Methods for selecting ecological compounds that can increase the skin tolerance threshold

Abstract

The present invention relates to a method for selecting at least one compound, preferably ecobiological, intended for the prevention and / or care of sensitive and / or sensitized skin, which involves identifying the modulation of the content of TAFA4 and / or the nucleic acid encoding it.

Description

Technical Field

[0001] The present invention relates to a method for selecting at least one compound capable of increasing the skin tolerance threshold by the activity of chemokine TAFA4. In particular, this is a method for selecting at least one, preferably ecobiological, compound intended for the prevention and / or care of sensitive skin and / or sensitized skin, which includes, in particular, a step of quantifying the content of TAFA4 and / or the nucleic acid encoding TAFA4, and a step of comparing this quantification with a reference value in order to identify whether there is a change in the content of TAFA4 and / or the nucleic acid encoding TAFA4.

Background Art

[0002] The skin constitutes a major barrier that separates the organism from the external environment and is composed of three main layers, from the deepest to the most superficial, namely the hypodermis, the dermis, and, in many cases, the stratum corneum of the epidermis where a microbiota, considered the ultimate skin layer, forms colonies. Due to its constant interaction with the environment, the skin undergoes multiple assaults that change its balance. In parallel with this exogenous hazard, each individual also has internal factors that affect skin health, namely genetic structure or hormones. These different exogenous and endogenous factors increase the proportion of the population that perceives their skin as hypersensitive or sensitive, and significant individual differences in skin tolerance and sensitivity levels occur.

[0003] The results of epidemiological studies indicate that sensitive skin syndrome is a global problem that is constantly increasing with changes in prevalence from one country to another (Misery et al., 2018b). In France, it has been shown that 59% of the population has sensitive skin (Misery et al., 2018a). More generally, in Europe in recent years, the prevalence of sensitive skin has increased significantly, namely +7% in 10 years (Misery et al., 2018a). Equivalent prevalences have also been found in Asia and Latin America.

[0004] Sensitive skin is characterized by increased skin responsiveness, that is, an overreaction to external and internal intrusions and irritants to which the skin would normally be sufficiently tolerable (Stander et al., 2009). In other words, sensitive skin reacts more rapidly due to its lowered tolerance threshold.

[0005] Sensitive skin is now recognized as a real syndrome by the medical community, with an established official definition by the International Forum for the Study of Itch (IFSI): "A syndrome characterized by the manifestation of discomfort (stinging, tension, heating-up, itching, and irritation) in response to stimuli that do not normally cause such sensations. The skin may appear normal or may be accompanied by erythema. Sensitive skin can affect all parts of the body, especially the face" (Misery et al., 2017).

[0006] According to the current model accepted by the scientific community, sensitive skin syndrome is the result of the cumulative effect of the various skin changes described below. • Changes in barrier function that make it easier for aggressive agents that cause skin inflammation to penetrate the skin; • Changes in cutaneous nerve endings of type C, particularly neuropathy of small nerve fibers (Huet et al., 2018); Nonspecific inflammation of keratinocytes, including pro-inflammatory mediators such as cytokines (IL-1, IL-8, TNFα), inflammatory lipid mediators (E2 prostaglandins, F2α prostaglandins, and leukotrienes), and histamine; and • Neurogenic inflammation is generated by the release of neuromediators that maintain skin inflammation and cause vasodilation (Stander et al., 2009).

[0007] Skin hypersensitivity significantly impacts overall quality of life, often leading individuals to reduce or discontinue the use of cosmetics to prevent worsening irritation. Indeed, cosmetics generally contain compounds that can trigger inflammatory reactions, such as surfactants or preservatives. These intolerance reactions can be individual-specific and limited to highly specific conditions and / or compound combinations, making them difficult to predict. Furthermore, for certain products, such as sunscreens and products targeting specific skin conditions, reducing the frequency of application and / or the amount of cosmetics applied at the risk of exacerbating skin hypersensitivity is not recommended.

[0008] Furthermore, it is important that by using non-medicinal cosmetic, advantageously ecobiological compounds, especially those presented in a form suitable for topical application, it is possible to control, for example, atmospheric factors (pollution, temperature, humidity, etc.), allergic phenomena, healing effects, or even skin sensitivity caused by sunburn.

[0009] For example, European Patent No. 1457780 describes a screening method that allows for the selection of a target ingredient capable of treating pruritus and dry or sensitive skin syndrome by reducing the production of stem cell factor (SCF), which is used as a specific biomarker. However, SCF is known to be primarily involved in melanocyte retention and melanin synthesis induction at the cutaneous level (Grabbe et al., 1994; Atef et al., 2019). Consequently, modulation of this factor may lead to serious side effects in terms of skin pigmentation.

[0010] Therefore, there remains a clear need for methods to identify compounds, preferably non-pharmaceutical cosmetic compounds, that can be advantageously ecological, adapted for topical administration and / or oral administration, and particularly present in galenic form, which can increase the skin tolerance threshold and / or reduce skin discomfort by acting on endogenous mechanisms for controlling pain and inflammation specific to human skin.

[0011] To date, the TAFA4 protein (TAFA chemokine-like family member 4), also known as FAM19A4 (sequence similarity 19 family (chemokine (CC motif)-like) member A4), a neurokine primarily produced in humans by certain nerve fibers (Wang et al., 2015; Yoo et al., 2021), has been identified as an anti-inflammatory and analgesic agent. In humans, the amino acid sequence of TAFA4 is encoded by mRNA nucleotide sequences that correspond to the sequences of SEQ ID NO: 1 and / or SEQ ID NO: 2, and / or partially or completely to the sequences of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.

[0012] The TAFA4 protein consists of two domains containing a signal peptide and a highly conserved chemokine-like sequence (Wang et al., 2015). In humans, TAFA4 is strongly expressed in the brain and weaker in other organs such as the colon, spleen, pancreas, prostate, liver, and lungs (Leeman et al., 2020; Liu et al., 2017; Tom Tang et al., 2004; Wang et al., 2015).

[0013] In mice, at the cutaneous level in vivo, TAFA4 is expressed and secreted by certain neurons whose free terminals are found in the skin, particularly DRG neurons (dorsal root ganglia), and by sensory nerve fibers C-LTMR (C-low threshold mechanoreceptors), the latter of which are involved in the transmission of sensory signals (Delfini et al., 2013; Hoeffel et al., 2021; Salio et al., 2021).

[0014] Studies have shown that knockout mice for TAFA4 can survive without apparent abnormalities and respond to sharp thermal and mechanical stimuli (Delfini et al., 2013). However, they exhibit impaired skin regeneration after UV exposure (Hoeffel et al., 2021) and hypersensitivity to pain (Delfini et al., 2013, Kambrun et al., 2018; Salio et al., 2021). In particular, TAFA4 knockout mice exhibit impaired inflammation resolution after 35 days of UVC irradiation in an actinic-like erythema model. This condition can be compensated for by intradermal injection of recombinant TAFA4 protein after irradiation (Hoeffel et al., 2021).

[0015] Furthermore, TAFA4 knockout mice exhibit a significantly increased incidence of hypersensitivity to pain in pathological conditions (either due to intradural injection of inflammatory pain agonists or partial sciatic nerve lesions) compared to control mice. Intradural injection of recombinant human TAFA4 compensates for the condition in TAFA4 knockout mice. In mice, TAFA4 also appears to play a role in recovery, particularly during the healing process after sunburn (Hoeffel et al., 2021). Moreover, it can control allergic phenomena, particularly by inducing the synthesis of IL-10, which then acts on immune-derived inflammation (Qiu et al., 2022).

[0016] In vivo injection of human TAFA4 protein subcutaneously or at the neuronal level in the bone marrow of mice reduces locally perceived pain / hypersensitivity in the skin up to 4 hours after injection, particularly in neuropathic pain models or postoperative pain models (Kambrun et al., 2018; Yoo et al., 2021).

[0017] The use of the TAFA4 protein, its constituent peptide fragments, or the nucleic acid encoding it as anti-inflammatory and analgesic agents has already been proposed. Accordingly, International Publication 2014 / 180853 discloses the use of TAFA4 in pharmaceutical compositions that enable the prevention, reduction, or treatment of pain. International Publication 2020 / 064907 describes the use of TAFA4 or the nucleic acid encoding it to combat skin inflammation. International Publication 2021 / 156310 discloses the use of TAFA4 or the nucleic acid molecule encoding it to treat inflammatory diseases.

[0018] Although TAFA4 is described as an anti-inflammatory and analgesic agent, its incorporation into cosmetic and dermatological compositions, particularly for topical use, is difficult due to issues such as its bioavailability, its molar mass of 15-20 kDa which inhibits skin penetration while maintaining biological activity, and metabolism by skin cells or microbiota bacteria.

[0019] Therefore, there is a clear need to identify compounds, preferably non-pharmaceutical cosmetic compounds, that can be presented in advantageously ecological, particularly topically applicable formulations, that enable the prevention and / or care of sensitive and / or sensitized skin by increasing the skin tolerance threshold and / or reducing skin discomfort, and further by acting on endogenous mechanisms of pain and inflammation control specific to human skin. [Overview of the project]

[0020] Remarkably, the applicant has continued these ecobiological methods, investigating the effects on the actual causes of skin problems and the means of providing the skin with means to strengthen itself, and has demonstrated that the regulation of the endogenous content of TAFA4 and / or the nucleic acid encoding it (i.e., regulation of TAFA4 transcription and / or protein expression) is possible, particularly at the skin level, and more specifically, through topical intervention (excluding exogenous supply of TAFA4 and / or the nucleic acid encoding it).

[0021] Even more surprisingly, the applicant demonstrated that major living cells of the skin, namely keratinocytes and fibroblasts, express TAFA4 in an endogenous manner and that they can regulate this expression.

[0022] These key findings, by working in respect to the natural mechanisms described in this way, open the way to entirely new management of skin sensitivity that is more respectful of the skin ecosystem, more effective, and more sustainable, particularly topically and especially cosmetically.

[0023] Accordingly, the applicant has identified that the modulation of the endogenous content of TAFA4 and / or the nucleic acid encoding it is an indicator that allows for the classification of candidate compounds according to whether or not they have the ability to prevent and / or treat sensitive and / or sensitized skin, particularly by increasing the skin tolerance threshold, especially through the use of relevant experimental models.

[0024] Based on this observation, the applicant has developed a reliable (i.e., acceptable sensitivity and / or specificity) means, particularly in the field of cosmetics, that is easy to decipher / interpret and can be incorporated into research and development routines, for determining whether to select preferably ecobiological compounds intended for the prevention and / or care of sensitive and / or sensitized skin, the means being based on the adjustment of the content of TAFA4 and / or the nucleic acid encoding it.

[0025] Therefore, a first object of the present invention is to identify a regulator, preferably an increase, of the content of TAFA4 and / or a nucleic acid encoding it (i.e., encoding TAFA4), preferably mRNA, and to identify a method for selecting at least one compound, preferably of a biologic origin, intended for the prevention and / or care of sensitive and / or sensitized skin after stress, more preferably after allergy. That is, this is a method that particularly includes the step of identifying a regulator, preferably an increase, of the content of TAFA4 and / or a nucleic acid encoding TAFA4, preferably mRNA.

[0026] In particular, the method according to the present invention 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, preferably messenger ribonucleic acid (mRNA); c) comparing the value obtained in step b) with a reference value; d) selecting the at least one compound that induces an increase in the content of TAFA4 and / or a nucleic acid encoding TAFA4 in step c). comprises. [[ID=_{16}]]

[0027] Within the scope of the present invention, the term "compound" means a substance or compound having biological and / or therapeutic properties underlying physiological effects and / or galenic effects. This can be an active ingredient, an excipient or a mixture thereof.

[0028] In the context of the present invention, the terms “ecobiological composition,” “ecobiological compound,” “ecobiological component,” “ecobiological activator,” or “ecobiological excipient” each mean a composition, compound, component, or excipient that is beneficial (respectful) to humans, their interactions with the world, and the planet. In particular, they mean a composition, compound, component, activator, or excipient that is beneficial to the population of living cells that make up the skin (i.e., skin microbiota, keratinocytes, fibroblasts, etc.) that constantly interact with each other and with their environment.

[0029] Within the scope of the present invention, “ecological biological method” means a specific method initiated and developed by the inventors / applicants that combines dermatobiology and dermatoecology to promote the long-term survival of skin according to its natural mechanisms.

[0030] Within the scope of the present invention, the term "TAFA4" means together and individually any protein, polypeptide, or peptide derived from mammalian, preferably human or mouse, more preferably human cells, as well as all peptide fragments comprising it, and all its biologically active derivatives, corresponding to the active peptide sequence of the said protein, including different, existing, or predicted isoforms produced by alternative mRNA. In particular, the amino acid sequence of TAFA4 corresponds to the sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2. More specifically, "derivative" or "fragment" means a protein sequence containing TAFA4 of a different origin (e.g., a non-human mammal) that is at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, or even further at least 90% identical to the sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2.

[0031] Within the scope of the present invention, the term "biologically active derivative" means isoforms, fragments, or modified isoforms or fragments, particularly those covalently modified by grafts of organic groups, such as capryloyl, stearoyl, palmitoyl fatty acid chains, or by acetylation, methylation, etc. The derivatives may have biological activity similar to or greater than that of the proteins from which they are derived.

[0032] Within the scope of the present invention, the term “nucleic acid encoding TAFA4” means, together and individually, any nucleic acid, preferably any ribonucleic acid, more preferably any messenger ribonucleic acid (mRNA) derived from mammalian cells encoding TAFA4, including its different isoforms. In particular, the nucleotide sequence of TAFA4 corresponds, partially or entirely, 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 to a sequence that is at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, or even more than 90% identical to one of SEQ ID NOs: 3-7, and therefore corresponds to a sequence containing a nucleotide sequence encoding the TAFA4 protein of a different origin (e.g., a non-human mammal). These complementary deoxyribonucleic acid (cDNA) corresponds to as many alternative mRNAs identified in humans.

[0033] Within the scope of this invention, the term "care" means topical care, in particular cosmetic and non-therapeutic care.

[0034] Within the scope of the present invention, the term “sensitized skin” means, advantageously as a result of stress, and more advantageously as a result of allergy, injured and / or invasive skin, and / or inflamed skin, and / or painful and / or impaired skin.

[0035] Within the scope of the present invention, the term "contact with at least one skin cell" means either direct (e.g., cells in culture) or indirect (e.g., by topical application) exposure to at least one candidate compound according to the present invention. In particular, indirect exposure by topical application may occur on a whole organism, preferably on human skin, on a skin explant, on reconstructed epidermis, or on reconstructed skin.

[0036] Within the scope of the present invention, the term "skin explant" means a biopsy material of human skin derived from surgical waste and comprising the whole epidermis and skin compartments and / or subcutaneous tissue compartments.

[0037] Within the scope of the present invention, the term “skin substitute” means, preferably, reconstructed human skin or epidermis, comprising differentiated cells distributed in several layers. In particular, within the meaning of the present invention, reconstructed skin comprises at least two compartments, namely, a skin compartment and an epidermal compartment. These models may be produced by 3D printing or cell differentiation and may include several cell types to approximate the full functionality (nerve innervation, immunization, pigmentation, angiogenesis, etc.) of human or animal epidermis as closely as possible. Examples of this type of adapted model have been commercialized by companies such as LabSkin Creations or Straticell, SkinEthic, Episkin, or Phenion.

[0038] Within the scope of the present invention, the term “reconstructed epidermis” means epidermis produced in vitro by conventional methods well known to those skilled in the art (e.g., Limat and Hunziker 2002; Poumay et al., 2004). The keratinocytes used in this epidermis are advantageously derived from sensitive human skin.

[0039] Within the scope of this invention, the term “reconstructed skin” means epidermal components comprising in vitro-generated keratinocytes and skin components comprising in vitro-generated fibroblasts, incorporated into skin matrix tissue. Reconstructed skin is commercially available. The keratinocytes and fibroblasts used in this skin may, advantageously, be derived from sensitive human skin.

[0040] Within the scope of the present invention, the term “skin cell” means any cell type that can be found at least partially in one of the three layers of skin, namely, epidermal cells (e.g., keratinocytes, melanocytes, sebocytes, Merkel cells, immune cells, e.g., Langerhans cells, nerve endings of type C fibers), dermal cells (e.g., mast cells, vascular smooth muscle cells, specialized muscle cells, fibroblasts and immune cells, dermal dendritic cells, fibrous cells, endothelial cells), hypodermal cells (e.g., adipocytes), nerve cells and macrophages. According to the present invention, it is clear that the term “skin cell” includes any nerve cell whose terminals or fibers are found in the skin. In particular, the skin cell is advantageously a human fibroblast or keratinocyte, and in particular, the skin cell is a macrophage, advantageously preferably a human M1 macrophage. For example, methods for obtaining M1 macrophage-like cells by differentiating THP-1 monocytes (Horiba et al., 2022) or PMBC monocytes (mononuclear cells from human peripheral blood; Wang et al., 2015) are known to those skilled in the art. In particular, these are GINIP+ type, C-type neurons (Salio et al., 2021; Jung et al., 2023).

[0041] Within the scope of the present invention, the terms “reference value,” “normal value,” “normal,” “standard,” “typical value,” or “reference biological sample” are used interchangeably in contrast to “tested value,” “test value,” or “test biological sample,” and mean a value or average of a parameter (e.g., gene expression, particularly at the mRNA level, etc.) that originates from one or more subjects and represents the underlying state of the measured values ​​considered. The reference value and the test value are obtained by performing the same detection method, quantification method, identification method, etc.

[0042] In certain embodiments, the reference value according to the present invention is a measured value obtained without contacting at least one skin cell according to the present invention with at least one compound according to the present invention. That is, the reference value according to the present invention is a measured value obtained under the same conditions (in particular, experimental, measured, etc.) as in step b) when step a) is absent in the method according to the present invention.

[0043] Preferably, the measurement of the reference value according to the present invention is performed prior to or concurrently with step b) according to the present invention, more advantageously under the same conditions and even more advantageously using the same culture of at least one skin cell.

[0044] Within the scope of this invention, the terms “content,” “amount,” and “level” are interchangeable.

[0045] Within the scope of the present invention, the term "content of TAFA4 and / or the nucleic acids (one or more) encoding it" means the levels present in the cell (i.e., the intracellular medium) and / or the extracellular medium after secretion by the cell.

[0046] Within the scope of the present invention, the term "skin content of TAFA4 and / or the nucleic acids encoding it (one or more)" means the levels present in skin cells (i.e., intracellular medium) and / or extracellular medium after secretion by said skin cells.

[0047] Within the scope of the present invention, the term "endogenous skin content of TAFA4 and / or nucleic acids encoding it" means the basal level present in skin cells (i.e., intracellular medium) and / or extracellular medium after secretion by said skin cells, without induction or deletion of TAFA4 expression under normal conditions, particularly under normal culture conditions.

[0048] According to a particular embodiment, the content of TAFA4 and / or the nucleic acid encoding TAFA4 according to the present invention is measured in the intracellular and / or extracellular medium of at least one skin cell according to the present invention.

[0049] Within the scope of this invention, the terms “intracellular medium” or “intracellular space” mean the internal environment within a cell in which intracellular biological processes occur. This includes the cytoplasm, nucleus, and organelles. The terms “extracellular medium” or “extracellular space” mean the space outside a cell, consisting of fluids such as blood, lymph, interstitial fluid, and, in particular, in the case of skin cells, the extracellular matrix or ECM secreted by skin cells. By definition, the secretion of intracellular components such as extracellular matrix precursors or cytokines occurs in the extracellular medium.

[0050] Within the scope of the present invention, the term "modification of the content of TAFA4 and / or nucleic acid encoding TAFA4" means any modification of the content of TAFA4 and / or nucleic acid encoding TAFA4, transcription, and / or expression of the TAFA4 protein.

[0051] According to a particular embodiment, the adjustment according to the present invention corresponds to an increase in the content of TAFA4 and / or the nucleic acid encoding it relative to a reference value according to the present invention. Preferably, the increase in the content of TAFA4 is at least 120%, more preferably 150%, and even more preferably 200% relative to the reference value according to the present invention, and / or the increase in the content of the nucleic acid encoding TAFA4 is at least 120%, more preferably 150%, even more preferably 200%, or even more than 1000% relative to the reference value according to the present invention.

[0052] Within the scope of the present invention, the term "change in expression" means overexpression or underexpression of a gene. In particular, the term "overexpression" means that the expression of a gene is increased relative to a reference value, and the term "underexpression" means that the expression of a gene is decreased relative to a reference value.

[0053] Unless otherwise specified, in the sense of this invention, "nucleotide sequence encoding an amino acid sequence" means all nucleotide sequences encoding the amino acid sequence, including degenerate nucleotide sequences that enable obtaining the amino acid sequence. Nucleotide sequences encoding proteins, RNA, or cDNA may optionally contain introns.

[0054] The terms "encoding" or "encoding for," and "encode" or "encode for" are used interchangeably and refer to the properties inherent in a particular sequence of nucleotides in a polynucleotide such as a gene, cDNA, or mRNA, and the biological properties arising therefrom, in order to serve as a matrix for the synthesis of polypeptides having a defined amino acid sequence. Thus, a gene codes for a protein if the transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or another biological system. Both coding strands, whose nucleotide sequences are identical to the mRNA sequence and are generally listed in sequence listings and databases, and non-coding strands used as a matrix for the transcription of a gene or cDNA, may be designated as encoding a protein or another product of that gene or cDNA.

[0055] In the meaning of the present invention, the terms “peptide,” “polypeptide,” and “protein” are interchangeable and refer to compounds consisting of amino acid residues covalently linked by peptide bonds. By definition, a protein contains at least two amino acids, without being limited with respect to the maximum number of amino acids. The polypeptides equally include several peptides and / or proteins, each containing two or more amino acids linked to one another by peptide bonds. As used in this context, the terms refer in the art to both short chains, also commonly designated as peptides, oligopeptides, and oligomers, and longer chains, generally designated as proteins, which have many types. The “polypeptide” includes, among other things, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. The polypeptides include native peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0056] The terms “identical” and “similar” refer to sequence similarity, measured as the percentage of sequence identity between two polypeptides or two nucleic acid molecules. The molecules are identical in this respect if the same amino acid monomer base or amino acid monomer subunit occupies a position in each of the two sequences being compared (for example, if adenine occupies a position in each of the two DNA molecules). The percentage of identity or similarity between two sequences depends on the number of corresponding positions shared by the two sequences, and corresponds to this number divided by the number of positions compared and multiplied by 100. For example, if six out of ten positions in two paired sequences are identical, the two sequences are 60% identical or similar. Generally, the comparison is performed by aligning the two sequences to obtain maximum identity with respect to the protein sequences, without considering conservative substitutions that form part of the sequence identity.

[0057] Within the scope of the present invention, the term “amplification primer” means a nucleotide fragment having specificity for hybridization with a target nucleotide sequence in an enzymatic amplification reaction of a target nucleotide sequence, for example, under conditions determined for the initiation of enzymatic polymerization, and which may contain 5 to 100 nucleotides, preferably 15 to 30 nucleotides. Generally, a “primer pair” consisting of two primers that hybridize to two complementary strands is used. When amplifying several different genes is sought, several pairs of different primers are preferably used, each preferably having the ability to specifically hybridize with a different gene.

[0058] Within the scope of the present invention, the term “hybridization probe” means a nucleotide fragment typically comprising 5 to 100 nucleotides, preferably 15 to 90 nucleotides, and more preferably 15 to 35 nucleotides, which has hybridization specificity under conditions determined to form a hybridization complex having a target nucleotide sequence. The probe also includes a reporter (e.g., a fluorophore, an enzyme, or any other detection system) that enables the detection of the target nucleotide sequence. In the present invention, the target nucleotide sequence may be a nucleotide sequence contained in messenger RNA (mRNA) or a nucleotide sequence contained in complementary DNA (cDNA) obtained by reverse transcription of the mRNA. When several different genes are being explored as targets, several different probes are preferably used, each preferably having the ability to specifically hybridize with a different gene.

[0059] Within the scope of the present invention, “hybridization” means a process in which, under appropriate conditions, two nucleotide fragments having sufficiently complementary sequences, such as a hybridization probe and a target nucleotide fragment, can form a double helix with stable and specific hydrogen bonds. A nucleotide fragment “capable of hybridizing” with a polynucleotide is a fragment capable of hybridizing with the polynucleotide under hybridization conditions that can be determined in any case by known methods. The hybridization conditions are determined by the precision of the operating conditions. Hybridization is all the more specific, as it is performed with greater precision. This is defined in particular according to the base composition of the probe / target double helix and the degree of mismatch between the two nucleic acids. All these data are well known, and appropriate conditions can be determined by those skilled in the art. Generally, depending on the length of the hybridization probe used, the temperature for the hybridization reaction is about 20–70°C, and in particular 35–65°C in a saline solution of about 0.5–1 M concentration. A step of detecting the hybridization reaction is then performed.

[0060] Within the scope of the present invention, the term “enzyme amplification reaction” means a process that generates multiple copies of a target nucleotide fragment by the action of at least one enzyme. Such amplification reactions are well known to those skilled in the art, and in particular, the following methods may be mentioned: polymerase chain reaction (PCR), ligase chain reaction (LCR), repair chain reaction (RCR), self-sustaining sequence replication (3SR) according to International Publication No. 90 / 06995, nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA) according to U.S. Patent No. 5399491, and loop-mediated isothermal amplification (LAMP) according to U.S. Patent No. 6410278. When the enzyme amplification reaction is PCR, more specifically, when the amplification step follows the step of reverse transcription of messenger RNA (mRNA) to complementary DNA (cDNA), and when the PCR is quantitative, after the step of qPCR or RT-qPCR, preferably RT-PCR (RT being “reverse transcription”).

[0061] Within the scope of the present invention, the terms “inducer” and “inducer for the synthesis of TAFA4 and / or nucleic acids encoding it” are interchangeable and mean agents, treatments or culture conditions capable of inducing a determined reaction or a programmed sequence of reactions in a biological system, i.e., the production of TAFA4 and / or nucleic acids encoding it (i.e., transcription and / or translation).

[0062] Within the scope of the present invention, the terms “inhibitor” and “inhibitor of the synthesis of TAFA4 and / or the nucleic acid encoding it” are interchangeable and mean agents, treatments or culture conditions capable of inducing partial or total inhibition of a determined reaction or programmed sequence of a reaction in a biological system, i.e., the production of TAFA4 and / or the nucleic acid encoding it (i.e., transcription and / or translation).

[0063] Within the scope of the present invention, the term "deletion of endogenous cutaneous content of TAFA4 and / or nucleic acids encoding it" means the application of agents, treatments, or culture conditions that can also induce a decrease or removal of the content of TAFA4 and / or nucleic acids encoding it in a biological system.

[0064] Within the scope of the present invention, the terms “sensitizer,” “irritant,” or “contaminant” are interchangeable and mean agents, treatments, or culture conditions that cause skin dysregulation or skin sensitization, i.e., inflammatory conditions, such as those observed during increased expression and / or concentration of IL-8, IL-1, TNFα, or other inflammatory cytokines, which have different physiological constants for the degree of deviation from normal values. Advantageously, such agents may be surfactants, more advantageously sodium lauryl sulfate (SDS), or further, preservatives, more advantageously methylisothiazolinone, or other substances that do not correspond to cosmetic ingredients but may be found as contaminants in cosmetic ingredients, more advantageously chromium salts, nickel salts, cobalt, or substances present in the environment, more advantageously particulate matter, pollen.

[0065] Within the scope of the present invention, the term "unsensitized cells" means cells derived from a donor that have not been subjected to stress that could sensitize sensitive skin and / or sensitized skin.

[0066] Preferably, the present invention aims to provide a method for selecting at least one compound having the following technical features, obtained individually or in combination, such as those defined above.

[0067] The adjustment of the content of TAFA4 and / or the nucleic acid encoding TAFA4 is an increase;

[0068] The aforementioned increase in the TAFA4 content corresponds to at least 120%, more preferably 150%, and even more preferably 200% of the reference value according to the present invention;

[0069] The aforementioned increase in the content of the nucleic acid encoding TAFA4 corresponds to at least 120%, more preferably 150%, even more preferably 200%, or even more than 1000% of the reference value according to the present invention;

[0070] • The at least one compound is not one of the TAFA4 protein and / or one of its biologically active derivatives and / or isoforms;

[0071] The method described above is an in vivo, in vitro, or ex vivo method;

[0072] The method is in vivo or ex vivo, characterized in that the contact in step a) is carried out by topical application of the at least one compound to the skin of a whole organism, preferably to the skin of a non-human whole organism, to a skin explant, to reconstructed epidermis, or to reconstructed skin;

[0073] The above method is an in vivo method for human subjects;

[0074] The aforementioned method is an in vivo method in non-human subjects, preferably in rodents (e.g., mice, rats, guinea pigs, or rabbits), cats, dogs, primates (e.g., chimpanzees), birds (e.g., chickens), reptiles, amphibians, or fish (e.g., zebrafish);

[0075] The method described above is an in vitro method characterized by performing the contact of step a) by adding to a culture medium a culture of a cell selected from the group consisting of, preferably, keratinocytes, fibroblasts, dendritic cells, Merkel cells, vascular smooth muscle cells, special muscle cells, fibroblasts, mast cells, lymphocytes, monocytes, dendritic cells, preferably cutaneous dendritic cells; Langerhans cells, adipocytes, nerve cells, macrophages, preferably M1-type macrophages or M2-type macrophages; endothelial cells; and mixtures thereof;

[0076] The at least one skin cell is at least one macrophage, preferably at least one on-M1 macrophage, and more preferably at least one human M1 macrophage;

[0077] The at least one skin cell is at least one keratinocyte, preferably at least one human keratinocyte, more preferably at least one normal human keratinocyte;

[0078] The at least one skin cell is at least one fibroblast, preferably at least one human fibroblast, more preferably at least one normal human fibroblast;

[0079] The method, prior to performing step a), involves the following by at least one skin cell: preferably, phorbol-12-myristate-13-acetate (Pam) at a concentration of 1-20 ng / ml; preferably, lipopolysaccharide (LPS) at a concentration of 0.1 μg / ml-2 mg / ml; preferably, calcium at a concentration of 0.1 mM-10 mM; preferably, forskolin at a concentration of 10 μM-200 μM; preferably, a combination of calcium at a concentration of 0.1 mM-10 mM and preferably, forskolin at a concentration of 10 μM-200 μM; preferably, epidermal growth factor (EGF) at a concentration of 10 ng / ml-200 ng / ml; preferably, fetal bovine serum (FBS) at a concentration of 0.1-5 wt% of the total culture medium; exposure to UV light, preferably 2-10 J / cm² for at least 5 minutes, preferably at least 10 minutes. 2 The further step includes exposure to UVA at doses between those doses and adding to the culture medium an inducer for the synthesis of TAFA4 and / or nucleic acids encoding it, selected from a mixture thereof;

[0080] The addition step is carried out for 30 minutes to 72 hours, preferably under the following culture conditions, namely, an atmosphere saturated with 37°C, 5% CO2, and / or humidity;

[0081] The method further includes, before carrying out step a), adding an inhibitor of the synthesis of TAFA4 and / or the nucleic acid encoding it to the culture medium by at least one skin cell;

[0082] The method further includes, before performing step a), deleting the endogenous skin content of TAFA4 and / or the nucleic acid encoding it, which is produced by the at least one skin cell;

[0083] Step b) above, which measures the content of TAFA4 and / or the nucleic acid encoding it, preferably mRNA, is performed at the protein level by ELISA, immunohistochemistry (including colorimetric, fluorescence, and luminescence), other colorimetric methods, or Western blotting, dot blotting, capillary immunoelectrophoresis (WES), and / or at the nucleic acid level by in situ hybridization, Northern blotting, sequencing, or RT-PCR, preferably RT-qPCR;

[0084] The content of TAFA4 and / or the nucleic acid encoding TAFA4, preferably mRNA, is measured in the intracellular and / or extracellular medium of at least one skin cell;

[0085] Before carrying out step a), preferably, at least one skin cell is brought into contact with a sensitizer, preferably a cosmetic sensitizer, more preferably a surfactant or preservative, by topical application or by addition to a culture medium;

[0086] The amino acid sequence of TAFA4 partially or entirely corresponds to the sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2, or corresponds to at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, or even more than 90% of the sequence of one of SEQ ID NOs: 1-2;

[0087] The nucleotide sequence of TAFA4 partially or entirely 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 corresponds to at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, or even more than 90% identical to one of SEQ ID NOs: 3-7;

[0088] The method includes, in step b), an additional measurement of the content of at least one indicator agent different from TAFA4 and / or the nucleic acid encoding it, such as IL-8, IL-1, TNF-α, or other anti-inflammatory cytokines, wherein the content of the indicator agent is compared to a reference value specific to step c);

[0089] The reference value is a measurement obtained without contacting the at least one skin cell with the at least one compound;

[0090] The reference value is a measurement obtained under the same conditions as in step b) but without step a);

[0091] • The measurement of the reference value is more advantageously performed under the same conditions, and even more advantageously, using the same culture of at least one skin cell, before or concurrently with step b) according to the present invention; • The selection step d) relates to another selection step based on the induction of an increase or decrease in the content of the indicator agent (a selection-dependent adjustment) to determine whether or not to select the at least one compound;

[0092] The method further comprises, after performing step d), step e), more advantageously, incorporating at least one compound selected from non-therapeutic, preferably ecobiological cosmetic and / or food compositions, which is presented in a formulation suitable for topical and / or oral administration;

[0093] Step e), which involves incorporating the at least one selected compound, advantageously corresponds to the addition of at least one active ingredient typically used in the cosmetic, food, and / or dermatological fields, selected from, for example, retinol, healing agents, preservatives, and essential oils; and

[0094] Step e), which involves incorporating the at least one selected compound, may preferably correspond to the addition of at least one excipient, selected from fats, emulsifiers, coemulsifiers, hydrophilic or lipophilic gelling agents, preservatives, antioxidants, solvents, exfoliating agents, fragrances, fillers, neutralizing agents, pro-penetrating agents, and polymers, which are commonly used in the cosmetic, food, and / or dermatological fields.

[0095] Accordingly, according to one aspect of the present invention, the method according to the present invention is an in vitro method, in which contact is carried out by adding to a culture medium, and at least one skin cell is selected from keratinocytes, melanocytes, sebocytes, Merkel cells, vascular smooth muscle cells, special muscle cells, fibroblasts, mast cells, lymphocytes, monocytes, dendritic cells, preferably cutaneous dendritic cells; Langerhans cells, adipocytes, nerve cells, preferably M1 or M2 type macrophages; endothelial cells; and mixtures thereof.

[0096] According to one embodiment, the at least one skin cell is preferably selected from human, more preferably normal keratinocytes and fibroblasts; and mixtures thereof.

[0097] According to one aspect of the present invention, the at least one skin cell is a mouse cell, preferably a cell derived from an immortalized mouse line of B16 melanocytes.

[0098] The content of TAFA4 and / or the nucleic acid encoding it, preferably mRNA, can be measured by any method known to those skilled in the art. Examples include Northern blotting, Southern blotting, PCR, RT-PCR, quantitative RT-PCR, SAGE and its derivatives, nucleic acid chips including cDNA chips, oligonucleotide chips and mRNA chips, tissue chips and RNA-Seq, immunohistological methods, immunoprecipitation, Western blotting, Western blotting simplified by capillary immunoelectrophoresis (WES), dot blotting, ELISA or ELISPOT, protein chips, antibody chips or tissue chips conjugated to immunohistochemical techniques, FRET or BRET techniques, microscopic or histochemical methods including confocal microscopy and electron microscopy, one or more excitations Methods based on the use of wavelength, appropriate optical methods, e.g., electrochemical methods (voltammetry and current titration techniques), atomic force microscopy, and radiofrequency methods, e.g., multipolar, confocal, and non-confocal resonance spectroscopy, fluorescence detection, luminescence, chemiluminescence, absorbance, reflectance, transmittance, and birefringence or refractive index (e.g., surface plasmon resonance, elliptic polarization, resonant mirror method, etc.), flow cytometry, isotopic or magnetic radio resonance imaging, polyacrylamide gel electrophoresis (SDS-PAGE) analysis; HPLC spectrophotometry, liquid chromatography / mass spectroscopy / mass spectrometry (LC-MS / MS) can be mentioned.

[0099] Advantageously, the quantification of TAFA4 content can be performed by immunohistochemistry or immunocytochemistry of skin or cell sections in culture and other methods; by Western blotting; by dot blotting; or by Western blotting (WES) simplified by capillary immunoelectrophoresis. In particular, protein dot blotting is based on the principle of hybridization between a probe, generally labeled antibody, and target of interest. It is simple and rapid and does not require any complex purification steps. To prepare a dot blot, small dots are formed by depositing a biological sample (cell supernatant or protein extract) onto a nitrocellulose or nylon membrane. After drying, the membrane is incubated with a probe to detect the target protein. Western blotting (WES) simplified by capillary immunoelectrophoresis combines the principles of Western blotting and capillary immunoelectrophoresis to detect and quantify specific proteins in biological samples without the cumbersome process of membrane transfer. In this method, the proteins are first separated by capillary electrophoresis according to their size and charge; then, the target protein is detected using a specific antibody directly labeled on the capillary, followed by automated analysis in a dedicated instrument. Alternatively, the TAFA4 content can be quantified by ELISA in cell culture supernatant or cell extract, for example, by interpolating the optical density of the sample on a calibration curve obtained by serial dilution of recombinant human TAFA4 protein (e.g., 0.1561 ng / ml to 10 ng / ml). In practice, viability tests can be performed on the cell layer using ((3-4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) or MTT, as is known to those skilled in the art. The TAFA4 assay is used only when cell viability is greater than 80% and is associated with this cell viability value.

[0100] The content of messenger ribonucleic acid (mRNA) encoding TAFA4 can be advantageously measured by in situ hybridization in cell sections; by Northern blotting; or further, by RT-qPCR. Advantageously, total RNA is extracted from the tissue using, for example, commercially available kits (such as Rneasy Plus Mini QIAGEN). After the extraction, a total RNA assay can be performed, which allows for the determination of the quantity and quality of RNA in the cell extract; this step can be performed, for example, using the Bioanalyser and RNA 6000 Nano kit. cDNA is generated using the QuantiTect Reverse Transcription Kit (QIAGEN), for example, before real-time quantitative PCR for the TAFA4 gene at each treatment using the QuantiFast SYBR Green PCR Kit (QIAGEN), and after the expression of a housekeeping gene (e.g., GAPDH) as a control and TAFA4 with specific primers based on SEQ ID NOs: 3, 4, 5, 6, or 7.

[0101] Therefore, according to a particular embodiment, the TAFA4 content is measured by ELISA, immunohistochemistry in cell sections, or Western blotting; the TAFA4 content is measured by immunohistochemistry, in situ hybridization in cell sections, Northern blotting, sequencing, or RT-PCR, preferably RT-qPCR.

[0102] The quantification of the content of at least one marker and / or nucleic acid encoding it, distinct from TAFA4, such as those mentioned above, may be performed according to the methods identified above.

[0103] According to a particular embodiment, a method for selecting at least one compound, preferably ecobiological, that is intended to prevent and / or care for sensitive and / or sensitized skin after stress, more advantageously, and more advantageously after an allergy, is as follows: a) In vitro contact of at least one skin cell by adding the at least one compound to a culture medium; b) Preferably, measure the content of TAFA4 and / or the nucleic acid encoding TAFA4 in the culture medium and / or in the intracellular medium of at least one skin cell; c) Preferably, compare the reference value obtained without bringing the at least one skin cell into contact with the at least one compound with the value obtained in step b); d) Selecting at least one compound that induces an increase in the content of TAFA4 and / or the nucleic acid encoding it in step c), preferably more than 120%, and advantageously more than 150%, relative to the reference value.

[0104] According to an alternative embodiment, a method for selecting at least one compound, preferably ecobiological, that is intended to prevent and / or care for sensitive and / or sensitized skin after stress, more advantageously, and more advantageously after an allergy, is as follows: a) Ex vivo contact of at least one skin cell with the at least one compound by topical application to a skin explant, skin substitute, reconstructed epidermis, or reconstructed skin; b) Preferably, measure the content of TAFA4 and / or the nucleic acid encoding it in the intracellular and / or extracellular medium of at least one skin cell; c) Preferably, compare the reference value obtained without contacting the at least one skin cell with the at least one compound with the value obtained in step b); d) Selecting at least one compound that induces an increase in the content of TAFA4 and / or the nucleic acid encoding it in step c), preferably more than 120%, and advantageously more than 150%, relative to the reference value.

[0105] According to yet another embodiment, a method for selecting at least one compound, preferably ecobiological, that is intended to prevent and / or care for sensitive and / or sensitized skin after stress, more advantageously, and more advantageously after an allergy, is as follows: a) Contacting at least one skin cell in vivo with the at least one compound by topical application to the skin of a whole organism, preferably human or non-human; b) Preferably, measure the content of TAFA4 and / or the nucleic acid encoding it in the intracellular and / or extracellular medium of at least one skin cell; c) Preferably, compare the reference value obtained without contacting the at least one skin cell with the at least one compound with the value obtained in step b); d) Selecting at least one compound that induces an increase in the content of TAFA4 and / or the nucleic acid encoding it in step c), preferably more than 120%, and advantageously more than 150%, relative to the reference value.

[0106] According to certain embodiments, the at least one skin cell used in the method according to the present invention is not sensitized.

[0107] According to an alternative embodiment, the at least one skin cell used in the method according to the present invention is sensitized.

[0108] According to another embodiment, the at least one cell is derived from a donor characterized by sensitive or sensitized skin, preferably hypersensitive or reactive skin.

[0109] According to an alternative embodiment, at least one cell is pre-contacted with a sensitizer, preferably a cosmetic sensitizer, and more preferably a surfactant and a preservative, either by topical application or by adding it to a culture medium.

[0110] According to a particular aspect of the present invention, the endogenous skin content of TAFA4 and / or the nucleic acid encoding it in the cells according to the present invention is deleted, and then the cells are contacted with at least one candidate compound.

[0111] In a particular embodiment, the compound according to the present invention does not correspond to the TAFA4 protein, one of its isoforms, one of its biologically active derivatives, TAFA4, the ribonucleic acid encoding it, the nucleic acid encoding it, or any combination thereof.

[0112] Another object of the present invention relates to a kit for performing a method for selecting at least one compound, preferably ecobiological, that is intended to prevent and / or care of sensitive skin and / or sensitized skin after stress, more advantageously after allergy, and more advantageously, the means for amplification and / or detection, wherein the means for amplification and / or detection are specific reagents for expression products of a TAFA4 target gene selected from among TAFA4, advantageously one of the TAFA4 protein and / or its biologically active derivatives, an amplification primer, a hybridization probe, or an antibody.

[0113] Another object of the present invention relates to compositions comprising at least one compound selected by the method described above.

[0114] Preferably, the present invention aims to provide compositions such as those described above, having the following technical features obtained individually or in combination.

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

[0116] The composition comprises at least one compound selected by the method described above;

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

[0118] The composition may be a liquid, or, if not a liquid, presented in the form of a cream, ointment, milk, lotion, serum, or gel;

[0119] The composition is preferably presented orally in at least one formulation suitable for non-therapeutic food application; and

[0120] The composition is either colored or uncolored, and preferably colored by a coloring agent.

[0121] Another object of the present invention relates to the use of modulating the content of TAFA4 and / or the nucleic acid encoding it, preferably mRNA, as an indicator that allows candidate compounds to be classified according to whether or not they have the ability to increase the skin tolerance threshold.

[0122] Another object of the present invention also relates to a method for producing cosmetic and / or food compositions presented in formulations suitable for non-therapeutic, advantageously ecobiological, and more advantageously topical and / or oral administration, comprising incorporating into the composition at least one compound selected by a selective method according to the present invention.

[0123] The following examples, though not limiting, are considered novel to the components of the present invention and the prior art, and in themselves form any of the features described in the claims as schematic means. [Brief explanation of the drawing]

[0124] [Figure 1] Figure 1 shows the effects of valproic acid, TGF-β, and LPS at different concentrations, as well as the effect of LPS at a concentration of 1 μg / ml, on TAFA4 synthesis in normal human fibroblasts (NHF), expressed as the induction rate. NS: p > 0.05; *: p < 0.05; **: p < 0.01; ***: p < 0.001. [Figure 2] Figure 2 shows the effect of EGF protein concentration (30 ng / ml) on TAFA4 synthesis in normal human keratinocytes (NHF), expressed as the induction rate. NS: p > 0.05; *: p < 0.05; **: p < 0.01; ***: p < 0.001. [Modes for carrying out the invention]

[0125] (Example I - Keratinocyte and fibroblast protocol) I.1 Introduction The objective of this study is to provide a simple and rapid protocol that enables the study of the regulation of TAFA4 expression or transcription in keratinocyte and fibroblast systems using cosmetic compounds and compositions.

[0126] It should be noted that, optionally, it is possible to realize a substance that induces TAFA4 expression, and in particular, when the amount of the gene encoding the TAFA4 protein or the amount of TAFA4 protein is below the detection limit imposed by a method realized by those skilled in the art, it is possible to enhance the expression of the gene encoding this protein and / or provide a reference TAFA4 production value, i.e., a positive control, which must be achieved to classify candidate compounds in the category of preferably ecological compounds that enable the prevention and / or care of sensitive and / or sensitized skin.

[0127] I.2 Cell culture In this study, normal human keratinocytes (NHK) and normal human fibroblasts (NHF) were used. These were obtained from Promocell (France) and Lonza (France).

[0128] The conditions tested for model development are as follows: ·Cell type: NHK, NHF • Culture medium: KBM + 1% P / S for NHK, DMEM + 1% P / S for NHF. Processing time: 48 hours and 72 hours • Seeding density: 15,000 seeds / well in a 96-well plate over a 48-hour period. 10,000 cells / well in a 96-well plate in 72 hours.

[0129] The inventors chose to test different induction conditions for two cell types. These inducers were selected as positive controls based on data from the literature. • 24-hour pretreatment with 10 ng / ml PMA (P1585-1MG, SIGMA) • 24-hour pretreatment with 50 ng / ml PMA • 1 mg / ml LPS (L2880-10MG, SIGMA) 10 ng / ml LPS + 1 mg / ml PMA (24-hour pretreatment for PMA) 1 mg / ml LPS + 50 ng / ml PMA (24-hour pretreatment for PMA) • 1.2 mM calcium (C7902-500G, SIGMA) 1.2 mM calcium + 10 μM forskolin • 100 μM forskolin (93049-10MG, SIGMA) • 10 μM forskolin

[0130] Specifically, regarding NHK: • 10 ng / ml EGF (AF100-15, Peprotech) • 30 ng / ml EGF • 10 ng / ml EGF + 100 μM forskolin • 30 ng / ml EGF + 100 μM forskolin

[0131] Specifically, regarding NHF ·0.5% FBS (S1900-500B, Dominique Dutscher) 2% FBS • 0.5% FBS + 100 μM forskolin 2% FBS + 100 μM forskolin

[0132] I.3 Screening of Compounds or Compositions Cells (NHK, NHF) are seeded in a 96-well plate in the corresponding complete culture medium. After incubation at 37°C for 24 hours in 5% CO2, the compound or composition to be evaluated is added to the culture medium at a selected concentration, and the cells are incubated at 37°C for 48 or 72 hours in 5% CO2. Positive induction controls are evaluated in parallel with the activator.

[0133] After different incubation times, the supernatant is collected and stored at -80°C, then assayed for TAFA4 by ELISA. The assay is performed only if the cell viability is greater than 80%.

[0134] To evaluate TAFA4 expression by RT-qPCR, cells are harvested after incubation with an activator or positive control for a shorter kinetic period (30 minutes to 24 hours).

[0135] I.4 Selection of Compounds or Compositions to be Evaluated Different pharmaceutical or cosmetic molecules can be pre-selected and tested. These molecules will be solubilized directly into the culture medium, or solubilized in ethanol or DMSO according to their properties.

[0136] I.5 Assay of TAFA4 in culture supernatant by ELISA The quantification of TAFA4 in the supernatant or cell extract is performed using ELISA, i.e., Enzyme-Linked Immunosorbent Assay (reference: abx522987, Cliniscience, France) with reagents recommended by the manufacturer.

[0137] The optical density (OD) is read using a spectrophotometer at 450 nm. The concentration of TAFA4 is calculated from the standard range established for recombinant human TAFA4 protein (0.1561 ng / ml to 10 ng / ml).

[0138] The results are expressed as ng / ml of TAFA4 per well. An MTT viability test is performed in the cell layer. The TAFA4 assay is used only when cell viability is greater than 80%, and is associated with this cell viability value.

[0139] I.6 Quantification of TAFA4 expression by RT-qPCR After incubation with an activator for 30 minutes to 24 hours, total RNA extraction is performed using the Rneasy Plus Mini kit, following the supplier's (Qiagen) recommendations.

[0140] Total RNA assays allow for the determination of the quantity and quality of RNA in cell extracts. These assays are performed using Bioanalyser and the RNA 6000 Nano kit.

[0141] Reverse transcription performed using the QuantiTect Reverse Transcription Kit (Qiagen) makes it possible to obtain cDNA.

[0142] Real-time quantitative PCR is performed on the TAFA4 gene at each treatment stage. The Qiagen QuantiFast SYBR Green PCR kit is used. • Housekeeping gene: GAPDH • Target gene: TAFA4 → Refer to the gene's specific primer.

[0143] I.7 MTT cell viability test After incubation and collection of the culture supernatant, the wells were rinsed with D-PBS. After removing the D-PBS, 100 μl of 1 mg / ml MTT solution was added per well and incubated at 37°C for 3 hours. Then, the MTT was removed and 100 μl of DMSO was added to each well to dissolve the formazan crystals. After homogenization of the color, the optical density was read at 540 nm using a spectrophotometer (Victor3, Perkin Elmer). The results were then expressed by the following formula: Viability (%) = (treated OD condition / OD (100% viability)) × 100. Cells representing 100% viability correspond to cells incubated at different time points in the control culture medium.

[0144] NB:MTT ((3-4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) is a soluble yellow coloring agent (succinate dehydrogenase) that is metabolized by mitochondrial enzymes to a deep blue compound, namely formazan. The formazan crystals are then solubilized in DMSO, and the optical density is measured by spectrophotometric method at 540 nm.

[0145] I.8 Statistical analysis Data were collected from three independent experiments conducted as a set. Quantitative analysis is presented in the form of mean ± standard deviation. Statistical significance is determined by the Student's Test. Differences are considered statistically significant if p < 0.05. (NS:p>0.05;*:p<0.05;**:p<0.01;***:p<0.001).

[0146] (Example II - Macrophage Protocol) II.1 Introduction The objective of this study is to provide a simple and rapid protocol for investigating the regulation of TAFA4 expression or transcription in inflammatory macrophage models using cosmetic compounds and compositions.

[0147] Furthermore, to provide a model in which the endogenous constitutive production of TAFA4 must be restored in order to classify candidate compounds in the category of preferably ecological compounds intended for the prevention and / or care of sensitive and / or sensitized skin, a deletion of TAFA4 content (deletion of the expression of the gene encoding the TAFA4 protein and / or the amount of TAFA4 protein) can be implemented.

[0148] II.2 Cell culture Macrophages are immune cells capable of infiltrating tissues. Macrophages themselves differentiate from monocytes (blood cells) that differentiate from bone marrow stem cells. Macrophages and monocytes are phagocytic cells. Their role is to engulf cellular debris and pathogens, and they contribute to a broader immune response by synthesizing different types of mediators.

[0149] There are three main types of macrophages. M0 is an inactivated macrophage. M1 has pro-inflammatory properties. At the skin level, they are particularly involved in the destruction of pathogens, especially during the healing process. M2 possesses anti-inflammatory and pre-decomposing properties. At the skin level, they are particularly involved in the recovery phase of the healing process.

[0150] At the in vitro level, there are different cell models that closely resemble the macrophage phenotype.

[0151] Within this range, these different models can be used. Human monocyte strains: THP-1, U-937, SC, AML-193, HL-60 / S4

[0152] THP-1 is the most documented and used cell type in the literature.

[0153] Regarding THP-1: Culture medium: RPMI 1640 (Roswell Park Memorial Institute) + 10% FBS (fetal bovine serum) (Wang et al., 2015) ± 100 mg / L gentamicin, 4.5 g / L glucose, 1 mM pirubate, 0.05 mM 2-mercaptoethanol and 2 mM L-glutamine (Shen et al., 2014) ± penicillin (100 units / mL) and streptomycin (100 μg / mL) (Kawano et al., 2015) in a humid environment at 37°C with 5% CO2 in the air.

[0154] THP-1 can be differentiated into M0 macrophages, M1 macrophages, and M2 macrophages according to the culture conditions described by Horiba et al., 2022.

[0155] Macrophages are derived from monocytes, which are derived from human PBMCs (human peripheral blood mononuclear cells) derived from human peripheral blood samples.

[0156] PMBCs are isolated from blood using Polymorphprep. Monocytes (CD14+) are then collected after being allowed to adhere to PMBCs for 4 hours. Culture medium: RPMI 1640 + 10% FBS. In vitro stimulation with M-CSF (macrophage colony-stimulating factor), followed by treatment with LPS (Escherichia coli O55:B5) combined with IFN-γ (gamma interferon) or IL-4 (interleukin-4), differentiates monocytes into M0 macrophages. These can then be differentiated into M1 or M2 macrophages, respectively, for 24 hours (Wang et al., 2015) in a humid environment with 5% CO2 in the air and at 37°C.

[0157] Macrophages are derived from CD34+ hematopoietic stem cells. These cells are recovered in vivo from samples derived from bone marrow, peripheral blood, and human umbilical cord blood (Clanchy and Hamilton (2013)). Conventionally, these cells are recovered from umbilical cord blood.

[0158] II.3 Selection of Compounds or Compositions Effective for Inducing TAFA4 Expression or Synthesis II.3.1. Protocol Cells are seeded into 96-well plates in the corresponding complete culture medium. After culturing at 37°C for 24 hours in 5% CO2, the activator to be evaluated is added to the culture medium at a selected concentration, and the cells are incubated at 37°C for 48 or 72 hours in 5% CO2. Positive induction controls are evaluated in parallel with the activator.

[0159] After different incubation times, the supernatant is collected and stored at -80°C, then assayed for TAFA4 by ELISA. The assay is performed only if the cell viability is greater than 80%.

[0160] To evaluate TAFA4 expression by RT-qPCR, cells are harvested after incubation with an activator or positive control for a shorter kinetic period (30 minutes to 24 hours).

[0161] II.3.2. Inducing factors (positive controls) In a literature search, two inducers, LPS and UV, were identified as capable of inducing TAFA4 synthesis at the cutaneous level (Hoeffel et al., 2021; Wang et al., 2015).

[0162] II.3.3. In the case of LPS stimulation Monocytes / macrophages are pre-treated with PMA (phorbol-12-myristate-13-acetate, 10 ng / ml) for 24 hours, followed by stimulation with LPS (1 μg / ml) for 12 or 24 hours (Wang et al., 2015).

[0163] II.3.4. In the case of UV stimulation After removing the culture medium and rinsing with D-PBS, the cells are irradiated with UV light.

[0164] Data regarding the macrophage exposure protocol described: • In a Biosun System including an irradiation chamber equipped with a 365nm illumination lamp, dosimeter / detector, calibrator, and software, 6.7 J / cm² was administered for 15 minutes. 2 UVA dose (Shen et al., 2014); or UVA was measured using a black lamp with a peak emission energy of 360 nm. The emitted dose was measured by radiometer (UVX-36; UVP, Inc., San Gabriel, California). UVB was not detected by the UVX-31 detector. The sample-level irradiation dose was 2.5 mW / cm². 2 (Kawano et al., 2015).

[0165] II.4 Selection of Compounds or Compositions to be Evaluated Different molecules can be pre-selected and tested. These molecules will either be solubilized directly into the culture medium or, depending on their properties, into ethanol or DMSO.

[0166] II.5 MTT Cell Survival Rate Test See point I.7.

[0167] II.6 Quantification of TAFA4 expression by RT-qPCR After incubation with an activator for 30 minutes to 24 hours, total RNA extraction is performed using the Rneasy Plus Mini Kit, following the supplier's (Qiagen) recommendations.

[0168] Total RNA assays allow for the determination of the quantity and quality of RNA in cell extracts. These assays are performed using Bioanalyser and the RNA 6000 Nano kit.

[0169] Reverse transcription performed using the QuantiTect Reverse Transcription Kit (Qiagen) makes it possible to obtain cDNA.

[0170] Real-time quantitative PCR is performed on the TAFA4 gene at each treatment stage. The Qiagen QuantiFast SYBR Green PCR Kit is used. • Housekeeping gene: GAPDH • Target gene: TAFA4 → Refer to the gene's specific primer.

[0171] II.7 Assay of TAFA4 in culture supernatant by ELISA The quantitative determination of TAFA4 in the supernatant is performed using ELISA, i.e., Enzyme-Linked Immunosorbent Assay (reference: abx522987, Cliniscience, France) with reagents recommended by the manufacturer.

[0172] The optical density (OD) is read using a spectrophotometer at 450 nm. The concentration of TAFA4 is calculated from the standard range established for recombinant human TAFA4 protein (0.1561 ng / ml to 10 ng / ml).

[0173] The results are expressed as ng / ml of TAFA4 per well. An MTT viability test is performed in the cell layer. The TAFA4 assay is used only when cell viability is greater than 80%, and is associated with this cell viability value.

[0174] II.8 Statistical analysis Data were collected from three independent experiments conducted as a set. Quantitative analysis is presented in the form of mean ± standard deviation. Statistical significance is determined by the Student's Test. Differences are considered statistically significant if p < 0.05. (NS:p>0.05;*:p<0.05;**:p<0.01;***:p<0.001).

[0175] (Example III - Differentiation of SH-SY5Y cells into sensory nerve cells and analysis of TAFA4 levels) III.1 Introduction The purpose of this study is to investigate the regulation of TAFA4 expression in neuronal cell types capable of producing TAFA4.

[0176] III.2 Materials and Methods III.2.1 SH-SY5Y cell cultures and differentiation The SH-SY5Y human neuroblastoma cells used in this study were obtained from Sigma Aldrich (94030304). The cells were thawed in a 37°C water bath with P13, and then immediately placed in DMEM. The cell suspension was centrifuged at 125×g for 7 minutes at 4°C, 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.

[0177] Cells were seeded into a 96-well plate at a density of 7000 cells per well and cultured in an incubator at 37°C with 95% air and 5% CO2. To avoid any edge effects, the first and last columns, as well as the first and last systems of the plate, were not used in this study. Empty wells were filled with water. The cells were allowed to grow to confluence (80%).

[0178] At confluence (after 1 day), cells 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 μM retinoic acid (RA), allowing for their differentiation. The medium was changed every 2 days. Only 60 wells were used from a 96-well plate.

[0179] III.2.2 Expression / release of TAFA4 at different time points (D8, D10, D12) after exposure to the compound Eight, ten, or twelve days after differentiation by RA, cells are stressed for 24 hours with two compounds individually diluted at different concentrations in the culture medium. Simultaneously, a control condition without stress is maintained at each differentiation stage (eight, ten, and twelve days; reference values).

[0180] Apply only 100 μL of culture medium.

[0181] III.2.3 qPCR:TAFA4 gene expression Several hours after applying the stressor (following the stressor and procedure), lyse the cells with the RNS XS nucleo spin kit (Macherey Nagel). Briefly, add 100 μL of lysis buffer per well according to the manufacturer's (Macherey Nagel) instructions. The procedure includes DNase treatment. Perform RNA extraction in a laboratory room dedicated to biomolecular experiments using sterile DNase / RNase-free solution and laboratory consumables (e.g., filter tips, tubing). Assess the total amount and purity of RNA present in each sample by spectrophotometry using a NanoDrop instrument (NanoDrop technologies LLC). Store RNA samples at temperatures below -70°C. Obtain cDNA by reverse transcription from 25 ng of total RNA using a major script reverse transcriptase (SensiFAST cDNA synthesis kit, Bioline meridian) according to the manufacturer's instructions. Store cDNA samples at -15 / -25°C. Perform the PCR reaction using the CFX96® real-time system (Biorad). Using 3 μM primers and SYBR Premix (ONEGreen Fast qPCR Premix, OZYME), 10 ng of initial RNA equivalents is subjected to PCR amplification. The PCR reaction is achieved using the following cycling method (30 seconds at 95°C, then 5 seconds at 95°C, then 30 seconds at 60°C), for a total of 40 cycles.

[0182] To measure the relative expression levels of mRNA encoding the selected genes, each sample is analyzed for the selected genes (GAPDH and TAFA4) using RT-qPCR. The gene expression analysis is evaluated using the 2-ΔΔCt method (Livak and Schmittgen (2001)) where Ct is the cycle threshold. A negative control (without cDNA) is performed for each pair of primers. The expression is standardized against a housekeeping gene (e.g., GAPDH).

[0183] III.2.4 Statistical analysis All values ​​are expressed as mean ± SEM (standard error of the mean). After one-way ANOVA, statistical analysis is performed using Fisher's LSD test. p < 0.05 is considered statistically significant.

[0184] III.3 Results and Conclusions These studies made it possible to identify regulatory substances, namely inhibitors and inducers of TAFA4 expression in SH-SY5Y cells.

[0185] (Example IV - Evaluation of the effect of a compound on TAFA4 synthesis in normal human fibroblast cultures) IV.1 Introduction The objective of this study is to develop a cell model that enables the quantification of TAFA4 synthesis in skin cells.

[0186] IV.2 Materials and Methods IV.2.1 Cell culture In this study, normal human fibroblasts (NHF) were used (Lonza, Switzerland). These cells were seeded in 24-well plates at a rate of 10,000 cells per well in DMEM + 1% penicillin-streptomycin (P / S). After 24 hours of incubation, the cells were incubated with the compounds listed in Table 1.

[0187] [Table 1]

[0188] After incubation at 37°C with 5% CO2 for 72 hours, the supernatant and protein extract (obtained from the cell layer) are collected and stored at -80°C.

[0189] Before assaying the TAFA4 protein, assess the toxicity of the compound being tested using the MTT test. Select only concentrations that allow for a survival rate of over 80% to be quantified by ELISA and dot blotting.

[0190] Data was collected from three independent experiments conducted in a triad.

[0191] IV.2.2 MTT cell viability test See point I.7.

[0192] IV.2.3 TAFA4 array by dot blotting Dot blotting is an immunoenzyme detection method for quantifying proteins in a sample after they have been deposited onto a nitrocellulose membrane in a well. Specific equipment (Biorad, France) is used in conjunction with the nitrocellulose membrane. The equipment and consumables used in this experiment are shown in Table 2 below.

[0193] [Table 2]

[0194] The apparatus is subjected to a vacuum pressure that allows the sample (supernatant or protein extract) to be aspirated, and thus the protein to bind to the nitrocellulose membrane. Taking the principle of gravity into account, the apparatus is positioned at a height relative to the vacuum pump.

[0195] Pre-hydrate the membrane with 100 μl of TBS buffer (Tris base + NaCl diluted with water, adjusted to pH 7.5) per well. Repeat this process twice to optimize membrane hydration for optimal binding of proteins in the sample. Attach the analyte (supernatant and protein extract) and different solutions of recombinant TAFA4 protein at different concentrations within the TAFA4 standard range (0.78–100 ng / ml) to the plate (100 μl / well volume is used for protein extract, and 500 μl / well volume for supernatant), then activate the aspirate flow. After aspirating all of the sample, add 100 μl of TBS per well to confirm that all of the sample passes through the membrane. Thus, the proteins in the sample will adhere to the nitrocellulose membrane, and 1 cm 2 It creates spots.

[0196] For labeling and marking, the nonspecific sites are saturated by incubating the membrane in a bath containing 5% skim milk in TBS buffer (vol: 30 ml) under stirring overnight at 4°C. The following day, the membrane is washed with 20 ml of TBS-Tween buffer (0.05% TBS + Tween 20) under stirring for 10 minutes. This washing is repeated three times, changing the washing buffer (vol: 30 ml). The membrane is incubated for 2 hours at ambient temperature under stirring in a bath containing primary antibody diluted to 1 / 200 in TTBS buffer + 1% skim milk (vol: 20 ml). The membrane is then washed in the same manner as above.

[0197] Next, the membrane is incubated for 2 hours at ambient temperature under stirring in a solution containing TTBS buffer + 1% skim milk (volume: 20 ml) diluted to 1 / 500 (antibody conjugated to HRP). The membrane is washed in the same manner as above. The secondary antibody alone is used as a negative control.

[0198] The presence of proteins in each deposit is indicated by incubating the membrane in a bath of pure ECL (a chemiluminescent substrate for detecting the activity of peroxidase (HRP, horseradish peroxidase) conjugated to a secondary antibody). The presence of TAFA4 protein in the membrane is indicated by the formation of gray to black spots that depend on the intensity of the reaction, and therefore on the amount of protein. The membrane is read by a Fusion FX instrument (Vilber Lourmat) coupled with VisionCapt software (Vilber Lourmat). Quantification of TAFA4 protein in the sample under test is performed using VisionCapt software, which allows for measurement of the intensity of various spots. Then, a sample volume is generated using a standard curve of TAFA4 standards established by recombinant protein.

[0199] IV.2.4 Statistical analysis Data were collected from three independent experiments conducted as a set. Quantitative analysis is presented in the form of mean ± standard deviation. Statistical significance is determined by the Student's Test. Differences are considered statistically significant if p < 0.05. (NS:p>0.05;*:p=0.05;**:p=0.01;***:p=0.001).

[0200] IV.3 Results and Conclusions Figure 1 shows the effects of valproic acid, TGF-β, and LPS on the regulation of TAFA4 in the NHF supernatant after 72 hours of incubation. Results are expressed as the percentage of TAFA4 expression, standardized to 100% (DMEM medium control). Data were obtained from two independent experiments conducted in triplicates.

[0201] Valproic acid and TGF-β induce TAFA4 synthesis in a dose-dependent manner (for TGF-β, both of the two doses showed statistically significant induction, and for valproic acid, one of the two doses showed statistically significant induction).

[0202] LPS inhibits TAFA4 synthesis (statistically significant inhibition) at the dose tested.

[0203] (Example V - Evaluation of the effect of a compound on TAFA4 synthesis in normal human keratinocyte cultures) V.1 Preface The objective of this study is to develop a cell model that enables the quantification of TAFA4 synthesis in skin cells.

[0204] V.2 Materials and Methods V.2.1 Cell culture In this study, normal human keratinocytes (NHK) were used. These were obtained from Promocell (Germany). Cells were seeded at a rate of 100,000 cells / well in 24-well plates using KGM. After 24 hours, the cells were incubated at 37°C with 5% CO2 in KBM+P / S medium with the compounds shown in the table below.

[0205] After 72 hours of incubation, the supernatant and protein extract (obtained from the cell layer) were collected and stored at -80°C. The test compounds are shown in Table 3 below.

[0206] [Table 3]

[0207] After incubation at 37°C with 5% CO2 for 72 hours, the supernatant and protein extract (obtained from the cell layer) are collected and stored at -80°C.

[0208] Before assaying the TAFA4 protein, assess the toxicity of the compound using the MTT test. Select only concentrations that allow for a survival rate of over 80% to be quantified by ELISA and dot blotting.

[0209] Data was collected from three independent experiments conducted in a triad.

[0210] V.2.2 MTT cell viability test See point I.7.

[0211] V.2.3 TAFA4 array by dot blotting See point IV.2.3.

[0212] V.2.4 Statistical analysis See point IV.2.4.

[0213] V.3 Results and Conclusions Figure 2 shows the effect of EGF on the regulation of TAFA4 in NHK supernatant after 72 hours of incubation. The results are expressed as the percentage of TAFA4 expression, standardized to 100% (KGM medium control). Data were obtained from three independent experiments.

[0214] EGF tested at 30 ng / ml inhibited TAFA4 synthesis (statistically significant).

[0215] literature Atef A., El-Rashidy MA, Abdel Azeem A., Kabel AM (2019) The Role of Stem Cell Factor in Hyperpigmented Skin Lesions. Asian Pac J Cancer Prev.;20(12):3723-3728.

[0216] Delfini, M.-C., Mantilleri, A., Gaillard, S., Hao, J., Reynders, A., Malapert, P., Alonso, S., Francois, A., Barrere, C., Seal, R., et al. (2013). TAFA4, a chemokine-like protein, modulates injury-induced mechanical and pain chemical hypersensitivity in mice. Cell Rep 5:378-388.

[0217] Grabbe J., Welker P., Dippel E., Czarnetzki BM (1994) Stem cell factor, a novel cutaneous growth factor for mast cells and melanocytes. Arch Dermatol Res. 287:78-84.

[0218] 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.

[0219] 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.

[0220] Huet F,Dion A,Batardiere A,Nedelec AS, Le Caer 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.

[0221] 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.

[0222] Kambrun,C.,Roca-Lapirot,O.,Salio,C.,Landry,M.,Moqrich,A.,and Le Feuvre,Y.(2018).TAFA4 Reverses Mechanical Allodynia through Activation of GABAergic Transmission and Microglial Process Retraction.Cell Rep 22:2886-2897.

[0223] 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.

[0224] Leeman,A.,Jenkins,D.,Del Pino,M.,Ordi,J.,Torne,A.,Doorbar,J.,Meijer,C.J.L.M.,van Kemenade,F.J.,and 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.

[0225] Livak KJ,Schmittgen TD.(2001)Analysis of relative gene term data using real-time quantitative PCR and the 2(-Delta C(T))Method.Methods.25(4):402-8

[0226] 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.

[0227] Misery L.,Stander S.,Szepietowski J.C.,Reich A.,Wallengren J,Evers A.W.,Takamori K.,Brenaut E.,Le Gall-Ianotto C.,Fluhr J.,Berardesca E.,Weisshaar E.(2017)Definition of Sensitive Skin:An Expert Position Paper from the Special Interest Group on Sensitive Skin of the International Forum for the Study of Itch.Acta Derm Venereol.97:4-6.

[0228] Misery L,Jourdan E, Huet F,Brenaut E,Cadars B,Virassamynaik 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.

[0229] Misery L,Jourdan E,Abadie S,Ezzedine K,Brenaut E,Huet F,Sayag M,Taieb C(2018b).Development and validation of a new tool to assess the Burden of Sensitive Skin(BoSS).J Eur Acad Dermatol Venereol.12:2217-2223.

[0230] 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.

[0231] 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.

[0232] 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.

[0233] 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.

[0234] Shen,C.,Turney,T.W.,Piva,T.J.,Feltis,B.N.,and Wright,P.F.A.(2014).Comparison of UVA-induced ROS and sunscreen nanoparticle-generated ROS in human immune cells.Photochem Photobiol Sci 13:781-788.

[0235] Stander,S.,Schneider S.W.,Weishaupt,C.,Luger,T.A.,and Misery,L.(2009)Putative neuronal mechanisms of sensitive skin Exp.Dermatol.,18:417 423.

[0236] 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.

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Claims

1. A method for selecting at least one compound, preferably ecobiological, intended for the prevention and / or care of sensitive and / or sensitized skin, which involves identifying the content of TAFA4 and / or the nucleic acid encoding TAFA4, preferably the messenger ribonucleic acid (mRNA), and preferably the content of the messenger ribonucleic acid (mRNA), with the intention of controlling or increasing its content.

2. The following steps: a) Contacting at least one of the compounds with at least one skin cell, b) Measuring the content of TAFA4 and / or the nucleic acid encoding TAFA4, c) Compare the value obtained in step b) with the reference value. d) Selecting at least one compound that induces an increase in the content of TAFA4 and / or nucleic acids in step c), The method according to claim 1, characterized by including

3. The method according to claim 2, characterized in that the reference value is a measurement value obtained without bringing the at least one skin cell into contact with the at least one compound.

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

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

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

7. The method according to any one of claims 1 to 6, wherein the method is an in vivo or ex vivo method, and the contact in step a) is carried out by topical application of the at least one compound to the skin of a whole organism, preferably to the skin of a whole non-human organism, a skin explant, reconstructed epidermis or reconstructed skin.

8. - The above method is an in vitro method, - The at least one skin cell is selected from keratinocytes, melanocytes, sebocytes, Merkel cells, vascular smooth muscle cells, special muscle cells, fibroblasts, mast cells, lymphocytes, monocytes, dendritic cells, preferably cutaneous dendritic cells; Langerhans cells, adipocytes, nerve cells, macrophages, preferably M1-type macrophages or M2-type macrophages; endothelial cells; and mixtures thereof; preferably selected from keratinocytes, fibroblasts and mixtures thereof; and - The contact in step a) is performed by adding to the culture medium. The method according to any one of claims 2 to 6, characterized in that

9. The aforementioned at least one skin cell, - At least one macrophage, preferably at least one M1 type macrophage, more preferably at least one human M1 type macrophage; and / or, - At least one keratinocyte, preferably at least one human keratinocyte, more preferably at least one normal human keratinocyte; and / or - At least one fibroblast, preferably at least one human fibroblast, preferably at least one normal human fibroblast; and / or • At least one nerve cell, The method according to claim 8, characterized in that...

10. The method according to any one of claims 2 to 9, characterized in that step b) measuring the content of TAFA4 and / or the nucleic acid encoding TAFA4, preferably mRNA, is carried out at the protein level by ELISA, immunohistochemistry, colorimetric method, Western blotting, dot blotting, or Western blotting simplified by capillary immunoelectrophoresis (WES), and / or carried out at the nucleic acid level by in situ hybridization, Northern blotting, sequencing, or RT-PCR, preferably RT-qPCR.

11. The method according to any one of claims 2 to 10, characterized in that, prior to carrying out step a), at least one skin cell is brought into contact with a sensitizer, preferably a cosmetic sensitizer, more preferably a surfactant or preservative, by topical application or by addition to a culture medium.

12. - The amino acid sequence of TAFA4 corresponds to the sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2, and / or corresponds to at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, or even more than 90% of the sequence of one of SEQ ID NOs: 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, and / or corresponds to at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, or even more than 90% of the same sequence as one of SEQ ID NOs: 3 to 7. The method according to any one of claims 1 to 11, characterized in that

13. A method for producing a cosmetic composition and / or food composition in a non-therapeutic, preferably ecobiological, and more preferably topical and / or orally administered formulation, comprising incorporating into the composition at least one compound selected by the method of any one of claims 1 to 12.

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