METHOD FOR PRODUCING AVATAR MOUSE AS ATOPIC DERMATITIS ANIMAL MODEL AND USE THEREOF

Abstract

The present invention relates to a method for producing an avatar mouse, which is an animal model of atopic dermatitis, and a method of screening a therapeutic agent for atopic dermatitis using the same. According to the present invention, it is possible to more effectively identify a patient-specific immune response by representing an immune response in an actual atopic dermatitis patient. Thus, the present invention is expected to be widely used in the development of a new immunotherapy system enabling tailored treatment.

Claims

1. A method for generating an atopic dermatitis avatar mouse, the method comprising steps of: (a) separating CD3+ T cells from peripheral blood mononuclear cells (PBMCs) isolated from a subject with atopic dermatitis; and (b) injecting the CD3+ T cells into an immunodeficient mouse.

2. The method of claim 1, wherein the atopic dermatitis is atopic dermatitis induced by house dust mites.

3. The method of claim 1, wherein the immunodeficient mouse is any one selected from the group consisting of a nude mouse, an NOD scid gamma (NSG) mouse, a non-obese diabetic (NOD) mouse, a severe combined immunodeficiency (SCID) mouse, an NOD-SCID mouse, and an NOG (NOD/SCID Il2rg.sup.−/−) mouse.

4. The method of claim 1, wherein the injecting in step (b) is performed by intravenous injection.

5. The method of claim 1, wherein step (a) further comprises separating CD3+ T cell-depleted peripheral blood mononuclear cells (CD3 depleted PBMCs), CD14+ monocyte cells or dendritic cells from the peripheral blood mononuclear cells (PBMCs).

6. The method of claim 1, wherein step (b) further comprises injecting CD3+ T cell-depleted peripheral blood mononuclear cells or injecting CD14+ monocyte cells and dendritic cells.

7. The method of claim 6, wherein the injecting of the CD3+ T cell-depleted peripheral blood mononuclear cells or the injecting of the CD14+ monocyte cells and the dendritic cells is performed by intradermal injection.

8. An atopic dermatitis avatar mouse generated by the method of claim 1.

9. A method for screening a substance for treating or alleviating atopic dermatitis, the method comprising steps of: (a) preparing a first subject and a second subject, each of the first subject and the second subject being a atopic dermatitis avatar mice of claim 8; (b) treating the first subject with a candidate substance for treating or alleviating atopic dermatitis; and (c) determining that the candidate substance is a substance for treating or alleviating atopic dermatitis, when atopic dermatitis in the first subject has been alleviated compared to that in the second subject.

10. The method of claim 9, wherein the atopic dermatitis is atopic dermatitis induced by house dust mites.

11. The method of claim 9, wherein the candidate substance is a pharmaceutical composition for preventing or treating atopic dermatitis.

12. The method of claim 9, wherein the candidate substance is a cosmetic composition for preventing or alleviating atopic dermatitis.

13. The method of claim 12, wherein the cosmetic composition is any one selected from the group consisting of lotion, nourishing lotion, nourishing essence, massage cream, cosmetic bath water additive, body lotion, body milk, bath oil, baby oil, baby powder, shower gel, shower cream, sunscreen lotion, sunscreen cream, suntan cream, skin lotion, skin cream, UV blocking cosmetics, cleansing milk, anti-hair loss cosmetics, face and body lotion, face and body cream, skin whitening cream, hand lotion, hair lotion, cosmetic cream, jasmine oil, bath soap, water soap, beauty soap, shampoo, hand sanitizer (hand cleaner), medicated soap for non-medical use, cream soap, facial wash, whole body cleanser, scalp cleanser, hair rinse, cosmetic soap, tooth whitening gel, and toothpaste.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0050] FIGS. 1A, 1B, 1C, 1D and 1E show the results of examining the engraftment rate of human peripheral blood mononuclear cells (PBMCs) in avatar mice.

[0051] FIGS. 2A, 2B, 2C and 2D show the results of analyzing human T cells in the skin lymph nodes, spleens, and skin lesion tissues of avatar mice by flow cytometry.

[0052] FIGS. 3A, 3B and 3C show the results of analyzing human T cells and dendritic cells in human PBMCs and the lymph nodes and skin lesion tissues of avatar mice by flow cytometry.

BEST MODE

[0053] One embodiment of the present invention relates to a method for generating an atopic dermatitis avatar mouse, the method comprising steps of: (a) separating CD3+ T cells, CD3+ T cell-depleted peripheral blood mononuclear cells (CD3 depleted PBMCs), CD14+ monocyte cells or dendritic cells from peripheral blood mononuclear cells (PBMCs) isolated from a subject with atopic dermatitis; and (b) injecting the cells into an immunodeficient mouse.

[0054] Another embodiment of the present invention relates to an atopic dermatitis avatar mouse generated by the above method.

[0055] Still another embodiment of the present invention relates to a method for screening a substance for treating or alleviating atopic dermatitis, the method comprising: (a) preparing a first subject and a second subject as the avatar mice with atopic dermatitis generated by the above method; (b) treating the first subject with a candidate substance for treating or alleviating atopic dermatitis; and (c) determining that the candidate substance is a substance for treating or alleviating atopic dermatitis, when atopic dermatitis in the first subject has been alleviated compared to that in the second subject.

Mode for Invention

[0056] Hereinafter, the present invention will be described in more detail with reference to examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention according to the subject matter of the present invention is not limited by these examples.

EXAMPLE 1

Recruitment of Atopic Dermatitis Patient Group and Preparation of Immunodeficient NSG Mice

[0057] The present inventors conducted all experiments with the approval of the Institutional Review Board (IRB) of the Yonsei University College of Medicine, and blood samples were obtained from atopic dermatitis patients sensitized to Dermatophagoides farinae (house dust mites (HDM)). In addition, immunodeficient NSG mice were purchased from JAX (USA), and six 6-week-old female mice were used in the experiment. The mice were stabilized in the laboratory animal room at a temperature of 23±2° C. and a humidity of 55±5% with a 12-hr dark/12-hr light cycle for 1 week.

EXAMPLE 2

Isolation of Peripheral Blood Mononuclear Cells (PBMCs) from Atopic Dermatitis Patients

[0058] In order to obtain human PBMCs (huPBMCs), GE Healthcare's Ficoll-Paque reagent was used. The Ficoll-Paque reagent and the blood sample obtained in Example 1 were placed in a 15-ml tube at a ratio of 1:1 so that they were not mixed together. The tube was placed in a centrifuge, followed by centrifugation at 2,500 rpm and RT for 15 minutes. Red blood cells were collected in the bottom layer and the buffy coat layer and plasma layer were present thereon. The plasma layer was discarded and the buffy coat layer was collected to obtain PBMCs. The obtained PBMCs were transferred to a fresh tube and mixed with an EDTA solution at a ratio of 1:1, and the mixture was placed in centrifuge and centrifuged at 1,500 rpm and RT for 10 minutes. Thereafter, the supernatant was discarded and the remaining pellet (PBMCs) was finally obtained. CD3+ T cells as pathogen-induced cells causing atopic dermatitis were separated from the PBMC. In addition, CD14+ cells as antigen presenting cells were separated from CD3-depleted PBMCs.

[0059] To separate the CD3+ T cells, Miltenyi Biotec's Pan T Cell Isolation Kit was used. According to the manufacturer's instructions of the kit, 400 μl MACS buffer per 10.sup.8 cells was added to the PBMCs, and 100 μl Pan T Cell Biotion Antibody Cocktail per 10.sup.8 cells was added thereto. Then, the mixture was incubated at 2 to 8° C. for 5 minutes, and then 300 μl MACS buffer per 10.sup.8 cells was added and 200 μl Pan T Cell MicroBead Cocktail per 10.sup.8 cells was added, followed by incubation incubated at 2 to 8° C. for 10 minutes. Thereafter, magnetic separation was performed using an LS column to obtain CD3+ T cells and the remaining CD3-depleted PBMCs. In addition, to obtain CD14+ monocyte cells and dendritic cells, the experiment was performed on the CD3 depleted PBMCs obtained in the above experiment using Miltenyi Biotec's CD14 MicroBeads, human kit, or Pan DC Enrichment kit according to the manufacturer's instructions of the kit, thus obtaining CD14+ monocyte cells or dendritic cells. A more detailed experimental method is as follows.

[0060] The CD3-depleted PBMCs were centrifuged at 300 g for 10 minutes, and the supernatant was discarded. 80 μl MACS buffer/10.sup.7 cells was added to and mixed well with the remaining pellet, and 20 μl CD14 MicroBeads per 10.sup.7 cells were added thereto, followed by incubation at 2 to 8° C. for 15 minutes. Thereafter, the cells were washed with 1 to 2 mL MACS buffer per 10.sup.7 cells and then centrifuged for 300×g for 10 minutes. The supernatant was discarded, and 500 μl of MACS buffer was added to and mixed well with the remaining pellet, and magnetic separation was performed using an LS column to obtain CD14+ monocyte cells.

[0061] To separate dendritic cells, CD3-depleted PBMCs were centrifuged at 300×g for 10 minutes, and then 350 μl MACS buffer per 10.sup.8 cells were added to and mixed well with the cells, and 50 μl FcR blocking reagent per 10.sup.8 cells was added to the cells. Next, 100 μl Pan DC Biotin-Antibody Cocktail per 10.sup.8 cells was added to the cells, followed by incubation at room temperature for 5 minutes. Thereafter, 400 μl MACS buffer per 10.sup.8 cells were added to the cells and 100 μl Pan DC MicoBead-Antibody Cocktail per 10.sup.8 cells were added thereto, followed by incubation at room temperature for 5 minutes. The cells were washed with 10 mL MACS buffer/10.sup.8 cells and centrifuged at 300×g for 10 minutes, and the supernatant was discarded. 500 μl MACS buffer/10.sup.8 cells was added to and mixed well with the remaining pellet, and magnetic separation was performed using an LS column, thus finally obtaining dendritic cells.

EXAMPLE 3

Generation of Avatar Mice Transplanted with huPBMCs by Intravenous Injection and Intradermal Injection

[0062] Human PBMC cells obtained from the blood of atopic dermatitis patients by the method of Example 2 were injected into immunodeficient NSG mice by two different routes of administration. 2×10.sup.6 pathogen-induced CD3+ T cells/20 μg were injected by intravenous injection, and in this case, CD3 T cells as well as whole PBMCs could be used. In order to sensitize intradermal dendritic cells as antigen-presenting cells, 3×10.sup.6 CD3-depleted PBMCs per 30 μg or 3×10.sup.6 CD14+ cells per 30 μg and 3×10.sup.6 dendritic cells per 30 μg were injected into the back of each mouse by intradermal injection. Avatar mice were generated by injecting PBMCs, derived from atopic dermatitis patient sensitized to house dust mites (HDMs), into the immunodeficient NSG mice. The avatar mice are meaningful in that they are mouse models that represent an atopic dermatitis patient-specific immune response using house dust mites, which are major allergens of atopic dermatitis.

[0063] After removing the hair on the back of the avatar mouse generated by the above-described method, dead skin cells were removed with 3M Tegaderm film once every 3 days, and 100 mg of a house dust mite ointment was applied to the back. The above procedure was carried out for 4 weeks. The house dust mite ointment used was an atopic dermatitis challenging ointment (Biostir, Osaka, Japan) which is an ointment containing a natural ingredient derived from mites. The ointment may be applied to induce house dust mite-specific allergic rhinitis, allergic asthma, or atopic dermatitis, and was used in the present invention to induce atopic dermatitis.

EXAMPLE 4

Flow Cytometry to Determine Human PBMC Engraftment Rate and Identify Humanized Mice

[0064] The engraftment rate of human PBMCs in the atopic dermatitis mouse model generated as described in Example 3 was examined over 4 weeks after PBMC injection. In order to confirm whether the avatar mouse was humanized, the results were observed at regular intervals every week. T cells were separated from the immune cells of the mouse animal model by orbital blood sampling, and the separated T cells were dispensed into a FACS tube at a density of 2.0×10.sup.6 cells/tube, and the content in each tube was washed twice with 1 mL FACS buffer at 100 rpm at 4° C. for 3 minutes. Each antibody to be checked below was mixed with FACS buffer, and 50 μl of the mixture was dispensed into each FACS tube and covered with a foil. After 20 minutes, the content in each tube was washed twice with 1 mL of FACS buffer, and then 0.5 mL of the buffer was placed in the FACS tube and the content in the tube was resuspended. Analysis was performed by flow cytometry. In this case, the cell surface was stained, and then the cells were fixed and permeabilized, and the sample was analyzed by flow cytometry in a Becton Dickinson FACS Canto instrument according to the manufacturer's protocol. The data were analyzed using FACS Diva software (V5.1). The results are shown in FIG. 1A. Referring to FIG. 1A, it was confirmed that the cells were negative for mouse CD45 while being positive for human CD45 over 1 to 4 weeks, and that the cells were positive for both human CD45 and human CD3. The process of changes in the engraftment rate of human PBMC cells over time was confirmed by comparing and confirming the correlation between human CD45 and human CD3 as human T cell markers from the mouse cells and the correlation between human CD45 and mouse CD45.

[0065] In addition, FIGS. 1B and 1C are graphs showing the results of analyzing the number of human CD3 and CD45 positive cells over 4 weeks by flow cytometry in order to confirm the engraftment rate of human PBMCs in the avatar mouse blood for each week. Referring to FIGS. 1B and 1C, it could be confirmed that the human T cells were much more in the avatar mouse having an inflammatory response induced using house dust mites than in the avatar mouse not treated with HDMs, and it was confirmed that the human CD3 positive percentage in the avatar mouse treated with house dust mites significantly increased over time, suggesting that the engraftment efficiency in the avatar mouse treated with house dust mites increased. In addition, FIGS. 1D and 1E show the results of analyzing human T cells in the spleens, lymph nodes, and skin lesions of the avatar mouse treated with house dust mites and the avatar mouse not treated with house dust mites. It can be seen that these results are the same as the results obtained in the blood.

[0066] Finally, after 4 weeks, flow cytometry was performed on lymph nodes, spleens, and skin lesion tissues in the same manner as above, and the results are shown in FIGS. 2A, 2B, 2C and 2D. It was confirmed that the lymph nodes were positive for human CD45, CD3, CD4 and CD8, and the same trend was also observed in spleens and skin lesions. As described above, it was confirmed that all the tissues were positive for human markers and negative for mouse CD45 as a mouse marker. In addition, FIGS. 3A, 3B and 3C show the results of analyzing human T cells and human antigen-presenting cells (APCs) in human PBMCs and the lymph nodes and skin lesion tissues of the avatar mouse. In FIGS. 3A, 3B and 3C, the non-lymphocyte portion corresponds to the upper portion in each figure, and the lymphocyte portion corresponds to the lower portion in each figure. As a result of separately analyzing lymphocytes and non-lymphocytes in FSC-SSC of human PBMCs, human T cells were identified in the lymphocytes, and APC cells were identified in the non-lymphocytes. Similarly, as a result of analyzing two divided populations (lymphocytes and non-lymphocytes) in the lymph nodes and skin lesions of the avatar mouse, the expression of cells as in human PBMC was confirmed. Thereby, it was confirmed that a patient-tailored atopic dermatitis animal model, that is, a humanized avatar mouse that represents the patient's immune response, was successfully generated. Only mice confirmed to have these characteristics may be selected and used for screening.

[0067] Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this detailed description is only of a preferred embodiment thereof, and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereto.

INDUSTRIAL APPLICABILITY

[0068] The present invention is directed to a human T cell-containing atopic dermatitis animal model that represents a specific immune response caused by house dust mites in an actual atopic dermatitis patient. When the present invention is used, it is possible to effectively identify a specific immune response depending on the cause of atomic dermatitis. Accordingly, the present invention may be widely used in the development of a patient-tailored preventive or therapeutic agent for atopic dermatitis.