Method and agent for modulating primary cilia of immune-related cells, method for evaluating test samples, and use 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 a regulator for regulating primary cilia of immune-related cells, a method for evaluating a test sample, and uses thereof. [Background technology]

[0002] Primary cilia are antenna-like organelles with microtubules as their 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 almost all cells have primary cilia, the relationship between primary cilia and immune responses in immune-related cells remains largely unclear.

[0003] Recent studies have suggested that primary cilia exist in immune-related cells, that primary cilia are associated with 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 indicators of immune-related diseases by observing primary cilia has been proposed (see Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. WO2019 / 172419 Summary of the Invention [Problem to be solved by the invention]

[0005] It is believed that regulating the primary cilia of immune-related cells (e.g., regulating the number and / or ratio of immune-related cells having primary cilia) can contribute to regulating the immune system and treating immune-related diseases. Therefore, there is a demand for the development of a technology for regulating the primary cilia of immune-related cells.

[0006] One embodiment of the present invention has been made in consideration of the above-mentioned problems, and its object is to provide a method and a regulator for regulating primary cilia of immune-related cells, a method for evaluating a test sample, and uses thereof. [Means for solving the problem]

[0007] As a result of intensive research to solve the above problems, the present inventors have discovered (i) the novel finding that the number and / or proportion of immune-related cells having primary cilia can be adjusted by adjusting the activation state of a signal transduction pathway of a specific cytokine in immune-related cells, and (ii) the novel finding that adjusting the primary cilia of immune-related cells changes the expression of a specific protein, and have completed the present invention. That is, the present invention includes the following configurations.

[0008] <1> A method for regulating primary cilia of immune-related cells, comprising a contacting step of contacting immune-related cells with a signal control substance that activates or inactivates the signal transduction 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, which comprises as one of the steps the method for regulating primary cilia of immune-related cells described in .

[0010] <3> An agent for regulating primary cilia of immune-related cells, comprising a signal control substance that activates or inactivates the signal transduction 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, comprising the regulator of primary cilia of immune-related cells described in .

[0012] <5> A method for evaluating a test sample, comprising: a first observation step of contacting an immune-related cell with a signal control substance that activates or inactivates a signal transduction 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 cell; a second observation step of contacting the immune-related cell with the signal control substance used in the first observation step and a test sample, and then observing the primary cilia of the immune-related cell; and an evaluation step of evaluating the effect of the test sample on the primary cilia of the immune-related cell 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, and (c) Th22 cytokine.

[0013] <6> and further 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 result in the evaluation step. <5> A method for evaluating a test sample according to claim 1. Effect of the Invention

[0014] According to the present invention, it is possible to provide a method and a regulator for regulating primary cilia of immune-related cells, as well as a method for evaluating a test sample. [Brief description of the drawings]

[0015] [Figure 1] 1 is a graph showing the effect of each cytokine on primary cilium of HaCat cells (human epidermal keratinocyte cell line) in an example of the present invention. [Diagram 2] 1 is a graph showing the effect of each cytokine on primary cilium of HaCat cells (human epidermal keratinocyte cell line) in an example of the present invention. [Diagram 3] 1 is a graph showing the effect of a mixture of cytokines on primary cilia of HaCat cells (human epidermal keratinocyte cell line) in an example of the present invention. [Figure 4]1 is a graph showing the effect of each cytokine on primary cilia of primary human epidermal keratinocytes in an example of the present invention. [Diagram 5] 1 is a graph showing the effect of each cytokine and a mixture of cytokines on primary cilia of primary human epidermal keratinocytes in an example of the present invention. [Figure 6] 1 is a graph showing the effect of cytokines on JNK protein expression in an example of the present invention. [Figure 7] 1 is a graph showing the effect of cytokines on the expression of CLDN4 protein in an example of the present invention. [Figure 8] 1 is a graph showing the effect of cytokines on the expression of DSG1 protein in an example of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0017] [1. Method for regulating primary cilia of immune-related cells] The method of the present invention for regulating primary cilia of immune-related cells includes a contacting step of contacting immune-related cells with a signal control substance that activates or inactivates the signal transduction pathway of at least one cytokine selected from the group consisting of (a) to (c) below: (a) Th2 cytokine, (b) Th17 cytokine, and (c) Th22 cytokine.

[0018] More specifically, the method of regulating primary cilia of immune-related cells according to the present invention may be (i) a method for increasing the number and / or proportion of immune-related cells having primary cilia, (ii) a method for inhibiting an increase in the number and / or proportion of immune-related cells having primary cilia, (iii) a method for decreasing the number and / or proportion of immune-related cells having primary cilia, or (iv) a method for inhibiting a decrease in the number and / or proportion of immune-related cells having primary cilia.

[0019] In the contact step, the method of contacting immune-related cells with a signal control substance is not particularly limited. For example, immune-related cells may be contacted with a signal control substance by adding the signal control substance to a medium in which the immune-related cells are cultured. In the contact step, the time for contacting immune-related cells with a signal control substance 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.

[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 may or may not be in contact with the signal control substance. The culture time is not particularly limited, and may be 1 to 3 days, 1 to 7 days, or 7 days or more.

[0021] Immune-related cells include immune cells that are mainly responsible for immune responses and immune function-bearing cells that are indirectly involved in immune cells. Immune function-bearing cells have, for example, the function of activating immune cells. Examples of immune cells include skin dendritic cells (e.g., Langerhans cells, dermal dendritic cells), lymphocytic immune cells (e.g., T cells, NK cells, B cells), and monocytic immune cells (e.g., conventional dendritic cells, monocytic dendritic cells (e.g., plasmacytoid dendritic cells)). On the other hand, examples of immune function-bearing cells include keratinocytes, fibroblasts, and epithelial cells. Immune-related cells may be immune-related cells collected from a living body, or may be established immune-related cells (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 simultaneously activated or inactivated. Examples of the cytokine signaling pathways to be activated or inactivated include (a) Th2 cytokine signaling pathway, (b) Th17 cytokine signaling pathway, (c) Th22 cytokine signaling pathway, (d) a combination of Th2 cytokine signaling pathway and Th17 cytokine signaling pathway, (e) a combination of Th17 cytokine signaling pathway and Th22 cytokine signaling pathway, (f) a combination of Th2 cytokine signaling pathway and Th22 cytokine signaling pathway, and (g) a combination of Th2 cytokine signaling pathway, Th17 cytokine signaling pathway, and Th22 cytokine signaling pathway.

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

[0024] In certain diseases (e.g., atopic dermatitis), the amount of a specific signal regulator (e.g., IL-4, IL-13, and IL-31) or the total amount of a combination of specific signal regulators (e.g., IL-4, IL-13, and IL-31) increases in cells. This suggests that adding a specific signal regulator to cells can produce model cells for a specific disease. Therefore, the method of regulating primary cilia in immune-related cells according to the present invention makes it possible to regulate primary cilia in model cells for a specific disease and to observe the effect of regulating primary cilia on a specific disease.

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

[0026] Examples of Th17 cytokines include, but are not limited to, 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, and more specific examples thereof include interleukin-22 (IL-22), interleukin-13 (IL-13), and interleukin-26 (IL-26).

[0028] The signal control substance is not particularly limited as long as it is capable of activating or inactivating the signal pathway of at least one cytokine selected from the group consisting of (a) Th2 cytokines, (b) Th17 cytokines, and (c) Th22 cytokines.

[0029] Specific examples of signal control substances include (i) substances that activate cytokine signaling pathways, (ii) substances that suppress activation of cytokine signaling pathways, (iii) substances that inactivate cytokine signaling pathways, and (iv) substances that suppress inactivation of cytokine signaling pathways.

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

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

[0032] The cytokines themselves can include, for example, 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, low molecular weight compounds, etc. Commercially available cytokine receptor inhibitors can be used.

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

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

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

[0037] Desmoglein 1 (DSG1) is a calcium-binding transmembrane protein that is a cell adhesion molecule that constitutes focal adhesions, a type of cell adhesion apparatus. DSG1 is involved in cell-cell adhesion in skin.

[0038] CLDN4 (claudin 4) is one of the proteins that constitute the tight junction. CLDN4 plays an important role in maintaining and forming the barrier function in the skin.

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

[0040] The method of adjusting the primary cilia of immune-related cells according to the present invention not only makes it possible to adjust the primary cilia of immune-related cells, but also makes it possible to adjust the expression level of a protein associated with the primary cilia of immune-related cells, and to observe the effect of the expression level of the protein on the cell.

[0041] [2. Regulators of primary cilia of immune-related cells] The agent for regulating primary cilia of immune-related cells of the present invention contains a signal control substance that activates or inactivates the signal transduction pathway of at least one cytokine selected from the group consisting of (a) to (c) below: (a) Th2 cytokine, (b) Th17 cytokine, and (c) Th22 cytokine.

[0042] More specifically, the agent for regulating primary cilia of immune-related cells according to the present invention may be one that (i) increases the number and / or proportion of immune-related cells having primary cilia, (ii) inhibits an 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 a decrease in the number and / or proportion of immune-related cells having primary cilia.

[0043] As the signal control substances have been explained above in [1. Method for regulating primary cilia of immune-related cells], detailed explanations thereof will be omitted here.

[0044] The agent for regulating primary cilia of immune-related cells according to the present invention may contain components other than the signal control substance, including, but not limited to, a buffer, a pH adjuster, an isotonicity agent, a preservative, an antioxidant, a high molecular weight polymer, an excipient, a carrier, a diluent, a solvent, a solubilizer, a stabilizer, a filler, a binder, a surfactant, a stabilizer, and the like.

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

[0046] The amount of substances other than the signal control substance contained in the regulator of primary cilia of immune-related cells according to the present invention is not particularly limited, and for example, when the regulator is taken as 100% by mass, the amount may be 0% by mass to 99.999% by mass, 0% by mass to 99.99% by mass, 0% by mass to 99.9% by mass, 5% by mass to 99.9% by mass, 10% by mass to 99.9% by mass, 20% by mass to 99.9% by mass, 30% by mass to 99.9% by mass, 40% by mass to 99.9% by mass, 50% by mass to 99.9% by mass, 60% by mass to 99.9% by mass, 70% by mass to 99.9% by mass, 80% by mass to 99.9% by mass, or 90% by mass to 99.9% by mass.

[0047] The amount of signal control substance contained in the agent for regulating primary cilia of immune-related cells according to the present invention is not particularly limited, and may be adjusted, for example, so that the amount of signal control substance contained in a solution (e.g., culture medium) in which immune-related cells are brought into contact with the signal control substance is 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 present inventors have newly found that the regulation of the primary cilia of immune-related cells changes the expression of specific proteins (specifically, JNK, DSG1, and CLDN4). This indicates that the regulator of the primary cilia of immune-related cells of the present invention can be used as a regulator of the expression of specific proteins.

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

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

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

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

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

[0054] 3. Evaluation method of 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 signal control substance that activates or inactivates a signal transduction 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 signal control 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 cytokines, (b) Th17 cytokines, and (c) Th22 cytokines.

[0055] A signal control substance activates or inactivates the signal transduction pathway of a specific cytokine. In the first observation step, a signal control substance is used to observe the effect of the signal control substance on the primary cilia of immune-related cells (corresponding to a control test). Meanwhile, in the second observation step, a signal control substance and a test sample are used to observe the effect of the test sample on the effect of the signal control substance on the primary cilia of immune-related cells. Then, by comparing the results of both observations, the effect of the test sample on the signal control substance (in other words, the effect of the test sample on the primary cilia of immune-related cells) can be evaluated.

[0056] Each step of the method for evaluating a test sample according to the present invention will be described below. Note that the configurations described in the above [1. Method for regulating primary cilia of immune-related cells] and [2. Regulator of primary cilia of immune-related cells] will not be described below.

[0057] <First observation step> In the first observation step, immune-related cells are contacted with a signal control substance that activates or inactivates the signal transduction pathway of at least one cytokine selected from the group consisting of (a) to (c) below, and then the primary cilia of the immune-related cells are observed; (a) Th2 cytokine, (b) Th17 cytokine, and (c) Th22 cytokine.

[0058] In the first observation step, the method of contacting immune-related cells with a signal control substance is not particularly limited. For example, immune-related cells may be contacted with a signal control substance by adding the signal control substance to a medium in which the immune-related cells are cultured. The time for contacting immune-related cells with a signal control substance 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, 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 may or may not be in contact with the signal control substance. The culture time is not particularly limited, and may be 1 to 3 days, 1 to 7 days, or 7 days or more.

[0060] In the first observation step, the primary cilia of the immune-related cells are observed using a microscope or the like, and the formation rate of the primary cilia of the immune-related cells is calculated. An example of a method for calculating the formation rate of the primary cilia and a method for observing the primary cilia will be described below.

[0061] In the first observation step, the primary cilia in immune-related cells can be observed as one of the indices to calculate the number of primary cilia. When calculating the number of primary cilia, for example, (a) the number of primary cilia per immune-related cell may be calculated, (b) the ratio 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 calculated. In addition, the total number of immune-related cells contained in a sample may be calculated as the total number of nuclei in the sample.

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

[0063] For example, in the case of (b) above, the number of primary cilia can be calculated as [the formation rate of primary cilia in immune-related cells] according to the following formula (II): [Formation rate of primary cilia in 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 Formula (II).

[0064] Observation of primary cilia can be performed, for example, by known immunohistochemical staining methods. Specifically, observation of primary cilia can be performed by contacting a test specimen-containing sample (for example, cells fixed with a fixative such as paraformaldehyde, or a test specimen embedded in paraffin and sectioned) with a substance that specifically binds to primary cilia (hereinafter referred to as a "first specific binding substance"), binding the first specific binding substance to primary cilia contained in the test specimen-containing sample, and detecting the labeling substance that has bound to the first specific binding substance. The method for preparing the test specimen-containing sample is not limited, and known methods can be used.

[0065] The first specific binding substance can be a substance (e.g., an antibody (primary antibody)) that specifically binds to a substance that serves as a marker for primary cilia. Examples of substances that serve as markers for primary cilia can include ADP ribosylation factor-like protein (Arl13B), acetylated tubulin, adenylate cyclase III, nephrocystin 3 (NPHP3), intraflagellar transport protein (IFT88), somatostatin receptor 3 (sstr3), polycystin-1 (TRPC1), transient receptor potential vanilloid 4 (TRPV4), platelet-derived growth factor receptor α (PDGFRα), and smoothened (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 the antibody include a monoclonal antibody, a polyclonal antibody, and fragments of these antibodies (e.g., a Fab fragment, a F(ab')2 fragment, and a single-chain antibody).

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

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

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

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

[0072] The labeling substance may be any substance (e.g., a fluorescent dye, an enzyme) capable of generating a detectable signal such as fluorescence or color. The labeling substance may be directly bound to the first specific binding substance, or may be directly bound to a substance that specifically binds to the first specific binding substance (hereinafter referred to as a "second specific binding substance"). Examples of the second specific binding substance include substances that specifically bind 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-xanthen-9-yl)-5-[[5-(2,5-dioxo-3-pyrrolin-1-yl)pentyl]carbamoyl]benzoic acid (e.g., Alexa Fluor 488 manufactured by 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 manufactured by Invitrogen); peroxidase; and alkaline phosphatase.

[0074] Detection of the labeled substance bound to the first specific binding substance can be carried out, for example, by visual detection 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 detection based on numerical values ​​such as fluorescence intensity.

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

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

[0077] The test sample used in the second observation step is not particularly limited, and may be, for example, an inorganic compound, an organic compound, a plant extract, a microbial extract, or a culture supernatant of various cells. The test sample may be used as it is, or may be used in a state dissolved in a solvent. The solvent may be, for example, physiological saline, phosphate buffered saline, or water.

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

[0079] <Evaluation process (1)> In the evaluation step, the observation results of the first observation step and the second observation step are compared to evaluate the effect of the test sample on the primary cilia of immune-related cells.

[0080] For example, suppose that the first observation step shows a result that the formation rate of primary cilia is increased, and the second observation step shows a result that the formation rate of primary cilia is higher than the observation result of the first observation step, in which case the test sample can be evaluated as having an effect of increasing the primary cilia of immune-related cells.

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

[0082] For example, suppose that the first observation step shows a result that the formation rate of primary cilia is increased, and the second observation step shows a result that the formation rate of primary cilia is lower than the observation result of the first observation step, in which case the test sample can be evaluated as having an effect of reducing the primary cilia of immune-related cells.

[0083] Suppose that the first observation step shows a result indicating that the formation rate of primary cilia is decreased, and the second observation step shows that the formation rate of primary cilia is decreased more than the observation result of the first observation step. In this case, the test sample can be evaluated as having an effect of decreasing the primary cilia of immune-related cells.

[0084] For example, suppose that the first observation step shows a result that the formation rate of primary cilia is increased, and the second observation step shows a result that the formation rate of primary cilia is substantially the same as the observation result of the first observation step. In this case, it can be evaluated that the test sample does not have an effect of increasing or decreasing the primary cilia of immune-related cells, or that the signal control substance suppresses a change (increase or decrease) in the number and / or proportion of immune-related cells having primary cilia.

[0085] Suppose that the first observation step shows that the formation rate of primary cilia does not change, and the second observation step shows that the formation rate of primary cilia is substantially the same as the observation 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 signal control substance suppresses changes (increases or decreases) in the number and / or proportion of immune-related cells having primary cilia.

[0086] Suppose that the first observation step shows a result that the formation rate of primary cilia decreases, and the second observation step shows a result that the formation rate of primary cilia is substantially the same as the observation result of the first observation step. In this case, it can be evaluated that the test sample does not have an effect of increasing or decreasing the primary cilia of immune-related cells, or that the signal control substance suppresses a change (increase or decrease) in the number and / or proportion of immune-related cells having primary cilia.

[0087] <Evaluation process (2)> The method may further comprise 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 result in the evaluation step. 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 result regarding the effect of the test sample on the primary cilia of the immune-related cells.

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

[0089] More specifically, for example, the method may be such that (i) if the evaluation result indicates that the test sample increases primary cilia, the test sample is evaluated as being capable of increasing the expression level of a specific protein (e.g., JNK); (ii) if the evaluation result indicates that the test sample increases primary cilia, the test sample is evaluated as being capable of decreasing the expression level of a specific protein (e.g., DSG1 and CLDN4); (iii) if the evaluation result indicates that the test sample decreases primary cilia, the test sample is evaluated as being capable of increasing the expression level of a specific protein (e.g., DSG1 and CLDN4); or (iv) if the evaluation result indicates that the test sample decreases primary cilia, the test sample is evaluated as being capable of decreasing the expression level of a specific protein (e.g., JNK).

[0090] This evaluation step makes it possible to evaluate not only the effect of the test sample on the primary cilia in immune-related cells, but also the effect on the expression level of proteins related to the primary cilia of immune-related cells. EXAMPLES

[0091] An embodiment of the present invention will now be described.

[0092] <1.Effects of cytokines on primary cilia of HaCat cells> 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 medium in which HaCat cells (human epidermal keratinocyte cell line) were cultured. The concentration of each cytokine in the medium was adjusted to the concentration shown in Figure 1. In addition, "ctrl" in Figure 1 corresponds to a test in which no cytokine was added to the medium. After adding each cytokine to the medium, HaCat cells were cultured in the presence of the cytokine 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, the HaCat cells were washed with PBS (phosphate buffered saline) solution, and then the HaCat cells were immersed in a PBS solution containing 4% paraformaldehyde for fixation treatment, and then the HaCat cells were immersed in a PBS solution containing a surfactant for permeabilization treatment.

[0095] The washed HaCat cells were reacted with an anti-Arl13B antibody (Proteintech, product name: Arl13B Polyclonal Antibody, product number: 17711-1-AP) overnight at 4°C.

[0096] After the reaction, the HaCat cells were washed with a surfactant-containing PBS solution. The washed HaCat cells were mixed with an anti-rabbit IgG antibody labeled with a fluorescent dye (manufactured by Thermo Fisher Scientific, product name: Donkey Anti-Rabbit IgG H&L (fluorescent dye: Alexa Fluor (registered trademark) 594, catalog number: A-21207)) and a nuclear staining agent Hoechst 33342 (manufactured by Thermo Fisher Scientific, product name: Hoechst 33342, HaCat cells were stained by reacting with 10 mg / mL trihydrochloride, trihydrate - solution in water, product number H3570 at room temperature (25°C) for 1 hour. After staining, the sample was washed with a PBS solution containing a surfactant.

[0097] The washed HaCat cells were observed using a confocal microscope (Olympus Corporation, product number: FV1200) to obtain images of five observation fields. The number of cell nuclei and the number of primary cilia contained in the images were measured. The formation rate of primary cilia in the HaCat cells was then 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] x 100 (Ia).

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

[0099] 2. Effects of cytokines on primary cilia of HaCat cells Th2 cytokines (IL-4, IL-13) or TNFα were added to the medium in which HaCat cells (human epidermal keratinocyte cell line) were cultured. The concentration of each cytokine in the medium was adjusted to 10 ng / mL, 20 ng / mL, 100 ng / mL, or 200 ng / mL, as shown in Figure 2. "ctrl" in Figure 2 corresponds to a test in which no cytokine was added to the medium. After adding each cytokine to the medium, HaCat cells were cultured in the presence of the cytokine for 48 hours.

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

[0101] The test results are shown in Figure 2. As is clear from Figure 2, IL-4, IL-13, and IL-31 significantly increased the formation rate of primary cilia. In particular, IL-13 and IL-31 increased the formation rate of primary cilia depending on the cytokine concentration. On the other hand, TNFα did not increase the formation rate of primary cilia.

[0102] In addition, IL-4, IL-13, IL-31, and TNFα are cytokines whose expression increases in atopic dermatitis, suggesting that there is a relationship between atopic dermatitis, the expression levels of specific cytokines, and the formation rate of primary cilia.

[0103] 3. Effect of cytokine mixture on primary cilia of HaCat cells A mixture of IL-4, IL-13, and TNFα was added to the medium in which HaCat cells (human epidermal keratinocyte cell 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. Note that "ctrl" in Figure 3 corresponds to a test in which no cytokines were 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 were performed as described above. <1> The same test method was followed, and after staining HaCat cells, the formation rate of primary cilia was calculated.

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

[0106] <4. Effects of cytokines on 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 medium in which primary human epidermal keratinocytes were cultured. The concentration of each cytokine in the medium was adjusted to the concentration shown in FIG. 4. In FIG. 4, "ctrl" corresponds to a test in which no cytokine was added to the medium. After adding each cytokine to the medium, primary human epidermal keratinocytes were cultured in the presence of the cytokine for 48 hours.

[0107] Subsequent tests were performed as described above. <1> The same test method was followed as in Example 1, and primary human epidermal keratinocytes were stained, after which the formation rate of primary cilia was calculated.

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

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

[0110] Subsequent tests were performed as described above. <1> The same test method was followed as in Example 1, and primary human epidermal keratinocytes were stained, after which the formation rate of primary cilia was calculated.

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

[0112] Furthermore, IL-4, IL-13, and IL-31 are cytokines whose expression levels increase in atopic dermatitis, suggesting that there is a relationship between atopic dermatitis, the expression levels of specific cytokines, and the formation rate of primary cilia.

[0113] 6. Effect of regulation of primary cilia on the expression of JNK, DSG1, and CLDN4 In this study, IL-13 was used to induce the formation of primary cilia. Meanwhile, in this study, siIFT88 (Thermo Fisher Scientific), an siRNA that inhibits the expression of IFT88 (intraflagellar transport protein 88) mRNA, was used to inhibit the formation of primary cilia. As a control for siIFT88, siCTRL (Silencer TM No. 2, Thermo Fisher Scientific) was used.

[0114] Normal human epidermal keratinocytes (NHEK) were seeded onto a well plate at a density that covered approximately 50 to 60% of the surface area of ​​the well plate.

[0115] 16 to 18 hours after seeding NHEKs, the medium for culturing NHEKs was replaced with a medium containing Lipofectamine (Thermo Fisher Scientific) and siIFT88 (final concentration 30 nM), and NHEKs were cultured in the medium for 6 hours. After 6 hours of culture, the medium was replaced with a medium not containing Lipofectamine and siIFT88. This treatment resulted in NHEKs with IFT88 knockdown. In addition, a test was performed using siCTRL instead of siIFT88 as a control.

[0116] One day after knockdown, the medium for culturing NHEKs 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] The cells were harvested 6, 12, 24, and 48 hours after the medium change. The harvested 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 a cell lysate.

[0118] Each cell lysate containing an equal amount of protein was loaded into a 10% polyacrylamide gel and subjected to electrophoresis, after which the proteins separated in the polyacrylamide gel were transferred onto a PVDF membrane.

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

[0120] Anti-phosphorylated JNK antibody was used as the primary antibody. Anti-GAPDH antibody was used to detect GAPDH, which serves as a standard for quantifying proteins. The PVDF membrane on which the target protein was transferred was contacted with each antibody overnight at 4°C, and the PVDF membrane was washed with TBST.

[0121] The secondary antibody used was an HRP-conjugated secondary antibody. After contacting the PVDF membrane washed with TBST with the secondary antibody, the PVDF membrane was washed with TBST.

[0122] The PVDF membrane was washed with TBST and then contacted with the HRP substrate to visualize the target protein transferred to the PVDF membrane as a band. The band intensity was quantified and statistically analyzed.

[0123] In addition, a similar test was performed using anti-DSG1 antibody or anti-CLDN4 antibody as the primary antibody. In this case, anti-actin antibody was used to detect actin as a standard for quantifying proteins. The NHEKs used in this study were those stimulated with a medium containing 100 ng / mL of IL-13 for 48 hours.

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

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

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

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

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

[0129] In addition, 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 those when the cells were not stimulated with siIFT88 or IL-13 (comparison of test results for "siCTRL" and "siIFT88+IL-13").

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

[0131] The present invention can be suitably used as a method and a regulator for adjusting the primary cilia of immune-related cells, and as a method for evaluating a test sample. As described in Patent Document 1, the primary cilia of immune-related cells are correlated with immune-related diseases, and therefore, according to the present invention for adjusting the primary cilia of immune-related cells, for example, it is possible to detect indicators of immune-related diseases, aid in diagnosis of the presence or absence of immune-related diseases or aid in diagnosis of the prognosis of immune-related diseases, evaluate the treatment of immune-related diseases or the suppressive effect of immune-related disease inhibitors on immune-related diseases, and evaluate whether a test sample is a substance having an immune function control effect. In addition, since the primary cilia of immune-related cells are involved in the proliferation of immune-related cells and immune-related diseases, it is also possible to control immune functions by controlling the formation of the primary cilia of immune-related cells. Therefore, the present invention is expected to be used in testing immune-related diseases, aid in diagnosis of the presence or absence of immune-related diseases or aid in diagnosis of the prognosis of immune-related diseases, development of therapeutic drugs for immune-related diseases, and development of quasi-drugs or cosmetic ingredients for suppressing immune-related diseases.

Claims

1. A method for regulating primary cilia of immune-related cells, the method comprising a contacting step of contacting immune-related cells with one or more selected from interleukin-4 (IL-4), interleukin-13 (IL-13), interleukin-31 (IL-31) and interleukin-33 (IL-33), The method for adjusting, wherein the immune-related cells are HaCat cells or epidermal keratinocytes.

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

3. An agent for regulating primary cilia of immune-related cells, comprising one or more selected from interleukin-4 (IL-4), interleukin-13 (IL-13), interleukin-31 (IL-31) and interleukin-33 (IL-33), The immune-related cells are HaCat cells or epidermal keratinocytes.

4. A regulator of expression of at least one protein selected from the group consisting of JNK, DSG1 and CLDN4 in immune-related cells, comprising the regulator of primary cilia of immune-related cells according to claim 3, The immune-related cells are HaCat cells or epidermal keratinocytes.

5. a first observation step of contacting immune-related cells with one or more signal control substances selected from interleukin-4 (IL-4), interleukin-13 (IL-13), interleukin-31 (IL-31), and interleukin-33 (IL-33), and then observing the primary cilia of the immune-related cells; a second observation step of contacting the immune-related cells with the signal control 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 an effect of the test sample on the primary cilia of the immune-related cells by comparing an observation result of the first observation step with an observation result of the second observation step, The evaluation method, wherein the immune-related cells are HaCat cells or epidermal keratinocytes.

6. The method for evaluating a test sample according to claim 5, further 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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