Atopic dermatitis improving agent, inhibitory T cell differentiation inducing agent, food composition, health food, cosmetics, and inhibitory T cell differentiation inducing method

Abstract

To provide a new atopic dermatitis improver or the like to solve problems such as side effects arising from prolonged use of topical steroids.SOLUTION: The present invention provides an atopic dermatitis improver that comprises a bird's nest-derived component as an active ingredient; specifically, a regulatory T-cell differentiation inducer that induces differentiation into regulatory T-cells, comprising a bird's nest-derived component as an active ingredient. According to aspects of the present invention, it is possible to provide an atopic dermatitis improver or the like that can be used without concern for side effects.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to an agent for improving atopic dermatitis, an agent for inducing differentiation of regulatory T cells, a food composition, a health food, a cosmetic product, and a method for inducing differentiation of regulatory T cells. [Background technology]

[0002] The immune system is a system that distinguishes between "self" and "non-self" and recognizes and eliminates infectious microorganisms and self-derived mutated cells as "non-self" while maintaining unresponsiveness to "self" (immune tolerance).

[0003] In the immune system, helper T cells receive antigen information from antigen-presenting cells such as macrophages and dendritic cells, differentiate according to the information they receive, and release various cytokines to control the immune response.

[0004] They are also called CD4-positive T cells because they express the CD4 antigen on their cell surface. One type of CD4-positive T cells, Tregs, are defined as "T cells that have the function of suppressing immune responses and are responsible for immune tolerance." In 1995, Sakaguchi et al. discovered that "CD4-positive T cells that constitutively express the CD25 molecule suppress immune responses," and these cells were later named regulatory T cells (Tregs). Subsequently, the transcription factor Foxp3 was identified as a specific molecular marker for Tregs, establishing their physiological significance and leading to rapid progress in elucidating the development, differentiation, and molecular mechanisms of Tregs.

[0005] The main role of Tregs is immune tolerance, which suppresses strong immune responses to self-antigens and non-self but non-pathogenic antigens. It has been reported that depletion of Tregs from normal mice results in type 1 diabetes, which closely resembles human autoimmune diseases, inflammatory bowel disease caused by an exaggerated immune response to intestinal bacteria, and allergies caused by an exaggerated immune response to environmental substances. Tregs are also known to control immune homeostasis and balance inflammation by interacting with effector T cells and antigen-presenting cells.

[0006] For this reason, activation of Tregs is thought to be an effective means of avoiding inflammation caused by immune hyperactivity, and active research is being conducted into the clinical application of Tregs in the treatment of allergies and autoimmune diseases, and in alleviating rejection reactions during organ transplants (Non-patent documents 1, 2, 3).

[0007] A typical example of an excessive immune response is atopic dermatitis, which is one of the most common skin diseases. Topical steroids are currently used as one of the main treatments for atopic dermatitis. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Tomohiro Fukaya et al., Crucial roles of B7-H1 and B7-DC expressed on mesenteric lymph node dendritic cells in the generation of antigen-specific CD4+Foxp3+ regulatory T cells in the establishment of oral tolerance, Blood. 116(13): 2266-2276, 2010 [Non-patent document 2] Akamatsu et al., Conversion of antigen-specific effector / memory T cells into Foxp3-expressing Treg cells by inhibition of CDK8 / 19. Science Immunology 25 Oct 2019:Vol. 4, Issue 40, eaaw2707 [Non-patent document 3] Di Ianni M et al., Tregs prevent GVHD and promote immune reconstitution in HLA-haploidentical transplantation. Blood,117: 3921-3928,2011 Summary of the Invention [Problem to be solved by the invention]

[0009] However, topical steroids have problems such as side effects that occur when used for a long period of time.

[0010] Therefore, an object of the present invention is to provide a new agent for improving atopic dermatitis. [Means for solving the problem]

[0011] A first aspect of the present invention is an agent for improving atopic dermatitis, which comprises a swallow's nest-derived ingredient as an active ingredient.

[0012] A second aspect of the present invention is a regulatory T cell differentiation inducer that induces differentiation into regulatory T cells, and comprises a swallow's nest-derived component as an active ingredient.

[0013] A third aspect of the present invention is the regulatory T cell differentiation inducer of the second aspect, wherein the active ingredient improves the ability to produce retinoic acid.

[0014] A fourth aspect of the present invention is the regulatory T cell differentiation inducer of the third aspect, wherein the active ingredient increases the transcription level of the RALDH2 gene.

[0015] A fifth aspect of the present invention is the regulatory T cell differentiation inducer of the second aspect, wherein the active ingredient increases the transcription level of the TGF-β gene.

[0016] A sixth aspect of the present invention is the regulatory T cell differentiation inducer according to the second aspect, wherein the active ingredient reduces the transcription level of the TNF-α gene and / or the IL-13 gene.

[0020] A tenth aspect of the present invention is a food composition comprising the active ingredient according to any one of the first to ninth aspects.

[0021] An eleventh aspect of the present invention is a health food comprising the active ingredient according to any one of the first to ninth aspects.

[0022] A twelfth aspect of the present invention is a cosmetic product comprising the active ingredient according to any one of the first to ninth aspects.

[0023] A thirteenth aspect of the present invention is a method for inducing differentiation of regulatory T cells, which induces differentiation into regulatory T cells, the method comprising the step of applying to a subject an external preparation containing a swallow's nest-derived component as an active ingredient. [Effects of the Invention]

[0024] According to each aspect of the present invention, it is possible to provide an agent for improving atopic dermatitis that can be used without worrying about side effects.

[0025] In particular, according to the second to ninth aspects of the present invention, it is possible to provide novel inducers of regulatory T cell differentiation.

[0026] According to the tenth to twelfth aspects of the present invention, it is possible to provide an agent for improving atopic dermatitis that can be taken orally. [Brief explanation of the drawings]

[0027] [Figure 1] FIG. 1 is a diagram showing an outline of the mechanism of onset of atopic dermatitis. [Figure 2] FIG. 10 shows the fluorescence values ​​of Raldh2 promoter-EGFP in Kaempferol, Quercetin, and bird's nest samples. [Figure 3] FIG. 1 shows the expression levels of the endogenous RALDH2 gene in Kaempferol, Quercetin, and bird's nest samples. [Figure 4] FIG. 1 shows the state of scabs in a mouse model induced with atopic dermatitis. [Figure 5] FIG. 1 shows changes in inflammation scores in atopic dermatitis-like induced model mice. [Figure 6] FIG. 1 shows changes in scab area in atopic dermatitis-like induced model mice. [Figure 7] FIG. 1 shows the state of the ear in an atopic dermatitis-like induced model mouse. [Figure 8] FIG. 1 shows the ear thickness in atopic dermatitis-like induced model mice. [Figure 9] 1 shows a micrograph of the dorsal tissue of a mouse model induced with atopic dermatitis. [Figure 10] FIG. 1 shows the epidermal thickness in the dorsal tissue of atopic dermatitis-like induced model mice. [Figure 11] FIG. 1 shows the dermis thickness in the dorsal tissue of an atopic dermatitis-like induced model mouse. [Figure 12] FIG. 1 shows the number of infiltrating cells in the dorsal tissue of atopic dermatitis-like induced model mice. [Figure 13] FIG. 1 shows the expression level of the TNF-α gene in the dorsal tissue of atopic dermatitis-like induced model mice. [Figure 14] FIG. 1 shows the expression level of the IL-13 gene in the dorsal tissue of atopic dermatitis-like induced model mice. [Figure 15] FIG. 1 shows the expression level of the Foxp3 gene in the dorsal tissue of atopic dermatitis-like induced model mice. [Figure 16] FIG. 1 shows the expression level of the TGF-β gene in the dorsal tissue of atopic dermatitis-like induced model mice. DETAILED DESCRIPTION OF THE INVENTION

[0028] Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. However, the present invention is not limited to the following examples. [Example]

[0029] 1. Background Figure 1 shows an overview of the mechanism of atopic dermatitis development. Th1, Th2, and Th17 are subsets of helper T cells that are closely related to Tregs. These cells release various cytokines to activate the immune system and play an important role in infection defense and other areas. It is believed that an imbalance of these four cells in the body can cause disease, and atopic dermatitis is a prime example of an excessive immune response.

[0030] Atopic dermatitis is one of the most common skin diseases. Topical steroids are currently used as one of the main treatments for atopic dermatitis, but they have problems such as side effects that occur with long-term use. The pathology of this disease includes immune response breakdown and barrier function abnormalities, and it has been reported that the enhancement and activation of Th2 responses and enhancement of IgE responses are major factors that induce atopic dermatitis.

[0031] During the acute phase of atopic dermatitis, Th2 cells, which produce cytokines such as IL-4 and IL-13, are concentrated in large numbers at the lesion site, suggesting that Th2 cells play an important role in the early inflammation of atopic dermatitis. During the chronic phase, mast cells and Th2 cells release cytokines such as IL-5, which act on eosinophils. Eosinophils are known to infiltrate the inflammatory site and secrete granule proteins and cytokines, further promoting the inflammatory response.

[0032] Transforming growth factor-β (TGF-β) and retinoic acid (RA) are essential for the induction of peripheral Tregs. Retinoic acid, in particular, has been reported to affect the function, differentiation, and proliferation of immune cells, promoting Treg differentiation and suppressing the differentiation of Th17, which releases inflammatory cytokines. Retinoic acid is a vitamin A metabolite responsible for the physiological function of vitamin A. Retinol, the basic form of vitamin A, is converted to retinoic acid via retinal. The enzyme that catalyzes the conversion of retinal to retinoic acid is called retinal dehydrogenase (RALDH), and there are three isoforms: RALDH1, RALDH2, and RALDH3. Dendritic cells in gut-related tissues are known to primarily express RALDH2.

[0033] The present inventors came up with the idea of ​​inducing immune tolerance by activating RALDH2 and increasing Treg through diet, which is a daily activity.

[0034] Therefore, the present inventors have focused on RALDH2 and constructed a screening system for RALDH2-activating food ingredients. In the search for and verification of candidate Treg-enhancing ingredients, it was revealed that kaempferol, a type of polyphenol, induces Treg differentiation and suppresses dextran sulfate sodium (DSS)-induced enteritis in mice.

[0035] The present inventors screened food ingredients to identify Treg-inducing foods, and then directly applied the identified Treg-inducing food ingredients to the back of a mouse model of atopic dermatitis to verify their functionality.

[0036] 2. Experimental Materials and Methods 2-1.Cell culture 2-1-1. Cultivation of THP-1 cells, a human acute monocytic leukemia-derived cell line This study used THP-1 cells, a human acute monocytic leukemia-derived cell line. THP-1 cells were subcultured in RPMI 1640 medium (Nissui, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS; Life Technologies, CA, USA) (complement inactivated by heating in a 56°C incubator for 30 minutes) in Petri dishes (FALCON, Tokyo, Japan) at 37°C in the presence of 5% CO2. RPMI 1640 medium was prepared by dissolving 5.1 g of RPMI 1640 powder in 500 mL of Milli-Q water, sterilizing by autoclaving, and then adding 0.5 mL of 0.1 g potency streptomycin sulfate (Meiji Seika Pharma, Tokyo, Japan), 1 mL of 100,000 U penicillin G potassium (Meiji Seika Farm, Tokyo, Japan), and 8 mL of 10% NaHCO3 (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). The medium was stored at 4°C.

[0037] 2-1-2. Cultivation of THP-1 (Raldh2p-EGFP) cells We used THP-1(Raldh2p-EGFP) cells, established in a previous study, transfected with a mouse Raldh2p-EGFP reporter vector. THP-1(Raldh2p-EGFP) cells are a stable cell line that expresses EGFP fluorescence in response to activation of the RALDH2 promoter. As with THP-1 cells, they were subcultured in RPMI 1640 medium supplemented with 10% FBS in a Petri dish at 37°C in the presence of 5% CO2.

[0038] 2-1-3. Differentiation induction of THP-1 cells THP-1 cells exhibit macrophage-like morphology upon differentiation. In this experiment, THP-1 cells were cultured for 48 hours in RPMI 1640 medium containing 10% FBS after adding phorbol 12-myristate 13-acetate (PMA; LC Laboratories, Germany) to a final concentration of 100 ng / mL.

[0039] 2-2. Samples and their preparation methods Kaempferol and Quercetin were tested with bird's nests (samples A to C). The bird's nests were provided by M-Style Japan Co., Ltd. The bird's nest samples were prepared at 150 μg / mL in 1x PBS. Each sample was stored at -20°C and thawed as needed before use. The bird's nests included at least swiftlet nests and petrel nests.

[0040] 2-3. Search for food ingredients that activate the Raldh2 promoter using the IN Cell Analyzer 2200 The Raldh2 promoter activation ability was evaluated using THP-1 (Raldh2p-EGFP) cells to which food ingredient samples had been added. THP-1 (Raldh2p-EGFP) cells were cultured at a final concentration of 6.0 × 10 5 The cells were seeded onto a 96-well black plate (GmbH, Kremsmünster, Austria) at a concentration of 15000 cells / mL and induced to differentiate for 48 hours. After differentiation induction, bird's nest was added to the cells at a final concentration of 150 μg / mL to 15,000 μg / mL, and the cells were cultured at 37°C in the presence of 5% CO2 for 24 hours. DMSO was used as a food component as a control, and 1x PBS was used for the bird's nest. After incubation, 100 μL / well of 8% paraformaldehyde was added to the culture medium to a final concentration of 4%. The cells were fixed by incubating at room temperature for 15 minutes. 8% paraformaldehyde was prepared by dissolving paraformaldehyde (FUJIFILM Wako, Tokyo, Japan) in 1x PBS to 8% paraformaldehyde, adding 5 μg / mL of 2 N mol / L sodium hydroxide solution, and dissolving at 60°C. After 15 minutes, the fixative was removed and the cells were washed twice with 1x PBS. Then, 100 μL / well of Hoechst 33342 solution (Dojindo, Kumamoto, Japan) diluted to a final concentration of 2 μg / mL was added and the cells were incubated at room temperature for 20 minutes to stain the nuclei. After 20 minutes, the Cellstain®-Hoechst 33342 solution was removed and the cells were washed twice with 1x PBS. 100 μL of PBS was added to each well, and EGFP fluorescence intensity was measured using an IN Cell Analyzer 2200. The protocol used was "HaCaT-eGFP 20190627_210203_rev."

[0041] 2-4. Measurement of endogenous RALDH2 gene expression by quantitative RT-PCR 2-4-1.Food ingredient processing The effects of Raldh2 promoter activity on endogenous RALDH2 expression were examined by quantitative RT-PCR using parent THP-1 cells in the swallow's nest samples selected by the IN Cell Analyzer 2200. THP-1 cells were cultured at a final concentration of 6.0 × 10 5 THP-1 cells were seeded into a 6-well plate at a final concentration of 150 μg / mL. After inducing differentiation of THP-1 cells for 48 hours, bird's nest samples were added to a final concentration of 150 μg / mL. DMSO was added instead of bird's nest samples as a control.

[0042] 2-4-2.Total RNA extraction Total RNA was prepared using the High Pure RNA Isolation Kit (Roche, Basel, Switzerland) according to the manufacturer's protocol. All reagents and equipment used from total RNA preparation to the completion of the reverse transcription reaction were RNase-free.

[0043] First, 6.0 × 10 5Cells were seeded at 0.1 cells / mL and cultured at 37°C in DMEM medium containing 10% FBS. After 24 hours, each sample was added to a concentration of 10 μM. After addition, the cells were cultured at 37°C for 48 hours in DMEM medium containing 10% FBS. After 48 hours, the medium was completely removed and the cells were washed with 200 μL of 1x PBS. Then, 200 μL of 1x PBS and 400 μL of the cell lysis solution (lysis / binding buffer) included in the High Pure RNA Isolation Kit were added. The cell lysate was lysed by spreading it throughout the dish. The entire cell lysate was collected into a 1.5 mL tube. The collected sample was thoroughly suspended in a vortex mixer for 60 seconds and briefly spun down. The High Pure Filter tubes and collection tubes included in the kit were assembled, and the cell lysate was added to the filter tubes. The samples were centrifuged at 10,000 × g for 15 seconds at room temperature, and the liquid discharged into the collection tubes was discarded. The High Pure Filtert tubes and collection tubes were reassembled. To each 1.5 mL tube, 90 μL of DNase Incubation buffer and 10 μL of DNase I were added and mixed. This mixture was added to the Filtert tubes and allowed to stand at room temperature for 15 minutes. After 15 minutes, 500 μL of Wash Buffer I (included in the kit) was added to the Filtert tubes and centrifuged at 10,000 × g for 15 seconds at room temperature. After centrifugation, the liquid discharged into the collection tubes was discarded, and the Filtert tubes and collection tubes were reassembled. 500 μL of Wash Buffer II was added to the Filtert tubes and centrifuged at 10,000 × g for 15 seconds at room temperature. After centrifugation, the liquid discharged into the collection tubes was discarded, and the Filtert tubes and collection tubes were reassembled. Further, 200 μL of Wash Buffer II was added, and the mixture was centrifuged at room temperature at 14,000 × g for 2 minutes.After centrifugation, the Filtert tubes were inserted into new 1.5 mL tubes, and 50 μL of elution buffer was added to the center of the tubes. The tubes were then left to stand at room temperature for 3 minutes. The tubes were then centrifuged at 10,000 × g for 1 minute at room temperature to elute the total RNA. The concentration of the eluted total RNA was measured based on the absorbance at 260 nm using a Nano Drop 2000c (Thermo Fisher Scientific, Waltham, USA).

[0044] 2-4-3. cDNA synthesis using reverse transcriptase ReverTra Ace 1.0 μg of total RNA extracted from cells was mixed with 5 pmol of Oligo(dT)20 primer (TOYOBO, Osaka, Japan), and RNase-free water was added to a total volume of 13 μL. The reaction was performed at 65°C for 5 minutes in a Thermal Cycler PTC-200 (MJ Research, Waltham, MA, USA) and immediately transferred to ice for rapid cooling. During this time, the reverse transcriptase reaction program was advanced to the 42°C step and then paused. After cooling the sample on ice for 5 minutes, a mixture of 4 μL of reverse transcriptase reaction buffer, 2 μL of 10 mM dNTPs (GE Healthcare), and 0.5 μL of reverse transcriptase ReverTra Ace (100 units / μL) (TOYOBO) was added per sample and mixed gently. cDNA was then synthesized by incubation at 42°C for 20 minutes, 99°C for 5 minutes, and 4°C for 5 minutes. This cDNA was used as a template for quantitative RT-PCR.

[0045] 2-4-4. Primer design The target genes whose expression levels were measured by quantitative RT-PCR were searched for on NCBI (http: / / www.ncbi.nlm.nih.gov / gene / ), and the primer sequences were determined and synthesized based on the sequences. Primer synthesis was outsourced to Takara. The primers used to detect the internal control β-actin and the target genes are shown in Table 1.

[0046] [Table 1]

[0047] 2-4-5. Quantitative RT-PCR reaction The cDNA prepared by the above method was used as a template. To a 0.2 mL tube, 49 μL of sterile water was added, along with 3.5 μL each of forward and reverse primers diluted to 10 pmol / mL, 7.0 μL of template cDNA (primers for the target gene diluted 1 / 5, β-actin diluted 1 / 50), and 24.5 μL of THUNDERBIRD SYBR qPCR Mix (TOYOBO)—a high-efficiency real-time PCR master mix (2x concentration). After thorough mixing, 25 μL of each sample was added to three wells of a 96-well plate. Quantitative RT-PCR was performed using a Thermal Cycler Dice Real Time System (TaKaRa BIO, Shiga, Japan). PCR reactions consisted of one cycle of 95°C for 30 seconds followed by 45 cycles of 95°C for 5 seconds, 55°C for 10 seconds, and 72°C for 20 seconds. Detection was performed by FAM. The expression level of the target gene was relatively quantified using the ΔΔCt method.

[0048] 2-5.RNA-seq analysis using THP-1 cells 6.0 × 10 THP-1 cells 5Cells were seeded into a 5 mL dish at 100 cells / mL and induced to differentiate with PMA for 48 hours. The bird's nest sample selected in Section 4 was then added to a final concentration of 150 μg / mL, and Kaempferol and Quercetin, also selected in Section 4, were added to a final concentration of 10 μM each. After 24 hours, the culture supernatant was removed and the cells were washed with 1x PBS. 2 mL of TRIzol reagent was added, and the dish was tilted and rocked simultaneously for 1 minute. The dish was scraped with a cell scraper (Sarstedt, Tokyo, Japan) for 30 seconds, and the cells were collected into a 1.5 mL sample tube. The collected samples were instantly cooled in liquid nitrogen and stored at -80°C. RNA-seq experiments were outsourced to Cell Innovator, Inc. Based on the data obtained, cells with a read count of 100 or more and a logFc value of 0.27 or greater or -0.27 or less were selected compared to the control. The selected genes were analyzed using DAVID (https: / / david.ncifcrf.gov / ). The EntrezGeneIDs of the extracted altered genes were entered into the DAVID database, and Functional Annotation Clustering was performed to cluster them by function. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis was also performed using DAVID.

[0049] 2-6.Animal testing 2-6-1. Animals and breeding methods C57BL / 6 mice (6 weeks old, n = 5) were purchased from CLEA Japan, Inc. (Tokyo, Japan). They were housed in small mouse cages (NATSUME SEISAKUSHO, Japan), one per cage, and fed solid food (MF, Oriental, Tokyo, Japan). They were housed in racks (Oriental, Tokyo, Japan) under a controlled temperature of 22–26°C, 50–60% humidity, and a 12-h light / dark cycle. Sterile water was available ad libitum. Experimental animals were divided into four groups: control group (5 mice), swallow's nest group (5 mice), kaempferol group (5 mice), and quercetin group (5 mice). All mouse procedures and manipulations were approved by the Kyushu University Animal Experiment Ethics Committee and in accordance with the Guide for the Care and Use of Laboratory Animals (approval number: A21-465-0, "Analysis of the Skin-Improvement Effect of Foods on Dermatitis").

[0050] 2-6-2. Creation of atopic dermatitis-like induced mouse model After 1 week of breeding, the dorsal hair of mice was clipped under anesthesia to a size of approximately 2.5 cm × 2.5 cm using electric clippers (THRIVE MODEL 2100, Daito Electric Machine Industry, Osaka, Japan) and then shaved with an electric shaver (Panasonic, Osaka, Japan). Experiments were performed the following day. Female 7-week-old C57BL / 6 mice (n = 5) were treated with a 1% solution of DNCB (2,4-dinitrochlorobenzene) (FUJIFILM Wako, Tokyo, Japan), a chemical that induces atopic dermatitis, in a 4:1 mixture of acetone (FUJIFILM Wako) and olive oil (FUJIFILM Wako). A 100 μL solution was applied to the back of each mouse on days 0 and 3 to induce atopic dermatitis-like inflammation. To prevent spontaneous inflammation suppression, 100 μL of 0.4% DNCB was applied to the back of each mouse every 3 days for 13 days. 10 μL of 0.4% DNCB was applied to the right ear of each mouse every three days for 13 days. At the end of the experiment, 20 days after the start of the experiment, the mice were euthanized by cervical dislocation and the skin on their backs was collected.

[0051] 2-6-3. Preparation and application of coating samples For the control group, 150 μL of 50% ethanol was applied to the back of the mice and 15 μL to the right ear. For the swallow's nest, Kaempferol, and Quercetin groups, 1% of each food ingredient was dissolved in 50% ethanol, and 150 μL of each food ingredient was applied to the back of the mice and 15 μL to the right ear.

[0052] 2-7. Functionality verification using atopic dermatitis-like induced mouse model 2-7-1. Measurement of inflammation score, scab area, and ear thickness The inflammation score was calculated by adding three types of food ingredients to atopic dermatitis-like induced model mice and assigning a score to indicate whether inflammation was suppressed. The inflammation score was categorized into six levels. Score changes were classified as follows: normal (score 0), mid-recovery (score 1), early recovery (score 2), mild inflammation (score 3), moderate inflammation (score 4), and severe inflammation (score 5). The area of ​​the scabs was calculated by measuring the area where the scabs formed. Ear thickness was calculated by measuring the right ear, the inflamed area, with a vernier caliper.

[0053] 2-7-2. Verification of gene expression in mouse skin RNA extraction from mouse skin tissue was performed according to the product protocol for the RNeasy Fibrous Tissue Mini Kit (QIAGEN, Hilden, Germany).

[0054] (1) Skin fracture A portion of the skin sample taken from a mouse was cut into a piece approximately 3 cm x 3 mm in size and placed in a 1.5 mL tube with a dimpled interior for a BioMasher II (Nippi, Tokyo, Japan). 10 μL of 2-mercaptoethanol (FUJIFILM Wako) was added to 1 mL of the kit's Buffer RLT, and 300 μL of this mixture was added on top of the tissue. Next, the skin tissue was quickly crushed using the attached stirrer. Homogenization was continued until clumps of skin tissue were no longer visible to the naked eye.

[0055] (2) Total RNA extraction The homogenized tissue lysate was centrifuged at 18,000 × g for 10 minutes at room temperature, and the lysate was transferred to a new 1.5 mL tube, taking care not to contaminate the pellet. 590 μL of RNase-free water and 10 μL of Proteinase K solution (included in the kit) were added to the lysate and mixed thoroughly by pipetting. The mixture was then incubated at 55°C for 10 minutes and centrifuged at 10,000 × g for 3 minutes at room temperature. The supernatant (approximately 900 μL) was pipetted into a new 1.5 mL tube. A 0.5-fold volume (approximately 450 μL in this case) of 99% ethanol (FUJIFILM Wako) was added to the cleared lysate and mixed by pipetting. A 700 μL sample was applied to an RNeasy Mini Spin Column placed in a 2 mL collection tube provided with the kit and centrifuged at ≥8,000 × g for 15 seconds at room temperature. The filtrate in the collection tube was discarded, and the same procedure was repeated with the remaining sample. 350 μL of Buffer RW1 was added to the RNeasy spin column and centrifuged at ≥8,000 × g for 15 seconds at room temperature to wash the membrane. 10 μL of DNase I stock solution, prepared by dissolving DNase I (1,500 Knitz units) in 550 μL of RNase-free water, was added to 70 μL of Buffer RDD. Because DNase I is sensitive to physical denaturation, mixing was performed by gently inverting the tube. A brief centrifugation was performed to collect any remaining solution on the tube walls. 80 μL of DNase I incubation solution per sample was pipetted directly onto the RNeasy spin column membrane and incubated for 15 minutes at room temperature. To avoid incomplete digestion by DNase I, the DNase I incubation reaction mixture was added directly to the RNeasy spin column membrane to avoid contact with the spin column walls. After 15 minutes, 350 μL of Buffer RW1 was added to the RNeasy Spin Column, and the column was centrifuged at room temperature at 8,000 × g or higher for 15 seconds.Next, 500 μL of 4× Buffer RPE was diluted with 99% ethanol to 1× Buffer RPE and added to each RNeasy spin column. The membrane was then washed by centrifugation at ≥8,000 × g at room temperature for 15 seconds. To prevent ethanol carryover during RNA elution, another 500 μL of 1× Buffer RPE was added to the RNeasy spin column, and the column and membrane were washed by centrifugation at ≥8,000 × g at room temperature for 2 minutes. After centrifugation, the RNeasy spin column was carefully removed from the collection tube, taking care not to contact the filtrate. The RNeasy spin column was then transferred to a new 2 mL collection tube and centrifuged at maximum speed for 1 minute. The RNeasy spin column was then placed in a new 1.5 mL collection tube (provided), and 50 μL of RNase-free water was added directly to the RNeasy spin column membrane. The tube was then closed and centrifuged at 8,000 × g or higher for 1 minute at room temperature. The resulting eluate was used as the RNA solution. The RNA concentration in the solution was calculated based on the absorbance at 260 nm using a NanoDrop 2000 / 2000c spectrophotometer and used in quantitative RT-PCR experiments.

[0056] (3) cDNA synthesis using reverse transcriptase Super Script RNase-free water was added to 0.8 μg of total RNA extracted from cells to a total volume of 16 μL, and 4 μL of Super Script IV VILO Master Mix (Invitrogen, Tokyo, Japan) was added. cDNA was synthesized by incubation in a Thermal Cycler PTC-200 at 25°C for 10 minutes, 50°C for 10 minutes, and 85°C for 5 minutes. This cDNA was used as a template for quantitative RT-PCR. The quantitative RT-PCR method is as described above.

[0057] 2-7-3. Preparation of paraffin-embedded sections (1) Formalin fixation and water washing Skin tissue from the test area on the back was collected using dissection scissors. To prevent shrinkage, the collected skin tissue was stretched thoroughly and stapled to filter paper. The tissue was then immersed in 10% neutral buffered formalin (FUJIFILM Wako) along with the filter paper and fixed at room temperature for 24 hours. After the tissue was fixed, it was washed with running water (tap water) for 1 hour. Care was taken not to let tap water come into direct contact with the fixed specimen.

[0058] (2) Alcohol dehydration and degreasing Because paraffin is insoluble in water, it is necessary to remove the water contained in the tissue fragments and the water contained in the intracellular fat using alcohol. First, 99% ethanol (FUJIFILM Wako) was diluted with ultrapure water to 70% ethanol, and the fixed specimens were soaked in the filter paper for 24 hours. After that, the filter paper was soaked in fresh 100% ethanol every two hours for a total of four times. At this time, the ethanol was thoroughly drained and the specimens were soaked in ethanol from a new container.

[0059] (3) Xylene penetration (dealcoholization) Because the alcohol used in the dehydration process is insoluble in paraffin, it was necessary to replace it with an intermediate agent that is soluble in both paraffin and ethanol. The required sections of the fixed specimens attached to the filter paper were cut with a cutter to fit into Uni-Cassettes for Paraffin-Embedded Tissue Sectioning (M498-2, Simport Scientific, Beloe, Canada), each approximately 3 cm x 3 mm in size. Each specimen was then placed in a separate cassette. The specimens were then immersed in fresh xylene twice, 30 minutes apart. The xylene was thoroughly drained before being immersed in a new container.

[0060] (4) Paraffin infiltration (embedding) After 1 hour of replacement with the intermediate solution at 60°C, the Uni-Cassette was infiltrated three times for 45 minutes in melted paraffin (Sakura Finetek, Tokyo, Japan) at 60°C in an incubator set at 60°C. After infiltration, a small amount of paraffin was poured into a stainless steel embedding dish. Using pre-warmed tweezers to prevent the paraffin from solidifying, the fixed specimen was placed on the bottom of the embedding dish in the direction of the desired cutting, i.e., perpendicular to the cutting direction (i.e., the bottom and cut surface were parallel). The bottom of the embedding dish was then cooled on ice, and the paraffin was allowed to solidify approximately halfway to fix the base. Further paraffin was then poured on top for embedding. Before the poured paraffin solidified, the Uni-Cassette used was placed on top of the specimen and solidified as a mount. The cooled and solidified paraffin block was stored at room temperature.

[0061] (5) Preparation of paraffin sections (thin sectioning and spreading) After securing the specimen stage of a rotary microtome (Histo Core AUTOCUT R, Leica, Wetzlar, Germany), the specimen was mounted on a paraffin block by clamping it between a uni-cassette (which acts as a standstock) attached to the microtome. The section thickness was set at 5 μm to prevent cells from overlapping, allowing for easy observation of tissue structure. The blade position and the angle of the specimen stage were adjusted to ensure that the underside of the paraffin block was parallel to the microtome blade, allowing for thin slices. The resulting ribbon-like paraffin specimens were cut into appropriate lengths, gently scooped with a brush to avoid tangling, and floated in distilled water in a stainless steel tray. Sections with the cleanest shapes were selected and floated in hot water at approximately 40°C for approximately 20 seconds to smooth out any wrinkles. The smoothed sections were then transferred to glass slides (PLATINUM PRO, Matsunami Glass, Osaka, Japan) and fully stretched to ensure a firm seal. The slides were then left to dry overnight on a paraffin spreader set at 40°C. The slides were then stored in a preparation box.

[0062] 2-7-4.H&E staining (1) Deparaffinization, dexyleneization, and water immersion To remove paraffin from the tissue, the slides with the paraffin-embedded sections were placed in a staining basket and immersed twice in xylene in a new staining vat, with 10-minute intervals between each. Next, to dexyleneize, the slides were immersed twice in 99% ethanol in a new staining vat, with 5-minute intervals between each. The slides were then immersed in 90%, 80%, and 70% ethanol for 3 minutes each. To minimize carryover, the slides were thoroughly wiped clean before transferring to the new staining vat. After washing, excess liquid was removed from the area around the specimen on the slide with Kimwipes, and a water-repellent circle was created by encircling the specimen with Super Pap Pen Liquid Blocker (Daido Sangyo, Japan). Next, 200 μL of Mayer's hematoxylin solution (Wako) was added to the water-repellent circle and allowed to stand at room temperature for 5 minutes. After 5 minutes, the waste liquid was discarded and the slides were washed with DW for 3 minutes.

[0063] (2) H&E staining To stain cell nuclei blue-purple with hematoxylin and other structures pink with eosin, 200 μL of Mayer's hematoxylin solution (FUJIFILM Wako) was dropped into the water-repellent circle and allowed to stand at room temperature for 5 minutes. After 5 minutes, the waste solution was discarded and the circle was washed with DW for 3 minutes. After washing, excess liquid around the specimen on the slide was wiped off with a Kimwipe or cotton swab, and 200 μL of eosin solution was dropped into the water-repellent circle and allowed to stand at room temperature for 3 minutes. The eosin solution was prepared by adding 0.1 g of eosin (FUJIFILM Wako) to 3 mL of DW, 7 mL of 99% ethanol, and 50 μL of acetic acid (FUJIFILM Wako). After 3 minutes, the waste solution was discarded and the circle was washed with DW for 2 minutes. The slide was then immersed in 60% ethanol for 2 minutes, followed by two immersions in 99% ethanol. Finally, the tissue was stained by immersing it in xylene for 2 minutes, and then in a 1:1 mixture of xylene and 99% ethanol for 2 minutes each time.

[0064] (3) Enclosure A cover glass (24 mm × 36 mm) was slowly tilted over from the left edge and sealed, taking care not to trap air bubbles. After sealing, the slide was allowed to dry on a mat.

[0065] (4) Detection The epidermal thickness, dermal thickness, and number of infiltrating cells were observed by observing the tissues under a fluorescence microscope (EVOS M5000 Imaging System, Thermo Fisher Scientific).

[0066] 2-8.Statistical Processing Statistical analysis was performed using Student's t-test and Tukey-Krammer method, and p<0.05 was considered to indicate a significant difference.

[0067] 3.Results 3-1. Search for Raldh2-activating food ingredients 3-1-1. Search for food ingredients that activate the Raldh2 promoter using the IN Cell Analyzer 2200 Figure 2 shows the Raldh2 promoter-EGFP fluorescence intensity in Kaempferol, Quercetin, and bird's nest samples. THP-1 (Raldh2p-EGFP) cells were induced to differentiate for 48 hours, and then bird's nest samples were added and cultured for 24 hours. After culture, changes in EGFP fluorescence intensity in THP-1 (Raldh2p-EGFP) cells were measured using an IN Cell Analyzer 2200. As shown in Figure 2, some bird's nest samples enhanced the Raldh2 promoter-EGFP fluorescence intensity (Sample B, final concentrations 450 μg / mL and 4500 μg / mL).

[0068] 3-1-2. Evaluation of endogenous RALDH2 gene expression by quantitative RT-PCR Figure 3 shows the expression levels of endogenous RALDH2 gene by Kaempferol, Quercetin, and bird's nest samples. After 48 hours of differentiation induction, wild-type THP-1 cells were added with bird's nest samples and cultured for 24 hours. Total RNA was then collected, cDNA was synthesized, and the effect on endogenous RALDH2 expression was examined by quantitative RT-PCR. As shown in Figure 3, some bird's nest samples (sample B) increased the transcription level of endogenous RALDH2.

[0069] RNA-seq These results demonstrate that bird's nest, Kaempferol, and Quercetin are all dietary ingredients that increase the Raldh2 promoter-EGFP fluorescence intensity in differentiated THP-1 (Raldh2p-EGFP) cells and increase the transcription level of endogenous RALDH2 in wild-type THP-1 cells. Therefore, in this section, we used RNA-seq to identify functional mRNAs among the three identified food ingredients. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis was performed using the DAVID database. The analysis revealed changes in KEGG pathways involved in immune regulation. Table 2 shows the KEGG Pathway classification of common changes in THP-1 cells treated with bird's nest, Kaempferol, and Quercetin.

[0070] [Table 2]

[0071] 3-3. Skin inflammation suppression effect of RALDH2-activating food ingredients 3-3-1. Verification of inflammation score, scab area, and ear thickness in atopic dermatitis-like induced mouse model Six-week-old female C57BL / 6 mice were acclimated for one week, then shaved. Atopic dermatitis-like inflammation was induced by applying 100 μL of 1% DNCB (2,4-Dinitrochlorobenzene) (FUJIFILM Wako) to the backs of the mice on days 0 and 3. From day 5, the treatment was switched to 0.4% DNCB, and 100 μL was applied to the backs and 10 μL to the right ears of the mice every three days to maintain the inflammation. From day 7, for 14 days, three food ingredients—bird's nest, Kaempferol, and Quercetin—dissolved in 50% ethanol were applied at 150 μL to the backs and 15 μL to the right ears at 1% concentration to test for their remission. Sample B, which had shown promising results above, was used as the bird's nest. During this period, the degree of inflammation on the backs of the mice was assessed using a six-point scale, and the area of ​​scabs on the backs of the mice was measured.

[0072] Figure 4 shows the state of scabs in a mouse model induced with atopic dermatitis. Figure 5 shows changes in inflammation scores in a mouse model induced with atopic dermatitis. As shown in Figures 4 and 5, the DNCB-treated swallow's nest group of 7-week-old mice showed a significantly reduced level of inflammation compared with the control group, DNCB-treated with 50% ethanol.

[0073] Figure 6 shows changes in scab area in atopic dermatitis-like induced model mice. As shown in Figure 6, the scab area of ​​7-week-old mice in the DNCB-treated swallow's nest group, the Kaempferol group, and the Quercetin group was significantly reduced compared to the DNCB-treated 50% ethanol group.

[0074] Next, the mice were dissected 20 days after the start of the experiment, and the thickness of the right ears of the mice was measured. Figure 7 shows the appearance of the ears in a mouse model induced with atopic dermatitis. Figure 8 shows the ear thickness in a mouse model induced with atopic dermatitis. As shown in Figures 7 and 8, the right ears of 7-week-old mice in the DNCB-treated swallow's nest group, Kaempferol group, and Quercetin group were significantly thinner than those in the DNCB-treated 50% ethanol group.

[0075] 3-3-2. Measurement of epidermal thickness, dermal thickness, and number of infiltrating cells in the dorsal tissue of atopic dermatitis-like induced model mice Figure 9 shows micrographs of the dorsal tissue of a mouse model of induced atopic dermatitis. Figures 10, 11, and 12 show the epidermal and dermal thicknesses and the number of infiltrating cells in the dorsal tissue of a mouse model of induced atopic dermatitis, respectively. Seven-week-old mice were dissected 20 days after the start of the experiment, and dorsal tissue from each group was collected. Paraffin sections were prepared and stained with hematoxylin and eosin to measure the epidermal and dermal thicknesses and the number of infiltrating cells in the dorsal tissue. As shown in Figures 9-11, the DNCB-treated swallow's nest group, Kaempferol group, and Quercetin group showed significantly reduced epidermal and dermal thickness compared to the DNCB-treated 50% ethanol group. Furthermore, as shown in Figure 12, the number of infiltrating cells was significantly reduced in the DNCB-treated swallow's nest group, the Kaempferol group, and the Quercetin group of 7-week-old mice compared to the DNCB-treated 50% ethanol group.

[0076] 3-3-3. Verification of gene expression in the dorsal tissue of atopic dermatitis-like induced mouse model Figures 13, 14, 15, and 16 show the expression levels of the TNF-α gene, IL-13 gene, Foxp3 gene, and TGF-β gene in the dorsal tissue of atopic dermatitis-like induced mouse models, respectively. RNA was collected from the dorsal tissue of 7-week-old mice, and the expression levels of genes related to inflammation suppression were measured by quantitative RT-PCR. As shown in Figure 13, the DNCB-treated swallow's nest group, Kaempferol group, and Quercetin group of 7-week-old mice showed significantly reduced gene expression levels of the inflammatory cytokine TNF-α compared to the DNCB-treated 50% ethanol group. Furthermore, as shown in Figure 14, the DNCB-treated swallow's nest group and Quercetin group of 7-week-old mice showed significantly reduced gene expression levels of the inflammatory cytokine IL-13 compared to the DNCB-treated 50% ethanol group. Next, we measured the gene expression level of Foxp3, a molecular marker of Tregs. As shown in Figure 15, the DNCB-treated Quercetin group of 7-week-old mice had significantly increased Foxp3 gene expression compared to the DNCB-treated 50% ethanol group. Furthermore, as shown in Figure 16, the DNCB-treated swallow's nest group, Kaempferol group, and Quercetin group of 7-week-old mice had significantly increased gene expression of TGF-β, an immunosuppressive cytokine, compared to the DNCB-treated 50% ethanol group.

[0077] 4. Discussion THP-1 cells can be induced to differentiate into macrophage-like cells. In this experiment, PMA was added to a final concentration of 100 ng / mL and the cells were cultured in RPMI 1640 medium containing 10% FBS for 48 hours.

[0078] In the search for RALDH2-activating food ingredients, we first created them by introducing a Raldh2p-EGFP vector into THP-1 cells. The fluorescence intensity of Raldh2p-EGFP-transfected THP-1 cells was tracked using an IN Cell Analyzer 2200. As shown in Figure 2, we confirmed an increase in the fluorescence intensity of food ingredients in multiple bird's nest samples (A150, B450, B4500). This method allows us to monitor the presence or absence of RALDH2 promoter activation by fluorescence intensity, so it was concluded that using these cells is effective when screening a large number of samples at once.

[0079] However, this screening merely evaluates promoter activity relative to the fluorescence value of the negative control, and is a screening stage that eliminates those with a low possibility of actually increasing the expression level of RALDH2, rather than searching for those with a high possibility of actually increasing the expression level of RALDH2.

[0080] Furthermore, the Raldh2 gene sequence incorporated into the Raldh2p-EGFP vector is only a portion of the Raldh2 promoter region, and measurements using the IN Cell Analyzer 2200 merely evaluate the activation ability of the Raldh2 promoter, which is made up of a partial region of the food component. Therefore, in samples that showed an increase in fluorescence intensity in the primary screening, we measured the transcription level of the RALDH2 gene using quantitative real-time PCR. The concentration of bird's nest samples used in this experiment was selected to be 450 μg / mL, as this concentration provided stable fluorescence intensity.

[0081] As a result, it was revealed that one of the bird's nest samples in this study increased the transcription level of the endogenous RALDH2 gene (sample B450).

[0082] Comparing the results of previous studies measuring the transcription levels of the RALDH2 gene revealed that Kaempferol, and now Quercetin, both commonly increased the transcription levels of the endogenous RALDH2 gene, and in this study they were identified as RALDH2-enhancing food ingredients. Kaempferol and Quercetin are both flavonoids. Flavonoids belong to the polyphenol family, a group of compounds with two or more phenolic hydroxyl groups in the molecule, and are particularly a group of phenyl compounds with a structure in which two benzene rings are connected by three carbon atoms. Both have numerous physiological activities, and it has been reported that Quercetin strengthens the intestinal barrier, while Kaempferol increases Foxp3 expression in Treg cells and suppresses collagen-induced arthritis.

[0083] Furthermore, when comparing the structural formulas of Kaempferol and Quercetin, the only difference is the presence or absence of one hydroxyl group. Therefore, it is possible that Kaempferol and Quercetin activate RALDH2 through some common action derived from their common structure. In addition, this study newly identified bird's nest sample as a Raldh2-enhancing food ingredient.

[0084] Based on the results obtained so far, in vivo testing has suggested that bird's nest, Kaempferol, and Quercetin enhance retinoic acid production in monocyte-derived cells, thereby promoting the differentiation of naive T cells into Treg cells.

[0085] However, it remains unclear what immune mechanisms these three compounds affect. RNA-seq analysis of THP-1 cells altered by bird's nest, Kaempferol, and Quercetin treatment revealed changes in pathways involved in immune regulation, such as lekocyte transendothelial migration. These findings suggest that some components secreted by bird's nest, Kaempferol, and Quercetin treatment activate pathways involved in immune regulation in THP-1 cells, providing valuable information on immune regulation.

[0086] Based on the above results, it has become clear that three types of food ingredients, bird's nest, Kaempferol, and Quercetin, increase the transcription level of the endogenous RALDH2 gene and contribute to regulating the immune system. Next, we conducted an in vivo test to verify whether they have the effect of suppressing atopic dermatitis, a type of excessive immune response, by directly applying them to mice.

[0087] In order to clarify the effects of Tregs, which have an immune response suppressing effect, in in vivo tests, it was thought necessary to activate immune cells, and so Kaempferol, Quercetin, and swallow's nest, which were selected in in vitro tests, were applied to inflamed mice.

[0088] In this study, atopic dermatitis-like disease was induced using DNCB (2,4-Dinitrochlorobenzene). In the DNCB-induced atopic dermatitis-like model, DNCB is used as a hapten. A hapten is a substance that lacks immunogenicity by itself and is thought to function only as a reactogenic substance, and therefore functions as an antigen by binding to high molecular weight proteins in the body.

[0089] It is said that applying DNCB several days after skin sensitization causes redness and swelling of the epidermis, mainly caused by macrophages and lymphocytes. It is known that in atopic dermatitis, the innate immune system is activated. Innate immunity is activated by recognizing the components of foreign substances and phagocytosing pathogens, with macrophages and dendritic cells playing key roles.

[0090] Indicators of the progression of atopic dermatitis include thickening of the epidermis and dermis, and an increase in the number of infiltrating cells. In this study, the degree of inflammation was compared by measuring the thickness of the epidermis and dermis, the number of infiltrating cells, and the thickness of the ear.

[0091] In this study, 7-week-old female C57BL / 6 mice were treated with DNCB to induce atopic dermatitis-like symptoms, and then swallow's nest, Kaempferol, and Quercetin were applied directly to the shaved backs of the mice for 14 days to measure the degree of inflammation, the area of ​​scabs that had formed on the backs, and the thickness of the ears.

[0092] The results showed that the degree of inflammation in the swallow's nest was significantly reduced, and that swallow's nest, Kaempferol, and Quercetin significantly reduced the area of ​​the scab.

[0093] Next, tissue from the backs of the mice was collected and stained with H&E to examine the anti-inflammatory effects of swallow's nest, Kaempferol, and Quercetin.

[0094] The results showed that, compared to the control, the three food ingredients suppressed thickening of the epidermis and dermis, which are the main lesions of atopic dermatitis, and suppressed an increase in the number of infiltrating cells.

[0095] Considering the results of in vitro studies, it is thought that promoting RALDH2 expression increases the amount of retinoic acid and promotes differentiation into Treg cells. As a result, the number of Treg cells increases, suppressing excessive immune responses and possibly suppressing inflammation.

[0096] Finally, quantitative RT-PCR was performed on mouse dorsal tissue, and an increase in the transcription levels of the genes for Foxp3, a master factor for Tregs that acts to suppress inflammation, and TGF-β, an immunosuppressive cytokine, was observed.

[0097] Since TGF-β has been reported to exhibit strong antiproliferative activity in epithelial cells and blood cells, it was suggested that the three food components have the ability to suppress atopic dermatitis-like inflammation.

[0098] It was also revealed that the transcription levels of TNF-α gene and IL-13 gene, which are cytokines that promote inflammation, were reduced.

[0099] It has been revealed that TNF-α is overexpressed at the site of inflammation in atopic dermatitis, and that IL-13 is involved in promoting the secretion of IgE antibodies, which are the basis for the secretion of histamine and leukotrienes that cause an immediate inflammatory response.

[0100] Considering the results of the three food ingredients in this study, combined with those obtained from the in vitro and in vivo tests mentioned above, it was suggested that the three food ingredients are Treg-regulating food ingredients that have the function of suppressing atopic dermatitis.

[0101] However, the mechanism by which these functionalities are realized remains unclear. Regarding the molecular basis of the functionality provided by food components, it has been reported that polyphenols bind to specific receptors on the cell membrane when they affect cells.

[0102] Therefore, it is possible that the functional food ingredients identified in this study also cause changes in gene expression through signals mediated by receptors on the cell membrane.

[0103] Sialic acid is also a major component of bird's nest. Cell surfaces are covered with glycans, the terminals of which are made up of acidic sugars called sialic acids. Cells recognize each other and molecules via this sialic acid, and it is known that intermolecular binding via sialic acid plays an important role in various cellular functions. It has been suggested that sialic acid may be involved in immune regulation through homeostasis and pathology through its interaction with sialic acid-binding Ig lectins, which are known to be primarily expressed in innate immune cells such as dendritic cells, macrophages, and B cells.

[0104] It has also been reported that antigens modified with sialic acid induce Tregs by imposing control on dendritic cells via Siglec-E. Based on these findings, we believe that sialic acid-binding proteins of the Siglec family are expressed in immune system cells, and that one of these Siglec family members may have some effect on intracellular signal transduction through modification of sialic acid, the main component of bird's nest, resulting in the suppression of inflammation.

[0105] In the above study, the functionality of three food ingredients, bird's nest, Kaempferol, and Quercetin, was examined by applying them directly to the area where inflammation was occurring.

[0106] However, currently, dietary therapy in daily life is expected to be a treatment for atopic dermatitis that has no side effects and is less burdensome.

[0107] The inventors have been conducting research with the aim of improving daily life through the application of functional ingredients. In the future, we will orally administer the three food ingredients we have obtained to mice with inflammation and verify their functionality, which we believe will enable the development of a more practical dietary therapy.

[0108] Furthermore, Treg activation can induce inflammation suppression, which may enable the suppression of various inflammatory diseases (autoimmune diseases, allergies, inflammatory bowel disease, microbial infections, etc.).

[0109] Overall, it was thought that swallow's nest has a skin-improving effect by acting on cells on the skin surface and cells of the immune system.

Claims

1. A regulatory T cell differentiation inducer that induces differentiation into regulatory T cells, A regulatory T cell differentiation inducer (excluding agents for improving atopic dermatitis) containing water-solubilized swallow's nest as an active ingredient.

2. The regulatory T cell differentiation inducer according to claim 1 (excluding agents for improving atopic dermatitis), wherein the active ingredient improves retinoic acid production ability.

3. The regulatory T cell differentiation inducer according to claim 2 (excluding agents for improving atopic dermatitis), wherein the active ingredient increases the transcription level of the RALDH2 gene.

4. The regulatory T cell differentiation inducer according to claim 1 (excluding agents for improving atopic dermatitis), wherein the active ingredient increases the transcription level of the TGF-β gene.

5. The regulatory T cell differentiation inducer according to claim 1 (excluding agents for improving atopic dermatitis), wherein the active ingredient reduces the transcription amount of the TNF-α gene and / or the IL-13 gene.

6. A food composition for inducing differentiation of regulatory T cells (excluding agents for improving atopic dermatitis), comprising the active ingredient of any one of claims 1 to 5.

7. A health food (excluding atopic dermatitis ameliorators) for inducing differentiation of regulatory T cells, comprising the active ingredient of any one of claims 1 to 5.

8. A cosmetic for inducing differentiation of regulatory T cells (excluding atopic dermatitis ameliorators), comprising the active ingredient of any one of claims 1 to 5.

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