HK40007908A - Pharmaceutical composition for preventing or treating hypersensitivity immune disease, and method for producing same - Google Patents

Description

Pharmaceutical composition for preventing or treating allergic immune diseases and preparation method thereof

Technical Field

The present invention relates to a composition for preventing, ameliorating or treating allergic immune diseases including allergic diseases and the like, and a composition prepared by the above method.

Background

In the immune system, immune responses are coordinated and balanced with immune tolerance. In the case of an excessive immune reaction, allergic diseases, autoimmune diseases, etc. may occur, which have a great influence on human survival and quality of life.

Allergy (Allergy) refers to a pathological allergic reaction, mediated by antibodies or cells, caused by the immune mechanisms of non-self substances such as allergens. The allergic reaction is expressed as a Th2(T-helper type 2) cellular reaction, which involves cytokines such as interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-13 (IL-13) and Tumor Necrosis Factor (TNF) -a, chemokines such as T cell expression and secretion (regulated uplink activation, normal T-cell expressed & secreted, RANTES), eotaxin (eotaxin) and macrophage chemotactic protein-3 (MCP-3) playing an important role in the allergic inflammatory response. Also, autoimmune diseases (autoimmunity) with pathological hypersensitivity to self substances are included.

The diseases caused by allergic reactions include asthma, rhinitis, urticaria, allergic reactions and the like, and some allergic reactions include allergic asthma, eczema and the like caused by genetic binding with specific allergic antigens. Such allergies are characterized in that they are usually caused by harmless substances (pollen, moulds, animal hair and dandruff, dust, mites and peanuts, etc.).

As a therapeutic method for treating allergic diseases which has been developed so far, there is a method of identifying an allergen of an allergic disease from which an allergic patient suffers, administering it in a small amount for several years, and gradually reducing the allergen. However, this treatment has disadvantages in that the treatment takes several years and there is a problem that anaphylactic shock or the like may be caused. In addition, there are a method using a DNA vaccine, a therapeutic method of blocking the binding of immunoglobulin e (ige) to a mast cell receptor, and a therapeutic method using an antibody against interleukin-4 as a cytokine which causes allergy, etc., but these methods have disadvantages in that they are expensive or their therapeutic effects have not been fully demonstrated.

On the other hand, dendritic cells are one of representative antigen-presenting cells that play a central role in innate immunity and acquired immune regulation due to acquired causal selectivity, and specifically, perform a function of presenting information of an antigen invaded mainly from the outside to T cells.

The dendritic cells are composed of various partial subgroups (subtypes) according to origin (origin), phenotype (phenotype) and function (function). Specifically, plasmacytoid dendritic cells (pDCs) can produce type I IFN at high levels in response to stimulation from various pathogenic bacterial sources. It is distinguished from conventional dendritic cells (cDCs) by these surface phenotypes. The above plasmacytoid dendritic cells expressed B220 and expressed CD11c at a low level, in contrast, conventional dendritic cells expressed CD11c and CD11B at a high level, but did not express B220. The above plasmacytoid dendritic cells and dendritic cells are obtained during the maturation of immature dendritic cells so that they can be used as effective stimulators of harmonious immune responses.

As described above, it is known that plasmacytoid dendritic cells play an important role in defense against viral infection by secreting a large amount of type I IFNs to activate immune cells when infected with DNA viruses or single-stranded RNA viruses. In particular, it helps to amplify T cell-based antiviral responses by strongly activating mature dendritic cells (mDCs) that can present antigen.

However, in fact, the differentiation or action of plasmacytoid dendritic cells in vivo is very little studied compared with other dendritic cells. There is an urgent need for a technique for producing an immune-tolerant dendritic cell in which immune tolerance is always maintained or enhanced by regulating the expression of a specific gene that governs the immunity of the dendritic cell or by discovering an immune tolerance-inducing substance. In particular, although many studies have been conducted to demonstrate that the above-described plasmacytoid dendritic cells play an important role in various autoimmune diseases or infectious diseases, a systematic and standardized differentiation method for preparing immune-tolerant plasmacytoid dendritic cells has not been clarified.

Disclosure of Invention

Technical problem

The present inventors have found that when a Toll-like receptor agonist (TLR agonist) is treated on immature dendritic cells (dendritic cells) and then induced to differentiate, or when the Toll-like receptor agonist is treated during differentiation of the immature dendritic cells, immune-tolerant plasmacytoid dendritic cells are induced, and when the immune-tolerant plasmacytoid dendritic cells are used, allergic immune diseases can be effectively prevented, ameliorated, or treated, thereby achieving the present invention.

It is an object of the present invention to provide a method for preparing a composition effective for preventing, ameliorating or treating an allergic immune disease.

It is still another object of the present invention to provide a composition prepared by the above method, which is effective for preventing, ameliorating or treating allergic immune diseases.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, the appended claims and the accompanying drawings.

Problem solving scheme

In order to achieve the above objects, the present invention provides a method for preparing a pharmaceutical composition for preventing or treating an allergic immune disease, comprising treating immature dendritic cells with a Toll-like receptor agonist (Toll-like agonist) to prepare immune-tolerant plasmacytoid dendritic cells.

Also, the present invention provides a pharmaceutical composition for preventing or treating an allergic immune disease, comprising an immune-tolerant plasmacytoid dendritic cell induced by treating an immature dendritic cell with a Toll-like receptor agonist.

Also, the present invention provides a prophylactic or therapeutic use of an immune-tolerant plasmacytoid dendritic cell for allergic immune diseases induced by treating an immature dendritic cell with a Toll-like receptor agonist.

Also, the present invention provides use of an immune-tolerant plasmacytoid dendritic cell induced by treating an immature dendritic cell with a Toll-like receptor agonist for preparing a pharmaceutical composition for preventing or treating an allergic immune disease.

Also, the present invention provides a cosmetic composition for preventing or improving an allergic immune disease, comprising an immune-tolerant plasmacytoid dendritic cell induced by treating an immature dendritic cell with a Toll-like receptor agonist.

Also, the present invention provides a food composition for preventing or improving an allergic immune disease, comprising toleroresistant plasmacytoid dendritic cells induced by treating immature dendritic cells with a Toll-like receptor agonist.

Effects of the invention

The present invention can induce immune-tolerant plasmacytoid dendritic cells from immature dendritic cells at a high yield by a simple and easy procedure, and can stably supply a large amount of immune-tolerant plasmacytoid dendritic cells.

Also, in the present invention, the immune-tolerant plasmacytoid dendritic cells obtained as described above can effectively prevent or treat various allergic immune diseases including those derived from food allergy by suppressing the expression of allergen-specific immunoglobulin and T-helper type 2 cytokine, and are safe when administered to a human body.

Drawings

FIG. 1 is a diagram showing a design diagram of a treatment method of a factor for inducing differentiation of immature dendritic cells (Flt 3L-stabilizing media) and Toll-like receptor agonists (TLRs agonists), according to an embodiment of the present invention.

FIG. 2 is a graph showing the isolation of plasmacytoid dendritic cells (TLRs-pDC) induced to differentiate after treatment of Toll-like receptor agonist (Pam3) according to an embodiment of the present invention in example 1.

Part (a) of fig. 3 is a diagram showing surface-expressed molecules specifically induced to the induced-differentiation plasmacytoid dendritic cells after treating a Toll-like receptor agonist (Pam3) according to an embodiment of the present invention in example 1.

Part (b) of fig. 3 is a graph showing, in a graph, the ratio of the number of induced-differentiation plasmacytoid dendritic cells after the Toll-like receptor agonist (Pam3) was treated according to the ratio of the number of immature dendritic cells to that of an example of the present invention in example 1.

FIG. 4 is a graph showing the results of comparing the expression forms of cytokines (IFN-. alpha., IFN-. beta., IL-12p70, IL-10) in the induced-differentiation plasmacytoid dendritic cells with those of general plasmacytoid dendritic cells after treating a Toll-like receptor agonist (Pam3) according to an embodiment of the present invention in example 2.

Fig. 5 is a graph showing the results of comparing the expression morphology of cytokine IL-10 in the induced-differentiation plasmacytoid dendritic cells with general plasmacytoid dendritic cells (non) after treating various Toll-like receptor agonists according to an embodiment of the present invention in example 3.

FIG. 6 is a graph showing the results of comparing the time points of the treatment of Toll-like receptor agonist (Pam3) by immature dendritic cells according to example 4 to the expression profiles of cytokines (IFN-. alpha., IFN-. beta., TNF-. alpha., IL-12p70, IL-10) according to an embodiment of the present invention.

Fig. 7 is a graph showing the results of comparing the expression patterns of MHC complex molecules, CD80, and CD86 in the induced-differentiation plasmacytoid dendritic cells and general plasmacytoid dendritic cells after treating various Toll-like receptor agonists (Pam3) according to an embodiment of the present invention in example 5.

Fig. 8 is a graph showing the results of comparing the expression patterns of IDO, CCR9 and PD-L1 in the induced-differentiation plasmacytoid dendritic cells and general plasmacytoid dendritic cells after treating various Toll-like receptor agonists (Pam3) according to an embodiment of the present invention in example 6.

Fig. 9, section (a), is a graph showing the results of treating induced differentiation-induced plasmacytoid dendritic cells or general plasmacytoid dendritic cells after treating T cells with a Toll-like receptor agonist (Pam3) according to an embodiment of the present invention in example 7, and then confirming whether to proliferate to regulatory T cells by flow cytometry LSRFortessa x-20.

Part (b) of fig. 9 is a graph showing the results of measuring the proliferation rate of regulatory T cells after treating the induced differentiation of plasmacytoid dendritic cells or general plasmacytoid dendritic cells after treating T cells with a Toll-like receptor agonist (Pam3) according to an embodiment of the present invention in example 7.

Fig. 10 is a graph showing the degree of binding of wild-type dendritic cells (WT) and dendritic cells isolated from TLR2 knock-out mice (TLR2-/-) to Rv1411c protein in example 8.

FIG. 11 is a graph showing the results of comparing the expression patterns of cytokines (IFN-. alpha., IFN-. beta., TNF-. alpha., IL-12p70, IL-10) in plasmacytoid dendritic cells (Rv1411c (0.1. mu.g/ml) -pDC, Rv1411c (0.5. mu.g/ml) -pDC) induced to differentiate after treating Toll-like receptor agonist (Rv1411c protein (0.1. mu.g/ml, 0.5. mu.g/ml)) according to an embodiment of the present invention in example 9 with those of general plasmacytoid dendritic cells.

Fig. 12 is a graph showing the results of comparing the expression patterns of MHC complex molecules, CD80, CD86 and immune tolerance inducing molecules (IDO, CCR9 and PD-L1) in the induced-to-differentiate plasmacytoid dendritic cells (Rv1411c-pDC) and general plasmacytoid dendritic cells (pDC) after the Toll-like receptor agonist (Rv1411c protein) was treated according to an embodiment of the present invention in example 10.

Fig. 13 is a graph showing the results of comparing the proliferation degree of T cells after mixing and culturing the plasmacytoid dendritic cells (Rv1411c-pDC) induced to differentiate with the general plasmacytoid dendritic cells (pDC) after treating the Toll-like receptor agonist (Rv1411c protein) according to an embodiment of the present invention in example 11.

FIG. 14 is a graph showing the results of confirming the secretion pattern of IFN-gamma in example 11 after mixing and culturing plasmacytoid dendritic cells (Rv1411c-pDC) induced to differentiate with general plasmacytoid dendritic cells (pDC) after treating a Toll-like receptor agonist (Rv1411c protein) according to an embodiment of the present invention.

Part (a) of fig. 15 is a graph showing the results of treating induced differentiation-induced plasmacytoid dendritic cells (Rv1411c-pDC) or general plasmacytoid dendritic cells (pDC) after treating T cells with a Toll-like receptor agonist (Rv1411c protein) according to an embodiment of the present invention, and then confirming whether to proliferate to regulatory T cells by flow cytometry or not with a flow cytometer lsrortessa x-20 in example 12.

Part (b) of fig. 15 is a graph showing the results of measuring the proliferation rate of regulatory T cells after treating the induced differentiation-induced plasmacytoid dendritic cells (RVc1411c-pDC) or general plasmacytoid dendritic cells (pDC) after treating T cells with a Toll-like receptor agonist (Rv1411c protein) according to an embodiment of the present invention in example 12.

Fig. 16 is a graph showing the results of the change in the proliferation potency of T cells relative to the ratio of CD 25-effector T cells to primed T cells after culturing T cells differentiated by induced plasmacytoid dendritic cells (Rv1411c-pDC) with CD4+, CD 25-effector T cells after treating a Toll-like receptor agonist (Rv1411c protein) according to an embodiment of the present invention in example 13.

Fig. 17 is a graph showing the results of comparing the numbers of plasmacytoid dendritic cells (TLR aginst) and Non-treatment-induced plasmacytoid dendritic cells (Non) after treating Toll-like receptor agonists (RYv1411c protein and Pam3) according to an embodiment of the present invention in example 14.

Fig. 18 is an experimental design drawing showing an animal model for preparing the yolk protein-induced food allergy in example 15.

Fig. 19 is a graph showing, in a graphical manner, the results of evaluating the symptoms of systemic anaphylaxis after injecting plasmacytoid dendritic cells (TLRs-pDC 14d i.v.) induced after treating Toll-like receptor agonist (Rv1411c protein) according to an embodiment of the present invention into the animal model of egg yolk protein-induced food allergy in example 15.

FIG. 20 is a graph showing the results of measuring the body temperature change of rectum after injecting plasma cell-like dendritic cells (TLRs-pDC 14 di.v.) induced after treating Toll-like receptor agonist (Rv1411c protein) according to an embodiment of the present invention to the animal model of yolk protein-induced food allergy in example 15.

Fig. 21 is a graph showing the results of measuring the incidence of diarrhea after injecting plasmacytoid dendritic cells (TLRs-pDC 14d i.v.) induced after treating Toll-like receptor agonist (Rv1411c protein) according to an embodiment of the present invention into the animal model of egg yolk protein-induced food allergy in example 15.

FIG. 22 is a graph showing the results of measuring immunoglobulin E (IgE) and immunoglobulin G1(IgG1) in blood after injection of plasmacytoid dendritic cells (TLRs-pDC 14d) induced after treatment of Toll-like receptor agonist (Rv1411c protein) according to an embodiment of the present invention into the animal model of egg yolk protein-induced food allergy in example 16.

FIG. 23 is a graph showing the results of measuring the concentrations of interleukin-4 and interleukin-5 in blood after injection of plasmacytoid dendritic cells (TLRs-pDC 14d) induced after treatment of Toll-like receptor agonist (Rv1411c protein) according to an embodiment of the present invention in the animal model of egg yolk protein-induced food allergy in example 17.

However, in fig. 1 to 23, P <0.05, P <0.01, and P <0.005 are indicated.

Detailed Description

Best mode for carrying out the invention

The present invention relates to a method for preparing a pharmaceutical composition for preventing or treating an allergic immune disease comprising the step of preparing an immune-tolerant plasmacytoid dendritic cell as described below.

In the present invention, the step of preparing the above-mentioned immunotolerant plasmacytoid dendritic cells may be performed by treating the dendritic cells with a Toll-like receptor agonist.

In the present specification, the term "Dendritic Cell (DC)" refers to a professional antigen presenting cell (professional antigen presenting cell) that presents various antigen samples to T cells together with MHC (major histocompatibility complex) class I complex or MHC class ii complex by uptake of antigen into the cell. The dendritic cells include immunogenic and tolerogenic antigen-presenting cells, and are classified into immature dendritic cells ("imdcs"), semi-mature dendritic cells ("smdcs") and mature dendritic cells ("mdcs") according to their maturity.

In the present specification, the term "immature dendritic cells" is found at the early stage of maturation of dendritic cells, does not express CD14, which is the surface phenotype of monocytes, and any of the co-stimulatory molecules CD40, CD54, CD80, CD86 and CD274 is expressed at a lower level than mature dendritic cells.

In the present specification, the term "semi-mature dendritic cell" as a dendritic cell which loses a part of the characteristics of an immature dendritic cell and has a part of the characteristics of a phenotype of a mature dendritic cell means a dendritic cell which partially or incompletely exhibits a mature morphology and a phenotypic characteristic.

In the present specification, the term "mature dendritic cell" refers to a cell formed by maturation of an immature dendritic cell. Mature dendritic cells are characterized by high expression of DC-LAMP and MHC class ii, CD40, CD54, CD80, CD86 and CD274, and have increased proliferation and/or increase production of dendritic cell cytokines in naive allogeneic T cells and allogeneic T cells (syngeneic T cells) upon release of anti-inflammatory cytokines (proinflammatory cytokines) and mixed lymphocyte reactions (mixed lymphocyte reactions). Mature dendritic cells typically express CCR7 and CXCR4 at high levels.

However, in the present invention, preferably, the dendritic cells treated with the above Toll-like receptor agonist are undifferentiated immature dendritic cells. The immature dendritic cells may include primary dendritic cells (naive dendritic cells), and may be isolated and obtained from bone marrow of a mammal or the like.

Also, in the present invention, the immune-tolerant dendritic cells induced to differentiate by treating immature dendritic cells with a Toll-like receptor agonist may be immune-tolerant plasmacytoid dendritic cells.

In the present specification, the term "Plasmacytoid dendritic cells" refers to a subset (subset) of dendritic cells, and in 1958, the morphology of Plasmacytoid cells (plasma cells) was first discovered histologically from lymph nodes of human body by dr.lennert and dr.remmelle and was disclosed, but since it does not express B cell specific marker (immunologlobulin), does not express CD3 which is a common marker of T cells and expresses lymphoid antigen (myocylindinge antigen) and MHC class ii, it was named Plasmacytoid T cells (Plasmacytoid monocytes). After that, with confirmation of its ability to induce allogeneic Mixed Lymphocyte Reaction (MLR), which is an inherent property of dendritic cells, the cell was named again as a plasmacytoid dendritic cell, and may also be simply referred to as "pDC".

In the present specification, the term "immunological tolerance" refers to a state in which an immune response is not shown to a specific antigen and a state in which an immune response is suppressed. Therefore, in the present invention, the "immune tolerant plasmacytoid dendritic cells" promote the secretion of anti-inflammatory cytokines such as IL-10 and inhibit the secretion of inflammatory cytokines such as IL-12p70 and TNF- α, and induce the differentiation of regulatory T cells (effector T cells) and inhibit the activity of effector T cells (effector T cells) because indoleamine-2,3dioxygenase (IDO) molecules, which are recently called immune tolerance-inducing molecules, and CCR9, which is a surface molecule of immune tolerance-inducing cells, are expressed at high levels.

In the present invention, the above Toll-like receptor agonist may be treated from before the above immature dendritic cells start to during differentiation to stably prepare an immune-tolerant plasmacytoid dendritic cell in a large amount. The "before the start of differentiation" may include a time point before the immature dendritic cells are treated with the differentiation inducing factors. The "differentiation period" may be a period from a time point when the immature dendritic cells are treated with the differentiation-inducing factor to a time point before the differentiation is completed by the differentiation-inducing factor. Preferably, a period from the time point of treating the differentiation-inducing factor to within 7 days after the above treatment may be included, but is not limited thereto.

In the present invention, the above Toll-like receptor agonist is not particularly limited to a specific time point as long as it is treated only before or during the initial differentiation of the immature dendritic cells as described above. However, in the present invention, in the case where the above-mentioned Toll-like receptor agonist is treated during differentiation of immature dendritic cells, the treatment may be performed within 7 days (168 hours), 5 days (120 hours), or 3 days (72 hours) from the time point of starting differentiation, but is not limited thereto. In the present invention, when the Toll-like receptor agonist is treated before the onset of differentiation of the immature dendritic cells, the differentiation of the immature dendritic cells may be initiated using the differentiation-inducing factor at a time point of the treatment of the agonist up to 36 hours, preferably within 24 hours, but the present invention is not limited thereto.

In the present invention, the Toll-like receptor agonist may be treated at least once, and although the number of times of treatment is not particularly limited, in the case where the Toll-like receptor agonist is treated during differentiation of immature dendritic cells, the Toll-like receptor agonist may be treated at least once within 7 days, 5 days or 3 days from the time point of starting differentiation of the immature dendritic cells. In the case where the Toll-like receptor agonist is treated before the immature dendritic cells are allowed to start differentiating, the immature dendritic cells may be treated with the Toll-like receptor agonist more than once, and then differentiated within 36 hours or 24 hours from any treatment time point, and optionally subjected to additional treatment more than once during the differentiation period.

The "initiation of differentiation" may be a step of adding a differentiation-inducing factor to a medium for immature dendritic cells or culturing the cells by inoculating immature dendritic cells into a medium containing a differentiation-inducing factor, but is not particularly limited as long as the differentiation of the immature dendritic cells can be induced by the differentiation-inducing factor.

In the present specification, the term "Toll-like receptor agonist" as a pathogen (pathogen) -derived preservation substance may be PAMPs (pathogen associated molecules). Wherein the pathogen is gram-positive bacteria, gram-negative bacteria, fungi or viruses. Also, the Toll-like receptor agonist may be damps (large associated molecular patterns) as an endogenous molecule released from injured or dead cells. In order to initiate an immune response and signal through TLR signaling, DAMPs or PAMPs collect adaptor molecules within the cytoplasm of the cell. The Toll-like receptor agonists described above, in combination with fragments, variants, analogues, homologues or Toll-like receptors, may be PAMPs or derivatives of DAMPs based on TLR-mediated activation for inducing activation of NF-. kappa.B activity, etc. The fragments, variants, analogues, homologues or derivatives as the Toll-like receptor agonists described above are at least 30-99% identical to the amino acids of the TLR agonist for inducing Toll-like receptor-mediated activation.

In the present invention, although the kind of the Toll-like receptor agonist to be treated for the above-mentioned immature dendritic cells is not particularly limited, it is preferably a PAMPs ligand, and more preferably a kind that signals a Myeloid differentiation factor primary response gene 88(Myeloid differentiation primary response gene 88) that can induce the differentiation of immune-tolerant plasmacytoid dendritic cells from the immature dendritic cells.

In the present invention, the Toll-like receptor agonist may include one or more selected from the group consisting of a TLR2 agonist, a TLR4 agonist, a TLR5 agonist, a TLR7 agonist, a TLR8 agonist, a TLR9 agonist, a TLR11 agonist, a TLR12 agonist, and a TLR13 agonist, and more specifically, may include one or more of the ligands shown in table 1 below, but is not limited thereto. Any ligand that binds to the Toll-like receptor of dendritic cells and is able to signal the primary response gene 88 of myeloid differentiation factors can be used.

In the present invention, when a TLR2 agonist is used as the Toll-like receptor agonist, the TLR2 agonist may further include one or more of a TLR1 agonist and a TLR6 agonist used as its co-receptor (co-receptor). Specific examples of the TLR1 agonist and TLR6 agonist include the ligands shown in table 1 below, but are not particularly limited as long as they are ligands that function as co-receptors of the TLR2 agonist.

TABLE 1

In the present invention, the above Toll-like receptor agonist may be derived from a microorganism, a virus, a plant or an animal, and may be synthetic, the origin of which is not particularly limited.

Also, in the present invention, the differentiation of the above-described immature dendritic cells may be performed by using a differentiation-inducing factor. Among them, it is preferable to use FMS-like tyrosine kinase3 ligand (FMS-like tyrosine kinase3, Flt3L) as the differentiation-inducing factor, but there is no particular limitation as long as it is capable of inducing and differentiating the immature dendritic cells into plasmacytoid dendritic cells and general dendritic cells.

In the present invention, the differentiation of the immature dendritic cells may be performed by culturing the immature dendritic cells in a medium containing a differentiation-inducing factor, or by adding the differentiation-inducing factor to the medium of the immature dendritic cells.

In the present invention, the differentiation-inducing factor may be added to the immature dendritic cells at least once during differentiation of the immature dendritic cells.

In the present specification, the term "FMS-like tyrosine kinase3 ligand (FMS-like tyrosine kinase3, Flt 3L)" corresponds to an endogenous low molecule that performs functions as a cytokine and a growth factor by activating hematopoietic progenitor cells (hematopoietic progenitors).

Also, in the present invention, the duration of differentiation of the above-mentioned immature dendritic cells is not particularly limited, and may vary depending on the kind of the medium used and the environment, and for example, may be performed for 5 to 15 days, preferably for 7 to 10 days, and more preferably for 8 to 9 days.

In the present invention, the tolerization of the above-mentioned plasmacytoid dendritic cells can be activated by further treating tolerizing plasmacytoid dendritic cells induced to differentiate from immature dendritic cells by treating the above-mentioned Toll-like receptor agonist.

In the present invention, as described above, the type of Toll-like receptor used for activating the immune-tolerant plasmacytoid dendritic cells that have completed differentiation is not particularly limited, and may include one or more selected from the group consisting of a TLR1 agonist, a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR6 agonist, a TLR7 agonist, a TLR8 agonist, a TLR9 agonist, a TLR11 agonist, a TLR12 agonist, and a TLR13 agonist, and preferably, a TLR9 agonist may be used.

In the present invention, the differentiation of immature dendritic cells into immune-tolerant plasmacytoid dendritic cells can be efficiently induced by the above-described procedure. Thus, a large amount of immune-tolerant plasmacytoid dendritic cells can be stably supplied by a simple and easy process using immature dendritic cells.

Also, in the present invention, the immune tolerant plasmacytoid dendritic cells obtained as described above inhibit the expression of inflammatory cytokines, promote the expression of anti-inflammatory cytokines, induce differentiation into regulatory T cells (regulatory T cells), and inhibit the activity of effector T cells (effector T cells), thereby effectively suppressing immune responses, and further, effectively preventing or treating various allergic immune diseases including those derived from food allergy by inhibiting the expression of allergen-specific immunoglobulin and T helper type 2 cytokines.

In the present invention, the above-mentioned "allergic immune diseases" refer to all diseases harmful to the human body which have excessive immune reactions to more than one exposure to antigens, for example, substances caused by the immune system of the human body, when pollen which does not cause any problem in the human body is contacted with the skin, taken into the body or caused by metabolism and lesions in the body, and repeatedly contacted with external substances to cause excessive immune reactions of memory cells.

Specifically, in the present invention, the allergic immune disease may be one or more selected from the group consisting of allergic urticaria, allergic rhinitis, allergic conjunctivitis, allergic asthma, allergic dermatitis, autoimmune hepatitis, allergic bronchopulmonary aspergillosis (allergic bronchus aspergillosis), and allergic stomatitis (allergic stomatitis), but is not limited thereto.

In the present invention, "prevention" refers to all actions that inhibit or delay the progression of allergic immune diseases by administering the composition of the present invention.

In the present invention, "treatment" and "amelioration" refer to all actions that result in an improvement or beneficial alteration of the symptoms of allergic immune diseases by administration of the compositions of the present invention.

In the present invention, the pharmaceutical composition may be in the form of a capsule, a tablet, a granule, an injection, an ointment, a powder, or a beverage, and the pharmaceutical composition may be administered to a human.

The pharmaceutical composition of the present invention is not limited to these, but can be formulated into oral dosage forms such as powder, granule, capsule, tablet, aqueous suspension, etc., external preparations, suppositories, and sterile injection solutions according to various conventional methods. The pharmaceutical compositions of the present invention may comprise a pharmaceutically acceptable carrier. When the pharmaceutically acceptable carrier is orally administered, a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a pigment, a perfume, etc. may be used, and in the case of using an injection, a buffer, a preservative, a painless agent, a solubilizer, an isotonic agent, a stabilizer, etc. may be used in combination, and in the case of topical administration, a base, an excipient, a lubricant, a preservative, etc. may be used. Dosage forms of the pharmaceutical compositions of the present invention can be prepared in a variety of ways by mixing with a pharmaceutically acceptable carrier as described above. For example, when administered orally, it can be prepared in the form of tablets, dragees, capsules, universal drugs (elixir), suspensions, syrups, wafers and the like, and in the case of injections, it can be prepared in the form of unit administration ampoules or in the form of multiple administrations. The dosage forms may be in other forms, solutions, suspensions, tablets, capsules, release formulations, and the like.

On the other hand, as examples of carriers and excipients suitable for formulation, diluents, lactose, glucose, sucrose, sorbose, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, and the like can be used. Further, a filler, an anticoagulant, a lubricant, a wetting agent, a perfume, an emulsifier, a preservative, and the like may be contained.

The route of administration of the pharmaceutical composition of the present invention is not limited thereto, but includes oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or rectal. Oral or non-oral administration is preferred.

In the present invention, "non-oral" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intradural, intralesional and intracranial injection or infusion techniques. The pharmaceutical compositions of the present invention may also be administered in the form of suppositories for rectal administration.

The pharmaceutical composition of the present invention may be varied depending on various factors such as the activity of the specific compound used, age, body weight, general health, sex, diet, administration time, administration route, excretion rate, drug combination and the severity of a specific disease to be prevented or treated, and the amount of the pharmaceutical composition to be administered may vary depending on the state, body weight, degree of disease, drug form, administration route and period of a patient, but may be appropriately selected by those skilled in the art and may be administered in the range of 0.0001mg/kg to 50mg/kg or 0.001mg/kg to 50mg/kg per day. The administration can be carried out once a day or in multiple times. The above amounts of administration are not intended to limit the scope of the invention in any way. The pharmaceutical compositions of the present invention may be formulated as pills, lozenges, capsules, liquids, gels, syrups, slurries, suspensions.

The compositions of the present invention may be used alone or in combination with methods of using surgery, radiation therapy, hormonal therapy, chemotherapy, and biological response modifiers.

Also, the present invention relates to a method for treating an allergic immune disease comprising the step of administering a pharmaceutically effective amount of the above-mentioned pharmaceutical composition of the present invention to a subject suffering from the allergic immune disease.

Also, the present invention relates to a method for preventing an allergic immune disease comprising the step of administering a pharmaceutically effective amount of the above-mentioned pharmaceutical composition of the present invention to a subject suffering from an allergic immune disease.

In the method of treatment or prevention of the present invention, the pharmaceutical composition to be administered overlaps with the aforementioned description, and therefore the following description is omitted in order to avoid undue complexity as described in the specification.

In the present invention, the method of administering the above pharmaceutical composition may be oral or non-oral administration, and when non-oral administration is performed, it may be administered by intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, epidural injection, intracerebroventricular (intrabrave) injection or intrapleural injection. The composition may be administered orally in clinical administration, and may be administered in the form of a general preparation.

In the present invention, the number of administration of the pharmaceutical composition is not particularly limited, and may be one or more, and more specifically, it may be administered once or more for the purpose of prevention and treatment or may be repeatedly administered when symptoms are present.

In this case, the pharmaceutically effective amount is 0.0001mg/kg to 50mg/kg or 0.001mg/kg to 50mg/kg, and is not limited thereto. The dosage form of the above-mentioned drugs may vary depending on the body weight, age, sex, health condition, diet, administration time, administration method, removal rate, severity of disease, etc. of a particular patient.

In the present specification, the term "administering" means providing a defined composition of the invention to an individual in any suitable way.

In the present specification, the term "subject" refers to all animals such as a patient, monkey, dog, goat, pig or mouse, having an allergic immune disease ameliorated by administration of the composition of the present invention.

In the present specification, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit or risk rate applicable to medical treatment, which can be determined by factors well known in the art and other medical fields, including the type of disease, severity, activity, sensitivity to drugs, administration time, administration route and excretion rate, treatment time of an individual, and drugs used concurrently.

Also, the present invention relates to a method for preparing a cosmetic composition for preventing or improving allergic immune diseases, comprising the step of preparing the above-described immune-tolerant plasmacytoid dendritic cells.

In the present invention, detailed description about a method for inducing immune-tolerant plasmacytoid dendritic cells by treating immature dendritic cells with a Toll-like receptor agonist overlaps with the description of the above pharmaceutical composition, and thus the following description is omitted to avoid the excessive complexity described in the specification.

In the present invention, the cosmetic composition can be prepared into lotions, nourishing creams, nourishing essences, massage creams, beauty bath additives, body lotions, base oils, baby powders, bath gels, body washes, sun screens, suntan creams, skin lotions, skin creams, uv-protection cosmetics, face cleansers, depilatories { make-up }, face and body lotions, face and body creams, skin whitening creams, skin creams, hair lotions, cosmetic creams, jasmine oils, bath soaps, water soaps, beauty soaps, shampoos, hand cleansers (hand lotions), medicinal soaps (non-medical), creams, facial cleansers, body cleansers, scalp cleansers, hair conditioners, cosmetic soaps, tooth whitening gels, toothpastes, and the like. To this end, the compositions of the invention may also comprise solvents, suitable carriers, excipients or diluents which are generally suitable for the preparation of cosmetic compositions.

The kind of solvent further included in the cosmetic composition of the present invention is not particularly limited, and for example, water, saline, dimethyl sulfoxide (DMSO), or a combination thereof may be used, and as a carrier, excipient, or diluent, purified water, oil, wax, fatty acid alcohol, fatty acid ester, surfactant, moisture absorbent (humectant), thickener, antioxidant, viscosity stabilizer, chelating agent, buffer, lower alcohol, and the like may be included, but not limited thereto. And, whitening agents, moisturizers, vitamins, sunscreen creams, perfumes, dyes, antibiotics, antibacterial agents, antifungal agents may be included as necessary.

The oil can be hydrogenated vegetable oil, castor oil, cotton seed oil, olive oil, palm oil, jojoba oil, avocado oil, etc., and the wax can be beeswax, spermaceti wax, carnauba wax, candelilla wax, montan wax, ozokerite, liquid paraffin, and lanolin.

Stearic acid, linoleic acid, linolenic acid, and oleic acid can be used as the fatty acid, cetyl alcohol, octyllauryl alcohol, oleyl alcohol, panthenol, lanolin alcohol, stearyl alcohol, and cetyl alcohol can be used as the fatty acid ester, and isopropyl myristate, and butyl stearate can be used as the fatty acid ester. As the surfactant, a cationic surfactant, an anionic surfactant, and a nonionic surfactant known in the art can be used, and a surfactant derived from a natural product can be preferably used.

In addition thereto, moisture absorbents, thickeners, antioxidants, and the like, which are well known in the cosmetic field, may be contained, and the kind and amount thereof are well known in the art.

The present invention also relates to a method for producing a food composition for preventing or ameliorating an allergic immune disease, which comprises the step of producing the above-described immune-tolerant plasmacytoid dendritic cells.

In the present invention, detailed description about a method for inducing immune-tolerant plasmacytoid dendritic cells by treating Toll-like receptor agonists with immature dendritic cells overlaps with the description in the above pharmaceutical composition, and thus the following description is omitted to avoid the excessive complexity described in the specification.

The food composition of the present invention can be prepared into various foods, for example, beverages, chewing gums, teas, vitamin complexes, powders, granules, tablets, capsules, desserts, rice cakes, breads, and the like. Since the food composition of the present invention is composed of a plant extract having little toxicity and side effects, it can be safely used for a long period of time even for the purpose of prevention.

When the microsporites of the present invention are included in a food composition, the amount thereof may be added in a proportion of 0.1% to 50% of the total weight.

In the case where the food composition is prepared in the form of a beverage, the treatment is not particularly limited except for containing the food composition in the suggested ratio, and for example, a conventional beverage may contain various flavors or natural carbohydrates as additional ingredients. That is, monosaccharides such as glucose, disaccharides such as fructose, polysaccharides such as sucrose, conventional sugars such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol may be contained as the natural carbohydrates. Examples of the flavoring agent include natural flavoring agents (thaumatin, stevia extracts (e.g., rebaudioside a, glycyrrhizin, etc.), and synthetic flavoring agents (saccharin, aspartame, etc.)).

In addition, the food composition of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colors, pectic acid and its salt, alginic acid, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonating agents used for carbonated beverages, and the like.

These components may be used alone or in combination. The proportions of these additives are not critical, but are generally selected in the range of 0.1 parts by weight to about 50 parts by weight per 100 parts by weight of the composition of the present invention.

Detailed description of the invention

The present invention will be described in more detail below with reference to examples. It will be apparent to those skilled in the art that these examples are merely for more specific description, and the scope of the present invention is not limited by these examples in light of the spirit of the present invention.

Examples

Example 1: toll-like receptor agonist regulating effect on plasmacytoid dendritic cell differentiation

In order to confirm whether differentiation into an immune-tolerant plasmacytoid dendritic cell is induced or not in the case of treating a Toll-like receptor agonist during differentiation of an immature dendritic cell, an experiment was performed as follows according to the design shown in fig. 1.

Specifically, bone marrow of the femoral part of the C57BL/6 mouse was sampled using a bone marrow sampling syringe. After the sampled bone marrow was washed with Phosphate Buffer (PBS), erythrocytes were removed by ammonium chloride. The isolated cells (3X 10)⁶cell/well) into a 6-well plate, 1ml of RPMI1640 containing 10% FBS (total bone serum), 2mM L-glutamine, 100U/ml penicillin/streptomycin, 50 μ M mercaptoethanol, 0.1mM non-essential amino acids, 1mM sodium pyruvate, and 250ng/ml FLT3L was added and medium differentiation was initiated. On the third day from the start of differentiation, Pam3 was treated at a concentration of 100ng/ml to 500 ng/ml. 5 days after the start of differentiation, 1ml of the above RPMI1640 was further supplemented, followed by culturingAnd culturing until the eighth day. Plasmacytoid dendritic cells were isolated from cells obtained by culture with a plasmacytoid dendritic cell isolation kit (Miltenyi Biotec, Auburn, Calif.) and a magnetic cell sorting system (Vario MACS: Miltenyi Biotec, Auburn, Calif.) at a purity of 85% or more (FIG. 2). Since the above-mentioned treatment with the Toll-like receptor agonist can affect the differentiation of the plasmacytoid dendritic cells, the surface expression molecule specifically induced in the plasmacytoid dendritic cells was confirmed on the eighth day, and the results thereof are shown in fig. 3 (a). Also, in the case of treating a Toll-like receptor agonist to plasmacytoid dendritic cells (TLRs-pDC), the ratio of the number of initial immature dendritic cells to the number of plasmacytoid dendritic cells obtained after 8 days had elapsed was measured, and the result thereof is shown in part (b) of FIG. 3. However, in order to confirm the differentiation regulating effect of the above Toll-like receptor agonist, the case of untreated Toll-like receptor agonist was represented as comparative example (pDC).

As shown in fig. 2, it was confirmed that differentiation into plasmacytoid dendritic cells was induced even when the Toll-like receptor agonist was treated, and that the differentiation efficiency was not different from that in the case of no treatment.

Example 2: confirmation of the stimulation of isolated plasmacytoid dendritic cells and the secretion pattern of cytokines

In order to confirm the cytokine secretion pattern of the plasmacytoid dendritic cells isolated in example 1, the isolated cells (5X 10)⁵cell/ml) were seeded into 48-well plates, and ODN1826(1 μ g/ml) was processed to generate stimulation. After 24 hours, the resulting supernatant was separated, and the cytokine secretion pattern was confirmed by Enzyme-Linked immunosorbent assay (ELISA), and the results thereof are shown in fig. 4. However, for the effect of the Toll-like receptor agonist treatment, the case where the Toll-like receptor agonist was not treated during the differentiation of the immature dendritic cells in example 1 described above is represented as comparative example (pDC).

As shown in FIG. 4, in general, type I interferons (IFN-. alpha.and IFN-. beta.) and TNF-. alpha.and IL-12p70, which are representative inflammatory cytokines, were strongly induced in plasmacytoid dendritic cells (pDCs). However, in the case of treating Toll-like receptor agonists (0.5. mu.g/ml) with plasmacytoid dendritic cells (TLRs-pDC) induced to differentiate, it was confirmed that the expression level thereof was sharply decreased when stimulated with ODN 1826. In contrast, it was confirmed that the expression level of IL-10 as an immunosuppressive cytokine was significantly increased in plasmacytoid dendritic cells (TLRs-pDC) induced after the treatment with a Toll-like receptor agonist.

Thus, the plasmacytoid dendritic cells induced to differentiate by treating immature dendritic cells with a Toll-like receptor agonist according to the present invention are immune-tolerant, unlike general plasmacytoid dendritic cells.

Example 3: confirmation of IL-10 in plasmacytoid dendritic cells based on treatment with various Toll-like receptor agonists Secretory form

In order to confirm the immune tolerance-inducing effects of various Toll-like receptor agonists using the ligands shown in table 2 below, differentiation of plasmacytoid dendritic cells was induced by the same method as in example 1 above. After 8 days of culture, plasmacytoid dendritic cells were sorted out with a purity of 85% or more using a plasmacytoid dendritic cell isolation kit (Miltenyi Biotec, Auburn, CA) and a magnetic cell sorting system (Vario MACS: Miltenyi Biotec, Auburn, CA). The isolated cells (5X 10)⁵cell/ml) were inoculated into 48-well plates, and after treating ODN1826 (1. mu.g/ml), cultured for 24 hours. Thereafter, only the supernatant was separated, and the secretory form of the immunosuppressive cytokine IL-10 was confirmed by the enzyme-linked immunosorbent assay, and the results thereof are shown in fig. 5. However, in order to confirm the effect of the Toll-like receptor agonist treatment, the case where the Toll-like receptor agonist was not treated during the differentiation of the immature dendritic cells was represented as comparative example (non).

TABLE 2

Non	TLR2 agonists	TLR3 agonists	TLR4 agonists	TLR7 agonists	TLR9 agonists
						Untreated	Pam3	Poly I：C	LPS	Imiquimod	CpG-ODN

As shown in fig. 5, it was confirmed that among various Toll-like receptor agonists stimulated with ODN1826, only the expression level of IL-10 of plasmacytoid dendritic cells induced to differentiate by treatment with TLR2 agonist, TLR4 agonist, TLR7 agonist and TLR8 agonist of primary response gene 88 signal of myeloid differentiation factor was significantly increased.

Thus, it was confirmed that the immune-tolerant plasmacytoid dendritic cells were induced when the Toll-like receptor that is a signal of the primary response gene 88 for myeloid differentiation factors in the pathway during differentiation of immature dendritic cells was treated.

Example 4: confirmation of fines in plasmacytoid dendritic cells based on time point of Toll-like receptor agonist treatment Form of cytokine secretion

To confirm the effect of inducing immune tolerance according to the treatment time point of the Toll-like receptor agonist, differentiation of plasmacytoid dendritic cells was induced by the same method as in example 1, and the Toll-like receptor agonist was treated on the day of initiation of differentiation (0day treatment) and 3 days after the day of initiation of differentiation (3day treatment)An animal agent. After 8 days of co-culture, plasmacytoid dendritic cells were isolated with a purity of 85% or more using a plasmacytoid dendritic cell isolation kit (Miltenyi Biotec, Auburn, CA) and a magnetic cell sorting system (Vario MACS: Miltenyi Biotec, Auburn, CA). The isolated cells (5X 10)⁵cell/ml) were inoculated into 48-well plates, and after treating ODN1826 (1. mu.g/ml), cultured for 24 hours. Thereafter, only the supernatant 24 was separated, and the secretion pattern of the cytokine was confirmed by the enzyme-linked immunosorbent assay, and the results are shown in FIG. 6. However, in order to confirm the effect of the Toll-like receptor agonist treatment, the case where the Toll-like receptor agonist is not treated during differentiation of the immature dendritic cells is represented as a comparative example (no-treatment, non).

As shown in FIG. 6, in the case of treating the Toll-like receptor agonist on the day of differentiation initiation (0day treatment) or on the day from the day of differentiation initiation to the third day (3day treatment), the expression of type I interferons (IFN-. alpha.and IFN-. beta.) and TNF-. alpha.and IL-12p70, which are representative inflammatory cytokines, was suppressed and the expression level of IL-10, which is an anti-inflammatory cytokine, was significantly increased, as compared with the case of no-treatment (no-treatment).

From this, it was confirmed that even when a Toll-like receptor agonist and FLT3L as a factor for inducing differentiation were treated simultaneously at the time point when the immature dendritic cells started to differentiate, the differentiation of the immune-tolerant plasmacytoid dendritic cells was induced as in the case of treatment during differentiation.

Example 5: confirmation of Co-stimulatory factors in plasmacytoid dendritic cells treated with a treatment Toll-like receptor agonist Expression of daughter and MHC molecules

In dendritic cells including plasmacytoid dendritic cells, when an external antigen is recognized, the antigen is presented by MHC molecules in order to activate T cells, and the interaction is promoted by expressing co-stimulatory factors such as CD80 and CD 86.

Thus, the plasmacytoid dendritic cells isolated in example 1 were stimulated with ODN1826, and then cultured for 24 hours, and then the expression level of the cell surface molecule was confirmed. First, in order to confirm the effect on the analysis of the expression of surface factors of plasmacytoid dendritic cells, anti-CD 11c (PE-Cy7, BD Biosciences), anti-PDCA-1 (PerCP-eFluor 710, ebiosciences) and cell surface molecules, which are specific markers for plasmacytoid dendritic cells, were treated with specific anti-CD 80(v450, BD Biosciences), anti-CD 86(APC, ebiosciences), anti-MHC-I (PE, ebiosciences) and anti-MHC-II (APC-eFluor 780, ebiosciences) antibodies at a temperature of 4 ℃ for 30 minutes, and then analyzed by a flow cytometer LSRFortesta x-20(BD Biosciences). The results are shown in FIG. 7. However, for the effect of the treatment with the Toll-like receptor agonist, the case where the Toll-like receptor agonist was not treated during the differentiation of the immature dendritic cells was represented as comparative example (pDC).

As shown in fig. 7, in general plasmacytoid dendritic cells (pDC), MHC class ii molecules for presenting CD86 and exogenous antigens as co-stimulatory factors were expressed at high levels, but in the case of plasmacytoid dendritic cells induced to differentiate by treatment with Toll-like receptor agonists (TLRs-pDC), no response or reduced response was shown. On the other hand, it was confirmed that CD80 and an endogenous antigen or cross-antigen MHC class I molecule for presentation as one of the other costimulators were expressed at a higher level than cells (pDC) with untreated plasmacytoid dendritic cells (TLRs-pDC) induced to differentiate, due to the treatment with a Toll-like receptor agonist.

Example 6: confirmation of immune tolerance-inducing component for the treatment of Toll-like receptor agonist on plasmacytoid dendritic cells Expression of the seed

In general, dendritic cells induce T cell responses by antigen presentation to activate acquired immune responses, and in the case of some immune-tolerant dendritic cells, have been reported to be able to suppress T cell responses. It is reported that the suppression of this immune response is recently induced by various immune tolerance-inducing molecules, for example, PD-L1 and IDO. Also, CCR9+ pDCs have been reported to induce various immune tolerance phenomena by modulating the strong induction of T cells.

Therefore, hereinafter, in order to confirm the expression of the immune tolerance-inducing molecules from the plasmacytoid dendritic cells isolated in the above example 1, the plasmacytoid dendritic cells were treated with cell surface factor antibodies of anti-CD 11c (PE-Cy7, BD Biosciences) and anti-PDCA-1 (PerCP-eFluor 710, ebioscience) as specific markers and anti-PD-L1 (PE, ebioscience) and anti-CCR 9(FITC, ebioscience) and the like as immune tolerance-inducing cell surface molecules at a temperature of 4 ℃ for 30 minutes. In order to measure the expression of IDO induced in cells, anti-IDO (eFluor660, ebioscience) was stained after 30 minutes of fixation/permeation (BD Bioscience) at a temperature of 4 ℃, and the expression level was analyzed using a flow cytometer lsrnoressa x-20, and the results thereof are shown in fig. 8. However, in order to confirm the effect of the Toll-like receptor agonist treatment, the case where the Toll-like receptor agonist was not treated during differentiation of the immature dendritic cells in example 1 described above was represented as comparative example (pDC).

As shown in fig. 8, in the case of the plasmacytoid dendritic cells treated with the Toll-like receptor agonist, when the plasmacytoid dendritic cells were stimulated with ODN1826, it was confirmed that the expression levels of CCR9 and IDO molecules were both significantly increased as compared to the untreated plasmacytoid dendritic cells, and CCR9 showed higher expression patterns than the untreated plasmacytoid dendritic cells even when the plasmacytoid dendritic cells were not stimulated.

Thus, when a Toll-like receptor agonist is treated at the time of differentiation of immature dendritic cells, plasmacytoid dendritic cells having immune tolerance are induced to differentiate.

Example 7: confirmation of Induction of regulatory T cells by treatment of Toll-like receptor agonists on plasmacytoid dendritic cells Capability of

It is reported that the immune-tolerant plasmacytoid dendritic cells (Tolerogenic pDC) induce differentiation into Regulatory T cells (tregs) and suppress the activity or proliferation of effector T cells (effector T cells).

Therefore, hereinafter, whether or not proliferation of regulatory T cells (Foxp3+ CD4+ T cells) based on plasmacytoid dendritic cells (TLRs-pDC) induced to differentiate by treatment with a Toll-like receptor agonist was confirmed. Specifically, T cells isolated from allogeneic mice and stained with celltrace (invitrogen) were compared with the plasmacytoid dendritic cells (TLRs-pDC) isolated in example 1 above at a ratio of 5: 1 ratio for 5 days. However, as the above-mentioned plasmacytoid dendritic cells, plasmacytoid dendritic cells stimulated with ODN1826 (ODN +) and plasmacytoid dendritic cells that are stimulated with ODN-, (ODN-) were used. After 5 days of culture, anti-CD 4(Percp-cy5.5, ebioscience) was treated at a temperature of 4 ℃ for 30 minutes, and Foxp 3/Transcription Factor Staining Buffer (transfer Factor stabilizing Buffer) (ebioscience) was treated at a temperature of 37 ℃ for 30 minutes. Thereafter, after treatment with anti-Foxp 3(PE, ebioscience), whether or not the T-regulatory cells proliferated was confirmed by flow cytometry, lsrortessa x-20, and the results thereof are shown in fig. 9 (a), and the proliferation rate of the T-regulatory cells was calculated and the results thereof are shown in fig. 9 (b). However, in order to confirm the effect of the Toll-like receptor agonist treatment, the general plasmacytoid dendritic cells stimulated (ODN +) with or without ODN1826 were represented as comparative example (pDC).

As shown in fig. 9, in the case of general plasmacytoid dendritic cells (pDC), the proliferation of regulatory T cells was not induced, but was instead confirmed to be inhibited when stimulated (ODN +) with ODN 1826. In contrast, in the case of plasmacytoid dendritic cells (TLRs-pDC) treated with a Toll-like receptor agonist to be induced to differentiate, it was confirmed that the proliferation of regulatory T cells was significantly higher in the case of stimulation with ODN1826 (ODN +) than in the case of non-stimulation (ODN-).

Thus, it was confirmed that in the case of further treating Toll-like receptor agonists to the tolerogenic plasmacytoid dendritic cells induced to differentiate according to the present invention, the tolerogenic activity was activated and also the differentiation into regulatory T cells was efficiently induced.

Example 8: confirmation of Toll-like receptor agonist Activity as Rv1411c protein

According to the existing report, the protein derived from tubercle bacillus can be combined with various Toll-like receptors. Therefore, hereinafter, the activity as a Toll-like receptor agonist of Rv1411c protein was confirmed by confirming the binding of dendritic cells differentiated from bone marrow of TLR2 gene knockout mice and wild type mice to Rv1411c protein.

Specifically, after isolating mouse bone marrow and removing erythrocytes, the dendritic cells were cultured for 8 days using RPMI1640 containing 10% FBS, 1% antibiotic and 100ng/ml GM-CSF (Granulocyte-macroporous-collagen stimulating factor) for differentiation. After 8 days of culture, after treating 5. mu.g/ml of Rv1411c protein with dendritic cells isolated from wild-type dendritic cells (WT) and TLR2 knock-out mice (TLR2-/-) respectively, the proteins were allowed to bind easily by mixing intermittently for 2 hours. Thereafter, an antibody having antigen-antibody specificity was stained for His molecule tagged to Rv1411c protein, the degree of binding was measured by flow cytometry, and the results thereof are shown in fig. 10.

As shown in fig. 10, it was confirmed that in wild-type dendritic cells (WT), binding to Rv1411c protein was increased compared to TLR2 knockout dendritic cells (TLR2-/-), but that Rv1411c protein was treated even in TLR2 knockout dendritic cells (TLR2-/-) with the same results as in the untreated experimental group.

Thus, Rv1411c protein binds to TLR2 receptors and is active as TL2 agonists.

Example 9: confirmation of the differentiation-inducing ability of the immune-tolerant plasmacytoid dendritic cell of the Rv1411c protein

The differentiation of the immunotolerant plasmacytoid dendritic cells was induced by the same procedure as in example 1, and Rv1411c protein (0.1. mu.g/ml, 0.5. mu.g/ml) in which the activity as a Toll-like receptor agonist in example 8 was confirmed was used as a Toll-like receptor agonist. After 8 days of culture, plasmacytoid dendritic cells were isolated with a purity of 85% or more using a plasmacytoid dendritic cell isolation kit (Miltenyi Biotec, Auburn, CA) and a magnetic cell sorting system (Vario MACS: Miltenyi Biotec, Auburn, CA). The isolated cells (5X 10)⁵cell/ml) were inoculated into 48-well plates, treated with ODN1826 (1. mu.g/ml), and cultured for 24 days. The supernatant obtained by the culture was separated, and the results of confirming the cytokine secretion morphology by Enzyme-Linked Immunosorbent assay (ELISA) are shown in the graph of fig. 11. The supernatant obtained by the culture was separated and purified by Enzyme-linked immunosorbent assay (ELISA)The cytokine secretion pattern was confirmed, and the results are shown in the graph of fig. 11. However, in order to confirm the treatment effect of Rv1411c protein, general plasmacytoid dendritic cells stimulated (ODN +) or unstimulated (ODN-) with ODN1826 as untreated Rv1411c protein were represented as comparative example (pDC).

As shown in FIG. 11, when the plasmacytoid dendritic cells (Rv1411c (0.1. mu.g/ml) -pDC, Rv1411c (0.5. mu.g/ml) -pDC) were treated with the Rv1411c protein to stimulate ODN1826, the expression of type I interferons (IFN-. alpha.and IFN-. beta.) and TNF-. alpha.and IL-12p70, which are representative inflammatory cytokines, strongly induced in general plasmacytoid dendritic cells abruptly decreased in a concentration-dependent manner, and the expression level of IL-10, which is an immunosuppressive cytokine, significantly increased in a concentration-dependent manner.

Thus, unlike the conventional plasmacytoid dendritic cells, it was confirmed that the plasmacytoid dendritic cells treated with Rv1411c protein had immune tolerance, and the degree of immune tolerance was increased in proportion to the treatment concentration of Rv1411c protein.

Example 10: confirming plasmacytoid dendritic cells induced to differentiate by treatment with Rv1411c protein Expression of co-stimulatory factors, MHC molecules and immune tolerance-inducing molecules

In the plasmacytoid dendritic cells induced to differentiate by Rv1411c protein in example 9, the expression patterns of surface molecules and enzymes were confirmed.

First, in order to analyze the influence of surface factor expression of plasmacytoid dendritic cells, plasmacytoid dendritic cells were treated with antibodies to cell surface factors such as anti-CD 11c (PE-Cy7, BD Biosciences) and anti-PDCA-1 (PerCP-eFluor 710, ebioscience) and anti-CD 80(v450, BD Biosciences), anti-CD 86(APC, ebioscience), anti-MHC-I (PE, ebioscience) and anti-MHC-II (APC-eFluor 780, ebioscience), anti-PD-L1 (PE, ebioscience) and anti-CCR 9(FITC, ebioscience) at a temperature of 4 ℃ for 30 minutes. In order to confirm the expression of IDO induced in the cells, anti-IDO (eFluor660, ebioscience) was treated at 4 ℃ for 30 minutes after fixation/permeation (BD Bioscience), and then analyzed using a flow cytometer lsrnoressa x-20, and the results thereof are shown in fig. 12. However, in order to confirm the effect of treatment with Rv1411c protein, general plasmacytoid dendritic cells untreated with Rv1411c protein and stimulated (ODN +) or unstimulated (ODN-) ODN1826 were shown as comparative example (pDC).

As shown in fig. 12, when ODN1826 was stimulated, MHC class ii molecules for presenting CD86 and exogenous antigens as co-stimulatory factors were expressed at high levels in general plasmacytoid dendritic cells (pDC) untreated with Rv1411c protein, whereas no response or reduced response characteristics were shown in the case of plasmacytoid dendritic cells (Rv1411c-pDC) induced to differentiate by treatment with Rv1411c protein.

In contrast, in the case of CD80 and an endogenous antigen or cross antigen MHC class I molecule as one for presentation as another costimulator, the plasmacytoid dendritic cells induced to differentiate by the Rv1411c protein (Rv1411c-pDC) were expressed at a higher level than the ordinary plasmacytoid dendritic cells (pDC).

In the case of the plasmacytoid dendritic cells (Rv1411c-pDC) induced to differentiate by treatment with Rv1411c protein, when ODN1826 was stimulated (ODN +), it was confirmed that the expression levels of PD-L1, CCR9, and IDO molecules were increased compared to the unstimulated (ODN-), and CCR9 and PD-L1 also showed higher expression patterns than the general plasmacytoid dendritic cells (pDC) in the unstimulated (ODN-).

Example 11: t cells of plasmacytoid dendritic cells induced to differentiate by treatment with Rv1411c protein Inhibition of the Activity of

The most important feature in the organism of immune-tolerant plasmacytoid dendritic cells is the activity of the inhibited T lymphocytes. Therefore, in order to confirm the effect of the plasmacytoid dendritic cells induced to differentiate by the treatment with Rv1411c protein in example 9 on the proliferation and activity of T cells, the following experiment was performed.

Specifically, PMA/Ionomycin (ebioscience) stimulation by 1X was isolated from allogeneic mice and used with CellTrace (Invitrogen) stained T cells (1.5X 10)⁵cell/well). This PMA/Ionomycin treatment increases the proliferation rate of T lymphocytes and promotes the secretion of interferon gamma (IFN-gamma). When these reactions were generated, T cells and Rv1411c proteins were mixed so that the ratio of plasmacytoid dendritic cells (Rv1411c-pDC) or general plasmacytoid dendritic cells (pDC) induced to differentiate became 1: 5, and culturing for 3 days. After 3 days, anti-CD 4(Percp-cy5.5, ebioscience), anti-CD 8(Percp-cy5.5, ebioscience) were treated and dyed at a temperature of 4 ℃ for 30 minutes. The proliferation degree of T cells was analyzed by a flow cytometer LSRFortessa x-20 and the results are shown in FIG. 13. Further, after isolating the supernatant obtained by culturing for 3 days, the cytokine secretion pattern was confirmed by Enzyme-linked immunosorbent assay (ELISA), and the results are shown in the graph of fig. 14. However, in order to confirm the treatment effect of Rv1411c protein, general plasmacytoid dendritic cells stimulated (ODN +) or unstimulated (ODN-) with ODN1826 as untreated Rv1411c protein were represented as comparative example (pDC).

As shown in fig. 13, although both the plasmacytoid dendritic cells induced to differentiate by treatment with Rv1411c protein (Rv1411c-pDC) and the general plasmacytoid dendritic cells (pDC) induced T cells, the plasmacytoid dendritic cells induced to differentiate by treatment with Rv1411c protein (Rv1411c-pDC) had a significantly lower proliferation-inducing ability for CD4+ T cells than the general plasmacytoid dendritic cells (pDC).

Also, as shown in fig. 14, the plasmacytoid dendritic cells (Rv1411c-pDC) induced to differentiate by treatment with Rv1411c protein induced secretion of IFN-gamma at a level lower than that of general plasmacytoid dendritic cells (pDC).

Example 12: regulatory T of plasmacytoid dendritic cells induced to differentiate by treatment with Rv1411c protein Modulation of differentiation of cells

T cells isolated from allogeneic mice and stained with celltrace (invitrogen) were cultured for 5 days with the plasmacytoid dendritic cells (Rv1411c-pDC) or general plasmacytoid dendritic cells (pDC) induced to differentiate by treatment with Rv1411c protein in example 9 above, so that the ratio thereof became 10: 1. 5: 1 or 1: 1. however, as the above-mentioned plasmacytoid dendritic cells, use was made of ODN 1826-stimulated (ODN +) and unstimulated (ODN-). On day 5 of culture, cells were treated with anti-CD 4(Percp-cy5.5, ebioscience) for 30 minutes at a temperature of 4 ℃ and Foxp 3/transcription factor staining buffer (ebioscience) for 30 minutes at a temperature of 37 ℃. Thereafter, anti-Foxp 3(PE, ebioscience) was stained and whether proliferation was confirmed as regulatory T cells was performed by flow cytometry, lsrortessa x-20, and the results thereof are shown in part (a) of fig. 15, and the proliferation rate of regulatory T cells was calculated and the results thereof are shown in part (b) of fig. 15.

As shown in fig. 15, in the case of general plasmacytoid dendritic cells (pDC), the proliferation of regulatory T cells was not induced, but when ODN1826 was used to stimulate (ODN +), it was confirmed that the proliferation of regulatory T cells was inhibited instead. In contrast, in the case of the plasmacytoid dendritic cells (Rv1411c-pDC) treated with Rv1411c protein to be induced to differentiate, it was confirmed that the proliferation of regulatory T cells was significantly higher in the case of stimulation with ODN1826 (ODN +) than in the case of non-stimulation (ODN-), and that the proliferation rate of regulatory T cells was increased as the ratio of T cells to plasmacytoid dendritic cells (Rv1411c-pDC) was increased.

Thus, in the case where Toll-like receptor agonists are further treated on the tolerogenic plasma cell-like cells induced to differentiate according to the present invention, the proliferation of regulatory T cells is also effectively induced by activating the immunotolerance.

Example 13: effector T based on plasmacytoid dendritic cells induced to differentiate by treatment with Rv1411c protein Inhibition of cellular activity

It is reported that regulatory T cells induce immune-tolerant dendritic cells and inhibit the proliferation of other T cells. Therefore, hereinafter, the activity of regulatory T cells as T cells induced to differentiate by Rv1411c protein was confirmed according to example 12 above.

First, the spleen of the mouse was isolated, red blood cells were removed, CD4+, CD 25-effector T cells were isolated using a magnetic cell sorting system (MACS), and then a purple proliferation dye (violet proliferation) was stained to identify the proliferation degree. Thereafter, differentiated T cells were cultured with CD4+, CD 25-effector T cells in wells coated with anti-CD 3e and anti-CD 28 antibodies for 2 days. As a result, the change in the proliferation potency of T cells was measured relative to the ratio of CD 25-effector T cells to priming T cells, and is graphically shown in fig. 16.

As shown in fig. 16, it was confirmed that, among the plasmacytoid dendritic cells induced to differentiate by treatment with Rv1411c protein, only T cells induced by cells stimulated with ODN1826 (Rv1411c-pdc (ODN)) reduced the degree of differentiation of effector T cells.

Thus, it was confirmed that the activity of effector T cells is effectively inhibited by activating immune tolerance in the case where tolerizing plasma cell-like cells induced to differentiate according to the present invention are further treated with a Toll-like receptor agonist.

Example 14: obtaining plasmacytoid dendritic cells induced to differentiate after treatment with Toll-like receptor agonists Yield of

Bone marrow from the thigh of C57BL/6 mice was sampled using a bone marrow sampling syringe. After the sampled bone marrow was washed with Phosphate Buffer (PBS), erythrocytes were removed by ammonium chloride. The isolated cells (5X 10)⁵cell/well) into a 6-well plate, 1ml of RPMI1640 containing 10% FBS (total bone serum), 2mM L-glutamine, 100U/ml penicillin/streptomycin, 50 μ M mercaptoethanol, 0.1mM non-essential amino acids, 1mM sodium pyruvate, and 250ng/ml FLT3L was added and medium differentiation was initiated. On the third day from the start of differentiation, Rv1411c protein and Pam3 were treated with Toll-like receptor agonist at concentrations of 100ng/ml to 500 ng/ml. 5 days after the start of differentiation, 1ml of the above RPMI1640 was further supplemented, and the culture was continued until the eighth day. Plasmacytoid dendritic cells were isolated from cells obtained by culture using a plasmacytoid dendritic cell isolation kit (Miltenyi Biotec, Auburn, Calif.) and a magnetic cell sorting system (Vario MACS: Miltenyi Biotec, Auburn, Calif.) and were 85% or more pure. The results of measuring the number of isolated plasmacytoid dendritic cells (TLR aginst) are shown in the graph of fig. 17. However,in order to confirm the effect of Toll-like receptor agonist treatment, the case where Toll-like receptor agonist was not treated during differentiation of immature dendritic cells was shown as comparative example (non).

As shown in fig. 17, it was confirmed that in the case of treating a Toll-like receptor agonist during differentiation of immature dendritic cells (TLR aginst), the number of plasmacytoid dendritic cells induced by differentiation was significantly increased as compared to the untreated case (Non).

Example 15: allergic diseases of plasmacytoid dendritic cells induced by differentiation following treatment with Toll-like receptor agonists Therapeutic effects

1. Preparation of animal model of food allergy disease induced by egg yolk protein

In order to create an animal model of food allergy disease induced by egg yolk albumin, the following experiment was performed according to the design shown in fig. 18.

Specifically, after 4-week-old BALB/c female mice were purchased and purified under Specific Pathogen Free (SPF) conditions for 1 week, experiments were performed when 5 weeks old. Mu.g of Ovalbumin (OVA) and 1mg of aluminum hydroxide (aluminum hydroxide, Alum, Sigma-Aldrich Korea LTD) were injected into the peritoneal cavity of each mouse on days 0 and 7, respectively, to sensitize food allergy (sensitization). From day 14 to day 21 of sensitization, boosting was performed by orally administering 10mg of egg yolk protein 4 times per one (boosting).

2. Preparation of immune-tolerant plasmacytoid dendritic cells

After treating Rv1411c protein to differentiate induced plasmacytoid dendritic cells and conventional plasmacytoid dendritic cells as described in example 9 above, they were stimulated with 1. mu.g/ml OVA peptides (OVA323-339 and OVA257-264) and 1. mu.g/ml ODN1826(TLR9 agonist) for 3 hours before being harvested (harvest).

To confirm the food allergy inhibitory effect by injection of the above activated immune-tolerant dendritic cells, on the 14 th day of sensitization, the vein of the animal mouse model prepared as described above was filled at a rate of 1X 10⁶Amount of cells/mouse plasmacytoid dendritic cells were injected intravenously (i.v.), and negative control group was injected only on day 14 of sensitizationAqueous Phosphorylation Buffer (PBS). Furthermore, 50mg of OVA was orally administered to each mouse on day 28 after the first sensitization to induce (challenge) anaphylaxis.

3. Evaluation of hypothermia and systemic anaphylaxis (anaphylaxis) symptoms caused by yolk protein-induced food allergy disease

For the evaluation of the symptoms of systemic anaphylaxis caused by anaphylaxis, the results were the same as the criteria in table 3 by visual observation, and the disease scores were given, the results of which are shown in fig. 19. Also, whether it is hypothermia or not was determined by measuring the change in body temperature of the rectum for 1 hour after induction of the allergic reaction using a digital thermometer, TESTO 925 (Tes to AG, germany), as shown in fig. 20, and the incidence of diarrhea was also evaluated as shown in fig. 21.

TABLE 3

Disease score	Symptoms of allergic reactions
		0	Without any symptoms (free jump)
1	Scratching or massaging the nose and around the head
		2	Swelling around eyes and mouth, decreased vitality, diarrhea
3	Cyanosis of asthma, dyspnea, mouth and tail
		4	No vitality (reaction), twitch or trembling after stimulation
5	Death was caused by death

As shown in fig. 19, when the yolk protein-derived food allergy was induced, the allergy was induced in the case of treating only the negative control group of phosphorylated aqueous buffer solution (PBS), so that the phenomena of reduction in activity amount and swelling and scratching around eyes and mouth, and dyspnea were observed. However, in the case of injecting the immune-tolerant plasmacytoid dendritic cells (TLRs-pDC 14d) differentiated and induced by the treatment of the Rv1411c protein into mice, there was no significant change, and only phenomena around the pars flexibilis of eyes and mouth were observed, and the symptoms thereof were greatly relieved as compared with the case of injecting the conventional plasmacytoid dendritic cells (pDS).

As shown in fig. 20, when the yolk protein-derived food allergy was induced, in the case of the negative control group treated with only phosphorylated buffer aqueous solution (PBS), the allergy caused rapid development of low temperature symptoms, but in the case of injecting the immune-tolerant plasmacytoid dendritic cells (TLRs-pDC) induced by differentiation by treatment with Rv1411c protein into mice, the rectal temperature decreased by a small amount, and rapid recovery of hypothermia was confirmed as compared with the above negative control group (PBS).

Also, as shown in fig. 21, the incidence of diarrhea was also shown to have diarrhea symptoms in all mice administered with only phosphorylated aqueous buffer solution (PBS), and 80% in the case of injection of conventional plasmacytoid dendritic cells (pDC), whereas the incidence of diarrhea was significantly reduced to around 40% in the case of injection of immune-tolerant plasmacytoid dendritic cells (TLRs-pDC 14d) induced by differentiation by treatment with Rv1411c protein into mice.

Thus, it was confirmed that the immune-tolerant plasma cell-like cells induced by differentiation according to the present invention had the effect of suppressing the initiation of the yolk protein-induced food allergy disease.

Example 16: immune tolerant plasmacytoid dendritic cells in animal models of food allergy induced by egg yolk albumin Influence of cells on immunoglobulins in blood

From the immunoglobulin E and immunoglobulin G1 antibodies, which are known to be antibodies involved in mediating T helper type 2 allergy, it was confirmed how the injection of immune-tolerant plasmacytoid dendritic cells in an egg yolk protein-induced food allergy disease animal model has an effect on the change in the concentration of immunoglobulin E and immunoglobulin G1 in blood.

Specifically, in the same manner as in example 15 above, after an egg yolk protein-induced food allergy disease animal model was prepared, 50mg of OVA (challenge) was administered after injection of Phosphate Buffered Saline (PBS) or differentiation of induced plasmacytoid dendritic cells by treatment of Rv1411c protein in example 9 above. After 1 hour had passed, serum was isolated from the extracted mouse blood, and the blood concentrations of OVA-specific immunoglobulin E and immunoglobulin G1 in the serum were confirmed, and the results are shown in FIG. 22.

As shown in fig. 22, when allergic reaction (PBS) was induced with yolk protein-derived food, the concentrations of OVA-specific immunoglobulin E and immunoglobulin G1 increased, but were confirmed to be significantly reduced in the case of injection of immune-tolerant plasmacytoid dendritic cells induced by differentiation after treatment with rv1411c protein, relative to Normal group (Normal).

Thus, the differentiation-induced immune-tolerant plasma cell-like cells according to the present invention are exposed to the egg yolk protein antigen, thereby decreasing the expression of increased OVA-specific immunoglobulin, thereby inhibiting the initiation of food allergy diseases.

Example 17: immune tolerant plasmacytoid dendritic cells in animal models of food allergy induced by egg yolk albumin The effect of cells on the production of T helper type 2 cytokines.

Generally, overexpression of T-helper type 2 cytokines is known to play an important role in the onset of allergic symptoms. Therefore, the change in the expression levels of interleukin-4 and interleukin-5 in serum was evaluated by injecting immune-tolerant plasmacytoid dendritic cells into an egg yolk protein-induced food allergy disease animal model, and an experiment for confirming the effect of suppressing allergy was performed.

Specifically, in the same manner as in example 15 above, after an egg yolk protein-induced food allergy disease animal model was prepared, 50mg of OVA (challenge) was administered after injection of Phosphate Buffered Saline (PBS) or differentiation of induced plasmacytoid dendritic cells by treatment of Rv1411c protein in example 9 above. After 1 hour had passed, serum was isolated from the extracted mouse blood, and the blood concentrations of OVA-specific immunoglobulin E and immunoglobulin G1 in the serum were confirmed, and the results are shown in FIG. 23.

As shown in fig. 23, when the egg yolk protein-derived food allergy reaction (PBS) was elicited, the expression levels of interleukin-4 and interleukin-5 as T helper type 2 cytokines were increased, but were significantly reduced in the case where immune-tolerant plasmacytoid dendritic cells (TLRs-pDC 14d) induced by differentiation by treatment of Rv1411c protein were injected into mice, relative to the Normal group (Normal).

Thus, it was confirmed that the immune-tolerant plasma cell-like cells induced by differentiation according to the present invention inhibit the initiation of food allergy diseases by inhibiting the expression of T-helper type 2 cytokines when food allergy is induced by egg yolk albumin.

While certain features of the invention have been described in detail above, it will be apparent to those skilled in the art that this detailed description is merely a preferred example, and should not be construed to limit the scope of the invention. Therefore, the actual scope of the invention will be defined by the appended claims and equivalents thereof.

Claims (21)

1. A method for preparing a pharmaceutical composition for preventing or treating an allergic immune disease,

comprising the step of treating a Toll-like receptor agonist on immature dendritic cells to prepare immunotolerant plasmacytoid dendritic cells.

2. The method for preparing the pharmaceutical composition for preventing or treating allergic immune diseases according to claim 1,

inducing differentiation into an immune-tolerant plasmacytoid dendritic cell by treating the above Toll-like receptor agonist before or during the initial differentiation of the above immature dendritic cell.

3. The method for preparing the pharmaceutical composition for preventing or treating allergic immune diseases according to claim 1,

the Toll-like receptor agonist is treated before the immature dendritic cells are differentiated to be completely differentiated.

4. The method for preparing the pharmaceutical composition for preventing or treating allergic immune diseases according to claim 2 or 3,

the differentiation of the above-described immature dendritic cells is performed by using a differentiation-inducing factor.

5. The method for preparing the pharmaceutical composition for preventing or treating allergic immune diseases according to claim 4,

the differentiation-inducing factor is an FMS-like tyrosine kinase3 ligand.

6. The method for preparing the pharmaceutical composition for preventing or treating allergic immune diseases according to claim 4,

treating the immature dendritic cells with the differentiation-inducing factor at least once.

7. The method for preparing the pharmaceutical composition for preventing or treating allergic immune diseases according to claim 1,

the immature dendritic cells are treated with the Toll-like receptor agonist and the differentiation-inducing factor at the same time.

8. The method for preparing the pharmaceutical composition for preventing or treating allergic immune diseases according to claim 1,

the Toll-like receptor agonist is treated more than once.

9. The method for preparing the pharmaceutical composition for preventing or treating allergic immune diseases according to claim 1,

the Toll-like receptor agonist pathway myeloid differentiation factor primary response gene 88 signals described above.

10. The method for preparing the pharmaceutical composition for preventing or treating allergic immune diseases according to claim 1,

the Toll-like receptor agonist comprises at least one selected from the group consisting of a TLR2 agonist, a TLR4 agonist, a TLR5 agonist, a TLR7 agonist, a TLR8 agonist, a TLR9 agonist, a TLR11 agonist, a TLR12 agonist and a TLR13 agonist.

11. The method for preparing the pharmaceutical composition for preventing or treating allergic immune diseases according to claim 10,

the Toll-like receptor agonist further comprises at least one of a TLR1 agonist and a TLR6 agonist.

12. The method for preparing the pharmaceutical composition for preventing or treating allergic immune diseases according to claim 2,

further comprising the step of further treating the tolerogenic plasmacytoid dendritic cells induced to differentiate with a Toll-like receptor agonist to activate the immunological tolerance.

13. The method for preparing the pharmaceutical composition for preventing or treating allergic immune diseases according to claim 12,

the Toll-like receptor agonist used for activating the immune tolerance is a TLR9 agonist.

14. The method for preparing the pharmaceutical composition for preventing or treating allergic immune diseases according to claim 1,

the allergic immune disease is at least one selected from the group consisting of allergic urticaria, allergic rhinitis, allergic conjunctivitis, allergic asthma, allergic dermatitis, autoimmune hepatitis, allergic bronchopulmonary aspergillosis, and allergic stomatitis.

15. A pharmaceutical composition for preventing or treating an allergic immune disease,

comprising immunologically tolerant plasmacytoid dendritic cells induced by treatment of immature dendritic cells with a Toll-like receptor agonist.

16. The pharmaceutical composition for preventing or treating allergic immune diseases according to claim 15,

inducing differentiation into the above-mentioned immune-tolerant plasmacytoid dendritic cells by treating the above-mentioned Toll-like receptor agonist before or during the initial differentiation of the above-mentioned immature dendritic cells.

17. The pharmaceutical composition for preventing or treating allergic immune diseases according to claim 15,

the Toll-like receptor agonist comprises at least one selected from the group consisting of a TLR2 agonist, a TLR4 agonist, a TLR5 agonist, a TLR7 agonist, a TLR8 agonist, a TLR9 agonist, a TLR11 agonist, a TLR12 agonist and a TLR13 agonist.

18. The pharmaceutical composition for preventing or treating allergic immune diseases according to claim 17,

the Toll-like receptor agonist further comprises at least one of a TLR1 agonist and a TLR6 agonist.

19. The pharmaceutical composition for preventing or treating allergic immune diseases according to claim 15,

the allergic immune disease is at least one selected from the group consisting of allergic urticaria, allergic rhinitis, allergic conjunctivitis, allergic asthma, allergic dermatitis, autoimmune hepatitis, allergic bronchopulmonary aspergillosis, and allergic stomatitis.

20. A cosmetic composition for preventing or ameliorating allergic immune diseases, characterized in that,

comprising immunologically tolerant plasmacytoid dendritic cells induced by treatment of immature dendritic cells with a Toll-like receptor agonist.

21. A food composition for preventing or improving allergic immune diseases,

comprising immunologically tolerant plasmacytoid dendritic cells induced by treatment of immature dendritic cells with a Toll-like receptor agonist.