Method for preparing primary cilia of immune-related cells, preparation agent, method for evaluating test samples, and utilization thereof.

Abstract

To provide a regulating method and a regulator for primary cilia of immune-related cells, and a method for evaluating a test sample.SOLUTION: A regulating method for primary cilia of immune-related cells includes a contact step of bringing an immune-related cell into contact with a signal controller that activates or inactivates a signal transduction pathway of at least one cytokine selected from the group consisting of (a) Th2cytokine, (b) Th17cytokine, and (c) Th22cytokine.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method and an agent for regulating primary cilia of immune-related cells, a method for evaluating a test sample, and their use.

Background Art

[0002] Primary cilia are antenna-like organs having microtubules as an internal skeleton. Non-motile primary cilia transmit extracellular signals into cells via various receptors present on the surface of the primary cilia. Although it is known that substantially all cells have primary cilia, there are many parts that have not been clarified regarding the relationship between primary cilia and immune responses in immune-related cells.

[0003] Recent studies have suggested that primary cilia are present in immune-related cells, that primary cilia are related to immune-related diseases, and that there is a correlation between the number of immune-related cells having primary cilia and the onset of immune-related diseases. Based on these findings, a method for detecting an index of immune-related diseases by observing primary cilia has been proposed (see Patent Document 1).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] It is considered that regulating primary cilia of immune-related cells (for example, regulating the number and / or ratio of immune-related cells having primary cilia) can contribute to the regulation of the immune system and the treatment of immune-related diseases. Therefore, the development of a technique for regulating primary cilia of immune-related cells is expected.

[0006] One embodiment of the present invention has been made in view of the above-mentioned problems, and its purpose is to provide a method for preparing primary cilia of immune-related cells, a preparing agent, a method for evaluating test samples, and the use thereof. [Means for solving the problem]

[0007] As a result of diligent research to solve the above problems, the inventors have discovered the novel finding that (i) the number and / or proportion of immune-related cells having primary cilia can be adjusted by adjusting the activation state of the signaling pathway of specific cytokines in immune-related cells, and (ii) the novel finding that adjusting the primary cilia of immune-related cells changes the expression of specific proteins, leading to the completion of the present invention. That is, the present invention includes the following configuration.

[0008] <1> A method for modifying the primary cilia of immune-associated cells, comprising a contact step of contacting immune-associated cells with a signaling regulatory substance that activates or inactivates the signaling pathway of at least one cytokine selected from the group consisting of (a) to (c) below: (a) Th2 cytokine, (b) Th17 cytokine, (c) Th22 cytokine.

[0009] <2> <1> A method for regulating the expression of at least one protein selected from the group consisting of JNK, DSG1, and CLDN4, comprising the method for regulating the primary cilia of immune-related cells described in [reference] as one of the steps.

[0010] <3> A regulator of primary cilia in immune-related cells, comprising a signaling regulator that activates or inactivates the signaling pathway of at least one cytokine selected from the group consisting of (a) to (c) below: (a) Th2 cytokine, (b) Th17 cytokine, (c) Th22 cytokine.

[0011] <4> <3> A regulator of the expression of at least one protein selected from the group consisting of JNK, DSG1, and CLDN4, which contains a primary cilia regulator of immune-related cells as described above.

[0012] <5> A method for evaluating a test sample, comprising: a first observation step of contacting immune-related cells with a signaling regulatory substance that activates or inactivates the signaling pathway of at least one cytokine selected from the group consisting of (a) to (c) below, and then observing the primary cilia of the immune-related cells; a second observation step of contacting the immune-related cells with the signaling regulatory substance used in the first observation step and a test sample, and then observing the primary cilia of the immune-related cells; and an evaluation step of evaluating the effect of the test sample on the primary cilia of the immune-related cells by comparing the observation results of the first observation step with the observation results of the second observation step; (a) Th2 cytokine, (b) Th17 cytokine, (c) Th22 cytokine.

[0013] <6> Furthermore, the system includes an evaluation step in which, based on the evaluation results in the evaluation step, the effect of the test sample on the expression of at least one protein selected from the group consisting of JNK, DSG1, and CLDN4 in the immune-related cells is evaluated. <5> Evaluation method for the test sample described. [Effects of the Invention]

[0014] According to the present invention, a method for preparing primary cilia of immune-related cells, a preparing agent, and a method for evaluating test samples can be provided. [Brief explanation of the drawing]

[0015] [Figure 1] This graph shows the effects of various cytokines on the primary cilia of HaCat cells (a human epidermal keratinocyte cell line) in an embodiment of the present invention. [Figure 2] This graph shows the effects of various cytokines on the primary cilia of HaCat cells (a human epidermal keratinocyte cell line) in an embodiment of the present invention. [Figure 3] This graph shows the effect of a cytokine mixture on the primary cilia of HaCat cells (a human epidermal keratinocyte cell line) in an example of the present invention. [Figure 4]In an embodiment of the present invention, it is a graph showing the effect of each cytokine on the primary cilium of primary human epidermal keratinocytes. [Figure 5] In an embodiment of the present invention, it is a graph showing the effect of each cytokine and a mixture of cytokines on the primary cilium of primary human epidermal keratinocytes. [Figure 6] In an embodiment of the present invention, it is a graph showing the effect of a cytokine on the expression of JNK protein. [Figure 7] In an embodiment of the present invention, it is a graph showing the effect of a cytokine on the expression of CLDN4 protein. [Figure 8] In an embodiment of the present invention, it is a graph showing the effect of a cytokine on the expression of DSG1 protein.

Mode for Carrying Out the Invention

[0016] The present invention will be described below, but the present invention is not limited thereto. The present invention is not limited to each configuration described below, and various modifications are possible within the scope shown in the claims. In addition, embodiments or examples obtained by appropriately combining the technical means disclosed in different embodiments or examples are also included in the technical scope of the present invention. Furthermore, by combining the technical means disclosed in each embodiment, new technical features can be formed. All academic documents and patent documents described in this specification are incorporated herein by reference as references. Unless otherwise specified in this specification, "X~Y" representing a numerical range is intended to mean "X or more and Y or less".

[0017] 〔1. Method for Adjusting Primary Cilium of Immune-Related Cells〕 The method for adjusting the primary cilium of immune-related cells according to the present invention has a contact step of bringing immune-related cells into contact with a signal control substance that activates or inactivates the signal transduction pathway of at least one cytokine selected from the group consisting of the following (a) to (c); (a) Th2 cytokine, (b) Th17 cytokine, (c) Th22 cytokine.

[0018] The method for regulating the primary cilium of immune-related cells according to the present invention is more specifically (i) a method for increasing the number and / or proportion of immune-related cells having a primary cilium, (ii) a method for suppressing an increase in the number and / or proportion of immune-related cells having a primary cilium, (iii) a method for decreasing the number and / or proportion of immune-related cells having a primary cilium, or (iv) a method for suppressing a decrease in the number and / or proportion of immune-related cells having a primary cilium.

[0019] In the contact step, the method of contacting immune-related cells with a signal control substance is not particularly limited. For example, the immune-related cells may be contacted with the signal control substance by adding the signal control substance to the medium in which the immune-related cells are cultured. In the contact step, the time for contacting the immune-related cells with the signal control substance is not particularly limited, and may be, for example, 0.1 hour to 100 hours, 0.1 hour to 75 hours, 0.1 hour to 50 hours, 0.1 hour to 25 hours, 0.1 hour to 10 hours, or 0.1 hour to 1 hour.

[0020] In the contact step, after contacting the immune-related cells with the signal control substance, the immune-related cells may be cultured for a desired time. During the culture, the immune-related cells and the signal control substance may or may not be in contact. The culture time is not particularly limited and may be 1 day to 3 days, 1 day to 7 days, or 7 days or more.

[0021] Immune-related cells include immune cells that are primarily responsible for immune responses and immune function-carrying cells that are indirectly involved with immune cells. Immune function-carrying cells, for example, have the function of activating immune cells. Examples of immune cells include cutaneous dendritic cells (e.g., Langerhans cells, dermal dendritic cells), lymphoid immune cells (e.g., T cells, NK cells, B cells), and monocyte immune cells (e.g., conventional dendritic cells, monocyte-derived dendritic cells (e.g., plasmacytoid dendritic cells)). On the other hand, examples of immune function-carrying cells include keratinocytes, fibroblasts, and epithelial cells. Immune-related cells may be immune-related cells collected from a living organism, or they may be immune-related cell lines (e.g., HaCaT cells).

[0022] In the contact step, one type of cytokine signaling pathway may be activated or inactivated in immune-related cells, or multiple types of cytokine signaling pathways may be activated or inactivated simultaneously. Examples of cytokine signaling pathways to be activated or inactivated include: (a) Th2 cytokine signaling pathways, (b) Th17 cytokine signaling pathways, (c) Th22 cytokine signaling pathways, (d) combinations of Th2 cytokine signaling pathways and Th17 cytokine signaling pathways, (e) combinations of Th17 cytokine signaling pathways and Th22 cytokine signaling pathways, (f) combinations of Th2 cytokine signaling pathways and Th22 cytokine signaling pathways, and (g) combinations of Th2 cytokine signaling pathways, Th17 cytokine signaling pathways, and Th22 cytokine signaling pathways.

[0023] In the contact step, when activating or inactivating each of the Th2 cytokine signaling pathways, Th17 cytokine signaling pathways, and Th22 cytokine signaling pathways, the activation or inactivation of each signaling pathway may be performed using one type of signal regulatory substance or using multiple types of signal regulatory substances. For example, each of the Th2 cytokines, Th17 cytokines, and Th22 cytokines has multiple subfamilies, as will be described later. In the contact step, the Th2 cytokine signaling pathways, Th17 cytokine signaling pathways, and Th22 cytokine signaling pathways may be activated or inactivated using a combination of multiple subfamilies (for example, (i) a combination of interleukin-4, interleukin-13, and interleukin-31, or (ii) a combination of an interleukin-4 inhibitor, an interleukin-13 inhibitor, and an interleukin-31 inhibitor).

[0024] In certain diseases (e.g., atopic dermatitis), the amount of specific signaling regulators (e.g., IL-4, IL-13, and IL-31), or the total amount of specific signaling regulators (e.g., IL-4, IL-13, and IL-31) combined, increases in cells. This suggests that adding specific signaling regulators to cells can create model cells for specific diseases. Therefore, the method for adjusting primary cilia in immune-related cells according to the present invention makes it possible to adjust primary cilia in model cells for specific diseases and to observe the effects of adjusting primary cilia on specific diseases.

[0025] Th2 cytokines are not particularly limited, but more specifically include interleukin-4 (IL-4), interleukin-13 (IL-13), interleukin-31 (IL-31), interleukin-33 (IL-33), and the gp130 / IL-6 cytokine family.

[0026] Th17 cytokines are not particularly limited, but more specifically include interleukin-17A (IL-17A), interleukin-17B (IL-17B), interleukin-17C (IL-17C), interleukin-17D (IL-17D), interleukin-17F (IL-17F), and interleukin-25 (IL-25).

[0027] Th22 cytokines are not particularly limited, but more specifically, they include interleukin-22 (IL-22), interleukin-13 (IL-13), and interleukin-26 (IL-26).

[0028] The signaling regulator is not particularly limited, as long as it can activate or inactivate the signaling pathway of at least one cytokine selected from the group consisting of (a) Th2 cytokines, (b) Th17 cytokines, and (c) Th22 cytokines.

[0029] Specifically, signal regulatory substances include (i) substances that activate cytokine signaling pathways, (ii) substances that suppress the activation of cytokine signaling pathways, (iii) substances that inactivate cytokine signaling pathways, or (iv) substances that suppress the inactivation of cytokine signaling pathways.

[0030] More specifically, signal regulatory substances include (i) substances that activate cytokine receptors, (ii) substances that suppress the activation of cytokine receptors, (iii) substances that inactivate cytokine receptors, or (iv) substances that suppress the inactivation of cytokine receptors.

[0031] Specific examples of signal regulatory substances include cytokines themselves, cytokine receptor inhibitors, and activators of cytokine receptors other than cytokines.

[0032] Examples of cytokines include Th2 cytokines (e.g., IL-4, IL-13, IL-31, IL-33, IL-5, IL-6, IL-10, GM-CSF), Th17 cytokines (e.g., IL-17A, IL-17B, IL-17C, IL-17D, IL-17F, IL-25), Th22 cytokines (e.g., IL-22, IL-13, IL-26), and combinations thereof.

[0033] Examples of cytokine receptor inhibitors include receptor neutralizing antibodies, antibody preparations, and small molecule compounds. Commercially available cytokine receptor inhibitors can be used.

[0034] The inventors have newly discovered that modifying the primary cilia of immune-related cells alters the expression of specific proteins (specifically, JNK, DSG1, and CLDN4). This indicates that the method for modifying the primary cilia of immune-related cells according to the present invention can be used as a method for modifying the expression of specific proteins.

[0035] The method for regulating the expression of JNK, DSG1, or CLDN4 according to the present invention comprises, as one of the steps, a method for regulating the primary cilia of immune-related cells according to the present invention (e.g., a contact step).

[0036] JNK (c-jun N-terminal kinase) is a phosphorylated protein involved in apoptosis, neurodegeneration, cell differentiation, cell proliferation, inflammation, cytokine production, and other processes.

[0037] DSG1 (desmoglein 1) is a cell adhesion molecule that constitutes adhesion plaques, one of the cell adhesion apparatuses, and is a calcium-binding transmembrane protein. In the skin, DSG1 is involved in cell-to-cell adhesion.

[0038] CLDN4 (claudin 4) is one of the proteins that make up tight junctions. In the skin, CLDN4 plays a major role in maintaining barrier function and forming barriers.

[0039] The method for regulating the expression of at least one protein selected from the group consisting of JNK, DSG1, and CLDN4 according to the present invention may more specifically be: (i) a method to increase the expression level of a specific protein (e.g., JNK) by increasing primary cilia; (ii) a method to decrease the expression level of a specific protein (e.g., DSG1 and CLDN4) by increasing primary cilia; (iii) a method to increase the expression level of a specific protein (e.g., DSG1 and CLDN4) by decreasing primary cilia; or (iv) a method to decrease the expression level of a specific protein (e.g., JNK) by decreasing primary cilia. The method for increasing or decreasing primary cilia is as described above.

[0040] The method for adjusting the primary cilia of immune-related cells according to the present invention not only allows for the adjustment of the primary cilia of immune-related cells, but also allows for the adjustment of the expression level of proteins related to the primary cilia of immune-related cells, and enables observation of the effect of the expression level of said proteins on the cells.

[0041] [2. Regulator for primary cilia of immune-related cells] The modifier for primary cilia of immune-related cells according to the present invention contains a signaling regulatory substance that activates or inactivates the signaling pathway of at least one cytokine selected from the group consisting of (a) to (c) below: (a) Th2 cytokine, (b) Th17 cytokine, (c) Th22 cytokine.

[0042] The primary cilia modifier of immune-related cells according to the present invention may more specifically be one that (i) increases the number and / or proportion of immune-related cells having primary cilia, (ii) inhibits the increase in the number and / or proportion of immune-related cells having primary cilia, (iii) decreases the number and / or proportion of immune-related cells having primary cilia, or (iv) inhibits the decrease in the number and / or proportion of immune-related cells having primary cilia.

[0043] Since the signal regulatory substances were explained above in [1. Method for regulating primary cilia of immune-related cells], a detailed explanation will be omitted here.

[0044] The primary cilia regulator of immune-related cells according to the present invention may contain components other than signal-controlling substances. These components other than signal-controlling substances are not particularly limited and include buffers, pH adjusters, isotonic agents, preservatives, antioxidants, high molecular weight polymers, excipients, carriers, diluents, solvents, solubilizers, stabilizers, fillers, binders, surfactants, and other similar substances.

[0045] The amount of signal-regulating substance contained in the primary cilia regulator of immune-related cells according to the present invention is not particularly limited. For example, when the regulator is considered as 100% by mass, the amount may be 0.001% to 100% by mass, 0.01% to 100% by mass, 0.1% to 100% by mass, 0.1% to 95% by mass, 0.1% to 90% by mass, 0.1% to 80% by mass, 0.1% to 70% by mass, 0.1% to 60% by mass, 0.1% to 50% by mass, 0.1% to 40% by mass, 0.1% to 30% by mass, 0.1% to 20% by mass, or 0.1% to 10% by mass.

[0046] The amount of substances other than the signal control substance contained in the primary cilia regulator of immune-related cells according to the present invention is not particularly limited, and may be 0% to 99.999% by mass, 0% to 99.99% by mass, 0% to 99.9% by mass, 0% to 99.9% by mass, 5% to 99.9% by mass, 10% to 99.9% by mass, 20% to 99.9% by mass, 30% to 99.9% by mass, 40% to 99.9% by mass, 50% to 99.9% by mass, 60% to 99.9% by mass, 70% to 99.9% by mass, 80% to 99.9% by mass, or 90% to 99.9% by mass.

[0047] The amount of signal-regulating substance contained in the primary cilia regulator of immune-related cells according to the present invention is not particularly limited, and for example, the amount of signal-regulating substance contained in the solution (e.g., culture medium) into which immune-related cells and the signal-regulating substance are brought into contact may be adjusted to be 0.01 ng / mL to 10 mg / mL, 0.01 ng / mL to 1 mg / mL, 0.01 ng / mL to 100 μg / mL, 0.01 ng / mL to 10 μg / mL, 0.01 ng / mL to 1 μg / mL, 0.01 ng / mL to 100 ng / mL, or 0.01 ng / mL to 10 ng / mL.

[0048] The inventors have newly discovered that modifying the primary cilia of immune-related cells alters the expression of specific proteins (specifically, JNK, DSG1, and CLDN4). This indicates that the primary cilia modifier of immune-related cells of the present invention can be used as a modifier of the expression of specific proteins.

[0049] The expression regulator for JNK, DSG1, or CLDN4 according to the present invention contains the primary cilia regulator for immune-related cells according to the present invention. The expression regulator for JNK, DSG1, or CLDN4 according to the present invention may consist of the primary cilia regulator for immune-related cells according to the present invention.

[0050] The expression regulator of at least one protein selected from the group consisting of JNK, DSG1, and CLDN4 according to the present invention may more specifically (i) increase the expression level of a specific protein (e.g., JNK) by increasing primary cilia, (ii) decrease the expression level of a specific protein (e.g., DSG1 and CLDN4) by increasing primary cilia, (iii) increase the expression level of a specific protein (e.g., DSG1 and CLDN4) by decreasing primary cilia, or (iv) decrease the expression level of a specific protein (e.g., JNK) by decreasing primary cilia. The regulators for increasing or decreasing primary cilia are as described above.

[0051] If the expression regulator for JNK, DSG1, or CLDN4 according to the present invention contains the primary cilia regulator for immune-related cells according to the present invention (hereinafter referred to as the active ingredient), the expression regulator for JNK, DSG1, or CLDN4 according to the present invention may contain ingredients other than the active ingredient. Examples of such ingredients other than the active ingredient (hereinafter referred to as minor ingredients) include buffers, pH adjusters, isotonic agents, preservatives, antioxidants, high molecular weight polymers, excipients, carriers, diluents, solvents, solubilizers, stabilizers, fillers, binders, surfactants, and stabilizers.

[0052] The amount of the active ingredient contained in the JNK, DSG1, or CLDN4 expression regulator according to the present invention is not particularly limited. For example, when the regulator is considered as 100% by mass, the amount may be 0.001% to 100% by mass, 0.01% to 100% by mass, 0.1% to 100% by mass, 0.1% to 95% by mass, 0.1% to 90% by mass, 0.1% to 80% by mass, 0.1% to 70% by mass, 0.1% to 60% by mass, 0.1% to 50% by mass, 0.1% to 40% by mass, 0.1% to 30% by mass, 0.1% to 20% by mass, or 0.1% to 10% by mass.

[0053] The amount of the by-components contained in the JNK, DSG1, or CLDN4 expression regulator according to the present invention is not particularly limited, and may be 0% to 99.999% by mass, 0% to 99.99% by mass, 0% to 99.9% by mass, 0% to 99.9% by mass, 5% to 99.9% by mass, 10% to 99.9% by mass, 20% to 99.9% by mass, 30% to 99.9% by mass, 40% to 99.9% by mass, 50% to 99.9% by mass, 60% to 99.9% by mass, 70% to 99.9% by mass, 80% to 99.9% by mass, or 90% to 99.9% by mass, with the regulator being 100% by mass.

[0054] [3. Evaluation Method for Test Samples] The method for evaluating a test sample according to the present invention comprises: a first observation step of contacting immune-related cells with a signaling regulatory substance that activates or inactivates the signaling pathway of at least one cytokine selected from the group consisting of (a) to (c) below, and then observing the primary cilia of the immune-related cells; a second observation step of contacting the immune-related cells with the signaling regulatory substance used in the first observation step and the test sample, and then observing the primary cilia of the immune-related cells; and an evaluation step of evaluating the effect of the test sample on the primary cilia of the immune-related cells by comparing the observation results of the first observation step with the observation results of the second observation step; (a) Th2 cytokine, (b) Th17 cytokine, (c) Th22 cytokine.

[0055] Signal-regulating substances activate or inactivate the signaling pathway of specific cytokines. In the first observation step, the effect of the signal-regulating substance on the primary cilia of immune-related cells is observed (equivalent to a control test). In the second observation step, the effect of the test sample on the effect of the signal-regulating substance on the primary cilia of immune-related cells is observed using both the signal-regulating substance and the test sample. By comparing the results of both observations, the effect of the test sample on the signal-regulating substance (in other words, the effect of the test sample on the primary cilia of immune-related cells) can be evaluated.

[0056] The following describes each step of the evaluation method for test samples according to the present invention. Note that the configurations described in [1. Method for preparing primary cilia of immune-related cells] and [2. Preparing agent for primary cilia of immune-related cells] above will not be described below.

[0057] <First observation step> In the first observation step, immune-associated cells are exposed to a signaling regulator that activates or inactivates the signaling pathway of at least one cytokine selected from the group consisting of (a) to (c) below, and then the primary cilia of the immune-associated cells are observed: (a) Th2 cytokine, (b) Th17 cytokine, (c) Th22 cytokine.

[0058] In the first observation step, the method of bringing immune-related cells into contact with the signaling molecule is not particularly limited. For example, the immune-related cells may be brought into contact with the signaling molecule by adding the signaling molecule to the culture medium in which the immune-related cells are cultured. The time for which the immune-related cells are in contact with the signaling molecule is not particularly limited and may be, for example, 0.1 to 100 hours, 0.1 to 75 hours, 0.1 to 50 hours, 0.1 to 25 hours, 0.1 to 10 hours, or 0.1 to 1 hour.

[0059] In the first observation step, immune-related cells may be brought into contact with a signaling regulatory substance, and then cultured for a desired time. During this culture, the immune-related cells and the signaling regulatory substance may or may not be in contact. The culture time is not particularly limited and may be 1 to 3 days, 1 to 7 days, or 7 days or longer.

[0060] In the first observation step, primary cilia of immune-related cells are observed using a microscope or similar device, and the primary cilia formation rate of immune-related cells is calculated. Below, an example of a method for calculating the primary cilia formation rate and a method for observing primary cilia are described.

[0061] In the first observation step, the number of primary cilia can be calculated by observing primary cilia in immune-related cells as one of the indicators. When calculating the number of primary cilia, for example, (a) the number of primary cilia per immune-related cell may be calculated, (b) the percentage of immune-related cells having primary cilia per a predetermined number of immune-related cells may be calculated, or (c) both (a) and (b) may be performed. In addition, the total number of immune-related cells contained in the sample may be calculated as the total number of nuclei in the sample.

[0062] For example, in the case of (a) described above, the number of primary cilia can be calculated as the [primary cilia formation rate of immune-related cells] according to the following formula (I); [Primary cilia formation rate of immune-related cells] = [[Total number of primary cilia present in immune-related cells in the sample] / [Total number of immune-related cells in the sample]] × 100 ... Equation (I).

[0063] For example, in the case of (b) described above, the number of primary cilia can be calculated as the [primary cilia formation rate of immune-related cells] according to the following formula (II); [Primary cilia formation rate of immune-related cells] = [[Total number of immune-related cells with primary cilia in the sample] / [Total number of immune-related cells in the sample]] × 100 ... Equation (II).

[0064] Primary cilia can be observed, for example, by known immunohistochemical staining methods. Specifically, primary cilia can be observed by contacting a sample containing the test specimen (for example, cells fixed with an immobilizing agent such as paraformaldehyde, or the test specimen prepared as paraffin-embedded sections) with a substance that specifically binds to primary cilia (hereinafter referred to as the "first specific binding substance"), thereby binding the first specific binding substance to the primary cilia contained in the sample containing the test specimen, and then detecting the labeling substance bound to the first specific binding substance. The method for preparing the sample containing the test specimen is not limited, and known methods can be used.

[0065] Examples of the first specific binding substance include substances that specifically bind to primary cilia markers (e.g., antibodies (primary antibodies)). Examples of primary cilia markers include ADP-ribosylation factor-like protein (Arl13B), acetylated tubulin, adenylyl cyclase III, nephrocystin 3 (NPHP3), flagellar transport protein (IFT88), somatostatin receptor 3 (sstr3), polycysten-1 (TRPC1), transient receptor potential vanilloid 4 (TRPV4), platelet-derived growth factor receptor α (PDGFRα), and Smoothund (Smo).

[0066] As described above, the first specific binding substance can be, for example, an antibody that specifically binds to a substance that serves as a marker for primary cilia. Examples of such antibodies include monoclonal antibodies, polyclonal antibodies, and fragments of these antibodies (e.g., Fab fragments, F(ab')2 fragments, and single-chain antibodies).

[0067] Monoclonal antibodies can be obtained, for example, by culturing a hybridoma that produces a monoclonal antibody against a primary cilia marker of immune-related cells in a desired medium to obtain the culture supernatant, and purifying the culture supernatant as needed. Hybridomas can be obtained by administering a primary cilia marker of immune-related cells intravenously, subcutaneously, or intraperitoneally to an animal (e.g., a mouse or rat), obtaining antibody-producing cells from the animal, and fusing these antibody-producing cells with myeloma cells.

[0068] Polyclonal antibodies can be obtained, for example, by administering a marker of the primary cilia of immune-related cells intravenously, subcutaneously, or intraperitoneally to an animal (e.g., a rabbit), obtaining serum from the animal, and purifying the serum as necessary.

[0069] Fab fragments can be obtained, for example, by digesting a monoclonal antibody against a marker of the primary cilia of immune-related cells with papain and purifying the digested product as needed.

[0070] The F(ab')2 fragment can be obtained, for example, by digesting a monoclonal antibody against a marker of primary cilia on immune-related cells with pepsin and purifying the digested product as needed.

[0071] Single-chain antibodies can be obtained, for example, by introducing a phagemide vector containing a nucleic acid construct in which a nucleic acid encoding the variable region of the light chain of a monoclonal antibody against a primary cilia marker of immune-related cells is linked to a nucleic acid encoding a linker and a nucleic acid encoding the variable region of the heavy chain of the monoclonal antibody into a host cell, expressing the polypeptide encoded by the nucleic acid construct within the host cell, and purifying the polypeptide as necessary.

[0072] The labeling substance may be any substance capable of generating a detectable signal such as fluorescence or color (e.g., a fluorescent dye or enzyme). The labeling substance may be directly bound to the first specific binding substance, or it may be directly bound to a specific binding substance for the first specific binding substance (hereinafter referred to as the "second specific binding substance"). An example of the second specific binding substance is a substance that specifically binds to the first specific binding substance (e.g., an antibody (secondary antibody)).

[0073] Examples of labeling substances include fluorescein isothiocyanate; 2-(3-iminio-4,5-disulfonato-6-amino-3H-xanthene-9-yl)-5-[[5-(2,5-dioxo-3-pyrroline-1-yl)pentyl]carbamoyl]benzoic acid (e.g., Alexa Fluor 488 from Invitrogen); 6-(2-carboxylato-4-carboxyphenyl)-1,2,10,11-tetrahydro-1,2,2,10,10,11-hexamethyl-4,8-bis-(sulfomethyl)-1,11-diaza-13-oxoniapentacene (e.g., Alexa Fluor 594 from Invitrogen); peroxidase; and alkaline phosphatase.

[0074] The label substance bound to the first specific binding substance can be detected by visual inspection using an optical microscope (e.g., a fluorescence microscope and a confocal laser microscope), an image analysis device (e.g., a fluorescence imaging analyzer), or a flow cytometry device (a fluorescence flow cytometer and an imaging flow cytometer), or by numerical detection such as fluorescence intensity.

[0075] <Second observation step> In the second observation step, immune-related cells are brought into contact with the signal-controlling substance used in the first observation step and the test sample, and then the primary cilia of the immune-related cells are observed.

[0076] In the second observation step, the method for contacting immune-related cells with the signaling substance and the test sample is not particularly limited, and the same method as in the first observation step may be used. The time for contacting immune-related cells with the signaling substance and the test sample is not particularly limited, and the same time as in the first observation step may be used. In the second observation step, similar to the first observation step, after contacting immune-related cells with the signaling substance and the test sample, the immune-related cells can be cultured for a desired time. In the second observation step, immune-related cells may be contacted with the signaling substance and the test sample simultaneously, or at different times.

[0077] The test sample used in the second observation step is not particularly limited and may include, for example, inorganic compounds, organic compounds, plant extracts, microbial extracts, and culture supernatants of various cells. The test sample may be used in its original state or dissolved in a solvent. Examples of such solvents include physiological saline, phosphate-buffered saline, and water.

[0078] In the second observation step, primary cilia of immune-related cells are observed using a microscope or similar device, and the formation rate of primary cilia of immune-related cells is calculated. The method for observing primary cilia and calculating the formation rate of primary cilia should be the same as in the first observation step.

[0079] <Evaluation Process (1)> In the evaluation step, the effect of the test sample on the primary cilia of immune-related cells is assessed by comparing the observation results from the first observation step with the observation results from the second observation step.

[0080] For example, suppose that in the first observation step, an increase in the primary cilia formation rate is observed, and in the second observation step, an even higher primary cilia formation rate is observed than in the first observation step. In this case, the test sample can be evaluated as having the effect of increasing primary cilia in immune-related cells.

[0081] Suppose that in the first observation step, a decrease in the primary cilia formation rate is observed, and in the second observation step, an increase in the primary cilia formation rate is observed compared to the results of the first observation step. In this case, the test sample can be evaluated as having the effect of increasing primary cilia in immune-related cells.

[0082] For example, suppose that in the first observation step, an increase in the primary cilia formation rate is observed, and in the second observation step, a decrease in the primary cilia formation rate is observed compared to the results of the first observation step. In this case, the test sample can be evaluated as having the effect of reducing primary cilia on immune-related cells.

[0083] Suppose that in the first observation step, a decrease in the primary cilia formation rate is observed, and in the second observation step, the primary cilia formation rate is observed to be even lower than the result of the first observation step. In this case, the test sample can be evaluated as having the effect of reducing primary cilia on immune-related cells.

[0084] For example, suppose the first observation step yields an increase in the primary cilia formation rate, and the second observation step yields a result where the primary cilia formation rate is approximately the same as the result of the first observation step. In this case, it can be concluded that the test sample does not have the effect of increasing or decreasing the primary cilia of immune-related cells, or that the signaling regulator suppresses changes (increases or decreases) in the number and / or proportion of immune-related cells that have primary cilia.

[0085] Suppose that in the first observation step, the result obtained is that the primary cilia formation rate does not change, and in the second observation step, the result obtained is that the primary cilia formation rate is approximately the same as the result of the first observation step. In this case, it can be evaluated that the test sample does not have the effect of increasing or decreasing the primary cilia of immune-related cells, or that the signaling regulatory substance suppresses changes (increases or decreases) in the number and / or proportion of immune-related cells that have primary cilia.

[0086] Suppose that in the first observation step, a decrease in the primary cilia formation rate is obtained, and in the second observation step, the primary cilia formation rate is found to be approximately the same as the result of the first observation step. In this case, it can be concluded that the test sample does not have the effect of increasing or decreasing the primary cilia of immune-related cells, or that the signaling regulatory substance suppresses changes (increases or decreases) in the number and / or proportion of immune-related cells that have primary cilia.

[0087] <Evaluation Process (2)> Furthermore, the evaluation step may include an evaluation step in which, based on the evaluation results in the evaluation step, the effect of the test sample on the expression of at least one protein selected from the group consisting of JNK, DSG1, and CLDN4 in the immune-related cells is evaluated. That is, in this evaluation step, the effect of the test sample on the expression of at least one protein selected from the group consisting of JNK, DSG1, and CLDN4 is evaluated based on the evaluation results regarding the effect of the test sample on the primary cilia of the immune-related cells.

[0088] As described above, since the expression of primary cilia in immune-related cells correlates with that of JNK, DSG1, and CLDN4, the effect of the test sample on the expression of these proteins can also be evaluated.

[0089] More specifically, for example, the method may involve: (i) if the test sample is evaluated to increase primary cilia, then the test sample is evaluated to be able to increase the expression level of a specific protein (e.g., JNK); (ii) if the test sample is evaluated to increase primary cilia, then the expression level of a specific protein (e.g., DSG1 and CLDN4) is evaluated to be able to decrease; (iii) if the test sample is evaluated to decrease primary cilia, then the test sample is evaluated to be able to increase the expression level of a specific protein (e.g., DSG1 and CLDN4); and (iv) if the test sample is evaluated to decrease primary cilia, then the test sample is evaluated to be able to decrease the expression level of a specific protein (e.g., JNK).

[0090] This evaluation process allows for the assessment of the effect of the test sample on primary cilia in immune-related cells, as well as its effect on the expression level of proteins related to primary cilia in immune-related cells. [Examples]

[0091] Examples of the present invention are described below.

[0092] <1. Effects of each cytokine on the primary cilia of HaCat cells> HaCat cells (human epidermal keratinocyte line) were cultured in a culture medium to which Th1 cytokines (IL-1β, TNFα, IFNγ), Th17 cytokines (IL-17A), Th22 cytokines (IL-22), or Th2 cytokines (IL-4, IL-13, IL-31, IL-33) were added. The concentrations of each cytokine in the culture medium were adjusted to the concentrations shown in Figure 1. In Figure 1, "ctrl" corresponds to the test in which cytokines were not added to the culture medium. After adding each cytokine to the culture medium, HaCat cells were cultured in the presence of the cytokines for 48 hours.

[0093] After 48 hours of culture, HaCat cells were stained with an antibody that recognizes Arl13B, a marker protein for primary cilia.

[0094] Specifically, HaCat cells were washed with PBS (phosphate-buffered saline) solution, and then immobilized by immersion in a PBS solution containing 4% paraformaldehyde. Subsequently, the HaCat cells were permeabilized by immersion in a PBS solution containing a surfactant.

[0095] Washed HaCat cells were reacted with anti-Arl13B antibody [proteintech, trade name: Arl13B Polyclonal Antibody, catalog number: 17711-1-AP] overnight at 4°C.

[0096] The reacted HaCat cells were washed with a surfactant-containing PBS solution. The washed HaCat cells were stained by reacting them with fluorescently labeled anti-rabbit IgG antibody [Thermo Fisher, trade name: Donkey Anti-Rabbit IgG H&L (fluorescent dye: Alexa Fluor® 594, catalog number: A-21207)] and nuclear stain Hoechst 33342 [Thermo Fisher, trade name: Hoechst 33342, Trihydrochloride, Trihydrate - 10 mg / mL Solution in Water, product number: H3570] at room temperature (25°C) for 1 hour. The stained samples were washed with a surfactant-containing PBS solution.

[0097] After washing, HaCat cells were observed using a confocal microscope (Olympus Corporation, model number: FV1200), and images were acquired from five observation fields. The number of cell nuclei and primary cilia in each image was measured. Subsequently, the primary cilia formation rate of HaCat cells was calculated according to formula (Ia), and the average value was calculated from the calculated values. [Primary cilia formation rate] = [Number of primary cilia in the image] / [Total number of cell nuclei in the image] × 100 ...(Ia).

[0098] Figure 1 shows the test results. As is clear from Figure 1, Th2 cytokines, Th17 cytokines, and Th22 cytokines significantly increased the rate of primary cilia formation.

[0099] <2. Effects of each cytokine on the primary cilia of HaCat cells> Th2 cytokines (IL-4, IL-13) or TNFα were added to the culture medium in which HaCat cells (human epidermal keratinocyte line) were cultured. The concentrations of each cytokine in the medium were adjusted to 10 ng / mL, 20 ng / mL, 100 ng / mL, or 200 ng / mL, as shown in Figure 2. In Figure 2, "ctrl" corresponds to the test in which cytokines were not added to the medium. After adding each cytokine to the medium, HaCat cells were cultured in the presence of the cytokines for 48 hours.

[0100] Subsequent tests are as described above. <1> The same test method was followed, and after staining the HaCat cells, the primary cilia formation rate was calculated.

[0101] Figure 2 shows the test results. As is clear from Figure 2, IL-4, IL-13, and IL-31 significantly increased the primary cilia formation rate. In particular, IL-13 and IL-31 increased the primary cilia formation rate in a cytokine concentration-dependent manner. On the other hand, TNFα did not increase the primary cilia formation rate.

[0102] Furthermore, IL-4, IL-13, IL-31, and TNFα are cytokines whose expression is elevated in atopic dermatitis. This suggests a correlation between atopic dermatitis, the expression levels of specific cytokines, and the rate of primary cilia formation.

[0103] <3. Effects of cytokine mixtures on the primary cilia of HaCat cells> A mixture of IL-4, IL-13, and TNFα was added to the culture medium in which HaCat cells (human epidermal keratinocyte line) were cultured. The concentration of each cytokine in the medium was adjusted to 1 ng / mL or 2 ng / mL, as shown in Figure 3. In Figure 3, "ctrl" corresponds to the test in which cytokines were not added to the medium. After adding the cytokine mixture to the medium, HaCat cells were cultured in the presence of cytokines for 48 hours.

[0104] Subsequent tests are as described above. <1> The same test method was followed, and after staining the HaCat cells, the primary cilia formation rate was calculated.

[0105] Figure 3 shows the test results. As is clear from Figure 3, the mixture of IL-4, IL-13, and TNFα significantly increased the primary cilia formation rate, even at low cytokine concentrations. This suggests that the synergistic effect of each cytokine significantly increased the primary cilia formation rate.

[0106] <4. Effects of various cytokines on the primary cilia of primary human epidermal keratinocytes> Th1 cytokines (IL-1β, TNFα, IFNγ), Th17 cytokines (IL-17A), Th22 cytokines (IL-22), or Th2 cytokines (IL-4, IL-13, IL-31, IL-33) were added to the culture medium containing primary human epidermal keratinocytes. The concentrations of each cytokine in the culture medium were adjusted to the concentrations shown in Figure 4. In Figure 4, "ctrl" corresponds to the test in which cytokines were not added to the culture medium. After adding each cytokine to the culture medium, primary human epidermal keratinocytes were cultured in the presence of the cytokines for 48 hours.

[0107] Subsequent tests are as described above. <1> Following a similar testing method, primary human epidermal keratinocytes were stained, and the primary cilia formation rate was calculated.

[0108] Figure 4 shows the test results. As is clear from Figure 4, Th2 cytokines, Th17 cytokines, and Th22 cytokines significantly increased the rate of primary cilia formation.

[0109] <5. Effects of individual cytokines and cytokine mixtures on the primary cilia of primary human epidermal keratinocytes> IL-4, IL-13, IL-31, or a mixture thereof, was added to the culture medium containing primary human epidermal keratinocytes. The concentrations of each cytokine in the medium were adjusted to those shown in Figure 5. In Figure 5, "ctrl" corresponds to the test in which no cytokines were added to the medium. After adding each cytokine to the medium, primary human epidermal keratinocytes were cultured in the presence of the cytokines for 48 hours.

[0110] Subsequent tests are as described above. <1> Following a similar testing method, primary human epidermal keratinocytes were stained, and the primary cilia formation rate was calculated.

[0111] Figure 5 shows the test results. As is clear from Figure 5, IL-4, IL-13, and IL-31 significantly increased the primary cilia formation rate.

[0112] Furthermore, IL-4, IL-13, and IL-31 are cytokines whose expression is elevated in atopic dermatitis. This suggests a correlation between atopic dermatitis, the expression levels of specific cytokines, and the rate of primary cilia formation.

[0113] <6. Effects of primary cilia regulation on JNK, DSG1, and CLDN4 expression> In this study, primary cilia formation was induced using IL-13. Conversely, primary cilia formation was inhibited using siIFT88 (Thermo Fisher Scientific), an siRNA that inhibits the expression of IFT88 (intraflagellar transport protein 88) mRNA. As a control for siIFT88, siCTRL (Silencer), which does not inhibit the expression of specific genes, was used. TM We used No. 2 (manufactured by Thermo Fisher Scientific).

[0114] Normal human epidermal keratinocytes (NHEKs) were seeded on a well plate at a density that covered approximately 50-60% of the well plate surface area.

[0115] 16–18 hours after seeding NHEK, the culture medium for NHEK was replaced with a medium containing Lipofectamine (Thermo Fisher Scientific) and siIFT88 (final concentration 30 nM), and NHEK was cultured in this medium for 6 hours. After 6 hours of culture, the medium was replaced with a medium that did not contain Lipofectamine or siIFT88. This treatment yielded NHEK in which IFT88 was knocked down. As a control, a test was also performed using siCTRL instead of siIFT88.

[0116] One day after knockdown, the culture medium for NHEK was replaced with medium containing IL-13 at a concentration of 50 ng / mL, medium containing IL-13 at a concentration of 100 ng / mL, or medium without IL-13.

[0117] Cells were collected 6, 12, 24, and 48 hours after the culture medium was changed. The collected cells were washed with chilled DPBS (Dulbecco's Phosphate Buffered Saline), and then lysed in RIPA buffer containing protease inhibitors and phosphatase inhibitors to obtain cell lysates.

[0118] Each cell lysate containing an equivalent amount of protein was packed into a 10% polyacrylamide gel, subjected to electrophoresis, and then the proteins separated within the polyacrylamide gel were transferred onto a PVDF membrane.

[0119] JNK, DSG1, and CLDN4 transferred onto the PVDF film were detected by Western blotting, and based on the detection results, JNK, DSG1, and CLDN4 were quantified.

[0120] An anti-phosphorylated JNK antibody was used as the primary antibody. An anti-GAPDH antibody was also used to detect GAPDH, which serves as a baseline for protein quantification. The PVDF membrane onto which the target protein was transferred was contacted with each antibody overnight at 4°C, and then the PVDF membrane was washed with TBST.

[0121] A secondary antibody conjugated with HRP was used as the secondary antibody. After contacting a PVDF membrane washed with TBST with the secondary antibody, the PVDF membrane was washed with TBST again.

[0122] By contacting a PVDF membrane washed with TBST with an HRP substrate, the target protein transferred to the PVDF membrane was imaged as a band. The intensity of the bands was quantified and statistically analyzed.

[0123] Furthermore, the same tests were performed using anti-DSG1 antibody or anti-CLDN4 antibody as the primary antibody. In this case, an anti-actin antibody was used to detect actin, which serves as a reference for protein quantification. The NHEK used in this test was NHEK that had been stimulated for 48 hours with a medium containing 100 ng / mL of IL-13.

[0124] Figure 6 shows the results of a test using anti-phosphorylated JNK antibody as the primary antibody. Figure 7 shows the results of a test using anti-CLDN4 antibody as the primary antibody. Figure 8 shows the results of a test using anti-DSG1 antibody as the primary antibody.

[0125] As is clear from the comparison of the test results for "siCTRL," "siCTRL + IL-13 50 ng / ml," and "siCTRL + IL-13 100 ng / ml" in Figure 6, increasing primary cilia increased JNK expression.

[0126] As is clear from the comparison of the test results for "siCTRL + IL-13 50 ng / ml" and "siIFT88 + IL-13 50 ng / ml" in Figure 6, and from the comparison of the test results for "siCTRL + IL-13 100 ng / ml" and "siIFT88 + IL-13 100 ng / ml" in Figure 6, reducing primary cilia resulted in a decrease in JNK expression.

[0127] As is evident from the comparison of the test results for "siCTRL" and "siCTRL+IL-13" in Figures 7 and 8, increasing primary cilia reduced the expression levels of DSG1 and CLDN4.

[0128] As is evident from the comparison of the test results for "siCTRL" and "siIFT88" in Figures 7 and 8, reducing primary cilia increased the expression levels of DSG1 and CLDN4.

[0129] Furthermore, in Figures 7 and 8, when cells were stimulated with both siIFT88 and IL-13, the expression levels of DSG1 and CLDN4 were approximately the same as when the cells were not stimulated with either siIFT88 or IL-13 (comparison of test results for "siCTRL" and "siIFT88+IL-13").

[0130] Therefore, it was shown that by adjusting primary cilia, it is possible to adjust not only the primary cilia of immune-related cells, but also the expression levels of proteins associated with primary cilia of immune-related cells. [Industrial applicability]

[0131] The present invention can be suitably used as a method and agent for adjusting primary cilia of immune-related cells, as well as a method for evaluating test samples. As described in Patent Document 1, there is a correlation between primary cilia of immune-related cells and immune-related diseases. Therefore, according to the present invention, which adjusts primary cilia of immune-related cells, it is possible to, for example, detect indicators of immune-related diseases, assist in diagnosing the presence or absence of immune-related diseases or assist in diagnosing the prognosis of immune-related diseases, evaluate the inhibitory effect on immune-related diseases by treatments or immune-related disease suppressors, and evaluate whether a test sample is a substance that has an immune function regulatory effect. Furthermore, since primary cilia of immune-related cells are involved in the proliferation of immune-related cells and immune-related diseases, immune function can also be controlled by controlling the formation of primary cilia of immune-related cells. Therefore, the present invention is expected to be used in the examination of immune-related diseases, assist in diagnosing the presence or absence of immune-related diseases or assist in diagnosing the prognosis of immune-related diseases, develop therapeutic drugs for immune-related diseases, and develop quasi-drugs or cosmetic ingredients to suppress immune-related diseases.

Claims

1. A method for preparing the primary cilia of immune-associated cells, comprising a contact step of bringing immune-associated cells into contact with interleukin-17A and / or interleukin-22, The preparation method wherein the immune-related cells are HaCat cells or epidermal keratinocytes.

2. A method for adjusting the expression of at least one protein selected from the group consisting of JNK, DSG1, and CLDN4 in immune-related cells, comprising the method for adjusting the primary cilia of immune-related cells as described in claim 1 as one of the steps.

3. A modifier for primary cilia of immune-associated cells, comprising interleukin-17A and / or interleukin-22, The aforementioned immune-related cells are HaCat cells or epidermal keratinocytes, which are the regulators.

4. A regulator for the expression of at least one protein selected from the group consisting of JNK, DSG1, and CLDN4 in immune-related cells, comprising the primary cilia regulator of immune-related cells described in claim 3.

5. A first observation step involves contacting immune-related cells with one or more signaling regulatory substances selected from interleukin-17A and interleukin-22, and then observing the primary cilia of the immune-related cells. A second observation step involves contacting the immune-related cells with the signal control substance used in the first observation step and the test sample, and then observing the primary cilia of the immune-related cells. A method for evaluating a test sample, comprising: an evaluation step of evaluating the effect of the test sample on the primary cilia of immune-related cells by comparing the observation results of the first observation step with the observation results of the second observation step, The method for evaluating whether the immune-related cells are HaCat cells or epidermal keratinocytes.

6. Furthermore, the method for evaluating a test sample according to claim 5, comprising an evaluation step of evaluating the effect of the test sample on the expression of at least one protein selected from the group consisting of JNK, DSG1, and CLDN4 in the immune-related cells, based on the evaluation results in the evaluation step.

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