding the TAFA4 protein or the amount of TAFA4 protein is less than or equal to the detection limit of the method implemented by the person skilled in the art and / or provides a reference value for the content of TAFA4, i.e. a positive control. In order to classify a candidate compound as a compound capable of preventing and / or caring for sensitive and / or sensitized skin, preferably an eco-biological type compound, this value must be reached.

[0150] I.2 Cell culture

[0151] The normal human keratinocytes (NHK) and normal human fibroblasts (NHF) used in this study were from Promocell (France) and Lonza (France).

[0152] The conditions tested to establish the model include

[0153] • Cell type: NHK, NHF

[0154] • Medium: KBM + 1% P / S for NHK, DMEM + 1% P / S for NHF

[0155] • Treatment time: 48h and 72h

[0156] • Seeding density: 15,000 cells / well for 48h, 96-well plate

[0157] 10,000 cells / well for 72h, 96-well plate.

[0158] We have chosen to test different induction conditions on both cell types:

[0159] These inducers have been chosen as positive controls based on reference data.

[0160] • 10 ng / ml of PMA (P1585-1MG, SIGMA) pre-treatment for 24h

[0161] • 50 ng / ml of PMA pre-treatment for 24h

[0162] • 1 mg / ml of LPS (L2880-10MG, SIGMA)

[0163] • 1 mg / ml of LPS + 10 ng / ml of PMA (PMA 24h pre-treatment)

[0164] • 1 mg / ml of LPS + 50 ng / ml of PMA (PMA 24h pre-treatment)

[0165] • 1.2 mM of calcium (C7902-500G, SIGMA)

[0166] • Calcium 1.2 mM + 10 mM forskolin

[0167] • 100 mM forskolin (93049-10MG, SIGMA)

[0168] • 10 mM forskolin

[0169] Specifically for NHKs:

[0170] • 10 ng / ml EGF (AF100-15, Peprotech)

[0171] • 30 ng / ml EGF

[0172] • 10 ng / ml EGF + 100 μΜ forskolin

[0173] • 30 ng / ml EGF + 100 μΜ forskolin

[0174] Specific for NHFs

[0175] • 0.5% FBS (S1900-500B, Dominique Dutscher)

[0176] • 2% FBS

[0177] • 0.5% FBS + 100 μΜ forskolin

[0178] • 2% FBS + 100 μΜ forskolin I.3 Screening of compounds or compositions

[0179] Cells (NHK, NHF) are seeded in 96-well plates and the corresponding complete medium is added. After 24 hours of incubation at 37°C, 5% CO2, the selected concentration of the compound or composition to be evaluated is added in the medium and then the cells are incubated for 48 hours or 72 hours at 37°C, 5% CO2. Positive induction controls are evaluated simultaneously with the active agent.

[0180] After different incubation times, the supernatant is collected and stored at -80°C and then the TAFA4 is detected by ELISA method. The detection is only performed if the cell viability is greater than 80%.

[0181] When evaluating the TAFA4 expression by RT-qPCR, the cells are recovered after a short incubation kinetics (30 minutes to 24 hours) with the active agent or the positive control.

[0182] I.4 Selection of compounds or compositions to be evaluated

[0183] Different pharmaceutical or cosmetic molecules are preselected and tested. These molecules are directly dissolved in the culture medium or in ethanol or dimethyl sulfoxide according to their nature.

[0184] I.5 Detection of TAFA4 in the culture supernatant by ELISA

[0185] TAFA4 in the supernatant or cell extract was quantitatively detected by ELISA test, enzyme-linked immunosorbent assay (ELISA) (reference number: abx522987, Cliniscience, France) using the reagents recommended by the manufacturer.

[0186] The optical density (OD) was read at a wavelength of 450 nm using a spectrophotometer. The concentration of TAFA4 was calculated based on the standard range determined for recombinant human TAFA4 protein (0.1561 ng / ml to 10 ng / ml).

[0187] The results for TAFA4 per well are expressed in ng / ml. An MTT viability assay is performed on the cell layer. The TAFA4 assay is only used when cell viability is greater than 80%, and this assay correlates with cell viability.

[0188] I.6 Quantification of TAFA4 expression by RT-qPCR

[0189] After incubation with the active agent for 30 minutes to 24 hours, total RNA was extracted using the RNeasyPlus Mini kit according to the supplier's (Qiagen) recommendations.

[0190] Total RNA analysis can assess the quantity and quality of RNA in cell extracts. This analysis is performed using a bioanalyzer and RNA6000Nano kit.

[0191] cDNA was obtained by reverse transcription using the QuantiTect Reverse Transcription Kit (Qiagen).

[0192] TAFA4 gene was detected by real-time quantitative PCR using Qiagen QuantiFast SYBR Green PCR Kit at each treatment time.

[0193] Housekeeping gene: GAPDH

[0194] Related gene: TAFA4 → See specific primers for this gene

[0195] I.7 MTT cell viability test

[0196] After incubation and collection of the culture supernatant, the wells were rinsed with D-PBS. After removal of the D-PBS, 100 μl of a 1 mg / ml MTT solution was added to each well and incubated at 37°C for 3 hours. Subsequently, the MTT solution was removed and 100 μl of DMSO was added to each well to dissolve the formazan crystals. After uniform colouring, the optical density at 540 nm was read using a spectrophotometer (Victor 3, Perkin Elmer). The results were expressed using the formula: survival rate = (OD value after treatment / OD value (100% survival)) x 100. Cells representing 100% survival correspond to cells incubated in control medium for different times.

[0197] Note: MTT ((3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is a soluble yellow colouring agent which, after metabolism by mitochondrial enzymes (succinate dehydrogenase), becomes a dark blue compound: formazan. The formazan crystals are then dissolved with dimethyl sulfoxide and the optical density is measured using spectrophotometry at a wavelength of 540 nm.

[0198] I.8 Statistical analysis

[0199] Data are from three independent experiments performed in triplicate. Quantitative analyses are expressed as mean ± standard deviation. Statistical significance was determined by Student's test. Differences were considered statistically significant from p < 0.05.

[0200] (NS: p > 0.05; *: p < 0.05; **: p < 0.01; ***: p < 0.001).

[0201] Example II: Macrophage protocol II.1 Introduction

[0202] The aim of the present study is to provide a simple, rapid protocol for studying the modulation of TAFA4 expression or transcription in a model of inflammatory macrophages by cosmetic compounds and compositions.

[0203] It must be noted that it is possible to provide a model by depleting the content of TAFA4 (depleting the expression of the gene encoding the TAFA4 protein and / or the number of TAFA4 proteins) in which the endogenous sustained production of TAFA4 must be restored in order to classify the candidate compounds as compounds for the prevention and / or care of sensitive and / or sensitized skin, preferably as eco-biotyped compounds.

[0204] II.2 Cell culture

[0205] Macrophages are immune cells capable of infiltrating tissues. Macrophages are differentiated from monocytes (blood cells), which are differentiated from bone marrow stem cells. Both macrophages and monocytes are cells with phagocytic capacity. Their role is to phagocyte debris and pathogens and contribute more broadly to the immune response by synthesizing different types of mediators.

[0206] There are mainly three types of macrophages:

[0207] • M0s are unactivated macrophages.

[0208] • M1s have pro-inflammatory properties. At the skin level, they are particularly involved in the destruction of pathogens, especially during wound healing.

[0209] • M2s have anti-inflammatory and pro-resolution of inflammation properties. At the skin level, they are particularly involved in the repair phase of healing.

[0210] At the in vitro level, there are different cell models that approach the macrophage type phenotypes.

[0211] These different models can be used in this scope:

[0212] • Human monocyte cell lines: THP-1, U-937, SC, AML-193, HL-60 / S4

[0213] THP-1s are the most described and used cells in the literature.

[0214] For THP-1s: 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), cultured in a humidified atmosphere containing 5% C02 in air and at a temperature of 37°C (Kawano et al., 2015).

[0215] According to the culture conditions described in Horiba et al., 2022, THP-1s can be differentiated into M0, M1 and M2 macrophages.

[0216] • Macrophages are derived from human PBMCs (human peripheral blood mononuclear cells) monocytes in human peripheral blood samples.

[0217] PMBCs were isolated from blood by Polymorphprep. Monocytes (CD14+) were recovered after 4 hours of adhesion to PMBCs. Culture medium: RPMI 1640 + 10% FBS. Monocytes can be differentiated in vitro into M0 macrophages by stimulation with M-CSF (Macrophage Colony-Stimulating Factor) and then treated with LPS (E. coli 055:B5) combined with IFN-gamma (Interferon-gamma) or IL-4 (Interleukin-4) for 24 hours at 37°C in a humidified atmosphere of 5% CO2 in air to differentiate them into M1 or M2 macrophages, respectively (Wang et al., 2015).

[0218] • Macrophages derived from CD34+ hematopoietic stem cells

[0219] These cells are extracted from bone marrow, peripheral blood and human umbilical cord blood (Clanchy and Hamilton, 2013). Usually, these cells are extracted from umbilical cord blood.

[0220] II.3.1. Protocol for selecting a compound or composition that is effective in inducing TAFA4 expression or synthesis

[0221] Cells are seeded in 96-well plates and the corresponding complete culture medium is added. After 24 hours of incubation at 37°C, 5% CO2, the selected concentration of the active agent to be evaluated is added to the culture medium, then incubated for 48 hours or 72 hours at 37°C, 5% CO2. Positive induction controls are evaluated simultaneously with the active agent.

[0222] After different incubation times, supernatants are collected and stored at -80°C, then TAFA4 is detected by ELISA. Detection is only performed if cell viability is greater than 80%.

[0223] When evaluating TAFA4 expression by RT-qPCR, cells are recovered after a short incubation kinetics (30 minutes to 24 hours) with the active agent or the positive control.

[0224] II.3.2. Inducing factors (positive controls)

[0225] During the literature search, it was found that two inducing factors are able to induce the synthesis of TAFA4 at the skin level: LPS and ultraviolet rays (Hoeffel et al., 2021 ; Wang et al., 2015).

[0226] II.3.3. LPS stimulation case

[0227] Monocytes / macrophages were pretreated with PMA (phorbol 12-myristate 13-acetate, 10 ng / ml) for 24 h and then stimulated with LPS (1 μg / ml) for 12 h or 24 h ( Wang et al., 2015 ).

[0228] II.3.4. Ultraviolet irritation

[0229] After removing the culture medium and rinsing with D-PBS, the cells were irradiated with ultraviolet light.

[0230] Data for the described macrophage irradiation protocol:

[0231] -In the Biosun system, 6.7 J / cm 2 The system consists of an irradiation chamber equipped with a 365 nm lamp, a dosimeter / sensor, a calibrator, and software (Shen et al., 2014); or

[0232] UVA was irradiated using a black light with peak emission energy at 360 nm. The emitted dose was measured by a radiometer (UVX-36; UVP Corporation, San Gabriel, CA). UVB was not detected by the UVX-31 sensor. The irradiance at the sample level was 2.5 mW / cm 2 (Kawano et al., 2015).

[0233] II.4 Selection of compounds or compositions to be evaluated

[0234] Different molecules are pre-selected and tested. Depending on their properties, these molecules are dissolved directly in the culture medium or in ethanol or dimethyl sulfoxide.

[0235] II.5MTT cell viability test

[0236] See point I.7.

[0237] II.6 Quantification of TAFA4 expression by RT-qPCR

[0238] After incubation with the active agent for 30 minutes to 24 hours, total RNA was extracted using the RNeasyPlus Mini Kit according to the supplier's (Qiagen) recommendations.

[0239] Total RNA analysis can assess the quantity and quality of RNA in cell extracts. This analysis is performed using a bioanalyzer and RNA6000Nano kit.

[0240] cDNA was obtained by reverse transcription using the QuantiTect Reverse Transcription Kit (Qiagen).

[0241] Real-time quantitative PCR of TAFA4 gene was performed using Qiagen QuantiFast SYBR Green PCR kit at each treatment time.

[0242] • Housekeeping gene: GAPDH

[0243] • Gene of interest: TAFA4 -> see specific primers for this gene

[0244] II.7 TAFA4 measurement in culture supernatant by ELISA

[0245] ELISA, Enzyme-Linked Immunosorbent Assay (Ref: abx522987, Cliniscience, France) was used to quantify TAFA4 in supernatant using the reagents recommended by the producer.

[0246] The optical density (OD) was read at 450 nm wavelength with a spectrophotometer. The concentration of TAFA4 was calculated according to the standard range determined for recombinant human TAFA4 protein (0.1561 ng / ml to 10 ng / ml).

[0247] Results are expressed in ng / ml of TAFA4 per well. MTT viability test was performed on the cell layer. TAFA4 measurement was used only if cell survival rate was greater than 80% and the test was related to cell survival rate value.

[0248] II.8 Statistical analysis

[0249] Data are from three independent experiments in triplicate. Quantitative analysis is expressed as mean ± standard deviation. Statistical significance was determined by Student's test. Differences were considered statistically significant from p < 0.05.

[0250] (NS: p > 0.05; *: p < 0.05; **: p < 0.01; ***: p < 0.001).

[0251] Example III: Differentiation of SH-SY5Y cells into sensory neurons and analysis of TAFA4 levels

[0252] III.1 Introduction

[0253] The aim of this study was to test the modulation of TAFA4 expression in neural cell types able to produce TAFA4.

[0254] III.2 Material and methods

[0255] III.2.1 SH-SY5Y cell culture and differentiation

[0256] SH-SY5Y human neuroblastoma cells used in this study were from Sigma Aldrich (94030304). Cells were thawed at 37°C in a water bath at P13 and immediately placed in DMEM. Cell suspension was centrifuged at 125xg, 4°C for 7 minutes and the pellet was suspended in a 1 : 1 mixture of Eagle's Minimum Essential Medium and F12 medium supplemented with 10% fetal bovine serum and PS solution. Cells were seeded in 96-well plates at a density of 7000 cells per well and incubated in an incubator at 37°C in 95% air, 5% carbon dioxide. To avoid edge effects, the first and last columns and the first and last rows of the plate were not used in the study. Empty wells were filled with water. Cells were allowed to grow to confluence (80%).

[0257] At confluence (1 day later), cells were developed in a 1 : 1 mixture of DMEM medium and F12 medium supplemented with 1% fetal bovine serum, NGF (5 ng / mL), PS solution (1%) and 10 μΜ retinoic acid (RA) to promote cell differentiation. The medium was changed every two days. Only 60 wells of the 96-well plate were used.

[0258] III.2.2 Expression / release of TAFA4 after different times of exposure to the compound (D8, D10, D12)

[0259] After 8, 10 or 12 days of differentiation with RA, cells were stressed for 24 hours with two compounds diluted separately in the culture medium to different concentrations. At the same time, non-stressed controls were performed at each differentiation time (8, 10 and 12 days; reference values).

[0260] Only 100 μί of culture medium was used.

[0261] III.2.3 qPCR: TAFA4 gene expression

[0262] After a few hours of stress application (depending on the stressor and the step), cells were lysed using the RNS XS nucleo spin kit (Macherey Nagel). Briefly, 100 μΙ_ of lysis buffer was added per well following the manufacturer's (Macherey Nagel) instructions. This procedure included a DNase treatment. RNA extraction was performed in a laboratory dedicated to biomolecular experiments, using sterile, DNase / RNase-free solutions and laboratory consumables (such as filter tips, tubes). The total amount and purity of RNA in each sample were evaluated by spectrophotometry using a Nanodrop device (NanoDrop technologies LLC). RNA samples were stored at a temperature lower than -70°C. cDNA was obtained from 25 ng of total RNA using the prime script reverse transcriptase (SensiFAST cDNA Synthesis Kit, Bioline meridian) according to the manufacturer's instructions. cDNA samples were stored at -15 / -25°C. PCR reactions were performed using the CFX96 TM Real-time system (Biorad). PCR amplification was performed on one equivalent of 10 ng of initial RNA using 3 μΜ of primers and SYBR Premix (ONE Green Fast qPCR Premix, OZYME). The PCR reaction was performed using the following cycle program: 30 seconds at 95°C, then 5 seconds at 95°C, 30 seconds at 60°C for 40 cycles.

[0263] Selected genes (GAPDH and TAFA4) were analyzed in each sample using RT-qPCR to measure the relative expression level of mRNA encoding the selected genes. Gene expression analysis was evaluated using the 2-AACt method (Livak and Schmittgen, 2001), where Ct is the threshold cycle. Negative controls were performed for each pair of primers (without cDNA). Expression was normalized to a housekeeping gene such as GAPDH.

[0264] III.2.4 Statistical analysis

[0265] All numerical values are expressed as mean ± SEM (standard error of the mean). Statistical analysis was performed using one-way ANOVA followed by Fisher's LSD test. p < 0.05 was considered significant.

[0266] III.3 Results and conclusions

[0267] These tests made it possible to identify modulators of TAFA4 expression in SH-SY5Y cells, i.e. inhibitors and inducers.

[0268] Example IV: Assessment of the effect of compounds on TAFA4 synthesis in normal human fibroblast cultures

[0269] IV.1 Introduction

[0270] The aim of this study was to establish a cellular model that would allow quantification of TAFA4 synthesis in skin cells.

[0271] IV.2 Materials and methods

[0272] IV.2.1 Cell culture

[0273] Normal human fibroblasts (NHFs) (Lonza - Switzerland) were used in this study. These cells were seeded at 10,000 cells per well in 24-well plates in DMEM + 1% penicillin-streptomycin (P / S). After 24 hours of culture, the cells were incubated with the compounds listed in Table 1.

[0274] [Table 1]

[0275]

[0276] After 72 hours of incubation at 37°C in 5% C02, the supernatant and the protein extract (extracted from the cell layer) were recovered and stored at -80°C.

[0277] Before testing the TAFA4 protein, the toxicity of the compounds tested was evaluated using the MTT test. Only concentrations that gave a survival rate of more than 80% were selected for quantification by ELISA and dot blot.

[0278] These data come from three independent experiments in triplicate.

[0279] IV.2.2 MTT cell viability test

[0280] See point I.7.

[0281] IV.2.3 Detection of TAFA4 arrays by dot blot

[0282] Dot blot is an immunoenzymatic detection method for quantitatively detecting proteins in a sample that has been deposited on a nitrocellulose membrane in a well. The specific equipment (Biorad, France) is used in conjunction with the nitrocellulose membranes. The equipment and consumables used in this experiment are shown in Table 2 below.

[0283] [Table 2]

[0284]

[0285]

[0286] The device is under vacuum pressure, which allows the sample (supernatant or protein extract) to be sucked in, thus allowing the proteins to bind to the nitrocellulose membrane. The device is placed at a height relative to the vacuum pump, so as to follow the principle of gravity.

[0287] The membrane has previously been hydrated with 100 μl of TBS buffer (Tris base + NaCl diluted in water, pH adjusted to 7.5) per well. This operation is repeated twice to ensure that the membrane is in an optimal state of hydration, thus achieving optimal binding of the proteins contained in the sample. The solutions of the sample to be analysed (supernatant and protein extract) and of the TAFA4 recombinant protein in different concentrations ranging from 0.78 to 100 ng / ml are deposited in the plate (100 μl of protein extract per well and 500 μl of supernatant per well), and the suction is started. When all the samples have been sucked in, 100 μl of TBS are added per well, so as to ensure that all the samples have passed through the membrane. The proteins contained in the sample are thus deposited on the nitrocellulose membrane in the form of 1 cm 2 spots.

[0288] In order to be labelled and revealed, the step of saturation of the non-specific sites is carried out by placing the membrane in a TBS buffer (volume: 30 ml) containing 5% skimmed milk, incubated with stirring at 4°C overnight. The next day, the membrane is washed with 20 ml of TBS Tween buffer (TBS + Tween 20 at a concentration of 0.05%) for 10 minutes with stirring. The washing is repeated 3 times by changing the washing buffer (volume: 30 ml). The membrane is placed in a tank containing the primary antibody diluted to 1 / 200 in TTBS buffer solution + 1% skimmed milk (volume: 20 ml), incubated with stirring at ambient temperature for 2 hours. Then, the membrane is washed in the same way as described above.

[0289] Then, the membrane is placed in a solution of secondary antibody (coupled to HRP) diluted to 1 / 500 in TTBS buffer solution + 1% skimmed milk (volume: 20 ml), incubated with stirring at ambient temperature for 2 hours. The membrane is washed in the same way as described above. The secondary antibody alone is used as a negative control.

[0290] The presence of proteins in each deposit is revealed by placing the membrane in a pure ECL (a chemiluminescent substrate used to detect the activity of the peroxidase (HRP, horseradish peroxidase) bound to the secondary antibody). The presence of TAFA4 protein on the membrane is materialized by the grey to black spots formed by the reaction, the formation of which depends on the intensity of the reaction and therefore on the quantity of protein involved. The membrane is read using a Fusion FX device (Vilber Lourmat) and VisionCapt software (Vilber Lourmat). The quantification of TAFA4 protein in the samples tested is done by the VisionCapt software, which can measure the intensity of the different spots. The quantity of sample is then calculated using the TAFA4 standard curve established with the recombinant protein.

[0291] IV.2.4 Statistical analysis

[0292] Data are from three replicates of independent experiments. Quantitative analyses are expressed as mean ± standard deviation. Statistical significance was determined by Student's test. Differences were considered statistically significant starting from p < 0.05.

[0293] (NS: p > 0.05; *: p = 0.05; **: p = 0.01; ***: p = 0.001).

[0294] IV.3 Results and conclusions

[0295] Figure 1 Modulation of TAFA4 in NHF supernatant by valproic acid, TGF-β and LPS after 72 hours of culture is shown. Results are expressed as percentage of TAFA4 expression, the control being normalized to 100% (DMEM medium control). Data are from two independent experiments in triplicate.

[0296] Valproic acid and TGF-β induce the synthesis of TAFA4 in a dose-dependent manner (statistically significant induction for 2 doses out of 2 for TGF-β; statistically significant induction for 1 dose out of 2 for valproic acid).

[0297] LPS inhibits the synthesis of TAFA4 at the doses tested (statistically significant inhibition).

[0298] Example V: Assessment of the effect of compounds on TAFA4 synthesis in normal human keratinocyte cultures

[0299] V.1 Introduction

[0300] The aim of this study was to establish a cellular model that would allow quantification of the synthesis of TAFA4 in skin cells.

[0301] V.2 Materials and methods

[0302] V.2.1 Cell culture

[0303] The normal human keratinocytes (NHK) used in this study were from Promocell (Germany). Cells were seeded at a density of 100,000 cells per well in KGM 24-well plates. After 24 hours, cells were incubated with the compounds indicated in the table below in KBM + P / S medium at 37°C, 5% C02.

[0304] After 72 hours of incubation, supernatants and protein extracts (obtained from the cell layer) were recovered and stored at -80°C. The compounds tested are shown in Table 3 below.

[0305] [Table 3]

[0306]

[0307]

[0308] After 72 hours of incubation at 37°C, 5% C02, supernatants and protein extracts (extracted from the cell layer) were recovered and stored at -80°C.

[0309] Before testing the TAFA4 protein, the toxicity of the compounds was evaluated using the MTT test. Only concentrations that allowed a survival rate greater than 80% were selected for quantification by ELISA and dot blot.

[0310] These data are from three independent experiments in triplicate.

[0311] V.2.2 MTT cell viability test

[0312] See point I.7.

[0313] V.2.3 TAFA4 array by dot blot

[0314] See point IV.2.3.

[0315] V.2.4 Statistical analysis

[0316] See point IV.2.4.

[0317] V.3 Results and conclusions

[0318] Figure 2 Example I: Keratinocyte and fibroblast protocol The modulation of TAFA4 in the supernatant of NHK by EGF after 72 hours of incubation is shown. The results are expressed as a percentage of TAFA4 expression, normalizing the control to 100% (KGM medium control). The data are from three independent experiments in triplicate.

[0319] EGF at 30 ng / ml tested inhibited the synthesis of TAFA4 (statistically significant).

[0320] References

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[0322] Delfini, M.-C., Mantilleri, A., Gaillard, S., Hao, J., Reynders, A., Malapert, P., Alonso, S., A., Barrere, C., Seal, R., et al. (2013). TAFA4, a chemokine-like protein, modulates injury-induced mechanical and chemical pain hypersensitivity in mice. Cell Rep 5: 378-388.

[0323] Grabbe J., Welker P., Dippel E., Czarnetzki B.M. (1994) Stem cell factor, a novel cutaneous growth factor for mast cells and melanocytes. Arch Dermatol Res. 287: 78-84.

[0324] Hoeffel G, Debroas G, Roger A, Rossignol R, Gouilly J, Laprie C, Chasson L, Barbon PV, Balsamo A, Reynders A, Moqrich A, Ugolini S. (2021). Sensory neuron-derived TAFA4 promotes macrophage tissue repair functions. Nature 594: 94-99.

[0325] Jung M, Dourado M, Maksymetz J, Jacobson A, Laufer BI, Baca M, Foreman O, Hackos DH, Riol-Blanco L, Kaminker JS. (2023) Cross-species transcriptomic atlas of dorsal root ganglia reveals species-specific programs for sensory function. Nat Commun. 14:1-366.

[0326] Huet F, Dion A, Batardière A, Nedelec AS, Le F, Bourgeois P, Brenaut E, Misery L. (2018) Sensitive skin can be small fibre neuropathy: results from a case-control quantitative sensory testing study. Br J. Dermatol. 179:1157-1162.

[0327] Horiba, S., Kami, R., Tsutsui, T., and Hosoi, J. (2022). IL-34 Downregulation-Associated M1 / M2 Macrophage Imbalance Is Related to Inflammaging in Sun-Exposed Human Skin. JID Innov 2: 100112.

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[0329] Kawano, A., Hayakawa, A., Kojima, S., Tsukimoto, M., and Sakamoto, H. (2015). Purinergic signaling mediates oxidative stress in UVA-exposed THP-1 cells. Toxicol Rep 2: 391-400.

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[0331] Limat A, Hunziker T. (2002) Use of epidermal equivalents generated from follicular outer root sheath cells in vitro and for autologous grafting of chronic wounds. Cells Tissues Organs. 172:79-85.

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[0333] 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.

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[0335] Misery L, Jourdan E, Huet F, Brenaut E, Cadars B, S., Sayag M, Taieb C (2018a) Sensitive skin in France: a study on prevalence, relationship with age and skin type and impact on quality of life. J Eur Acad Dermatol Venereol. 32:791-795.

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[0337] Poumay Y, Dupont F, Marcoux S, Leclercq-Smekens M, Herin M, Coquette A (2004) A simple reconstructed human epidermis: preparation of the culture model and use in vitro studies. Arch Dermatol Res. 296:203-11.

[0338] Qiu S, Luo X, Mo L, Zhang S, Liao Y, Guan L, Yang L, Huang Q, Liu D, Yang P. (2022) TAFA4-IL-10 axis potentiate immunotherapy for airway allergy by induction of specific regulatory T cells. npj Vaccines 7, 133.

[0339] Quint, W. G. V. (2020). Expression of p16 and HPV E4 on biopsy samples and methylation of FAM19A4 and miR124-2 on cervical cytology samples in the classification of cervical squamous intraepithelial lesions. Cancer Med 9: 2454-2461.

[0340] Salio, C., Aimar, P., Malapert, P., Moqrich, A., and Merighi, A. (2021). Neurochemical and Ultrastructural Characterization of Unmyelinated Non-peptidergic C-Nociceptors and C-Low Threshold Mechanoreceptors Projecting to Lamina II of the Mouse Spinal Cord. Cell Mol Neurobiol 41: 247-262.

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[0343] Tom Tang, Y., Emtage, P., Funk, W. D., Hu, T., Arterburn, M., Park, E. E. J., and Rupp, F. (2004). TAFA: a novel secreted family with conserved cysteine residues and restricted term in the brain. Genomics 83: 727-734.

[0344] Wang, W, Li, T., Wang, X, Yuan, W, Cheng, Y, Zhang, H, Xu, E., Zhang, Y., Shi, S, Ma, D, et al. (2015) FAM19A4 is a novel cytokine ligand of formyl peptide receptor 1 (FPR1) and is able to promote the migration and phagocytosis of macrophages. Cell Mol Immunol 12, 615-624.

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Claims

1. A method for selecting at least one compound, preferably an eco-biotype compound, for preventing and / or caring for sensitive and / or sensitized skin, wherein the method comprises identifying a regulation (preferably an increase) of the content of TAFA4 and / or nucleic acid (preferably messenger ribonucleic acid (mRNA) encoding TAFA4).

2. The method according to claim 1, characterized in that The method comprises the following steps: a) contacting the at least one compound with at least one skin cell; b) measuring the content of TAFA4 and / or a nucleic acid encoding TAFA4; c) comparing the value obtained in step b) with a reference value; d) selecting the at least one compound that induces an increase in TAFA4 and / or nucleic acid content in step c).

3. The method according to claim 2, characterized in that The reference value is a measurement value obtained when the at least one skin cell is not contacted with the at least one compound.

4. The method according to any one of claims 2 or 3, characterized in that The level of TAFA4 and / or a nucleic acid encoding TAFA4 is measured in the intracellular and / or extracellular medium of the at least one skin cell.

5. The method according to any one of claims 2 to 4, characterized in that: - the TAFA4 content is increased by at least 120%, more preferably 150%, even more preferably 200% compared to the reference value; and / or; - The content of the nucleic acid encoding TAFA4 is increased by at least 120%, more preferably 150%, even more preferably 200%, or even more than 1000% compared to a reference value.

6. The method according to any one of claims 1 to 5, characterized in that The at least one compound is not a TAFA4 protein or one of its biologically active derivatives.

7. The method according to any one of the preceding claims, characterized in that This is an in vivo or ex vivo method, the contacting in step a) being carried out by topically applying the at least one compound to the skin of a whole organism, advantageously the skin of a whole non-human organism, a skin explant, a reconstructed epidermis or a reconstructed skin.

8. The method according to any one of claims 2 to 6, characterized in that - the method is an in vitro method; - the at least one skin cell is selected 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 M1 or M2 type macrophages), endothelial cells; and mixtures thereof; preferably keratinocytes, fibroblasts and mixtures thereof; and - The contacting in step a) is performed by adding to the culture medium.

9. The method according to claim 8, characterized in that At least one skin cell is: - at least one macrophage, advantageously at least one M1 macrophage, preferably at least one human M1 macrophage; 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. The method according to any one of claims 2 to 9, characterized in that Step b) TAFA4 and / or nucleic acid encoding TAFA4 (preferably mRNA) content is determined at the protein level by ELISA test, immunohistochemistry, colorimetry, Western blotting, dot blot or Western blotting simplified by capillary immunoelectrophoresis (WES), and / or at the nucleic acid level by in situ hybridization, Northern blotting, sequencing or reverse transcription-polymerase chain reaction (RT-PCR), advantageously reverse transcription-quantitative polymerase chain reaction (RT-qPCR).

11. The method according to any one of claims 2 to 10, characterized in that Before performing step a), the at least one skin cell has been contacted with a sensitizer, advantageously a cosmetic sensitizer, more advantageously a surfactant or a preservative, by topical application or addition to the culture medium.

12. The method according to any one of the preceding claims, characterized in that: - the amino acid sequence of TAFA4 corresponds to the sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2 and / or a sequence that is at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, or even at least 90% identical to one of the sequences of SEQ ID NO: 1 to 2; and / or -The nucleotide sequence of TAFA4 corresponds to the sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7, or a sequence that is at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, or even at least 90% identical to one of the sequences of SEQ ID NO:3 to 7.

13. Method for formulating a non-therapeutic cosmetic and / or food composition, advantageously of the eco-biotype, more advantageously in the form of a pharmaceutical dosage form suitable for topical and / or oral administration, comprising incorporating into said composition at least one compound selected according to the method according to any of the preceding claims.

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