Use of lipid metabolites as agents promoting th17 cell differentiation

Abstract

To identify lipid metabolites that control Th17 cell differentiation and lipid biosynthetic enzymes involved therein, and based on that, to provide a screening method for a therapeutic agent for diseases caused by excessive Th17 cell responses.SOLUTION: Provided is 1-oleoyl-lysophosphatidylethanolamine (LPE(1-18:1)) as a lipid metabolic product that controls Th17 cell differentiation. Since LPE(1-18:1) binds to RORγt with high specificity and promotes differentiation into Th17 cells, a candidate compound for a therapeutic agent for diseases caused by excessive Th17 cell responses can be obtained by selecting a compound that inhibits the binding of LPE(1-18:1) to RORγt. Also provided are five lipid metabolic enzymes (Gpam, Gpat3, Lplat1, Pla2g12a, and Scd2) that are responsible for the biosynthesis of LPE(1-18:1) and control Th17 cell differentiation. By selecting a compound that inhibits the activity or expression of these lipid metabolic enzymes, a candidate compound for a therapeutic agent for diseases caused by excessive Th17 cell responses can be obtained.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to the use of lipid metabolites as promoters of Th17 cell differentiation. The present invention also relates to a method for screening a therapeutic agent for a disease caused by the activation of Th17 cells using the lipid metabolite or a lipid metabolism enzyme involved in its biosynthesis, and the like.

Background Art

[0002] Differentiation of naive T cells into Th17 cells can be controlled by a combination of cytokines such as IL-6 and TGFβ. Th17 cells contribute to host defense against fungi and extracellular bacteria. However, it has been revealed by genome-wide association studies that link Th17 cells, which are involved in the pathogenesis of various autoimmune diseases such as multiple sclerosis, psoriasis, inflammatory bowel disease, and type 2 diabetes, with genes preferentially expressed in these diseases and genes encoding STAT3 and IL23R.

[0003] Strict control of cell metabolism is required for the differentiation of Th17 cells. Differentiation of Th17 cells greatly depends on the involvement of de novo fatty acid biosynthesis (Non-Patent Documents 1 and 2). Acetyl-CoA carboxylase 1 (ACC1) encoded by the Acaca gene catalyzes the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA and controls the first step of cellular fatty acid metabolism (Non-Patent Document 3). When fatty acid biosynthesis is inhibited by using the pharmacological inhibitor 5-tetradecyloxy-2-furoic acid (TOFA) or by CD4 + T cell-specific deletion of ACC1, the differentiation of Th17 cells is inhibited. Furthermore, deletion of ACC1 reduces the proportion and number of IL-17A-producing memory phenotype CD4 + T cells (Non-Patent Document 1).

[0004] Interestingly, fatty acid flux has also emerged as an important regulator of Th17 plasticity (Non-Patent Documents 1, 2, and 4). Pharmacological inhibition or genetic deletion of ACC1 in CD4 cultured under conditions biased towards Th17+ In T cells, the expression of the transcription factor Foxp3, which is characteristic of regulatory T cells, was increased (Non-Patent Documents 1 and 2). Notably, the shift towards regulatory T cell differentiation in these ACC1-deficient cells could be overcome by the addition of exogenous fatty acids (16:0 or 18:1). This suggests that fatty acids or their metabolites may play an important role in regulating the balance between Th17 cells and regulatory T cells. Consistent with these in vitro results, administration of TOFA or genetic deletion of ACC1 protected mice from the pathology of experimental autoimmune encephalomyelitis (EAE) and reduced the proportion of IL-17A-producing CD4 + T cells in the central nervous system (Non-Patent Documents 1 and 2). Furthermore, administration of TOFA also improved the EAE pathology in diet-induced obese mice. In addition, inhibition of fatty acid biosynthesis by inhibiting the activity of fatty acid synthase, an enzyme that functions downstream of ACC1, reduces the inflammatory potential of Th17 cells (Non-Patent Document 5).

[0005] Retinoid-related orphan receptor γt (RORγt), one of the nuclear hormone receptors (NHRs), is a master transcription factor that plays an essential role in inducing the differentiation of Th17 cells (Non-Patent Document 6). Previously, the present inventors and other researchers have found that fatty acid metabolism is associated with RORγt activity during the differentiation process of Th17 cells (Non-Patent Documents 1 and 7). Two isoforms, RORg and RORγt, are transcribed from the Rorc gene. NHRs are transcription factors that direct a wide range of developmental, reproductive, and immune response programs. NHRs share a common modular structure composed of an N-terminal DNA-binding domain and a C-terminal ligand-binding domain. For transactivation, most NHRs require the interaction of the ligand-binding domain with a ligand. Several classes of lipophilic small molecules, such as hormones, vitamins, steroids, retinoids, and fatty acids, have been identified as NHR ligands (Non-Patent Document 8). Identifying the natural ligands of orphan NHRs is an important step in understanding how these receptors are regulated by dietary factors and endogenous metabolites. RORγt is a nuclear receptor consisting of three domains (Non-Patent Documents 9 and 10), which includes a conserved DNA-binding domain with two zinc finger motifs responsible for DNA binding and a conserved ligand-binding domain with a carboxy-terminal AF2 motif that recruits a family of steroid receptor transcriptional coactivators to stimulate gene expression (Non-Patent Documents 9 and 11).

[0006] Single-cell RNA sequencing (scRNA-seq) approaches have led to a deeper understanding of factors such as RORγt that control Th17 cell differentiation and function. Indeed, scRNA-seq of T cells from EAE mice has shown that low levels of CD5 molecule-like (CD5L) expression correlate with Th17 pathogenicity (Non-Patent Documents 7, 12). CD5L controls flux through the de novo fatty acid biosynthesis pathway. Lipidomic analysis of CD5L-deficient Th17 cells has revealed that the fatty acid composition of both phospholipids and neutral lipids is globally different, with increased levels of intracellular saturated and monounsaturated fatty acids and a corresponding decrease in polyunsaturated fatty acids such as arachidonic acid (Non-Patent Document 7). Furthermore, addition of exogenous polyunsaturated fatty acids to Th17 cells decreased RORγt binding at the Il17 and Il23r loci, whereas addition of exogenous saturated or monounsaturated fatty acids increased RORγt binding to the target loci (Non-Patent Document 7). Thus, the intracellular balance of fatty acid composition appears to affect Th17 function, at least in part, through the recruitment and / or activity of RORγt at loci associated with Th17 signature cytokines.

[0007] The important role of fatty acids in Th17 cell differentiation via lipid modification of transcription factors has been reported (Non-Patent Document 13). One example is STAT3, where palmitoylation via palmitoyltransferase DHHC7 promotes STAT3 membrane recruitment and its activation (Non-Patent Document 13). Once activated, phosphorylated STAT3 enters the nucleus and transcriptionally activates many important genes required for Th17 cell differentiation (Non-Patent Document 13).

[0008] In addition to fatty acid metabolism, metabolic flux through the cholesterol biosynthesis pathway is also involved in Th17 cell differentiation and may affect the control of RORγt activity (Non-Patent Documents 14-16). Genetic deletion of specific enzymes in the distal branch of the cholesterol biosynthesis pathway results in CD4 +IL-17 production by T cells is decreased (Non-Patent Document 14). Furthermore, treating Th17 cells with statins, which are pharmacological inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase in the cholesterol biosynthesis pathway, affects the balance between Th17 cells and regulatory T cells (Non-Patent Document 17). Oxysterols containing lanosterol and cholesterol biosynthesis intermediates increase RORγt activity (Non-Patent Documents 14 to 16), but it remains unclear which sterol metabolites act as physiological ligands for RORγt during the differentiation process of Th17 cells. Interestingly, neither pharmacological inhibition nor genetic deletion of cholesterol biosynthesis was able to completely suppress IL-17 production (Non-Patent Documents 14, 17). The molecular mechanism linking lipid homeostasis and the function of RORγt has not been fully elucidated, but these previous findings suggest that metabolites of fatty acids and cholesterol may function together as physiological modulators of RORγt.

Prior Art Documents

Non-Patent Documents

[0009]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Non-Patent Document 17

Summary of the Invention

Problems to be Solved by the Invention

[0010] An object of the present invention is to identify lipid metabolites that control Th17 cell differentiation and lipid biosynthetic enzymes involved therein, and based on this, provide therapeutic targets for diseases caused by excessive Th17 cell responses.

Means for Solving the Problems

[0011] The present inventors intensively studied to solve the above problems, and using a CRISPR-based screening system, identified five lipid metabolic enzymes (Gpam, Gpat3, Lplat1, Pla2g12a, and Scd2) that induce RORγt transcriptional activity and establish a gene expression program related to Th17 cells. Also, by non-target lipidome analysis, it was found that 1-oleoyl-lysophosphatidylethanolamine (hereinafter sometimes referred to as "LPE(1-18:1)") can act as a lipid modulator of RORγt activity in Th17 cells. Since LPE(1-18:1) directly binds to RORγt and promotes IL17a promoter activity, it was suggested that LPE(1-18:1) can function as a physiological ligand of RORγt. Furthermore, Pla2g12a, which is a gene encoding an orphan-type phospholipase A2 (PLA2) isoform and one of the five metabolic enzymes identified as controlling RORγt transcriptional activity, was gene-deficient, and the differentiation of Th17 cells disappeared and the EAE pathology was alleviated. From these results, it was found that LPE(1-18:1) and the five lipid metabolic enzymes involved in its biosynthesis can be potential targets for the treatment of chronic inflammatory diseases accompanied by a persistent Th17 cell response. Based on these findings, the present inventors further studied and completed the present invention.

[0012] That is, the present invention relates to the following. [1]A method for producing Th17 cells, which comprises inducing the differentiation of naive CD4+ T cells into Th17 cells by culturing the naive CD4+ T cells in a medium containing 1-oleoyl-lysophosphatidylethanolamine, IL-6 and TGF-β. [2]The production method according to [1], wherein the naive phenotype CD4+ T cells have a deletion, mutation or under-expression of at least one lipid metabolism enzyme gene selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, Scd2 and ACC1, or the expression of at least one lipid metabolism enzyme gene selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, Scd2 and ACC1 is suppressed in T cells during the process of differentiating into Th17 cells. [3]A Th17 cell differentiation promoter containing 1-oleoyl-lysophosphatidylethanolamine. [4]A cell preparation containing 1-oleoyl-lysophosphatidylethanolamine and Th17 cells. [5]A combination containing 1-oleoyl-lysophosphatidylethanolamine and RORγt. [6]A method for screening a candidate substance for a prophylactic or therapeutic agent for a disease caused by the activation of Th17 cells, which comprises selecting a compound that inhibits the interaction between 1-oleoyl-lysophosphatidylethanolamine and RORγt. [7]A method for screening a candidate substance for a prophylactic or therapeutic agent for a disease caused by the activation of Th17 cells, which comprises selecting a compound that inhibits the activity of any lipid metabolism enzyme selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, and Scd2. [8]The lipid metabolism enzyme is Pla2g12a, In the presence of a test compound, incubate 1-oleoylphosphatidylethanolamine with Pla2g12a, quantify the generated 1-oleoyl-lysophosphatidylethanolamine, and when the generated amount is lower than the generated amount in the absence of the test compound, select the test compound as a candidate substance for a prophylactic or therapeutic agent for a disease caused by the activation of Th17 cells, according to the method of [7]. [9]The method according to [7] or [8], wherein the disease caused by the activation of Th17 cells is an autoimmune disease, an allergic disease or arteriosclerosis.

[10] A nucleic acid that hybridizes under physiological conditions to DNA or mRNA encoding any lipid metabolism enzyme selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, and Scd2, thereby inhibiting its transcription and / or translation, or an effective amount of an expression vector of the nucleic acid, a prophylactic or therapeutic agent for a disease caused by the activation of Th17 cells.

[11] The prophylactic or therapeutic agent according to claim 10, wherein the disease caused by the activation of Th17 cells is an autoimmune disease, an allergic disease or arteriosclerosis.

Advantages of the Invention

[0013] The present invention provides LPE(1-18:1) as a lipid metabolite that controls Th17 cell differentiation. Since LPE(1-18:1) binds to RORγt with high specificity and promotes differentiation into Th17 cells, it can be a good therapeutic target for diseases caused by excessive Th17 cell responses. For example, by selecting a compound that inhibits the binding of LPE(1-18:1) to RORγt, a candidate compound for a therapeutic agent for diseases caused by excessive Th17 cell responses can be obtained. Furthermore, the present invention provides five lipid metabolism enzymes (Gpam, Gpat3, Lplat1, Pla2g12a, and Scd2) that are responsible for the biosynthesis of LPE(1-18:1) and control Th17 cell differentiation. These five lipid metabolism enzymes can also be good therapeutic targets for diseases caused by excessive Th17 cell responses, and by selecting a compound that suppresses the activity or expression of these five lipid metabolism enzymes, a candidate compound for a therapeutic agent for diseases caused by excessive Th17 cell responses can be obtained. Therefore, the present invention promotes the development of therapeutic agents and treatment methods for Th17-related diseases based on a new mechanism.

Brief Description of the Drawings

[0014]

Figure 1A

Figure 1B

Figure 1C

Figure 1D

Figure 1E

Figure 1F

Figure 1G

Figure 1H

Figure 2A

Figure 2B

Figure 2C

Figure 2D

Figure 2E

Figure 2F

Figure 2G

Figure 2H

Figure 2I

Figure 2J

Figure 2K

Figure 3A

Figure 3B

Figure 3C

Figure 3D

Figure 3E

Figure 3F

Figure 3G

Figure 4A

Figure 4B

Figure 4C

Figure 4D

Figure 4E

Figure 4F

Figure 4G

Figure 4H

Figure 5A

Figure 5B

Figure 5C

Figure 5D

Figure 5E

Figure 5F

Figure 5G

Figure 5H

Figure 5I

Figure 6A

Figure 6B

Figure 6C

Figure 6D

Figure 6E

Figure 6F

Figure 6G

Figure 6H

Figure 7A

Figure 7B

Figure 7C

Figure 7D

Figure 7E

Figure 7F

Figure 8

Figure 9A

Figure 9B

Figure 9C

Figure 9D

Figure 10

Figure 11A

Figure 11B

Figure 11C

Figure 11D

Figure 11E

Figure 11F

Figure 12

Figure 13A

Figure 13B

Figure 13C

Figure 14A

Figure 14B

Figure 15A

Figure 15B

Figure 16

Mode for Carrying Out the Invention

[0015] 1. Use of 1-oleoyl-lysophosphatidylethanolamine as an RORγt ligand and a promoter for Th17 cell differentiation As will be described in detail in the following examples, 1-oleoyl-lysophosphatidylethanolamine (LPE(1-18:1)) functions as a physiological ligand for RORγt, promotes the expression of Th17-related genes, and promotes differentiation into Th17 cells. Therefore, the present invention provides various uses of LPE(1-18:1) as an RORγt ligand and a promoter for Th17 cell differentiation. (1) Production of Th17 cells In one aspect, the present invention provides a method for producing Th17 cells (hereinafter referred to as "the production method of the present invention"), which includes inducing the differentiation of naive CD4+ T cells into Th17 cells by culturing the naive CD4+ T cells in a medium containing LPE(1-18:1), IL-6 and TGF-β. Since 1-oleoyl-lysophosphatidylethanolamine promotes the differentiation into Th17 cells, it is useful as a Th17 cell differentiation promoter, and by adding this to the medium, Th17 cells can be produced with high efficiency.

[0016] As used herein, naive CD4+ T cells refer to CD4+ T cells that have migrated from the thymus and have not had any experience of antigen stimulation. In one aspect, naive CD4+ T cells are CD62L positive (CD62L + ). Naive CD4+ T cells can be isolated from the peripheral blood, spleen, lymph nodes, bone marrow, thymus, etc. of mammals using antibodies against cell surface markers (e.g., anti-CD4 antibody, anti-CD62L antibody, etc.) by a cell sorter or magnetic beads. The cell purity of the naive CD4+ T cells (e.g., CD4 + CD62L - T cells) used in the production method of the present invention is usually 50% or more, preferably 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more.

[0017] As used herein, when the phenotype of a cell is represented by the presence or absence of the expression of a marker molecule (antigen), unless otherwise specified, the phenotype of the cell is represented by the presence or absence of specific binding by an antibody against the marker molecule. The determination of the cell phenotype by the presence or absence of the expression of the marker molecule is usually performed by flow cytometry analysis using a specific antibody against the marker molecule or the like. The expression of a marker molecule being "positive" means that the marker molecule is expressed on the cell surface (or inside the cell) and specific binding by an antibody against the marker molecule can be confirmed.

[0018] In this specification, Th17 cells are one subset of CD4-positive T cells and have the ability to produce IL-17A.

[0019] The naive CD4-positive T cells used in the production method of the present invention are derived from mammals. Examples of mammals include rodents such as mice, rats, hamsters, guinea pigs, rabbits of the order Lagomorpha, ungulates such as pigs, cows, goats, horses, sheep, carnivores such as dogs, cats, and primates such as humans, monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, chimpanzees, etc. The mammal is preferably a rodent (such as a mouse) or a primate (such as a human).

[0020] The concentration of LPE(1-18:1) in the medium is an effective concentration for promoting the differentiation into Th17 cells, usually 0.1 μM or more, preferably 1.0 μM or more. The upper limit of the LPE(1-18:1) concentration is theoretically the solubility, but if the lipid concentration in the medium is too high, it cannot be denied that unexpected side reactions may occur. Therefore, the concentration of LPE(1-18:1) in the medium is usually 100 μM or less, preferably 50 μM or less. In one aspect, the concentration of LPE(1-18:1) in the medium is 1.0 - 30 μM.

[0021] The concentration of IL-6 in the medium is an effective concentration for promoting the differentiation into Th17 cells, usually 0.1 ng / ml or more, preferably 1 ng / ml or more, more preferably 10 ng / ml or more. The upper limit of the IL-6 concentration is not particularly limited theoretically, but in order to reduce the reagent cost, it is usually 100 ng / ml or less.

[0022] As TGFβ, TGFβ1 or TGFβ3 can be used. Th17 cells induced by TGFβ1 and Th17 cells induced by TGFβ3 differ in phosphorylated Smad and pathogenicity. Th17 cells induced by TGFβ1 are accompanied by preferential phosphorylation of Smad2 and Smad3 and have low pathogenicity, while Th17 cells induced by TGFβ3 are reported to have high pathogenicity (Lee, Y. et al., Nat Immunol. 13: 991-999(2012)). The TGFβ concentration in the medium is an effective concentration that promotes differentiation into Th17 cells, usually 0.01 ng / ml or more, preferably 0.1 ng / ml or more, more preferably 1 ng / ml or more. The upper limit of the TGFβ concentration is not particularly limited theoretically, but is usually 100 ng / ml or less in order to reduce reagent costs.

[0023] In order to promote differentiation into Th17 cells, the culture may be carried out while stimulating the T cell receptor (TCR). Stimulation of the T cell receptor can be added, for example, by an anti-TCR antibody, an anti-CD3 antibody, co-culture with an antigen-presenting cell presenting an antigen recognized by the TCR, etc. Anti-TCR antibody and anti-CD3 antibody can be added to the medium to stimulate the TCR as long as they have agonist activity. Also, stimulation of the TCR can be added by culturing T cells on an anti-TCR antibody or anti-CD3 antibody immobilized on a plate.

[0024] In order to promote differentiation into Th17 cells, the culture may be carried out while adding stimulation to CD28 in addition to stimulation of the TCR. Stimulation of CD28 can be added by an anti-CD28 antibody having agonist activity, co-culture with an antigen-presenting cell, etc. An anti-CD28 antibody having agonist activity may be added to the medium, or T cells may be cultured on a plate immobilized with an anti-CD28 antibody having agonist activity.

[0025] In order to suppress the differentiation into Th1 cells and Th2 cells, 1, 2, or 3 antibodies selected from the group consisting of an anti-IL-2 antibody having inhibitory activity, an anti-IL-4 antibody having inhibitory activity, and an anti-IFN-γ antibody having inhibitory activity may be added to the medium.

[0026] As the basal medium of the medium used for culture, those well-known in the technical field of lymphocyte culture can be used, and it is not particularly limited as long as Th17 cells can be produced by the production method of the present invention. Examples include DMEM, EMEM, RPMI-1640, α-MEM, F-12, F-10, M-199, HAM, etc. A medium modified for lymphocyte culture or the like may be used, or a mixture of the above basal media may be used.

[0027] The medium can contain additives known per se. The additives are not particularly limited as long as Th17 cells can be produced by the production method of the present invention. Examples include serum, organic acids (such as sodium pyruvate, etc.), amino acids (such as L-glutamine, etc.), reducing agents (such as 2-mercaptoethanol, etc.), buffers (such as HEPES, etc.), antibiotics (such as streptomycin, penicillin, gentamicin, etc.), etc. The additives are preferably contained within the concentration ranges known per se for each.

[0028] For culturing naive CD4-positive T cells, the culture conditions usually used in the technical field of lymphocyte culture can be used. For example, the culture temperature is usually in the range of about 30 to 40 °C, and preferably about 37 °C is exemplified. CO 2 The concentration is usually in the range of about 1 to 10%, and preferably about 5% is exemplified. The humidity is usually in the range of about 70 to 100%, and preferably about 95 to 100% is exemplified. The culture period is a period sufficient for the differentiation of naive CD4-positive T cells into Th17 cells and is not particularly limited, but is usually about 3 days, and as a result of the culture, Th17 cells are obtained in the culture. For example, the culture is continued until the differentiation of naive CD4-positive T cells into Th17 cells is confirmed.

[0029] It may be confirmed whether the obtained cells have the function as Th17 cells. For example, the obtained cells may be cultured under stimulation to TCR, and it may be confirmed that IL-17A is produced. Alternatively, by RT-PCR or the like, it may be confirmed that the expression of genes differentially expressed in Th17 cells (such as Il17a, Il17f, Il21, Il22, Ltb4r1, Ccr6, Il23r, Il1r1, etc.) is enhanced.

[0030] Here, LPE(1-18:1) is a physiological endogenous RORγt ligand, and in wild-type mammals, it can be contained as a normal lipid metabolite in cells. Therefore, in the production method of the present invention, the Th17 cell differentiation promoting effect obtained when LPE(1-18:1) is externally added to the medium will be observed as the difference from the effect by endogenous LPE(1-18:1). On the contrary, when the expression of at least one selected from the group consisting of lipid metabolism enzymes (Pla2g12a, Lplat1, Gpam, Gpat3, Scd2, and ACC1) involved in the biosynthesis of LPE(1-18:1) is lost or suppressed, the activation of RORγt by endogenous LPE(1-18:1) is impaired, and Th17 cell differentiation is suppressed. In such a situation, when LPE(1-18:1) is externally added to the medium, the activation of RORγt and Th17 cell differentiation are restored, so that the contribution of LPE(1-18:1) to Th17 cell differentiation can be analyzed in detail. Therefore, in one aspect, naive phenotype CD4-positive T cells having a deletion, mutation, or insufficient expression of at least one lipid metabolism enzyme gene selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, Scd2, and ACC1 are used for culture, or the expression of at least one lipid metabolism enzyme gene selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, Scd2, and ACC1 is suppressed in T cells during the process of differentiating into Th17 cells.

[0031] Only one lipid metabolism enzyme gene selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, Scd2, and ACC1 may be deleted, mutated, or have reduced expression or suppressed expression, or multiple (2, 3, 4, 5, or 6) lipid metabolism enzyme genes may be deleted, mutated, or have reduced expression or suppressed expression. Since Pla2g12a is an enzyme that directly generates LPE(1-18:1), it is preferable to at least delete, mutate, or have reduced expression or suppressed expression of Pla2g12a. In one aspect, naive phenotype CD4-positive T cells having a deletion, mutation, or reduced expression of at least one lipid metabolism enzyme gene selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, and Scd2 are used for culture, or the expression of at least one lipid metabolism enzyme gene selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, and Scd2 is suppressed in T cells during the process of differentiating into Th17 cells. In one aspect, naive phenotype CD4-positive T cells having a deletion, mutation, or reduced expression of all lipid metabolism enzyme genes selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, and Scd2 are used for culture, or the expression of all lipid metabolism enzyme genes selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, and Scd2 is suppressed in T cells during the process of differentiating into Th17 cells. In one aspect, naive phenotype CD4-positive T cells having a deletion, mutation, or reduced expression of three lipid metabolism enzyme genes, Scd2, Lplat1, and Gpat3, are used for culture, or the expression of the three lipid metabolism enzyme genes, Scd2, Lplat1, and Gpat3, is suppressed in T cells during the process of differentiating into Th17 cells. In one aspect, naive phenotype CD4-positive T cells having a deletion, mutation, or reduced expression of only one lipid metabolism enzyme gene selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, and Scd2 are used for culture, or the expression of only one lipid metabolism enzyme gene selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, and Scd2 is suppressed in T cells during the process of differentiating into Th17 cells.

[0032] Examples of naive phenotype CD4-positive T cells having a deficiency, mutation, or insufficient expression of a lipid metabolism enzyme gene include the following. (1) Naive phenotype CD4-positive T cells isolated from a knockout mammal of a specific lipid metabolism enzyme gene. (2) Naive phenotype CD4-positive T cells isolated from a conditional knockout mammal in which a specific lipid metabolism enzyme gene is deficient in naive phenotype CD4-positive T cells. Examples of (2) include mammals (Non-Patent Document 1) in which a part or all of the genomic region of a specific lipid metabolism enzyme gene is flanked by LoxP sequences and the lipid metabolism enzyme gene is specifically deleted in CD4-positive cells by expressing Cre under the control of the CD4 promoter using the Cre-loxp system.

[0033] Examples of methods for suppressing the expression of a lipid metabolism enzyme gene in T cells during the process of differentiating into Th17 cells include the following. (1) Introducing a nucleic acid (siRNA, antisense nucleic acid, etc.) that hybridizes to DNA or mRNA encoding a specific lipid metabolism enzyme under physiological conditions and thereby inhibits its transcription and / or translation, or an expression vector for the nucleic acid, into T cells during the process of differentiating into Th17 cells. (2) Introducing a complex of a single-guide RNA targeting the genomic sequence of a specific lipid metabolism enzyme and Cas9 protein into T cells during the process of differentiating into Th17 cells.

[0034] (2) A cell preparation containing Th17 cells The present invention also provides a cell preparation containing LPE(1-18:1) and Th17 cells (hereinafter referred to as "the cell preparation of the present invention"). The cell preparation of the present invention can be prepared by the above-described production method of the present invention. The cell preparation can be Th17 cells suspended in a medium containing LPE(1-18:1). The concentration of LPE(1-18:1) in the cell preparation is usually 0.1 μM or more, preferably 1.0 μM or more. The concentration of LPE(1-18:1) in the cell preparation is usually 100 μM or less, preferably 50 μM or less. In one aspect, the concentration of LPE(1-18:1) in the cell preparation is 1.0 to 30 μM.

[0035] The cell purity of Th17 cells contained in the cell preparation of the present invention is, for example, 5% or more (preferably 10% or more, 15% or more, 20% or more, 25% or more, or 30% or more) as the ratio of the number of IL-17A positive cells to all cells when the cells contained in the cell preparation are stimulated with PMA (10 ng / ml), ionomycin (500 nM) and monensin (2 μM) for 6 hours and the production of IL-17A is analyzed by flow cytometry. In one aspect, the cell purity of Th17 cells contained in the cell preparation of the present invention is, for example, 50% or more (preferably 60% or more, 70% or more, 80% or more, or 90% or more) as the ratio of the number of RORγt positive cells to all cells when the production of RORγt is analyzed by flow cytometry.

[0036] The cell preparation of the present invention may contain IL-6 and TGFβ. The concentrations of IL-6 and TGFβ in the cell preparation can be the concentrations used in the production method of the present invention. Further, the cell preparation of the present invention can contain other components that can be contained in the medium used in the production method of the present invention within the range of concentrations that can be used in the production method of the present invention. The cell preparation of the present invention can be a culture containing Th17 cells obtained by culturing in the production method of the present invention.

[0037] The cell preparation of the present invention is useful for evaluating the effect of LPE(1-18:1) on the differentiation and function of Th17 cells.

[0038] (3) Combination with RORγt protein The present invention also provides a combination (hereinafter referred to as "the combination of the present invention") comprising LPE(1-18:1) and RORγt protein. The LPE(1-18:1) contained in the combination of the present invention is purified, and its lipid purity (the percentage of the weight of LPE(1-18:1) to the total lipid weight) is, for example, 50% or more (preferably 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more). The RORγt protein contained in the combination of the present invention is purified, and its protein purity (the percentage of the weight of RORγt protein to the total protein weight) is, for example, 50% or more (preferably 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more). In one aspect, the combination of the present invention is a liquid composition comprising LPE(1-18:1) and RORγt protein. In another aspect, the combination of the present invention is a kit, wherein LPE(1-18:1) and RORγt protein are contained in separate containers, and these containers are packaged together in one package. In one aspect, one of LPE(1-18:1) and RORγt protein is immobilized on a solid-phase carrier, and the other is a dry powder or a solution dissolved in a suitable solvent. Examples of the solid-phase carrier include ELISA plates, sensor chips for surface plasmon resonance measurement such as Biacore, etc. The combination of the present invention can be used for evaluating the interaction between LPE(1-18:1) and RORγt protein.

[0039] (4) Screening method for candidate substances for preventive or therapeutic agents for diseases caused by activation of Th17 cells The present invention provides a screening method for candidate substances for preventive or therapeutic agents for diseases caused by activation of Th17 cells. The screening method includes the following step 1) or 2): 1) Selecting a compound that inhibits the interaction between LPE(1-18:1) and RORγt; 2) Selecting a compound that inhibits the activity of any lipid metabolism enzyme selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, and Scd2. The screening method including 1) is referred to as "the screening method 1 of the present invention", the screening method including 2) is referred to as "the screening method 2 of the present invention", and these are collectively referred to as "the screening method of the present invention".

[0040] The screening method 1 of the present invention specifically includes the following steps. (1) Contacting LPE(1-18:1) with RORγt protein in the presence of a test compound. (2) Measuring the interaction between LPE(1-18:1) and RORγt protein, and (3) Selecting a test compound that inhibits the interaction between LPE(1-18:1) and RORγt as a candidate substance for a prophylactic or therapeutic agent for diseases caused by the activation of Th17 cells.

[0041] The interaction between LPE(1-18:1) and RORγt protein can be measured by appropriately combining various known methods. For example, either one of LPE(1-18:1) and RORγt protein is immobilized on a solid-phase carrier, and in the presence of a test compound, the other one labeled with a fluorescent substance or a radioisotope (used as a labeled reagent) is contacted. After removing the unreacted labeled reagent, the amount of the label bound to the solid-phase carrier is measured, whereby the interaction between LPE(1-18:1) and RORγt protein can be measured. Alternatively, either one of LPE(1-18:1) and RORγt protein is immobilized on a sensor chip, and in the presence of a test compound, the other one is contacted with the sensor chip, and then the interaction between the two is measured using the surface plasmon resonance (SPR) method, whereby the interaction between LPE(1-18:1) and RORγt protein can also be measured.

[0042] Alternatively, a vector encoding a reporter gene linked downstream of the IL-17a promoter is introduced into RORγt-expressing cells (e.g., T cell lines) in which the IL-17a promoter can function, cultured in the presence of LPE(1-18:1) and the test compound, and the interaction between LPE(1-18:1) and the RORγt protein can also be measured by measuring the expression of the reporter gene. Here, in order to suppress the background due to endogenous LPE(1-18:1), as the cells into which the vector is introduced, cells having a deficiency, mutation, or hypofunction of at least one lipid metabolism enzyme gene selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, Scd2, and ACC1, or cells in which the expression of at least one lipid metabolism enzyme gene selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, Scd2, and ACC1 is suppressed are preferably used.

[0043] The screening method 2 of the present invention specifically includes the following steps. (1) Incubating any lipid metabolism enzyme selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, and Scd2 with the corresponding substrate in the presence of the test compound; (2) Quantifying the enzyme reaction product generated by the lipid metabolism enzyme; and (3) Selecting a test compound that reduces the amount of the enzyme reaction product generated as a candidate substance for a prophylactic or therapeutic agent for diseases caused by the activation of Th17 cells.

[0044] Pla2g12a (phospholipase A2 group XIIA) is a secreted PLA 2 (sPLA 2)It is one of the subtypes and hydrolyzes the phospholipid that is the substrate to produce lysophospholipid having sn-1 fatty acid. The activity of Pla2g12a can be measured by incubating phospholipids (e.g., phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), etc.) and Pla2g12a near the optimal temperature of Pla2g12a (e.g., 37 °C) and quantifying the produced lysophospholipids (lyso-PE, lyso-PC, lyso-PS, etc.). For example, as the phospholipid of the substrate 14 C-labeled phospholipid is used, and the produced 14 C-labeled lysophospholipid is quantified using LC-MS / GC-MS, etc. Therefore, when using Pla2g12a as a lipid-metabolizing enzyme, in the presence of the test compound, Pla2g12a is incubated with phospholipids, and the produced lysophospholipid having sn-1 fatty acid is quantified. Considering that the active ingredient promoting Th17 differentiation is 1-oleoyl-lysophosphatidylethanolamine (LPE(1-18:1)), it is preferable to use 1-oleoylphosphatidylethanolamine as the phospholipid of the substrate and quantify the produced 1-oleoyl-lysophosphatidylethanolamine. The activity of Pla2g12a can also be evaluated using a commercially available phospholipase A2 activity measurement kit.

[0045] Lplat1, also called AGPAT1 (1-acylglycerol-3-phosphate O-acyltransferase 1), is an acyltransferase that uses fatty acid (acyl)-CoA, which is an activator of fatty acids, as a substrate and introduces a fatty acid chain to the sn-2 position of lysophosphatidic acid (LPA), which is the other substrate, in an ATP-dependent manner to produce phosphatidic acid (PA). The activity of Lplat1 can be measured by incubating a mixture containing lysophosphatidic acid (LPA), acyl-CoA, ATP, and Lplat1 near the optimal temperature of the enzyme (e.g., 37 °C) and quantifying the produced phosphatidic acid (PA). For example, either lysophosphatidic acid or acyl-CoA 14 is labeled with C, and the produced 14The C-labeled PA is quantified using LC-MS / GC-MS or the like. Therefore, when using Lplat1 as a lipid-metabolizing enzyme, in the presence of a test compound, Lplat1 is incubated with acyl-CoA, lysophosphatidic acid, and ATP, and the generated phosphatidic acid is quantified. Considering that the active ingredient promoting Th17 differentiation is 1-oleoyl-lysophosphatidylethanolamine (LPE(1-18:1)), it is preferable to use 1-oleoyl lysophosphatidic acid as the substrate lysophosphatidic acid and quantify the generated 1-oleoyl phosphatidic acid.

[0046] Gpam (glycerol-3-phosphate acyltransferase, mitochondrial), also called GPAT1 (glycerol-3-phosphate acyltransferase 1), is an acyltransferase that uses fatty acid (acyl)-CoA, an activator of fatty acids, as a substrate and introduces a fatty acid chain to the sn-1 position of the glycerol backbone of glycerol-3-phosphate (G3P) in an ATP-dependent manner to generate lysophosphatidic acid (LPA). Gpat3 (glycerol-3-phosphate acyltransferase 3) is also an acyltransferase that uses fatty acid (acyl)-CoA, an activator of fatty acids, as a substrate and introduces a fatty acid chain to the sn-1 position of the glycerol backbone of glycerol-3-phosphate (G3P) in an ATP-dependent manner to generate lysophosphatidic acid (LPA). The activities of Gpam and Gpat3 can be measured by incubating a mixture containing acyl-CoA, glycerol-3-phosphate (G3P), ATP, and the enzyme (Gpam or Gpat3) near the optimal temperature of the enzyme (e.g., 37°C) and quantifying the generated lysophosphatidic acid (LPA). For example, either acyl-CoA or G3P is 14 labeled with C, and the generated 14Quantify the C-labeled LPA using LC-MS / GC-MS, etc. (Jingsong Cao et al., Proc. Natl. Acad. Sci. USA, vol.103, 52, 19695-19700 (2006); Yet SF et al., Biochemistry 32:9486-9491 (1993); Haldar D. et al., Methods Enzymol 209:64-72 (1992)). Therefore, when using Gpam as a lipid-metabolizing enzyme, incubate Gpam with acyl-CoA, glycerol-3-phosphate, and ATP in the presence of the test compound, and quantify the generated lysophosphatidic acid. Also, when using Gpat3 as a lipid-metabolizing enzyme, incubate Gpat3 with acyl-CoA, glycerol-3-phosphate, and ATP in the presence of the test compound, and quantify the generated lysophosphatidic acid. Considering that the active ingredient promoting Th17 differentiation is 1-oleoyl-lysophosphatidylethanolamine (LPE(1-18:1)), it is preferable to use oleoyl-CoA as the acyl-CoA of the substrate and quantify the generated 1-oleoyl lysophosphatidic acid.

[0047] Scd2 (stearoyl-CoA-desaturase 2) is one of the unsaturated fatty acid biosynthetic enzymes, which introduces a double bond at the C9-C10 position of saturated fatty acids to generate monounsaturated fatty acids. Preferred substrates are stearoyl-CoA (18:0) and palmitoyl-CoA (16:0), which are converted to oleoyl-CoA (18:1) and palmitoleoyl-CoA (16:1), respectively. The activity of Scd2 is, for example, 14 Incubate a mixture of C-labeled saturated acyl-CoA, NADH, and Scd2 near the optimal temperature of Scd2 (e.g., 25 °C) to generate the 14The C-labeled unsaturated fatty acids can be measured by quantification using LC-MS / GC-MS etc. (Miyazaki, M. et al., J. Lipid Res. 47: 700-704 (2006); Miyazaki, M. et al., J. Nutr. 131: 2260-2268 (2001)). Therefore, when using Scd2 as a lipid-metabolizing enzyme, in the presence of a test compound, Scd2 is incubated with saturated acyl-CoA and NADH, and the generated monounsaturated acyl-CoA is quantified. Considering that the active ingredient promoting Th17 differentiation is 1-oleoyl-lysophosphatidylethanolamine (1-18:1), it is preferable to quantify the generated oleic acid (18:1) using stearoyl-CoA (18:0) as the saturated acyl-CoA of the substrate.

[0048] Examples of the test substance used in the screening method of the present invention include, for example, proteins, peptides, lipids, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc. These substances may be novel or known.

[0049] Furthermore, it may be confirmed in vitro whether the candidate substance selected in the screening method of the present invention suppresses the differentiation of Th17 cells. For example, in the presence of the candidate substance, naive CD4-positive T cells are cultured in a medium containing IL-6 and TGF-β, and the degree of induction of differentiation of naive CD4-positive T cells into Th17 cells is compared with that in the absence of the candidate substance. Then, based on the comparison results, a candidate substance that suppresses differentiation into Th17 cells in vitro can be selected as a candidate substance for a prophylactic or therapeutic agent for diseases caused by the activation of Th17 cells.

[0050] Alternatively, the in-vivo effect of the candidate substance selected in the screening method of the present invention may be confirmed. For example, the candidate substance is administered to a non-human mammalian model of a disease caused by the activation of Th17 cells, and the symptoms of the disease in the non-human mammal are compared with those of a control non-human mammal (the breeding conditions other than not administering the candidate substance are the same as those of the non-human mammalian model administered with the candidate substance). Examples of the non-human mammalian model of a disease caused by the activation of Th17 cells include mice induced with experimental autoimmune encephalomyelitis (EAE). Then, based on the comparison results, a candidate substance that suppresses the symptoms in the non-human mammal can be selected as a candidate substance for a prophylactic or therapeutic agent for a disease caused by the activation of Th17 cells whose in-vivo efficacy has been confirmed.

[0051] Examples of diseases caused by the activation of Th17 cells include autoimmune diseases such as multiple sclerosis, psoriasis, inflammatory bowel disease, type 2 diabetes, rheumatoid arthritis, etc., allergic diseases such as bronchial asthma, atopic dermatitis, allergic rhinitis, etc., arteriosclerosis, etc., but are not limited thereto.

[0052] (5) Prophylactic or therapeutic agent for diseases caused by the activation of Th17 cells The present invention also provides a prophylactic or therapeutic agent for a disease caused by Th17 cell activation (the prophylactic or therapeutic agent of the present invention), which contains a nucleic acid that hybridizes under physiological conditions to DNA or mRNA encoding at least one lipid metabolism enzyme selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, and Scd2 and thereby inhibits its transcription and / or translation, or an effective amount of an expression vector of the nucleic acid. By suppressing the expression of at least one lipid metabolism enzyme selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, and Scd2, the production of LPE(1-18:1) is suppressed, the activation of RORγt is suppressed, and the differentiation into Th17 cells is inhibited, and as a result, the disease caused by Th17 cell activation is prevented or treated. By administering to a subject (preferably a human) in need thereof an effective amount of a nucleic acid that hybridizes under physiological conditions to DNA or mRNA encoding at least one lipid metabolism enzyme selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, and Scd2 and thereby inhibits its transcription and / or translation, or an expression vector of the nucleic acid, the onset of a disease caused by Th17 cell activation in the subject can be prevented, or the disease caused by Th17 cell activation can be treated. The present invention provides a nucleic acid that hybridizes under physiological conditions to DNA or mRNA encoding at least one lipid metabolism enzyme selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, and Scd2 and thereby inhibits its transcription and / or translation, or an expression vector of the nucleic acid for use in the prevention or treatment of a disease caused by Th17 cell activation.

[0053] As used herein, the term "effective amount" means an amount that brings about the desired effect (e.g., therapeutic effect) in a subject. For example, in a subject administered this amount, compared to a subject not administered this amount, the symptoms or conditions of the disease are alleviated, reduced, or eliminated, or the progression thereof is delayed or suppressed. The effective amount can be appropriately determined by a physician according to factors such as the age, weight, gender, and severity of symptoms of the subject.

[0054] As used herein, "prevention" means, with respect to a certain disease or disorder (for example, a disease caused by the activation of Th17 cells), before such a state occurs, preventing such a state from occurring, reducing the risk of such a state, or making such a state alleviated or reduced.

[0055] Nucleic acids that hybridize under physiological conditions to DNA or mRNA encoding at least one lipid metabolism enzyme selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, and Scd2 and thereby inhibit its transcription and / or translation include siRNA or its precursor, antisense nucleic acid, etc. that target at least one lipid metabolism enzyme selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, and Scd2.

[0056] Examples of siRNA that targets at least one lipid metabolism enzyme selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, and Scd2 include (A) RNA containing a nucleotide sequence complementary to the nucleotide sequence of mRNA (mature mRNA or primary transcript) encoding at least one lipid metabolism enzyme selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, and Scd2 or a partial sequence thereof of 18 bases or more, and (B) RNA containing a nucleotide sequence of 18 bases or more that can specifically hybridize in cells of a target animal (preferably human or mouse) with mRNA (mature mRNA or primary transcript) encoding at least one lipid metabolism enzyme selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, and Scd2, and that suppresses the transcription and / or translation of at least one lipid metabolism enzyme selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, and Scd2 by hybridizing.

[0057] As used herein, "specific hybridization" means that a nucleic acid hybridizes more strongly to a target nucleotide than to other nucleotides.

[0058] Examples of the nucleotide sequence of mRNA encoding human Pla2g12a include, for example, the nucleotide sequence represented by SEQ ID NO: 1 (GenBank accession number: NM_030821.5). Examples of the nucleotide sequence of mRNA encoding human Lplat1 include, for example, the nucleotide sequence represented by SEQ ID NO: 3 (NM_001371437.1). Examples of the nucleotide sequence of mRNA encoding human Gpam include, for example, the nucleotide sequence represented by SEQ ID NO: 5 (NM_001244949.2). Examples of the nucleotide sequence of mRNA encoding human Gpat3 include, for example, the nucleotide sequence represented by SEQ ID NO: 7 (NM_001256421.1). Examples of the nucleotide sequence of mRNA encoding human Scd5 (corresponding to Scd2 in mice) include, for example, the nucleotide sequence represented by SEQ ID NO: 9 (NM_001037582.3).

[0059] In this specification, the nucleotide sequence is described as a DNA sequence unless otherwise specified. When the polynucleotide is RNA, thymine (T) shall be appropriately replaced with uracil (U).

[0060] The phenomenon called RNA interference (RNAi), in which when siRNA such as short double-stranded RNA is introduced into cells, the mRNA complementary to the RNA is degraded, has been known in nematodes, insects, plants, etc. for a long time, and it has been confirmed that this phenomenon also occurs in mammalian cells [Nature, 411(6836): 494-498 (2001)].

[0061] siRNA is typically a double-stranded oligonucleotide RNA composed of an RNA having a sequence complementary to the nucleotide sequence of the target gene's mRNA or a partial sequence thereof (hereinafter referred to as the target nucleotide sequence) and its complementary strand. The length of the portion complementary to the target nucleotide sequence contained in siRNA is usually about 18 bases or more, preferably 19 bases or more, more preferably about 21 bases or more, but is not particularly limited as long as it can specifically suppress the expression of the target gene. When the siRNA is longer than 23 bases, the siRNA can be degraded in cells to produce siRNA of about 20 bases or so. Therefore, theoretically, the upper limit of the length of the portion complementary to the target nucleotide sequence is the full length of the nucleotide sequence of the target gene's mRNA (mature mRNA or primary transcript). However, considering the avoidance of interferon induction, ease of synthesis, antigenicity issues, etc., the length of the complementary portion is, for example, about 50 bases or less, preferably about 25 bases or less, and most preferably about 23 bases or less. That is, the length of the complementary portion is usually about 18 to 50 bases, preferably about 19 to about 25 bases, more preferably about 21 to about 23 bases.

[0062] Also, the length of each RNA strand constituting the siRNA is usually about 18 bases or more, preferably 19 bases or more, more preferably about 21 bases or more, but is not particularly limited as long as it can specifically suppress the expression of the target gene, and theoretically there is no upper limit to the length of each RNA strand. However, considering the avoidance of interferon induction, ease of synthesis, antigenicity issues, etc., the length of the siRNA is, for example, about 50 bases or less, preferably about 25 bases or less, and most preferably about 23 bases or less. That is, the length of each RNA strand is, for example, usually about 18 to 50 bases, preferably about 19 to about 25 bases, more preferably about 21 to about 23 bases.

[0063] The target nucleotide sequence and the sequence complementary to it contained in the siRNA are preferably completely complementary. However, for base mutations at positions away from the center of the siRNA (which can be within a range of at least 90% or more, preferably 95% or more identity), the cleavage activity by RNA interference is not completely lost, but partial activity can remain. On the other hand, mutations in the central part of the siRNA have a great impact, and the cleavage activity of mRNA by RNA interference can be extremely reduced.

[0064] Examples of the siRNA precursor include shRNA. shRNA is cleaved intracellularly to generate siRNA. shRNA is a single-stranded RNA in which a sequence complementary to the target nucleotide sequence (the first sequence) and its complementary sequence (the second sequence) are linked via a hairpin loop portion, and by adopting a hairpin loop structure, the first sequence forms a double-stranded structure with the second sequence. The length of the portion complementary to the target nucleotide sequence contained in shRNA is usually at least about 30 bases, preferably at least 40 bases, more preferably at least about 50 bases, but is not particularly limited as long as it can specifically suppress the expression of the target gene. Since shRNA can be degraded intracellularly to generate siRNA of about 20 bases in length, theoretically, the upper limit of the length of the portion complementary to the target nucleotide sequence is the full length of the nucleotide sequence of the mRNA of the target gene (mature mRNA or primary transcript). However, considering the avoidance of interferon induction, ease of synthesis, antigenicity issues, etc., the length of the complementary portion is, for example, about 100 bases or less, preferably about 50 - 70 bases. The overall length of shRNA is also usually 100 bases or less, preferably about 50 - 70 bases. Note that the length of shRNA is indicated as the length of the double-stranded portion when it forms a double-stranded structure.

[0065] The length of the loop portion of the hairpin loop of shRNA is not particularly limited as long as it can specifically suppress the expression of the target gene, but is usually about 5 to 25 bases. The nucleotide sequence of the loop portion is not particularly limited as long as it can form a loop and the shRNA can specifically suppress the expression of the target gene.

[0066] siRNA and shRNA may have additional bases that do not form base pairs at the 5' and / or 3' ends. The length of the additional bases is not particularly limited as long as the siRNA or shRNA can specifically suppress the expression of the target gene, but is usually 5 bases or less, for example, 2 to 4 bases. The additional bases may be DNA or RNA, but using DNA can improve the stability of siRNA or shRNA. Examples of such sequences of additional bases include sequences such as ug-3', uu-3', tg-3', tt-3', ggg-3', guuu-3', gttt-3', ttttt-3', uuuuu-3', etc., but are not limited thereto.

[0067] Examples of the antisense nucleic acid that can specifically suppress the expression of at least one lipid metabolism enzyme selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, and Scd2 include (A) a nucleic acid containing a nucleotide sequence complementary to the nucleotide sequence of mRNA (mature mRNA or primary transcript) encoding at least one lipid metabolism enzyme selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, and Scd2 or a partial sequence thereof of 12 bases or more, and (B) a nucleic acid containing a nucleotide sequence of 12 bases or more that can specifically hybridize with mRNA (mature mRNA or primary transcript) encoding at least one lipid metabolism enzyme selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, and Scd2 in cells of a treated animal (preferably a human), and that can inhibit translation into the p38 polypeptide in the hybridized state, etc. can be mentioned.

[0068] The length of the portion that hybridizes with the target mRNA in the antisense nucleic acid is not particularly limited as long as it can specifically suppress the expression of the target gene, and is usually about 12 bases or more, and in some cases, it can be as long as the full-length sequence of the mRNA (mature mRNA or primary transcript). Considering the specificity of hybridization, the length is preferably about 15 bases or more, more preferably 18 bases or more. Also, considering issues such as ease of synthesis and antigenicity, the length of the portion that hybridizes with the target mRNA is usually about 200 bases or less, preferably about 50 bases or less, more preferably about 30 bases or less. That is, the length of the portion that hybridizes with the target mRNA is, for example, about 12 to about 200 bases, preferably about 15 to about 50 bases, more preferably about 18 to about 30 bases.

[0069] The target nucleotide sequence of the antisense nucleic acid is not particularly limited as long as it can specifically suppress the expression of the target gene, and it can be the full-length sequence of the mRNA (mature mRNA or primary transcript) of the target gene, a partial sequence (for example, about 12 bases or more, preferably about 15 bases or more, more preferably about 18 bases or more), or even the intron portion of the primary transcript.

[0070] The nucleotide sequence of the portion that hybridizes with the target mRNA in the antisense nucleic acid varies depending on the base composition of the target sequence, but in order to hybridize with the target mRNA under physiological conditions, it usually has an identity of about 90% or more (preferably 95% or more, most preferably 100%) with the complementary sequence of the target sequence.

[0071] The size of the antisense nucleic acid is usually about 12 bases or more, preferably about 15 bases or more, more preferably about 18 bases or more. Considering ease of synthesis and antigenicity issues, etc., the size is usually about 200 bases or less, preferably about 50 bases or less, more preferably about 30 bases or less.

[0072] Natural nucleic acids are easily degraded by nucleases present in cells at their phosphodiester bonds. Therefore, siRNA or its precursor, miRNA or its precursor, and antisense nucleic acids used in the present invention may be modified to be resistant to the nuclease and used as modified forms. Examples of modifications in the modified forms include, for example, those in which the sugar moiety is modified (e.g., 2'-O-methyl, 2'-fluoro, 2'-O-methoxyethyl), those in which the base moiety is modified, those in which the phosphate moiety or hydroxyl moiety is modified (e.g., biotin, amino group, lower alkylamine group, acetyl group, etc.), phosphorothioate modification, morpholino nucleic acid, etc., but are not limited thereto.

[0073] siRNAs and antisense nucleic acids targeting at least one lipid metabolism enzyme selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, and Scd2 can be prepared by determining the target sequence based on the mRNA sequence or chromosomal DNA sequence of the target gene and synthesizing a nucleotide sequence complementary thereto using a commercially available DNA / RNA automatic synthesizer (such as Applied Biosystems, Beckman, etc.). siRNA can be prepared by synthesizing the sense strand and the antisense strand respectively with a DNA / RNA automatic synthesizer, denaturing them at about 90 to about 95 °C for about 1 minute in an appropriate annealing buffer, and then annealing them at about 30 to about 70 °C for about 1 to about 8 hours. Also, longer double-stranded polynucleotides can be prepared by synthesizing complementary oligonucleotide strands so as to overlap alternately, annealing them, and then ligating them with ligase.

[0074] The prophylactic or therapeutic agent of the present invention can also contain, as an active ingredient, an expression vector capable of expressing (encoding) a nucleic acid that hybridizes under physiological conditions to DNA or mRNA encoding at least one lipid metabolism enzyme selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, and Scd2, thereby inhibiting its transcription and / or translation. In the expression vector, a nucleic acid that hybridizes under physiological conditions to DNA or mRNA encoding at least one lipid metabolism enzyme selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, and Scd2, thereby inhibiting its transcription and / or translation, or a nucleic acid (preferably DNA) encoding the same, is functionally linked to a promoter capable of exhibiting promoter activity in cells (e.g., naive CD4 T cells, Th17 cells, etc.) of a mammal (preferably a human) to which it is applied.

[0075] The promoter to be used is not particularly limited as long as it can function in cells (e.g., naive CD4 T cells, Th17 cells, etc.) of a mammal (preferably a human) to which it is applied. As the promoter, a polI promoter, a polII promoter, a polIII promoter, etc. can be used. Specifically, viral promoters such as the SV40-derived early promoter and the cytomegalovirus LTR, mammalian constitutive protein gene promoters such as the β-actin gene promoter, and RNA promoters such as the tRNA promoter are used.

[0076] When intending to express siRNA, it is common to use a polIII promoter as the promoter. Examples of polIII promoters include the U6 promoter, H1 promoter, tRNA promoter, and the like. When intending to express shRNA, in addition to the polIII promoter, a polII promoter can be used. In naive CD4 T cells or cells that are differentiating from naive CD4 T cells into Th17 cells, in order to specifically inhibit the expression of at least one lipid metabolism enzyme selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, and Scd2, the promoters of genes (CD4, IL-17a) that are specifically expressed in CD4 T cells may be used.

[0077] The above expression vector preferably contains a transcription termination signal, that is, a terminator region, downstream of the above-mentioned polynucleotide or the nucleic acid encoding the same. Furthermore, it can also contain a selection marker gene for cell transformation and a fluorescent protein gene.

[0078] The type of vector used in the expression vector in the present invention is not particularly limited, but examples of vectors suitable for administration to mammals such as humans include plasmid vectors; viral vectors such as retroviruses, adenoviruses, adeno-associated viruses, and lentiviruses.

[0079] A nucleic acid that hybridizes under physiological conditions to DNA or mRNA encoding one lipid metabolism enzyme selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, and Scd2 and thereby inhibits its transcription and / or translation, or only the expression vector of the nucleic acid may be used, or nucleic acids that inhibit the transcription and / or translation of a plurality of lipid metabolism enzymes with different target lipid metabolism enzyme types, or expression vectors of the nucleic acids may be used in combination. That is, (1) A nucleic acid that hybridizes under physiological conditions to DNA or mRNA encoding Pla2g12a and thereby inhibits its transcription and / or translation, or an expression vector of said nucleic acid; (2) A nucleic acid that hybridizes under physiological conditions to DNA or mRNA encoding Lplat1 and thereby inhibits its transcription and / or translation, or an expression vector of said nucleic acid; (3) A nucleic acid that hybridizes under physiological conditions to DNA or mRNA encoding Gpam and thereby inhibits its transcription and / or translation, or an expression vector of said nucleic acid; (4) A nucleic acid that hybridizes under physiological conditions to DNA or mRNA encoding Gpat3 and thereby inhibits its transcription and / or translation, or an expression vector of said nucleic acid; and (5) A nucleic acid that hybridizes under physiological conditions to DNA or mRNA encoding Scd2 and thereby inhibits its transcription and / or translation, or an expression vector of said nucleic acid may be used alone, selected from any one of the group consisting of, or may be used in combination of 2, 3, 4 or 5 selected from (1)-(5). In one embodiment, (1), (2), (3), (4) and (5) are all used in combination. In another embodiment, (2), (4) and (5) are used in combination.

[0080] The prophylactic or therapeutic agent of the present invention may contain, in addition to a nucleic acid that hybridizes under physiological conditions to DNA or mRNA encoding at least one lipid metabolism enzyme selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, and Scd2 and thereby inhibits its transcription and / or translation, or an expression vector of said nucleic acid, an arbitrary carrier, for example, a pharmaceutically acceptable carrier.

[0081] Examples of pharmaceutically acceptable carriers include excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate, etc.; binders such as cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, starch, etc.; disintegrants such as starch, carboxymethylcellulose, hydroxypropyl starch, sodium - glycol - starch, sodium hydrogen carbonate, calcium phosphate, calcium citrate, etc.; lubricants such as magnesium stearate, aerosil, talc, sodium lauryl sulfate, etc.; fragrances such as citric acid, menthol, glycyrrhizin ammonium salt, glycine, orange powder, etc.; preservatives such as sodium benzoate, sodium bisulfite, methyl paraben, propyl paraben, etc.; stabilizers such as citric acid, sodium citrate, acetic acid, etc.; suspending agents such as methylcellulose, polyvinylpyrrolidone, aluminum stearate, etc.; dispersing agents such as surfactants; diluents such as water, physiological saline, orange juice, etc.; base waxes such as cocoa butter, polyethylene glycol, white kerosene, etc., but are not limited thereto.

[0082] In order to facilitate the introduction of the nucleic acid or its expression vector into cells, the prophylactic or therapeutic agent of the present invention can further contain a nucleic acid introduction reagent. Examples of nucleic acid introduction reagents include cationic lipids such as lipofectin, lipofectamine, Lipofectamine RNAiMAX, Invivofectamine, DOGS (transfectam), DOPE, DOTAP, DDAB, DHDEAB, HDEAB, polybrene, or poly(ethyleneimine) (PEI). When using a retrovirus as an expression vector, reagents such as retronectin, fibronectin, polybrene, etc. can be used as introduction reagents.

[0083] Examples of the administration unit form of the prophylactic or therapeutic agent of the present invention include, but are not limited to, injections (solutions, suspensions, etc.), inhalants, creams, sprays, nasal drops, aerosol solutions, suppositories, enemas, patches, ointments, jellies, pastes, tablets, pills, drinking solutions, powders, suspensions, emulsions, granules, extracts, fine granules, syrups, infusions, decoctions, eye drops, troches, poultices, liniments, lotions, eye ointments, plasters, capsules, etc.

[0084] The content of the nucleic acid that hybridizes under physiological conditions to the DNA or mRNA encoding at least one lipid metabolism enzyme selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, and Scd2 in the pharmaceutical composition and thereby inhibits its transcription and / or translation, or the expression vector of the nucleic acid, is not particularly limited and can be appropriately selected within a wide range. For example, it is about 0.001% by weight to 99.999% by weight of the whole pharmaceutical composition.

[0085] The prophylactic or therapeutic agent of the present invention is administered to a mammal (preferably a human) so that the nucleic acid that hybridizes under physiological conditions to the DNA or mRNA encoding at least one lipid metabolism enzyme selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, and Scd2 and thereby inhibits its transcription and / or translation, or the expression vector of the nucleic acid, is delivered to naive CD4-positive T cells, Th17 cells, and cells that are differentiating into Th17 cells in vivo, and can suppress the differentiation into Th17 cells and the activation of Th17 cells in vivo.

[0086] Examples of the diseases caused by the activation of Th17 cells to be prevented or treated include, but are not limited to, autoimmune diseases such as multiple sclerosis, psoriasis, inflammatory bowel disease, type 2 diabetes, rheumatoid arthritis, allergic diseases such as bronchial asthma, atopic dermatitis, allergic rhinitis, and arteriosclerosis.

[0087] The prophylactic or therapeutic agent of the present invention is administered by a method corresponding to various forms upon its use. For example, in the case of an injection, it is administered intravenously, intraarterially, intramuscularly, intradermally, subcutaneously, intraarticularly, intramedullarily, within lymphoid tissue or intraperitoneally. In the case of a topical agent, it is directly sprayed, affixed or applied to a required site such as the skin or mucosa. In the case of tablets, pills, oral liquids, suspensions, emulsions, granules and capsules, it is administered orally. In the case of suppositories, it is administered rectally.

[0088] The dosage of the prophylactic or therapeutic agent of the present invention varies depending on the severity of the disease, the type of the disease, the activity and type of the active ingredient, the administration mode (e.g., oral, parenteral), the drug receptivity of the administration subject, body weight, age, etc., and cannot be generally stated. Usually, it is about 0.001 mg to about 1.0 g as the amount of the active ingredient per day for an adult.

[0089] All references cited in this specification, including publications, patent documents, etc., are incorporated herein by reference to the extent that they are individually and specifically incorporated by reference and the entire content thereof is specifically described as if it were set forth herein in full.

[0090] Hereinafter, the present invention will be described more specifically by way of examples, but the present invention is not limited thereto.

Examples

[0091] [Example 1] 1. Materials and Methods Study Design The objective of this study is to identify specific lipids that can control the activity of RORγt during the differentiation process of Th17 cells. For this purpose, CRISPR screening, transcriptome analysis by RNA-seq and qPCR, epigenetic analysis by ATAC-seq and chromatin immunoprecipitation, lipidomics analysis, immunofluorescence analysis, and in vivo functional assays were used. The number of samples and the number of experimental replicates are shown in the figure legends. Mice were randomly assigned to experimental groups. The sample size was selected according to equivalent studies previously conducted in the inventors' laboratory and the availability of animals, and all animals within one experiment were processed simultaneously. Adult mice in the control group and the experimental group were matched in age, background, and gender. Both male and female mice were used for cell quantification and in vitro cell culture, but there were no statistical gender differences. Except for cell sorting and RNA sequence analysis, the experiments were not blinded.

[0092] Mice Acaca on a C57BL / 6 background fl / fl Mice (Proc. Natl. Acad. Sci. U.S.A. 103, 8552-8557 (2006)) or Pla2g12a fl / fl (The International Knockout Mouse Consortium (IKMC) project ID 79088) was backcrossed 10 times with Cd4-cre mice (Jackson Laboratory) and maintained on a C57BL / 6 background. C57BL / 6 mice were purchased from CLEA Japan, Inc. (Tokyo, Japan). All mice were used at 6-10 weeks of age and were housed under specific pathogen-free (SPF) conditions. Animal experiments were conducted using a protocol (registration number: 30-1-002) approved by the Kazusa DNA Research Institute's Institutional Animal Care and Use Committee. The experiments and animal housing were conducted in accordance with the guidelines of the Kazusa DNA Research Institute.

[0093] Mouse T cell culture Naive (CD44 lo CD62L hi ) CD4+ T cells were purified from the spleens of mice. After lysing red blood cells, CD4 T cells were obtained using a CD4 T cell isolation kit with anti-CD4 microbeads (Miltenyi Biotec). + Naive CD44 lo CD62L hi cells were sorted to a purity of over 99.5% using a FACS Melody cell sorter (BD Biosciences). In some experiments, CD44 hi CD62L lo CD4 T cells with a memory phenotype were also sorted. Naive CD4 +T cells were plated in 24-well tissue culture plates (Costar) pre-coated with 10 μg / ml agonistic anti-TCRβ antibody (clone H57-597) and 1 μg / ml agonistic anti-CD28 antibody (clone 37.51, Biolegend). Anti-IL-2 (BD biosciences), recombinant mouse IL-6 (Peprotech) (10 ng / ml), recombinant human TGFβ1 (1 ng / ml) (Peprotech), anti-IL-4 antibody and anti-IFNγ antibody were included in Th17 cell culture. Anti-IL-2 (BD biosciences), recombinant mouse IL-6 (Peprotech) (10 ng / ml), recombinant human TGFβ3 (1 ng / ml) (Peprotech), anti-IL-4 antibody and anti-IFNγ antibody were included in pathogenic Th17 cell culture. Recombinant mouse IL-2 (Peprotech) (25 U / ml), recombinant IL-12 (Peprotech) (10 ng / ml), anti-IL-4 antibody (Biolegend) were included in Th1 cell culture. Recombinant IL-2 (Peprotech) (25 U / ml), recombinant IL-4 (Peprotech) (5 ng / ml), anti-IFNγ antibody (Biolegend) were included in Th2 cell culture. In some experiments, naive CD4 T cells were cultured under Th17 conditions in the presence of indicated concentrations of lipids containing LPE (1-18:1), LPI (1-18:1), LPA (1-18:1), LPC (1-18:1), LPE (2-18:1), PE (1,2-18:1), cholesterol, 25-OHC, 27-OHC, 7α,27-OHC, 7β,27-OHC, FA (18:1), or FA (20:4) on day 0. TOFA (Millipore) or curcumin (Sigma-Aldrich) was also added to the Th17 cell cultures on day 0. Th17 cells cultured for 3 days were used in almost all experiments including intracellular staining, qRT-PCR, ChIP assay, and lipidome analysis. Th1 and Th2 cells cultured for 5 days were used in intracellular staining, qRT-PCR, proliferation assay, and apoptosis assay. For retroviral infection, naive CD4 +After culturing T cells under Th17 conditions for 1 day, they were infected with retroviruses containing Scd2-, Gpam-, and Gpat3-IRES-hNGFR, GFP, or BFP.

[0094] Genome editing via Cas9 Short guide RNAs were designed using the online tool provided by CHOPCHOP (http: / / chopchop.cbu.uib.no) (Nat. Commun. 9, 4231 (2018)). Freshly isolated splenic CD44 lo CD62L hi Naive CD4 + T cells were activated with plate-bound anti-TCRβ antibody and CD28 antibody under Th17 cell culture conditions. Cas9 protein was prepared immediately before the experiment by incubating 1 μg of Cas9 with 0.3 μg of sgRNA in transfection buffer for 10 minutes at room temperature. Twenty-four hours after T cell activation under Th17 culture conditions, these cells were electroporated with a mixture of Cas9 / sgRNA complexes using the Neon transfection kit and device (Thermo Fisher Scientific). A non-targeting sgRNA sequence that does not recognize any sequence in the mouse genome was used as a negative control for the CRISPR Cas9 editing experiment. The sequences of the sgRNAs are shown in Table 1.

[0095]

Table 1

[0096] CRISPR screening By electroporation, a plasmid encoding Cas9 with an sgRNA cassette and eGFP fluorescent protein was transduced into the EL4 T cell line. Two days after electroporation, the transfected cells (eGFP +were sorted into single cell clones by FACS. After culturing these clones for two weeks, each clone was evaluated for RORγt reporter activity.

[0097] Lipidome analysis Non-target lipid profiling was performed as previously reported by Kazusa DNA Research Institute (Commun. Biol. 4, 820 (2021)). Briefly, 4 million cells were spin-down at the end of cell culture, and the pellet was washed with PBS and then redissolved in 150 μl of chloroform:methanol (1:2) containing EquiSPLASH (Avanti Polar Lipids) for internal standard. After sonication for 30 seconds, 10 μL of water was added, and the mixture was vigorously stirred at 750 rpm at 20 °C for 20 minutes. Centrifugation was performed at 1670 g at 20 °C for 10 minutes to collect the supernatant, which was transferred to an LC vial. LC-MS / MS analysis was performed using a quadrupole time-of-flight (Q TOF) / MS (TripleTOF 6600; SCIEX) combined with an ACQUITY UPLC system (Waters). LC separation was performed by gradient elution with mobile phase (A) [methanol / acetonitrile / water (1:1:3, v / v / v) containing 5 mM ammonium acetate (Wako Chemicals) and 10 nM EDTA (Tongrentang)] and mobile phase (B) [isopropanol (Wako Chemicals) containing 5 mM ammonium acetate and 10 nM EDTA]. Elution was performed at a flow rate of 300 μl / min at 45 °C using an L-column3 C18 (50 x 2.0 mm i.d., particle size 2.0 μm; National Institute of Biomedical Innovation). The solvent composition was started at 100% (A) for the first 1 minute, linearly changed to 64% (B) at 7.5 minutes, and held there for 4.5 minutes. The gradient was linearly increased to 82.5% (B) at 12.5 minutes, 85% (B) at 19 minutes, 95% (B) at 20 minutes, 100% (A) at 20.1 minutes, and 100% (A) at 25 minutes. The raw data files of Q TOF / MS were converted to MGF files using the SCIEX MS converter program, and quantitative analysis was performed using 2DICAL (Mitsui Information & Engineering). Identification of molecular species was achieved by comparison with retention time and MS / MS spectral data from information-dependent acquisition (IDA) mode (Nat. Biotechnol. 38, 1159-1163 (2020)).

[0098] Preparation of 3′ mRNA-seq library TRIzol reagent (Thermo Fisher Scientific) was used for the extraction of total cellular RNA, and Quantus Fluorometer (Promega) was used for the measurement of RNA concentration. A total of 500 ng of RNA was used for 3′ mRNA library preparation according to the manufacturer's protocol using the QuantSeq 3′ mRNA-Seq Library Prep Kit FWD (LEXOGEN). After the PCR step, the size distribution and yield of the library were measured using a D1000 high sensitivity tape station (Agilent) or an Agilent High Sensitivity DNA kit (Agilent) on a Bioanalyzer (Agilent). The pooled libraries were loaded onto an Illumina Nextseq500 platform and analyzed with 75 bp single reads.

[0099] Analysis of 3′ mRNA-seq data Fastp was used to trim adapter sequences from raw RNA-seq reads (Bioinformatics 34, i884-i890 (2018)). The trimmed reads for each sample were mapped to the reference mouse genome mm10 using STAR (Curr. Protoc. Bioinformatics 51, 11.14.11-11.14.19 (2015)) and normalized to 1 million reads of the original library. Genes with an average of more than 5 reads in any group were subjected to further analysis. Gene set enrichment analysis (GSEA) was performed to determine the statistical significance of the enrichment of known transcriptional signatures in the ranked gene list (Nat. Genet. 34, 267-273 (2003); Proc. Natl. Acad. Sci. U.S.A. 102, 15545-15550 (2005)). PCA analysis and heatmap were drawn using R software (https: / / cran.r-project.org / ) and amap (https: / / CRAN.R-project.org / package=amap).

[0100] ATAC-seq Sample Preparation 100,000 cells were pelleted, washed with 50 μL of PBS, and then treated with 50 μL of lysis buffer. The nuclei were resuspended in 40 μL of transposition reaction buffer together with 2 μL of Tn5 transposase (Active motif) and the accessible chromatin was tagged and fragmented. The reaction mixture was incubated at 37 °C for 30 minutes with shaking at 300 rpm. The fragmented DNA was purified using the column of the ATAC-seq kit and amplified by PCR for 11 or 12 cycles based on the amplification curve. After the PCR step, the size distribution and yield of the library were measured using a bioanalyzer (Agilent) with the Agilent High Sensitivity DNA kit. To determine the library concentration, qRT-PCR was performed using the GenNext NGS Library Quantification Kit (Toyobo). The pooled library was loaded onto the Illumina Nextseq500 platform and analyzed with 75 bp single reads.

[0101] Analysis of ATAC-seq Data Adapter sequences were trimmed from the raw ATAC-seq reads using fastp. The trimmed reads of each sample were mapped to the reference mouse genome mm10 using Bowtie2 (Nat. Methods 9, 357-359 (2012)), and peak calling was performed using MACS2 (Genome Biol. 9, R137 (2008)). The generated files were used to draw heatmaps with DiffBind (Nature 481, 389-393 (2012)). The BED file recording the ATAC-seq signal was converted to a BigWig file using deeptools (Nucleic Acids Res. 42, W187-W191 (2014)) and normalized by the number of reads per genomic coverage. The Integrative Genomic Viewer software program (Cancer Res. 77, e31-e34 (2017)) was used for visualization of the BigWig file.

[0102] Chromatin immunoprecipitation (ChIP) assay The ChIP assay was performed as previously described (Cell Rep. 37, 109921 (2021)). Briefly, 1 x 10 7 individual Th17 cells were fixed in 1% formaldehyde at 25 °C for 10 minutes, followed by the addition of 1.25 M glycine. The cells were pelleted, washed, and lysed in lysis buffer (50 mM HEPES (pH 7.9), 140 mM NaCl, 1 mM EDTA (pH 8.0), 10% Glycerol, 0.5% NP-40, 0.25% Triton X-100, 1 mM PMSF, 1 mg / ml aprotinin, and 1 mg / ml leupeptin). The lysate was sonicated using Covaris (M&S Instruments Inc.) to fragment the DNA to a length of 200 to 1,000 bp. The soluble fraction was diluted with ChIP dilution buffer and incubated overnight at 4 °C with antibodies conjugated to Dynabeads protein A and G. The immunocomplexes were then captured using a magnet and washed with low salt, high salt, LiCl, and Tris-EDTA wash buffers. The concentrated chromatin fragments were eluted with elution buffer (0.1 M NaHCO 3 ) containing 1% SDS. The eluate was incubated at 65 °C for 6 hours to reverse the formaldehyde crosslinking and treated with RNase A (10 mg / ml) and proteinase K (40 mg / ml). DNA was extracted using the QIAquick PCR purification kit (Qiagen). The antibodies used for the ChIP assay were as follows: anti-RORγt (H-190: Santa Cruz), anti-p300 (E8S2V: CST).

[0103] Experimental autoimmune encephalomyelitis (EAE) EAE was induced in mice by subcutaneous injection of 100 mg of MOG emulsified in complete Freund's adjuvant 35-55Peptides were immunized subcutaneously in mice, and then induced by injecting 200 ng of pertussis toxin intraperitoneally on day 0 and day 2 according to the manufacturer's protocol (Hooke Laboratories). To quantify the severity of the disease, scores were given on a scale of 0 - 5 daily; 0: no paralysis, 0.5: clumsy walking, 1: tail dragging, 2: tail dragging and partial hind limb paralysis, 3: complete hind limb paralysis, 4: quadriplegia, 5: moribund state. Animals that reached grade 4 or above were euthanized. To analyze the cells infiltrating the central nervous system, cell suspensions were prepared from the brain and spinal cord. Cells were stimulated with PMA (10 ng / ml) and ionomycin (500 nM), and cytokine production was analyzed by flow cytometry.

[0104] Reporter assay On day 0, EL4 T cells were transiently transfected by electroporation with the IL-17 promoter and CNS2 enhancer region plasmids together with RORγt. Furthermore, as an internal control for transfection efficiency, 20 ng of the Renilla luciferase reporter vector, pRL-TK (Promega), was added to each transfection. In some experiments, cells were cultured in the presence of lipids at the indicated concentrations. For the evaluation of IL-17 promoter activity, 20 hours after transfection, the transfected cells were stimulated with PMA (30 ng / ml) + ionomycin (500 nM). 16 hours after stimulation, cell extracts were prepared and luciferase assays were performed according to the instructions of the Dual Luciferase Reporter. For the binding of RORγt and NCOA1, EL4 T cells were transfected with the RORγt-LgBit and SmBit-NCOA1 plasmids. The transfected cells were treated with NanoBit Nano-Glo Live Cell Reagents for 1 hour. The IL-17a promoter used in the assay is shown as SEQ ID NO: 44, and the CNS2 enhancer region is shown as SEQ ID NO: 45, respectively.

[0105] Thermal shift assay The thermal shift assay was performed using a 20 μl sample containing human RORγt ligand binding domain (LBD) (5 mM), a given lipid metabolite (0.3 - 3.0 mM), and 2.5x SYPRO Orange (Sigma) in Protein Thermal Shift buffer (Thermo Fischer). The sample was heated from 25°C to 99°C at a rate of 1% in a StepOne Real-Time PCR System (Thermo Fischer). Using the ROX reporter, the reported melting value was calculated as the minimum in the negative derivative of the obtained melting curve using Protein Thermal Shift Software (Thermo Fischer). The DT m value was determined as the mean from four independent experiments performed in sextuplicate and normalized to DMSO.

[0106] Statistical analysis Data were analyzed using the GraphPad Prism software program (version 8). Differences were evaluated using one-way ANOVA with Tukey's post hoc analysis for parametric analysis of variance between groups. For non-parametric analysis of variance between groups, the Kruskal-Wallis with Dunn's test was used, and for pairwise comparisons, the Mann-Whitney test was used. For EAE data, the P value of the clinical score was calculated by two-way ANOVA followed by Sidak's multiple comparisons. Differences with a P value < 0.05 were considered significant. The sample size for animal experiments was selected based on past experience with similar models of brain or lung inflammation. There was no data excluded from the experimental analysis. The data showed similar variance between groups and were normally distributed when parametric tests were used. Statistical significance is shown as follows: N.S.: not significant, *P < 0.05, **P < 0.01, ***P < 0.005.

[0107] 2. Results Pooled CRISPR screening was used to identify essential enzymes in the lipid biosynthesis pathway that control RORγt activity. The inventors previously found that deletion or pharmacological inhibition of ACC1 suppresses the nuclear localization of RORγt without affecting the total protein level or mRNA abundance of nuclear RORγt (Non-Patent Document 1). Under these conditions, the binding of RORγt and p300 to the Il17 and Il23r loci decreased (Non-Patent Document 1). The change in the binding of RORγt and p300 in cells with inhibited ACC1 activity could be restored by the addition of oleic acid (OA). Therefore, the inventors hypothesized that specific OA-containing lipids could control RORγt activation and induce Th17 cell differentiation.

[0108] To examine the requirement for ACC1 and OA in the transcription of RORγt, Il17a promoter activity was evaluated using the EL4 T cell line. Consistent with the inventors' previous data, deletion or pharmacological inhibition of ACC1 suppressed Il17a promoter activity, and the activity was dose-dependently restored by exogenous addition of OA (Figs. 1A - C). Furthermore, genetic deletion of ACC1 decreased the binding of RORγt and nuclear receptor coactivator 1 (NCOA1) (Fig. 1D). Next, it was considered to identify the major lipid metabolic enzymes that are downstream components of ACC1 and are essential for activating RORγt in differentiating Th17 cells. Thirty-one candidate enzymes, including desaturases, lipases, and acyltransferases, were selected based on their preferential expression in Th17 cells compared to naive CD4 + T cells. To elucidate the phenotypic and functional effects of the 31 candidate enzymes, a pooled CRISPR-based screening approach was developed (Fig. 8). Briefly, by electroporation, a plasmid encoding Cas9, an sgRNA cassette, and the eGFP fluorescent protein was transfected into the EL4 cell line. Two days after electroporation, the transfected cells (eGFP + ) were sorted into single cell clones by FACS.

[0109] After culturing these clones for two weeks, the Il17a promoter activity was evaluated for more than 100 clones. When the Il17a promoter activity was quantified three times for each single-cell clone, a decrease in activity at a level almost equivalent to the decrease seen in the ACC1-deficient clones (clones #4, #18, #19, Figure 1E) was detected in several clones. To identify the sgRNAs that control RORγt activity, qPCR was used to selectively detect the sites targeted by each gene and examine the gene deletions in the clones with decreased Il17a promoter activity and those unaffected. As a result, it was found that the sgRNAs targeting Gpam, Gpat3 / Agpat9, Lplat1 / Agpat1, Pla2g12a, and Scd2, which are essential components of the lipid metabolism pathway, were genetically deleted in all clones with decreased Il17a promoter activity (Figure 1F). Furthermore, in the quintuple knockout (QKO), multiple sgRNAs that selectively target these five enzymes showed a phenotype similar to that of clone #4 with the lowest levels of the five enzymes and the ACC1-deficient clones, strengthening the conviction that the decrease in Il17a promoter activity was not due to off-target effects (Figure 1G). To confirm the contribution of sterol metabolites to RORγt activation, Hmgcr-deficient EL4 cells (Hmgcr - / - ) were included in the study (Non-Patent Document 16); no obvious changes were detected in the reporter assay (Figure 1G). It was also found that deleting these five enzymes decreased the nuclear co-localization of RORγt with NCOA1 or the p300 cofactor (Figure 1H).

[0110] Five specific lipid metabolism enzymes induce a core gene signature associated with Th17 cell differentiation. To evaluate the relevance of these specific enzymes in the control of Th17 cell differentiation, we performed multiplex gene targeting of the enzymes and examined whether their deficiency affected Th17 cell differentiation in vitro. Consistent with the functional findings of RORγt, in QKO-Th17 cells, a dramatic decrease in the proportion of IL-17A-producing cells was observed without a major effect on RORγt protein levels (Figs. 2A and 9A-C). In addition, single deletion of Scd2 partially affected the differentiation of Th17 cells, and triple targeting of Scd2, Lplat1, and Gpat3 was found to more strongly reduce IL-17A production (Figs. 2B and 9A). Targeting all five selected enzymes had the strongest effect on IL-17A production, and the level was similar to that in RORγt-deficient cells (Figs. 2A, B). Similarly, in QKO-Th17 cells, a decrease in IL17a and IL17f mRNA expression and IL-17A production was detected (Figs. 2C, D). In contrast, overexpression of three enzymes - Scd2, Gpam, and Gpat3 - increased the proportion of IL-17A-producing cells (Figs. 2E, F).

[0111] To examine the programming of Th17 cells by five lipid enzymes in vitro, RNA sequencing of Th17 cells differentiated under control, TOFA, QKO, or sgRorc conditions was performed. Notably, principal component analysis revealed that QKO-Th17 cells formed a different cluster from control Th17 cells and showed a molecular profile relatively close to that of TOFA-treated Th17 cells (Figure 2G). Unexpectedly, the transcriptional profile of QKO-Th17 cells was different from that of sgRorc-Th17 cells (Figure 2G). Next, the gene expression profiles of Th17 cells cultured with TOFA, QKO, or sgRorc were compared in detail. Compared with control cells, TOFA-, QKO-, and sgRorc-Th17 cells each showed a significant decrease in chronic inflammatory disease-related genes including Il23r and Il1r1 (Figure 2H and Figure 9D). Furthermore, according to the Venn diagram, 35 differentially expressed genes including RORγt-related genes were common among the three groups and decreased compared with the control group (Figure 2I). Gene set enrichment analysis revealed a significant inverse correlation with RORγt-related genes in QKO-Th17 cells by gene set enrichment analysis using a previously determined Th17 dataset (32) (Figure 2J). The 35 differentially expressed genes with reduced expression in QKO-Th17 cells included Il17a, Il17f, Il21, Il22, Ltb4r1, Ccr6, Il23r, Il1r1, and S100a1 (Figure 2K). These data suggest that five lipid metabolic enzymes are required to extract the core of the transcriptional profile related to Th17 polarization.

[0112] Oleic acid-containing phospholipids are decreased in QKO-Th17 cells. Next, in QKO-Th17 cells, it was examined whether changes in lipid metabolites are associated with dysregulation of RORγt function and disruption of the Th17-related transcriptome signature. Scd2 is required for the biosynthesis of monounsaturated fatty acids, and glycerol phosphate acyltransferases such as Gpam, Gpat3, and Lplat1 contribute to the production of phospholipids such as phosphatidic acid and lysophosphatidic acid. The secreted PLA 2 (sPLA 2 ) subtype Pla2g12a may be involved in the production of lysophospholipids with sn-1 fatty acids by hydrolysis of phospholipids (Figure 10). The inventors profiled the lipidomes of WT EL4 cells, Acaca - / - , clone #4, and QKO EL4 cell lines. By non-targeted cell lipidome analysis, a total of 524 lipids consisting of 262 glycerophospholipids, 99 glycerolipids, 55 sphingolipids, 43 cholesterol esters, 39 lysophospholipids, and 26 free fatty acids were identified in control cells. It was found that control cells were rich in lipid groups such as phospholipids and glycerolipids including acylglycerol. Using principal component analysis, the lipid profiles of each group were visualized (Figure 3A). As a result of the analysis, it was revealed that the QKO group formed a different cluster from control cells; this cluster was similar to the clusters of the Acaca - / - and clone #4 groups (Figure 3A). Using this robust lipidome profile, unsupervised hierarchical clustering was performed, and all four cell line groups were separated based on the similarity of the spectra measured for 524 lipids. This analysis generally classifies cell types by lipid phenotype; as a result, it was found that each group exhibited a different lipidome profile (Figure 3B). Acaca - / -, The overall composition of cellular lipids in each group of Clone #4 and QKO was different from that of WT cells (Figures 3B and S3B). Among them, the ratio of acylglycerol to phospholipid was significantly different (Figures 3B and S3B-D). Compared with control cells, the ratio of triacylglycerol (TG) to diacylglycerol (DG) decreased in the three types of cells lacking lipid biosynthetic enzymes (Figure S3D). In contrast, several lipid groups including phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and cholesterol ester (ChE) increased in the group lacking lipid biosynthetic enzymes. Furthermore, the content of the fatty acid side chains of total reduced lipids differed between the three different knockout groups and control cells (Figure S3E). Compared with control cells, the three types of knockout cells showed a decrease in the content of lipids containing saturated fatty acids and monounsaturated fatty acids. OA (25.1, 24.8, 25.3%), palmitic acid (12.9, 13.1, 11.0%), and stearic acid (10.2, 8.56, 6.61%) were found to be the main components of cellular lipids in the Acaca - / - , Clone #4, and QKO-EL4 cell groups, respectively. To identify lipid species responsible for RORγt activity, we focused on lipid groups that changed similarly in the Acaca - / - , Clone #4, and QKO groups compared with control cells. From the Venn diagram of the changed lipid species, three groups (Acaca - / -In all of Clone #4 and QKO, it was revealed that the content of 181 lipids decreased compared to control cells (Figure 3C). To more deeply understand the differences in specific lipid species, the fatty acid content of these 181 lipids was analyzed. Compared to control cells, QKO-EL4 cells showed a decrease in the content of lipids containing saturated fatty acids and monounsaturated fatty acids. In QKO-EL4 cells, it was found that OA (25.2%), palmitic acid (12.0%), palmitoleic acid (6.6%), and stearic acid (6.3%) were the main components of cellular lipids (Figure 3D). Since OA is important in controlling the function of RORγt (Figure 1C), individual lipid species were classified based on the OA content (Figure 3E). OA-containing lipids that decreased across all three groups were equally distributed in most compartments of phospholipids, lysophospholipids, and neutral lipids, while the corresponding OA-free lipids either did not change or had an increased abundance in the QKO group (Figure 3E). Most ceramides, sphingomyelins, lysophosphatidic acids, and glycosphingolipids including monohexosylceramide (G1Cer) and dihexosylceramide (G2Cer) did not contain OA (Figure 3E). The amounts of cholesterol esters, cardiolipin (CL), and carnitine containing OA did not consistently change compared to control cells in the three groups lacking lipid biosynthetic enzymes (Figure 3E). Furthermore, the contents of cholesterol, cholesterol sulfate, and acylhexosylcholesterol were similar in all four groups. Additionally, the amount of lysophosphatidylethanolamine (LPE) containing OA was lower than that in control cells in all three cell types lacking lipid biosynthetic enzymes, but this was not the case for LPE containing saturated or polyunsaturated fatty acids (Figure 3F, left). Similarly, the level of lysophosphatidylinositol (LPI) containing OA was also observed to be low in the three groups (Figure 3F, right). In contrast, lysophosphatidylcholine (LPC) and lysophosphatidylserine (LPS) containing OA were not greatly affected by the gene deletion of ACC1 and five enzymes. Furthermore, the lipidomes of control, TOFA-treated, and QKO-Th17 cells were compared.Consistent with the results obtained in the EL4 cell line, the amounts of LPE and LPI containing OA were significantly decreased in TOFA-treated or QKO-Th17 cells compared to control cells (Figure 3G). Collectively, these data indicated that the amounts of LPE and LPI containing OA were affected by the deletion of five enzymes involved in lipid biosynthesis.

[0113] 1-Oleoyl-LPE is required for the differentiation of Th17 cells and is a core molecular signature of Th17 cells We previously reported that the addition of exogenous fatty acids restores the function of Th17 cells in CD4-Cre + Acaca fl / fl mice (referred to as Acaca ΔT mice in this specification) and improves the proliferation, survival, and metabolic reprogramming of TOFA-treated activated CD4 + T cells (Non-Patent Document 1; J. Exp. Med. 218, e20210639 (2021); Nat. Commun. 7, 13683 (2016); Nat. Metab. 1, 261-275 (2019)). Therefore, we investigated whether the addition of exogenous lipids affects TOFA treatment or Acaca ΔTWe analyzed whether the function of Th17 cells could be restored. Surprisingly, it was found that when 1-oleoyl-LPE [LPE(1-18:1)] was added to TOFA-treated Th17 cells, the production of IL-17A was completely restored (Figure 4A and Figure 11A). In contrast, the addition of LPC(1-18:1) had no effect on IL-17A-producing cells. The addition of LPI(1-18:1) or lysophosphatidic acid [LPA(1-18:1)] to TOFA-treated Th17 cells partially restored IL-17A production. The addition of LPE(1-18:1) increased the proportion of IL-17A-producing cells in TOFA-treated Th17 cells in a dose-dependent manner (Figure 4B). In addition, the ability of LPE containing different fatty acids, such as palmitic acid (16:0), stearic acid (18:0), and heptadecenoic acid (17:1), to restore the differentiation of TOFA-treated Th17 cells was evaluated (Figure 4C). LPE(1-17:1) partially restored the phenotype of TOFA-treated Th17 cells, while LPE(1-16:0) or LPE(1-18:0) did not (Figure 4C). Furthermore, Acac ΔT We examined the effect of PE(1,2-18:1) on IL-17A production by Th17 cells, but no substantial increase in IL-17A-producing cells was detected (Figure 4D).

[0114] Next, focusing on the position of OA in LPE, Acaca ΔTThe effects of LPE(1-18:1) and LPE(2-18:1) on IL-17A production in Th17 cells were compared (Figure 11B). Although LPE(1-18:1) and LPE(2-18:1) have the same chemical formula, the different linking sites of oleic acid may account for the differences in physical properties. LPE(2-18:1) mildly restored cellular IL-17A production, but its effect was much weaker compared to LPE(1-18:1) (Figure 11B). At the optimal concentration of anti-TCRβ (10 μg / ml) at which Th17 cell differentiation is fully induced, LPE(1-18:1) did not affect Th17 cell differentiation. However, in cultures with suboptimal anti-TCRβ (1 μg / ml), the addition of LPE(1-18:1) increased the proportion of IL-17A-producing cells (Figures 11C-11F). It was also confirmed that at a lower concentration of anti-TCRβ (1 μg / ml), sufficient expression of the five enzymes could not be induced (Figure 11D). Correspondingly, in Th17 cells stimulated with 1 μg / mL anti-TCRβ, the amount of LPE(1-18:1) was lower compared to the levels detected in cells stimulated with 10 μg / mL anti-TCRβ (Figure S5E). Furthermore, it was confirmed that the protein level of RORγt was equivalent between cells stimulated with 1 mg / mL and 10 mg / mL anti-TCRβ (Figure 11F). Some studies have suggested that the cholesterol biosynthetic pathway is involved as an important regulator of RORγt activity in normal and pathological conditions (Non-Patent Documents 14-16). Therefore, cholesterol metabolites including 25-hydroxycholesterol (25-OHC) and 27-OHC, 7α,27-OHC or 7β,27-OHC were added to Acaca ΔT Th17 cell cultures, but no substantial restoration of IL-17A production was detected. Furthermore, it became clear that the amount of LPE(1-18:1) was lower than that in sgControl cells across three groups including sgScd2, sgScd2 / Gpat3 / Lplat1, or QKO-cells (Figure 4E). Also, as expected, the lipid reduction was most prominent in the QKO group, moderate in the sgScd2 / Gpat3 / Lplat1 group, and milder in the sgScd2 group.

[0115] Next, multiplex gene targeting of five enzymes was performed to examine whether their deficiencies affect the differentiation of Th1 and Th2 cells in vitro. In contrast to the strong effects of these enzymes on Th17 cell differentiation, the deficiencies of these enzymes did not affect IFNγ production or T-bet expression in Th1 cells. Deletion of lipid biosynthesis enzymes moderately decreased IL-4 production by Th2 cells but did not change GATA3 expression. Also, the effects of LPE(1-18:1) on the differentiation of Th1 and Th2 cells were examined, but no substantial changes were observed. Furthermore, proliferation and apoptosis assays were performed using control and QKO Th cells. Compared with control cells, QKO cells showed a slight decrease in proliferation, which was not restored by LPE(1-18:1) treatment. Neither knockout of lipid biosynthesis enzymes nor treatment of QKO cells with LPE(1-18:1) affected cell survival compared with control cells. Thus, deletion of the five enzymes partially affected cell proliferation or IL-4 production, but the effects were much weaker than those on Th17 cell differentiation. Furthermore, treatment with LPE(1-18:1) did not affect cell proliferation and IL-4 production.

[0116] The inventors performed RNA sequence analysis using Th17 cells treated with TOFA in the presence or absence of LPE(1-18:1) to clarify the contribution of LPE(1-18:1) to the core molecular signature of Th17 programming. Principal component analysis and hierarchical clustering analysis showed that each group exhibited a different transcriptome profile, and it was revealed that LPE(1-18:1) treatment was insufficient to restore the overall gene activity of TOFA-treated cells to the level seen in control Th17 cells (Figure 4F). Next, the expression of representative genes of the Th17 cell core molecular signature, such as Il17a, Il17f, Il23r, Ltb4r1, and Ccr6, was analyzed in control, TOFA-treated Th17 cells, and TOFA-treated cells supplemented with LPE(1-18:1) (Figure 4G, H, and Figure 12). Treatment of TOFA-treated cells with LPE(1-18:1) restored the expression of the Th17 core transcriptional signature to levels similar to those found in control Th17 cells. Furthermore, gene set enrichment analysis of the gene signature identified in a previously measured Th17 dataset (Nat. Immunol. 13, 991-999 (2012)) revealed that genes forming the core Th17-related signature were enriched by adding LPE(1-18:1) to Th17 cell cultures treated with TOFA. From the above results, it was shown that LPE(1-18:1) produced by ACC1 and a set of five lipid biosynthetic enzymes is required for the production of IL-17A and the induction of the transcriptional signature associated with Th17 cells.

[0117] LPE(1-18:1) regulated the function of RORγt in Th17 cell differentiation Next, we analyzed whether LPE(1-18:1) could control the binding and function of RORγt. From our previous immunofluorescence analysis, Acaca ΔTThe co-localization of RORγt and p300 in the nucleus of Th17 cells was suggested to be rescued by the added OA. Consistent with previous results, immunofluorescence microscopy showed that the co-localization of RORγt and p300 in QKO-Th17 cells was rescued by the addition of LPE(1-18:1) (Figure 5A). Similarly, the IL17a promoter activity in the QKO-EL4 cell line was also restored by the addition of LPE(1-18:1) (Figure 5B). To further evaluate the effect of LPE(1-18:1) on RORγt-dependent Th17 cell differentiation, the RORγt inverse agonist (SR1001) was added to the TOFA-treated Th17 cell culture supplemented with LPE(1-18:1). As previously found, the addition of LPE(1-18:1) restored IL-17A production in TOFA-treated Th17 cells, which was reversed by SR1001 (Figure 5C).

[0118] Next, ATAC-sequencing was used to detect transposase-accessible chromatin in control cells treated or not treated with LPE(1-18:1) and TOFA-treated Th17 cells. As a result of hierarchical clustering analysis, it was revealed that each group exhibited distinct regulome profiles (Figure 5D). Many open regulatory elements detected in control Th17 cells were deleted in TOFA-treated cells but were present in TOFA-treated cells supplemented with LPE(1-18:1). In TOFA-treated cells treated with LPE(1-18:1), the loci of Il17, Ltb4r1, Ccr6, Il1r, and Il21 were accessible (Figure 5D, E). Next, it was investigated whether the binding of RORγt to genomic regions could be controlled by treating Th17 cells with inhibited ACC1 with LPE(1-18:1) (Figure 5F, G). Since the decreased binding of RORγt to the Il17 and Il23r loci in TOFA-treated Th17 cells was restored by the addition of LPE(1-18:1), it was suggested that LPE(1-18:1) could regulate the binding of RORγt to DNA target regions in differentiating Th17 cells (Figure 5G and Figure 13A). A ChIP assay using an anti-p300 antibody was also performed. As a result, consistent with the results obtained by RORγt ChIP analysis, it was revealed that the binding of p300 to the Il17 and Il23r loci was decreased in TOFA-treated Th17 cells. Furthermore, since the binding of p300 to these regions was detected in TOFA-treated cells supplemented with LPE(1-18:1), it was suggested that LPE(1-18:1) might control the binding of p300 and RORγt at specific target loci during the differentiation process of Th17 cells (Figure 13B). Furthermore, when evaluating the effect of p300 inhibition on LPE(1-18:1)-induced Th17 cell differentiation, it was found that the p300 inhibitor (curcumin) effectively suppressed LPE(1-18:1)-induced IL-17A production in TOFA-treated cells (Figure 13C).

[0119] To investigate whether LPE(1-18:1) can directly bind to RORγt, the interaction between several lysophospholipids and the ligand-binding domain (LBD) was measured using the RORγt protein thermal denaturation assay. Ligand binding was indicated by the melting temperature T m of RORγt. Consistent with the results obtained from the cell-based reporter assay (Figure 5B), LPE(1-18:1) directly binds to the human RORγt LBD, and T m increased by 0.97 °C (0.3 mM), 3.88 °C (1.0 mM), and 7.48 °C (3.0 mM) compared to the control (Figure 5H, I). In contrast, the binding of other lipids including LPI(1-18:1), LPA(1-18:1), and LPC(1-18:1) to the RORγt LBD was considerably weaker than that of LPE(1-18:1), and the increases in T m were 1.97 °C, 1.36 °C, and 0.97 °C, respectively (Figure 5I). These series of results indicated that LPE(1-18:1) synthesized through the activities of five lipid metabolic enzymes is necessary for the proper functioning of RORγt in Th17 cell differentiation.

[0120] Pla2g12a regulated the differentiation of Th17 cells both in vitro and in vivo. If LPE(1-18:1) is a physiological regulator of RORγt activity in Th17 cells, it was thought that deleting the enzymes of this biosynthetic pathway genetically would mimic the phenotype of RORγt deficiency. Therefore, the inventors investigated whether the deficiency of Pla2g12a, a downstream enzyme of ACC1 responsible for the hydrolysis of phospholipids and thus the production of lysophospholipids, inhibits RORγt-dependent Th17 cell differentiation. Pla2g12a encodes the sPLA 2 subtype, and its in vivo role was completely unknown (37, 38). The inventors crossed Pla2g12a fl / fl mice with CD4-cre transgenic mice, and conditionally deleted the Pla2g12a gene in CD4 + T cells by the expression of Cre recombinase driven by the Cd4 promoter (herein Pla2g12aΔT (referred to as mouse). Pla2g12a ΔT In the mouse, the proportion of CD4 + T cells in the thymus and spleen was normal. The frequency and number of IL-17A-producing memory phenotype CD4 + T cells in the lung were lower in Pla2g12a ΔT mice than in control mice with an intact Pla2g12a gene even under steady-state conditions (Figures 6A, B). The number of IL-17A-producing cells in the spleen was slightly decreased in Pla2g12a ΔT mice, but the decrease was smaller than the difference observed in the lung. Furthermore, Th17 cells in the jejunum and ileum were also analyzed. Under steady-state conditions, a small number of IL-17A-producing cells were detected in the jejunum, and slightly more were detected in the ileum. From the overall data, it was shown that there was no change in Th17 cells between Pla2g12a fl / fl mice and Pla2g12a ΔT mice.

[0121] Next, it was examined whether the deficiency of Pla2g12a affects the differentiation of Th17 cells in vitro (Figure 6C). It was observed that the proportion of IL-17A-producing cells in Pla2g12a ΔT Th17 cells was decreased by approximately 50% compared to that of control mice (Figure 6D). Similarly, a decrease in the expression of IL17a and IL17f mRNA was detected in Pla2g12a ΔT Th17 cells (Figure 6E). Next, lipidome analysis of Pla2g12a ΔT Th17 cells was performed, and it was found that the level of LPE(1-18:1) was decreased by approximately 50% - 70% in Pla2g12a ΔT Th17 cells (Figures 6F, G). A similar trend was also detected in the supernatant of Pla2g12a ΔT Th17 cell culture (Figure 14A). Correspondingly, in Pla2g12a ΔT Th17 cells, Pla2g12a fl / flAn increase in OA-containing PE such as PE(18:1 / 20:4), PE(18:1 / 20:6), PE(18:1 / 18:2), PE(18:1 / 18:25) or PE(18:1 / 20:3) was observed compared to Th17 cells (Figure 6H). Furthermore, lysophospholipids containing LPA and LPI were ΔT decreased in Th17 cells compared to the control (Figure 14B). Furthermore, Pla2g12a ΔT When LPE(1-18:1) was added to Th17 cell cultures, the generation of IL-17A-producing cells recovered to approximately the same proportion as seen in Th17 cells (Figure 7A). Pla2g12a fl / fl Exogenous supplementation of FA(18:1) or FA(20:4) to Th17 cell cultures did not restore Th17 cell differentiation. Addition of these fatty acids to Th17 cell cultures ΔT did not affect the overall lipid profile. Consistent with the effect on Th17 cell differentiation, the level of LPE(1-18:1) was ΔT substantially unchanged when Th17 cell cultures were supplemented with 18:1 or 20:4. These data strongly suggest that Pla2g12a is likely to be the catalyst that cleaves PE (and possibly other phospholipids) to generate LPE(1-18:1) in differentiating Th17 cells. Also, to mimic Th17 cell differentiation under pathogenic conditions, naive Pla2g12a ΔT and Pla2g12a fl / fl and Pla2g12a ΔT CD4 + T cells were induced to differentiate with a cytokine cocktail of TGFβ3 + IL-6 + IL-23, and a significant decrease in GM-CSF production was observed in the Pla2g12a ΔT group (Figure 7B). Furthermore, deletion of Pla2g12a under pathogenic differentiation conditions also decreased IL-17 production (Figure 7B). Pla2g12a ΔTExogenous supplementation of LPE(1-18:1) to cell cultures restored the production of IL-17A and GM-CSF under pathogenic conditions (Figure 7B). Furthermore, qRT-PCR analysis confirmed that Pla2g12a-mediated LPE(1-18:1) regulated the expression of effector molecules related to Th17-mediated pathogenicity, including Il23r, Csf2, and Il3 (Figures 7C and 15A). The expression of Ifng was not affected by either genetic deletion of Pla2g12a or supplementation of LPE(18:1).

[0122] To examine the relevance of Pla2g12a to Th17 cell pathology in vivo, mice were immunized with MOG 35-55 / CFA to induce EAE. The disease state of EAE was less severe in Pla2g12a fl / fl mice than in Pla2g12a ΔT mice (Figure 7D). The protection against EAE onset in Pla2g12a ΔT mice was associated with suppression of IL-17A-producing cells in the central nervous system (Figure 7E). Notably, in Pla2g12a ΔT mice, the frequency of IL-17A + IFNγ + cells was also decreased (Figure 7E). Similarly, CD4 ΔT T cells from the draining lymph nodes and spleens of Pla2g12a + mice produced less IL-17A (Figures 7F and 15B). Collectively, these data indicated that Pla2g12a controls Th17 cell differentiation and Th17 cell-mediated pathology through the production of LPE(1-18:1).

[0123] 3. Discussion Th17 cells are a subset of helper cells of inflammatory T cells and have diverse functions ranging from neutrophilic inflammatory responses to pathogens to the driving force of several autoimmune diseases (Immunity 43, 1040-1051 (2015); Biomolecules 10, 1457 (2020)). Evidence has been accumulating that specific cellular lipid metabolic pathways, including fatty acids and cholesterol, play an essential role in the regulation of Th17 cell differentiation and function (Curr. Opin. Immunol. 46, 121-126 (2017); Cell. Mol. Life Sci. 74, 1231-1245 (2017)). However, it has been unclear how lipid metabolism and the function of RORγt are associated by what molecular mechanisms during the differentiation process of Th17 cells. By combining CRISPR-based screening and global lipidome analysis, we aimed to identify specific lipid metabolites essential for Th17 cell differentiation via RORγt. As a result, we identified five lipid enzymes necessary for the expression of RORγt activity and the core gene expression signature associated with Th17 cells. Furthermore, comprehensive lipidome analysis revealed that LPE(1-18:1) controls the function of RORγt in Th17 cell differentiation. It was also found that genetic deletion of Pla2g12a, one of the five lipid metabolic enzymes identified by CRISPR screening, halted Th17 cell differentiation and alleviated the pathology of EAE. Thus, we discovered that LPE(1-18:1), synthesized depending on five lipid enzymes, is an important fatty acid-containing lipid metabolite involved in RORγt-dependent Th17 cell differentiation and Th17 cell pathogenicity.

[0124] Mechanistically, it has been suggested that LPE(1-18:1) acts as a physiological ligand of RORγt during Th17 cell differentiation. In this study, when de novo fatty acid biosynthesis was inhibited by targeting either ACC1 or any of the five lipid metabolism enzymes identified by CRISPR screening, RORγt activity and Th17 cell differentiation disappeared. Further supporting this view, SCD2, one of the five enzymes, is required for the biosynthesis of OA, and (acyl)glycerolphosphatase acyltransferases such as GPAM, GPAT3, and LPLAT1 contributed to the production of glycerophospholipids. Lysophospholipids with sn-1 fatty acids are generated by hydrolysis of phospholipids via PLA 2 (J. Biol. Chem. 275, 39823-39826 (2000)). In this study, Pla2g12a ΔT The level of LPE(1-18:1) decreased in Th17 cells. Correspondingly, Pla2g12a ΔT In Th17 cells, an increase in PE containing OA such as PE(18:1 / 20:4), PE(18:1 / 20:6), PE(18:1 / 18:2), PE(18:1 / 18:25), PE(18:1 / 20:3) was observed. Consistent with this, Pla2g12a ΔTIn mice, the differentiation and pathogenicity of Th17 cells were impaired. Thus, our data revealed the in vivo biological role of Pla2g12a, a structurally unique sPLA2 subtype (Annu. Rev. Pathol. 8, 477-512 (2013); J. Biol. Chem. 276, 18321-18326 (2001)). Furthermore, Th17 cell differentiation via RORγt can be induced in media containing limited levels of fatty acids or lipids (Cell Metab. 21, 286-298 (2015)). From these observations, it is suggested that exogenous lipids are not required for proper RORγt activity and that de novo biosynthesis of endogenous lipids is sufficient for most of the physiological functions of RORγt in Th17 cell differentiation. De novo fatty acid biosynthesis was inhibited by genetic deletion of ACC1 or any of the five enzymes identified by screening, resulting in impaired production of LPE(1-18:1) and, consequently, defective Th17 cell differentiation. Consistent with this observation, IL-17A production restored by supplementation with LPE(1-18:1) was decreased to levels comparable to those in TOFA-treated cells by the RORγt inverse agonist SR1001.

[0125] Two studies using single-cell analysis of EAE mice also suggested that fatty acid metabolism controls the function of RORγt in Th17 cells (Non-Patent Documents 7 and 12). From these studies, it was shown that CD5L controls the flux through de novo fatty acid biosynthesis and affects the pathogenicity of Th17 cells. CD5L inhibits de novo fatty acid biosynthesis by directly binding to fatty acid synthase and inhibiting its enzyme activity. Cd5l - / -Analysis of Th17 cells revealed an increase in saturated and monounsaturated fatty acids, accompanied by a decrease in polyunsaturated fatty acid content. Furthermore, supplementation of pathogenic Th17 cells with exogenous polyunsaturated fatty acids decreased the binding of RORγt to the Il17 and Il23r loci, while exogenous saturated fatty acid treatment increased the RORγt occupancy of the target loci (Non-Patent Documents 7 and 12). These studies indicate that polyunsaturated fatty acids limit the ligand-dependent function of RORγt. The results of the present inventors clarified that LPE(1-18:1) restores the binding of RORγt to the Il17 and Il23r loci in TOFA-treated Th17 cells. Although the exact manner has not been clarified, polyunsaturated fatty acids may inhibit the production of LPE(1-18:1) and regulate RORγt activity in Th17 cells.

[0126] The data of the present inventors suggest that LPE(1-18:1) is an important regulator of RORγt activity. There are precedents for the interaction between derivatives of phospholipids and some members of the nuclear receptor superfamily (J. Biol. Chem. 285, 40409-40415 (2010)). 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine has been shown to bind to PPARα and control hepatic gene expression (Cell 138, 476-488 (2009)). Lysophosphatidic acid is also a very abundant phospholipid present intracellularly and in serum, but has been shown to bind to and activate PPARγ (Proc. Natl. Acad. Sci. U.S.A. 100, 131-136 (2003)). LPE is thought to be involved in cell signaling via the cell membrane and the activation of other enzymes, but there are still many unclear points regarding the physiological role of LPE (Int. Rev. Cell Mol. Biol. 321, 29-88 (2016)). The studies of the present inventors support the hypothesis that the biological activity of LPE is independent of its role as a membrane component.

[0127] Previous studies have shown that cholesterol biosynthesis controls RORγt activity, and thus RORγ-dependent thymocyte development, Th17 cell differentiation, and Lin - CD45 + IL-7Ra + have been shown to play important roles in the differentiation of LTi cells (Non-Patent Document 14). Lipidome analysis has revealed that in Th17 cells, the intracellular pool sizes of metabolites from the distal branch of the cholesterol biosynthesis pathway and specific oxysterols are increased (Non-Patent Document 16). In an add-back experiment using sterol metabolites, it has been shown that both sterol biosynthesis intermediates and cholesterol-derived metabolites can restore RORγt activity and Th17 cell differentiation (Non-Patent Documents 14, 16). On the other hand, in the add-back experiments of the present inventors using sterol metabolites including cholesterol, 25-OHC, 27-OHC, 7α, 27-OHC, and 7β, 27-OHC, Acaca ΔT was unable to restore the phenotype of Th17 cells. These results suggest that the defects in RORγt activity and Th17 cell differentiation due to inhibition of de novo fatty acid biosynthesis may not be caused by a decrease in cholesterol-derived metabolites. Correspondingly, based on the detection by the present inventors, genetic deletion of ACC1 or five metabolic enzymes that inhibit Il17a promoter activity in CRISPR screening did not seem to change cholesterol metabolites. Furthermore, it has been reported that neither pharmacological inhibition nor genetic deletion of cholesterol biosynthesis can completely abolish IL-17 production (Non-Patent Documents 14, 17). Combining these series of studies and the data of the present inventors, it is suggested that both LPE(1-18:1) and sterol derivatives coordinately regulate the function of RORγt during the differentiation process of Th17 cells.

[0128] An interesting finding in this study is that LPE(1-18:1) was very selective in controlling the function of RORγt during the process of Th17 cell differentiation. Lysophospholipids containing OA other than LPE, and LPE containing fatty acids other than OA, TOFA treatment, or Acaca ΔTThe Th17 phenotype of Th17 cells was not restored. Furthermore, even when using LPE(2-18:1), the IL-17A-producing cells only slowly regained activity, and the effect was much weaker than that of LPE(1-18:1). Consistent with this, the binding of LPE(1-18:1) to the RORγt LBD was much stronger than that of other lipids such as LPI(1-18:1), LPA(1-18:1), and LPC(1-18:1). It is interesting that while fatty acid metabolites have high specificity, a relatively broad range of cholesterol metabolites can control RORγt activity. Therefore, further studies to elucidate the detailed molecular mechanisms underlying the contribution of LPE(1-18:1) and cholesterol-derived metabolites to RORγt activity may lead to a complete understanding of the exact functional mode of lipid ligands in Th17 cell differentiation.

[0129] In summary, LPE(1-18:1) is synthesized by five metabolic enzymes, and it has been shown that this lipid controls the differentiation of Th17 cells and the severity of EAE. Importantly, LPE(1-18:1) is required for proper RORγt binding and function, and the core transcriptome signature of Th17 cells. This is ΔT the level of LPE(1-18:1) is decreased in Th17 cells, and, consistent with this, ΔT it has been shown that in mice, the differentiation and pathogenicity of Th17 cells are impaired. Furthermore, by supplying LPE(1-18:1) downstream of the lipid biosynthesis pathway, it was demonstrated that Pla2g12a plays a biological role in vivo as a regulator of Th17 immunity. In conclusion, LPE(1-18:1) and Pla2g12a may be potential targets for the treatment of chronic inflammatory diseases involving a persistent Th17 cell response.

[0130] [Example 2] Exosomes containing various lipids were treated with 4 mM CaCl 2After sonication in 100 mM Tris-HCl containing it for 5 minutes, recombinant Pla2g12a protein was added and incubated at 37 °C for 30 minutes. After incubation, the generated lipids were mixed with an internal standard substance, extracted, and analyzed using LC-MS for the detection of lysophospholipids. As a result, it was confirmed that various lipids including LPE(1-18:1) were produced (Figure 16).

Industrial Applicability

[0131] According to the present invention, LPE(1-18:1) is provided as a lipid metabolite that controls Th17 cell differentiation. Since LPE(1-18:1) binds to RORγt with high specificity and promotes differentiation into Th17 cells, it can be a good therapeutic target for diseases caused by excessive Th17 cell responses. For example, by selecting a compound that inhibits the binding of LPE(1-18:1) and RORγt, a candidate compound for a therapeutic agent for diseases caused by excessive Th17 cell responses can be obtained. Furthermore, according to the present invention, five lipid metabolic enzymes (Gpam, Gpat3, Lplat1, Pla2g12a, and Scd2) that are responsible for the biosynthesis of LPE(1-18:1) and control Th17 cell differentiation are provided. These five lipid metabolic enzymes can also be good therapeutic targets for diseases caused by excessive Th17 cell responses. For example, by selecting a compound that suppresses the activity or expression of the above five lipid metabolic enzymes, a candidate compound for a therapeutic agent for diseases caused by excessive Th17 cell responses can be obtained. Therefore, according to the present invention, the development of therapeutic agents and treatment methods for Th17-related diseases based on a new mechanism is promoted.

Claims

1. A method for producing Th17 cells, comprising inducing differentiation of naive CD4-positive T cells into Th17 cells by culturing the naive CD4-positive T cells in a medium containing 1-oleoyl-lysophosphatidylethanolamine, IL-6 and TGF-β.

2. The method of claim 1, wherein the naive phenotype CD4 positive T cells have a deletion, mutation or defective expression of at least one lipid metabolic enzyme gene selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, Scd2 and ACC1, or the expression of at least one lipid metabolic enzyme gene selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, Scd2 and ACC1 is suppressed in T cells in the process of differentiating into Th17 cells.

3. A Th17 cell differentiation promoter containing 1-oleoyl-lysophosphatidylethanolamine.

4. A cell preparation comprising 1-oleoyl-lysophosphatidylethanolamine and Th17 cells.

5. A combination comprising 1-oleoyl-lysophosphatidylethanolamine and RORγt.

6. A method for screening a candidate substance for a preventive or therapeutic agent for a disease caused by activation of Th17 cells, comprising selecting a compound that inhibits the interaction between 1-oleoyl-lysophosphatidylethanolamine and RORγt.

7. A method for screening candidate substances for preventing or treating diseases caused by activation of Th17 cells, comprising selecting a compound that inhibits the activity of any lipid metabolic enzyme selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, and Scd2.

8. The lipid metabolism enzyme is Pla2g12a, The method according to claim 7, comprising incubating 1-oleoylphosphatidylethanolamine with Pla2g12a in the presence of a test compound, quantifying the amount of 1-oleoyl-lysophosphatidylethanolamine produced, and selecting the test compound as a candidate for a preventive or therapeutic agent for a disease caused by activation of Th17 cells if the amount produced is lower than the amount produced in the absence of the test compound.

9. The method according to claim 7 or 8, wherein the disease caused by activation of Th17 cells is an autoimmune disease, an allergic disease, or arteriosclerosis.

10. A preventive or therapeutic agent for a disease caused by the activation of Th17 cells, comprising a nucleic acid that hybridizes under physiological conditions to DNA or mRNA encoding any lipid metabolic enzyme selected from the group consisting of Pla2g12a, Lplat1, Gpam, Gpat3, and Scd2, thereby inhibiting its transcription and / or translation, or an effective amount of an expression vector for the nucleic acid.

11. The preventive or therapeutic agent according to claim 10, wherein the disease caused by activation of Th17 cells is an autoimmune disease, an allergic disease, or arteriosclerosis.

Images