Marker for detection of multinucleated giant cells or for diagnosis of giant cell arteritis

Abstract

To provide a technique that enables the detection of multinucleated giant cells (MNGCs) present in inflammatory tissue of giant cell arteritis (GCA), and the conclusive diagnosis of GCA.SOLUTION: The present invention relates to markers for detecting MNGCs, markers for diagnosing GCA, and the like, enabling the detection of MNGCs present in inflammatory tissue of GCA, and the conclusive diagnosis of GCA.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a marker for detecting multinucleated giant cells, for example, a marker for detecting multinucleated giant cells present in inflamed tissues of giant cell arteritis. The present invention also relates to a marker for diagnosing giant cell arteritis. [Background technology]

[0002] Vasculitic syndromes comprise a heterogeneous group of inflammatory diseases, historically classified according to the size of the affected vessel. Some vasculitides involve granuloma-like structures, but the infiltrating cells vary from disease to disease. The molecular pathology and exact etiology of most vasculitides remain to be elucidated. Giant cell arteritis (GCA), which is classified as a large-vessel arteritis along with Takayasu's arteritis (TAK), has recently been subdivided into cranial type GCA (C-GCA), large vessel type GCA (LV-GCA), and mixed type GCA (M-GCA). Temporal artery occlusion leads to blindness, and coronary artery tissue damage is often accompanied by aneurysms and occasionally causes myocardial infarction. The pathology of GCA and TAK is shared in terms of massive infiltration of macrophages and scattered multinucleated giant cells (MNGCs) within the tunica media and adventitia. Furthermore, intimal hyperplasia may cause myofibroblast hyperplasia, leading to vascular occlusion. Therefore, the pathology of GCA consists of at least two pathological aspects: tissue destruction of the tunica media and hypertrophy of the intima due to accumulation of myofibroblasts (Non-Patent Documents 1 and 2).

[0003] Cell membrane fusion and multinucleation induce the formation of MNGCs in physiological environments. For example, osteoclasts in bone homeostasis, skeletal muscle cells in striated muscle formation, and syncytiotrophoblasts in placentation. On the other hand, MNGC formation is also induced in pathological conditions such as granulomatous lesions, and it has been proposed that the cause is abnormal division due to continuous inflammatory stimulation. Representative granulomas associated with tuberculosis and sarcoidosis are composed of MNGCs, epithelioid cells, and M2-type macrophages. MNGCs appear in vasculitis such as GCA, TAK, and GPA (granulomatosis with polyangiitis), as well as in chronic inflammatory diseases such as Crohn's disease. These disease-derived MNGCs have been classified as Langhans giant cells mainly based on their morphological appearance. However, their functionality and relationship with osteoclasts remain unclear, and their role in disease pathology, especially in terms of their osteoclast-like characteristics, i.e., their function in resorbing hardened tissue, is unclear. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Watanabe R, Berry GJ, Liang DH, et al. Pathogenesis of Giant Cell Arteritis and Takayasu Arteritis-Similarities and Differences. Curr Rheumatol Rep 2020; 22:68. [Non-Patent Document 2] Greigert H, Genet C, Ramon A, et al. New Insights into the Pathogenesis of Giant Cell Arteritis: Mechanisms Involved in Maintaining Vascular Inflammation. J Clin Med Res 2022;11. doi:10.3390 / jcm11102905 [Non-Patent Document 3] van Sleen Y, Jiemy WF, Pringle S, et al. A Distinct Macrophage Subset Mediating Tissue Destruction and Neovascularization in Giant Cell Arteritis: Implication of the YKL-40 / Interleukin-13 Receptor α2 Axis. Arthritis Rheumatol 2021; 73:2327-37. [Non-Patent Document 4] Burja B, Feichtinger J, Lakota K, et al. Utility of serological biomarkers for giant cell arteritis in a large cohort of treatment-naive patients. Clin Rheumatol2019; 38:317-29. [Non-Patent Document 5] Krausgruber T, Redl A, Barreca D, et al. Single-cell and spatial transcriptomics reveal aberrant lymphoid developmental programs driving granuloma formation. Immunity 2023; 56:289-306.e7. Summary of the Invention [Problem to be solved by the invention]

[0005] Under these circumstances, there was a need for the development of a method for detecting multinucleated giant cells (MNGCs) present in the inflamed tissue of giant cell arteritis (GCA), and thus for the definitive diagnosis of GCA. [Means for solving the problem]

[0006] The present invention has been made in consideration of the above-mentioned circumstances, and provides the following: a marker for detecting multinucleated giant cells (MNGC), a marker for diagnosing giant cell arteritis (GCA), an agent or composition for detecting MNGC, a diagnostic agent or composition for diagnosing GCA, a microarray for detecting MNGC, a microarray for diagnosing GCA, a kit for detecting MNGC, a kit for diagnosing GCA, a method for detecting the presence or absence of MNGC, and a method for diagnosing GCA.

[0007] [1] A marker for detecting multinucleated giant cells, comprising mRNA of two or more genes selected from the group consisting of MRC1, LRRC15, ACP5, ATP6V0D2, VDR, TREM2, HLA-DRA, and MMP12, or comprising proteins encoded by said two or more genes. [2] The marker according to [1] above, which is used in a method for diagnosing giant cell arteritis. [3] A diagnostic marker for giant cell arteritis, comprising mRNA of two or more genes selected from the group consisting of MRC1, LRRC15, ACP5, ATP6V0D2, VDR, TREM2, HLA-DRA, and MMP12, or comprising a protein encoded by said two or more genes.

[0008] [4] A detection agent or composition for detecting multinucleated giant cells, comprising a nucleic acid probe capable of binding to the mRNA of two or more genes selected from the group consisting of MRC1, LRRC15, ACP5, ATP6V0D2, VDR, TREM2, HLA-DRA, and MMP12, or comprising a substance capable of detecting proteins encoded by said two or more genes. [5] The detection agent or composition for detection described in [4] above, wherein the detectable substance is a substance capable of staining the protein encoded by the two or more genes, and / or an antibody against the protein encoded by the two or more genes. [6] The detection agent or composition for detection according to [4] or [5] above, which is used in a method for diagnosing giant cell arteritis. [7] A diagnostic agent or composition for giant cell arteritis, comprising a nucleic acid probe capable of binding to mRNA of two or more genes selected from the group consisting of MRC1, LRRC15, ACP5, ATP6V0D2, VDR, TREM2, HLA-DRA, and MMP12, or comprising a substance capable of detecting proteins encoded by said two or more genes. [8] The diagnostic agent or diagnostic composition according to [7] above, wherein the detectable substance is a substance capable of staining the protein encoded by the two or more genes, or an antibody against the protein encoded by the two or more genes.

[0009] [9] A microarray for detecting multinucleated giant cells, comprising a nucleic acid probe capable of binding to the mRNA of two or more genes selected from the group consisting of MRC1, LRRC15, ACP5, ATP6V0D2, VDR, TREM2, HLA-DRA, and MMP12.

[10] The microarray according to [9] above, for use in a method for diagnosing giant cell arteritis.

[11] A microarray for diagnosing giant cell arteritis, comprising nucleic acid probes capable of binding to the mRNA of two or more genes selected from the group consisting of MRC1, LRRC15, ACP5, ATP6V0D2, VDR, TREM2, HLA-DRA, and MMP12.

[0010]

[12] A kit for detecting multinucleated giant cells, comprising a nucleic acid probe capable of binding to the mRNA of two or more genes selected from the group consisting of MRC1, LRRC15, ACP5, ATP6V0D2, VDR, TREM2, HLA-DRA, and MMP12, or comprising the probe immobilized on a carrier, or comprising a substance capable of detecting proteins encoded by the two or more genes.

[13] The kit described in

[12] above, wherein the detectable substance is a substance capable of staining the proteins encoded by the two or more genes and / or an antibody against the proteins encoded by the two or more genes.

[14] The kit according to

[12] or

[13] above, which is used in a method for diagnosing giant cell arteritis.

[15] A diagnostic kit for giant cell arteritis, comprising a nucleic acid probe capable of binding to mRNA of two or more genes selected from the group consisting of MRC1, LRRC15, ACP5, ATP6V0D2, VDR, TREM2, HLA-DRA, and MMP12, or comprising the probe immobilized on a carrier, or comprising a substance capable of detecting proteins encoded by the two or more genes.

[16] The kit described in

[15] above, wherein the detectable substance is a substance capable of staining the proteins encoded by the two or more genes, or an antibody against the proteins encoded by the two or more genes.

[0011]

[17] Detecting mRNA of two or more genes selected from the group consisting of MRC1, LRRC15, ACP5, ATP6V0D2, VDR, TREM2, HLA-DRA, and MMP12 in a sample collected from a biological tissue, or reacting a sample collected from a biological tissue with a substance capable of detecting a protein encoded by the two or more genes, thereby detecting the protein; A method for detecting the presence or absence of multinucleated giant cells using the detection results obtained as an indicator.

[18] The method according to

[17] above, wherein the detectable substance is a substance capable of staining the proteins encoded by the two or more genes and / or an antibody against the proteins encoded by the two or more genes.

[19] The method according to

[17] or

[18] , used in a method for diagnosing giant cell arteritis.

[20] The method according to

[17] or

[18] above, wherein the sample taken from the biological tissue is a tissue sample derived from an artery.

[21] Detecting mRNA of two or more genes selected from the group consisting of MRC1, LRRC15, ACP5, ATP6V0D2, VDR, TREM2, HLA-DRA, and MMP12 in a sample collected from a biological tissue, or reacting a sample collected from a biological tissue with a substance capable of detecting a protein encoded by the two or more genes, thereby detecting the protein; A method for diagnosing giant cell arteritis using the obtained detection results as an indicator.

[22] The method according to

[21] above, wherein the detectable substance is a substance capable of staining the proteins encoded by the two or more genes and / or an antibody against the proteins encoded by the two or more genes.

[23] The method according to

[21] or

[22] above, wherein the sample taken from the biological tissue is a tissue sample derived from an artery. Effect of the Invention

[0012] According to the present invention, it is possible to provide a marker for detecting multinucleated giant cells (MNGC) present in inflamed tissues of giant cell arteritis (GCA) and a marker for diagnosing GCA, which enable detection of MNGC and definitive diagnosis of GCA. Furthermore, according to the present invention, it is also possible to provide a detection agent or composition for detecting MNGC, a diagnostic agent or composition for diagnosing GCA, a microarray for detecting MNGC, a microarray for diagnosing GCA, a kit for detecting MNGC, a kit for diagnosing GCA, a method for detecting the presence or absence of MNGC, and a method for diagnosing GCA. [Brief description of the drawings]

[0013] [Figure 1] Figures showing the results of histological observations of GCA and non-GCA arteries subjected to transcriptome analysis. (A) Representative histological images of temporal artery (TA) biopsies (TAB) obtained from the same subjects as in the microarray analysis, stained with HE. Scale bar: 500 μm. (B) Enlarged images of the boxed areas in each figure in (A). Scale bar: 100 μm. (C) EVG-stained photographs of the same TAB as in (A). Scale bar: 500 μm. (D) Enlarged images of the boxed areas in each figure in (C). Disorders of elastin (stained black) and collagen (stained pink) fibers were observed. Arrows indicate typical multinucleated giant cells (MNGCs), and the areas surrounded by dotted lines indicate areas where multinucleated giant cells are accumulated (Figures 1B and 1D). Scale bar: 100 μm. [Diagram 2]Figure 1 shows the results of transcriptome analysis of temporal artery biopsies. (A) Principal component analysis of the transcriptome of TAB from GCA or non-GCA specimens. Each dot represents a specimen. K-means clustering (k=2) was employed, and specimens classified into two clusters are shown in red (typical GCA) and blue (non-GCA / atypical GCA). Closed squares (■) indicate typical GCA and open squares (□) indicate atypical cases of specimens diagnosed with GCA. Closed circles (●) indicate typical non-GCA specimens, and open circles (○) indicate non-GCA specimens with a PAN (polyarteritis nodosa) diagnosis. (B) Volcano plot of differentially expressed genes (DEGs) between typical GCA and non-GCA / atypical GCA clusters. Grey: no significant expression difference; light pink: increased expression in typical GCA (log2 fold change>1, p<0.05); light blue: decreased expression in typical GCA (log2 fold change<-1, p<0.05). P values ​​were calculated by false discovery rate (FDR) corrected empirical Bayes moderated T statistic in the R package Limma. For ease of visualization, only protein-coding genes are shown.The positions and names of representative genes are shown in black and red (specifically, MARCO, Macrophage receptor with collagenous structure; MT1H, Metallothionein 1H; CHI3L1, Chitinase 3 like 1; IGLL5, Immunoglobulin lambda like polypeptide 5; CCL18, CC motif chemokine ligand 18; FBP1, Fructose-bisphosphatase 1; SOST, Sclerostin; ACTA1, Actin alpha 1, skeletal muscle; ITLN, Intelectin 1; MYOC, Myocilin. Dark red dots and names showed the genes the expression of which were validated by immunohistochemistry (figure 3). MMP12, Matrix metallopeptidase 12; LRRC15, Leucine rich repeat containing 15; MRC1, Mannose receptor C-type 1; ACP5, Acid phosphatase 5, tartrate resistant; TREM2, Triggering receptor expressed on myeloid cells 2; VDR, Vitamin D receptor; HLA-DRA, Major histocompatibility complex, class II, DR alpha; ATP6V0D2, ATPase H+ transporting V0 subunit d2). The genes in red were confirmed to be expressed by immunohistochemistry (specifically, MMP12, LRRC15, MRC1, ACP5, TREM2, VDR, HLA-DRA, ATP6V0D2). (C) Osteoclast marker genes overlaid on the volcano plot.Fourteen osteoclast-related marker genes (specifically, MMP9, matrix metallopeptidase 9; DCSTAMP, dendrocyte expressed seven transmembrane protein; CA2, carbonic anhydrase 2; VAV3, Vav guanine nucleotide exchange factor 3; CTSK, cathepsin K; CLCN7, chloride voltage-gated channel 7; TCIRG1, T cell immune regulator 1, ATPase H+ transporting V0 subunit a3; ACP5, acid phosphatase 5; ATP6V0D2, ATPase H+ transporting V0 subunit d2; TNFRSF11A, TNF receptor superfamily member 11a; CALCR, calcitonin receptor; NFATC1, nuclear factor of activated T cells 1; ITGAV, integrin subunit alpha V; ITGB3, integrin subunit beta) were identified. 3) Genes whose expression was significantly increased (adjp<0.05 and log2fc>1) are shown in red. [Diagram 3] Figure 1 shows the results of immunohistochemical analysis of DEG expression. (A) FFPE specimens of TAB from a GCA patient (G7) were stained with the indicated antibodies. (B) Magnification of the boxed areas in each panel in (A). [Figure 4] FIG. 1 shows the experimental conditions, the pH of the buffer used for epitope retrieval, the origin of the antibody, and the concentration used. [Diagram 5] FIG. 1 shows the results of a control experiment using an isotype antibody. [Figure 6] FIG. 13 shows results using other GCA vessels. [Figure 7] FIG. 1 shows the results of a control experiment using non-GCA blood vessels. [Figure 8] Figure 1 shows the results of a comparative transcriptome analysis between C-GCA and other inflammatory diseases. (A) Enrichment analysis of overlapping DEGs. Fisher's exact test was used to evaluate the enrichment of DEGs between GCA and other diseases. (B) Enrichment analysis of downregulated overlapping DEGs. (C) Venn diagram showing DEGs commonly upregulated in GCA, varicella zoster virus (VZV) antigen-negative GCA (VZV-GCA), and large vessel GCA (LV-GCA) derived from inflammatory aortic aneurysm (LV). Osteoclast marker genes are shown in red. (D) Venn diagram showing common DEGs regulated in GCA, Kawasaki disease (KD), Takayasu's arteritis (TAK), and atherosclerosis (AT). Osteoclast marker genes are shown in red. (E) Venn diagram showing common DEGs upregulated in GCA, rheumatoid arthritis (RA), tuberculosis (TB), and sarcoidosis (SA). Osteoclast marker genes are shown in red. (F) Enrichment analysis of osteoclast differentiation pathway (KEGG:04380). Upregulated DEGs (log2fc>1, adjp<0.05) from GCA and other diseases were analyzed using the g:Profiler online tool. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present invention will be described in detail below. The scope of the present invention is not limited to these descriptions, and other than the following examples, the present invention can be appropriately modified and implemented without departing from the spirit of the present invention. All publications cited in this specification, such as prior art documents, publications, patent publications, and other patent documents, are incorporated herein by reference.

[0015] 1. Overview of the Invention Giant cell arteritis (GCA) is one of the most common vasculitis in large blood vessels. Chronic arterial inflammation is often accompanied by thickening of the arterial wall, impairing blood flow. It may eventually cause blindness, one of the most serious complications of GCA, and increase the risk of myocardial infarction and aortic aneurysm. However, it remains intractable because its molecular pathogenesis is unknown.

[0016] The temporal artery (TA) is the most frequently affected artery in cranial-type GCA (C-GCA). The pathological features of arterial lesions are massive macrophage infiltration and the appearance of multinucleated giant cells (MNGCs) scattered in the tunica media, and the smooth muscle layer and elastic fiber layer are often destroyed. In addition, C-GCA often shows thickening of the intimal layer due to myofibroblast hyperplasia. We aimed to clarify the comprehensive molecular profile of GCA as a basis for understanding the pathogenesis at the molecular and cellular levels. To address this issue, we performed microarray analysis using fresh TA biopsies (TABs) to evaluate differentially expressed genes. Specimens were collected from patients suspected of GCA, consisting of GCA (n = 16) and non-GCA (n = 10), and finally, GCA (n = 10) and non-GCA (n = 6) samples were selected for analysis. RNA was extracted from the isolated TAB specimens and comprehensive gene expression profiling was performed using microarrays. After data analysis, immunohistochemical staining was performed. As a result, it was found that gene expression profiles obtained from GCA arteries showed an inflammatory signature and were clearly distinguished from those obtained from non-GCA arteries. The involvement of the microglial pathogen phagocytosis pathway and the TYROBP pathway, as well as immune-related pathways and macrophages, was shown. Of note, osteoclast-specific genes were found among the GCA signature genes, and their expression was confirmed in MNGCs adjacent to damaged extracellular matrix using immunohistochemistry. The GCA signature genes show considerable overlap with genes in other inflammatory diseases, including tuberculous granulomatosis, but osteoclast marker genes were excluded in Kawasaki disease and rheumatoid arthritis. The present inventors have revealed that tissue-destructive MNGCs in GCA have osteoclast-like characteristics. This finding indicates the possibility that osteoclast-like MNGCs endowed with immune-regulatory and hard tissue-resorbing functions may cause GCA pathology. The present invention was discovered based on the above findings and the like.

[0017] 2. A marker for detecting multinucleated giant cells (MNGC) and for diagnosing giant cell arteritis (GCA) As described above, the marker for detecting MNGC of the present invention comprises mRNA of two or more genes selected from the group consisting of MRC1 (Mannose receptor C-type 1), LRRC15 (Leucine rich repeat containing 15), ACP5 (Acid phosphatase 5, tartrate resistant), ATP6V0D2 (ATPase H+ transporting V0 subunit d2), VDR (Vitamin D receptor), TREM2 (Triggering receptor expressed on myeloid cells 2), HLA-DRA (Major histocompatibility complex, class II, DR alpha), and MMP12 (Matrix metallopeptidase 12), or comprises proteins encoded by the two or more genes. The marker for detecting MNGC of the present invention is used, for example, in a method for diagnosing GCA. Therefore, the marker for detecting MNGC of the present invention can also be used as a marker for diagnosing GCA.

[0018] When the presence of mRNA (preferably, increased mRNA amount) of the above two or more genes or the expression (preferably, increased expression) of the protein encoded by the above two or more genes is observed in a sample collected from the biological tissue of a subject, it can be determined or predicted that the subject is more likely to have MNGC or has a high risk of suffering from or suffering from GCA, compared with a case where such a presence is not observed. The sample collected from the biological tissue of a subject is not limited, and for example, a tissue sample derived from an artery (preferably, a tissue sample derived from the temporal artery) as well as blood (preferably, peripheral blood) can be used (hereinafter the same in the present invention), but a tissue sample derived from the temporal artery is more preferable.

[0019] In the marker for detecting MNGC and the marker for diagnosing GCA of the present invention, the two or more types of genes (combination of genes) are not limited, but may be, for example, a gene comprising MRC1 and at least one selected from the group consisting of LRRC15, ACP5, ATP6V0D2, VDR, TREM2, HLA-DRA, and MMP12; a gene comprising MRC1 and ACP5 and / or ATP6V0D2; and Genes including MRC1, ACP5, and ATP6V0D2 and the like are preferred. Here, MRC1 is a marker gene characteristic of macrophages, and ACP5 and ATP6V0D2 are marker genes characteristic of osteoclasts. Other genes LRRC15, VDR, TREM2, HLA-DRA, and MMP12 are genes whose expression is observed in various cells including macrophages, and their functions have been elucidated. It has been known that multinucleated giant cells (MNGCs) are differentiated from macrophages, but the present inventors have found for the first time that in MNGCs, not only the expression of marker genes characteristic of macrophages, but also the expression of marker genes characteristic of osteoclasts and other genes whose functions have been elucidated, are observed.

[0020] In the present invention, the giant cell arteritis (GCA) to be judged or predicted as above is not limited, and includes GCA that can occur in all vascular tissue sites, such as cranial GCA (C-GCA), large vessel GCA (LV-GCA), and mixed GCA (M-GCA). Among these, C-GCA is preferred because it allows for more accurate judgment or prediction of the presence or high risk of developing C-GCA.

[0021] 3. Detection agent or composition for MNGC, diagnostic agent or composition for GCA As described above, the MNGC detection agent or detection composition of the present invention comprises a nucleic acid probe capable of binding to the mRNA of two or more genes selected from the group consisting of MRC1, LRRC15, ACP5, ATP6V0D2, VDR, TREM2, HLA-DRA, and MMP12, or comprises a substance capable of detecting a protein encoded by the two or more genes. The MNGC detection agent or detection composition of the present invention is used, for example, in a method for diagnosing GCA. Therefore, the MNGC detection agent or detection composition of the present invention can also be used as a GCA diagnostic agent or diagnostic composition.

[0022] By using the MNGC detection agent or detection composition, or the GCA diagnostic agent or diagnostic composition of the present invention, when the presence of mRNA (preferably, increased mRNA amount) of the above-mentioned two or more genes, or the expression (preferably, increased expression) of proteins encoded by the two or more genes is observed in a sample taken from a subject's biological tissue, particularly in a tissue sample derived from an artery (preferably, a tissue sample derived from the temporal artery), it can be determined or predicted that the subject is more likely to have MNGC, or that the subject is suffering from or at high risk of suffering from GCA, compared to when such presence is not observed.

[0023] The explanation in Section 2 above is similarly applicable to preferred combinations of the above-mentioned two or more genes in the MNGC detection agent or composition for detection, or the GCA diagnostic agent or composition for diagnosis of the present invention, and to the giant cell arteritis (GCA) to be diagnosed.

[0024] In the present invention, the nucleic acid probe capable of binding to the mRNA of the two or more genes is not limited in terms of its nucleic acid sequence or structure, and any nucleic acid probe capable of binding to a part or all of the mRNA sequence in a sequence-specific manner can be appropriately designed and selected for use. Specifically, a nucleic acid probe capable of detecting (quantifying a certain amount of) the mRNA of the two or more genes contained in a sample collected from a subject's biological tissue can be used, and the probe may be designed for each of a plurality of regions (e.g., 3 to 10 regions per gene) in the target mRNA sequence (within the gene sequence), and only the probe sequence that is complementary to the target mRNA may be amplified. By preparing a plurality of probes for one gene, specificity for the target mRNA can be guaranteed, and the target mRNA can be quantified specifically and with high sensitivity from an mRNA sample of reduced quality or a sample containing a trace amount of mRNA. Such a quantification method can be used, for example, in a method for analyzing the expression of a large number of genes (e.g., hundreds of types) on a formalin-fixed tissue slide (quantitatively analyzing the target mRNA). The nucleic acid probe may be prepared as a probe that can be used in quantitative PCR (qPCR, real-time PCR). When a sufficient amount of target mRNA is expected to be present in a sample, the nucleic acid probe may be prepared as a nucleic acid probe to be mounted on a microarray, for example.

[0025] In the present invention, the substance capable of detecting the protein encoded by the above two or more genes is not limited, but may be, for example, a substance capable of detecting the protein (target protein) present in a sample collected from a subject's biological tissue using a known detection means or detection technique (for example, immunohistochemical staining, Western blotting, etc.), and may include, for example, an antibody capable of specifically binding to the target protein, and an antibody fragment thereof, but is not particularly limited thereto. In addition, the detectable substance may also include, for example, various known substances capable of staining the target protein, and may be used in staining by an immunostaining method (antibody staining method) together with the above-mentioned antibody or antibody fragment.

[0026] As a detection method using the detectable substance, a known detection method, for example, an immunoassay method, an immunostaining method, etc., can be used according to the usual method. For example, the immunoassay method includes a labeled immunoassay method (including an immunostaining method) and a turbidimetric immunoassay (TIA). In the former case, preferred examples include an enzyme immunoassay (EIA) such as an ELISA, as well as a radioimmunoassay (RIA) and a fluorescent immunoassay (FIA). In addition, preferred examples of the labeled immunoassay method include a Western blot method (combined with an electrophoresis method) that utilizes a combination with another separation method. Among these, ELISA and / or Western blot methods are preferred.

[0027] The antibody capable of specifically binding to the target protein may be a polyclonal or monoclonal antibody, or may be a recombinant antibody (e.g., a chimeric antibody, a humanized antibody, and a human antibody (fully human antibody)). The antibody fragment refers to a partial region of the various antibodies (i.e., an antibody fragment derived from the various antibodies), and examples thereof include Fab, Fab', F(ab')2, Fv (variable fragment of antibody), single-chain antibody (H chain, L chain, H chain V region, L chain V region, etc.), scFv, diabody (scFv dimer), dsFv (disulfide stabilized V region), and peptides containing at least a portion of a complementarity determining region (CDR). The various antibodies and antibody fragments described above can be prepared by various known methods.

[0028] As the nucleic acid probe capable of binding to the mRNA of the above-mentioned two or more genes, and the substance capable of detecting the protein encoded by the above-mentioned two or more genes, those that have been independently synthesized, prepared, extracted, purified, etc. may be used, but there is no limitation thereto, and known or commercially available substances may also be used.

[0029] In the MNGC detection agent or composition, or the GCA diagnostic agent or composition of the present invention, the blending ratio of the nucleic acid probe or the detectable substance is not particularly limited, but is preferably, for example, 1 to 100% by weight, and more preferably 10 to 100% by weight. The MNGC detection agent or composition, or the GCA diagnosis agent or composition of the present invention may contain other components in addition to the nucleic acid probe or the detectable substance, as long as the effects of the present invention are not significantly impaired. For example, the other components may include a primary antibody detection reagent, a color-developing substrate, etc.

[0030] In addition, the present invention also includes a method for detecting MNGC and a method for diagnosing GCA, which are characterized by using a nucleic acid probe capable of binding to the mRNA of the two or more genes, or a substance capable of detecting the proteins encoded by the two or more genes, and the use of a nucleic acid probe capable of binding to the mRNA of the two or more genes, or a substance capable of detecting the proteins encoded by the two or more genes, for producing a drug for use in the method for detecting MNGC or the method for diagnosing GCA.

[0031] More specifically, the above-mentioned method for detecting MNGC and the method for diagnosing GCA are as follows: Detecting mRNA of two or more genes selected from the group consisting of MRC1, LRRC15, ACP5, ATP6V0D2, VDR, TREM2, HLA-DRA, and MMP12 in a sample collected from a biological tissue, or reacting a sample collected from a biological tissue with a substance capable of detecting a protein encoded by the two or more genes, thereby detecting the protein; Examples of the method include a method for detecting the presence or absence of MNGC and a method for diagnosing GCA, using the obtained detection result as an index.

[0032] 4. MNGC detection kit, GCA diagnosis kit As described above, the MNGC detection kit and GCA diagnosis kit of the present invention include a nucleic acid probe capable of binding to the mRNA of two or more genes selected from the group consisting of MRC1, LRRC15, ACP5, ATP6V0D2, VDR, TREM2, HLA-DRA, and MMP12, or the probe is immobilized on a carrier, or includes a substance capable of detecting a protein encoded by the two or more genes. The MNGC detection kit of the present invention is used, for example, in a method for diagnosing GCA. Therefore, the MNGC detection kit of the present invention can also be used as a diagnostic kit for GCA.

[0033] The explanation in the above section 2. can be similarly applied to preferred combinations of the above two or more types of genes in the MNGC detection kit and GCA diagnosis kit of the present invention, and to the giant cell arteritis (GCA) to be diagnosed.

[0034] The nucleic acid probe capable of binding to the mRNA of the two or more genes and the substance capable of detecting the protein encoded by the two or more genes, which are included in the MNGC detection kit and GCA diagnosis kit of the present invention, are similarly applicable to the explanation in the above section 3. The nucleic acid probe immobilized on a carrier is not limited to a particular type, and various carriers that can be used in microarrays, panel assays, and the like can be used.

[0035] By using the MNGC detection kit or GCA diagnosis kit of the present invention, when the presence of mRNA (preferably, increased mRNA amount) of the above-mentioned two or more genes or the expression (preferably, increased expression) of the protein encoded by the two or more genes is observed in a sample taken from a subject's biological tissue, particularly in a tissue sample derived from an artery (preferably, a tissue sample derived from the temporal artery), it can be determined or predicted that the subject is more likely to have MNGC or is suffering from or at high risk of suffering from GCA, compared to when such presence is not observed.

[0036] In the kit of the present invention, when the kit contains a nucleic acid probe capable of binding to the mRNA of the two or more genes or a substance capable of detecting a protein encoded by the two or more genes, the nucleic acid probe or the detectable substance may be stored, for example, in a powdered state or in a solution state (dissolved in pure water, buffer, physiological saline, or the like) under refrigerated or frozen conditions (-30°C to -80°C) in consideration of its stability (storage property), ease of use, etc. In particular, when the kit contains an antibody or an antibody fragment as the detectable substance, it is preferably provided in a dissolved state in consideration of its stability (storage property), ease of use, etc.

[0037] The kit of the present invention may contain other components in addition to the nucleic acid probe, or the probe immobilized on a carrier, or the detectable substance. Examples of other components include a primary antibody detection reagent and a chromogenic substrate that can be used in ELISA or Western blot. The kit may contain at least the nucleic acid probe, or the probe immobilized on a carrier, or the detectable substance as a component. Therefore, all of the components essential for detecting MNGC or diagnosing GCA may or may not be included together with the nucleic acid probe, or the probe immobilized on a carrier, or the detectable substance, and are not particularly limited.

[0038] 5. Others, pharmaceutical compositions, etc. The present invention can also provide a pharmaceutical composition for preventing or treating giant cell arteritis (GCA). By suppressing or inhibiting the generation or proliferation of multinucleated giant cells (MNGCs) that may be present in inflamed tissues of GCA, the effect of preventing or treating GCA is expected. Specifically, the pharmaceutical composition for preventing or treating GCA according to the present invention is preferably characterized by comprising, as an active ingredient, a compound that inhibits or suppresses the expression (e.g., protein synthesis from the mRNA of the gene) of two or more genes selected from the group consisting of MRC1, LRRC15, ACP5, ATP6V0D2, VDR, TREM2, HLA-DRA, and MMP12, or a compound having inhibitory or suppressive activity against a protein encoded by the two or more genes, or a prodrug of the compound, or a pharmacologically acceptable salt thereof, or a hydrate or solvate thereof (hereinafter, for convenience, these are referred to as "inhibitor A"). The pharmaceutical composition of the present invention is not limited to those used for preventing or treating GCA, and may also be used for preventing or treating various vascular disorders and vascular diseases caused by GCA. In addition, in the present invention, the treatment of GCA, etc., specifically includes, for example, recovery of vascular disorder symptoms in GCA, or the continuation and / or resumption of the recovery action, and also includes inhibition of the progression of GCA and improvement of prognosis.

[0039] In the present invention, for example, (i) a pharmaceutical composition for use in a method for preventing or treating GCA in a subject (patient), the pharmaceutical composition comprising an inhibitor A as an active ingredient; (ii) a preventive or therapeutic agent for GCA, comprising inhibitor A; (iii) a method for preventing or treating GCA, comprising using inhibitor A, specifically administering to a subject an effective amount of inhibitor A; (iv) use of inhibitor A for the manufacture of a medicament for the prevention or treatment of GCA; (v) use of inhibitor A for the prevention or treatment of GCA; and (vi) Inhibitor A for the prevention or treatment of GCA The present invention may also include the above-mentioned inventions.

[0040] Inhibitor A, which is an active ingredient of the pharmaceutical composition of the present invention, may be synthesized, extracted, purified, or the like, but is not limited thereto, and known or commercially available inhibitors may also be used.

[0041] In the present invention, the compound that inhibits or suppresses the expression of the two or more genes (for example, protein synthesis from the mRNA of the gene) as the inhibitor A is not limited, but may be, for example, a compound containing a nucleic acid that contains a sequence complementary to at least a part of the base sequence of the gene. Here, information on the sequence of each target gene is registered and published on the website of GenBank of the National Center for Biotechnology Information (NCBI) (https: / / www.ncbi.nlm.nih.gov / genbank / ) and can be referred to as appropriate.

[0042] In the present invention, the type of nucleic acid containing a sequence complementary to at least a part of the base sequence of each target gene is not limited, and may have a single-stranded structure or a double-stranded structure, and examples thereof include antisense nucleic acids (RNA degrading antisense nucleic acids (Gapmer antisense nucleic acids), splicing control antisense nucleic acids, etc.), short interfering RNA (siRNA), and short hairpin RNA (shRNA). The base length of these nucleic acids can be appropriately designed according to the type of nucleic acid and the mode of use, and is not limited, for example, as long as it is 5 to 50 bases. The nucleic acid used in the present invention may be DNA or RNA, and the nucleotides constituting the nucleic acid may be natural nucleotides or modified or artificial nucleotides (e.g., nucleotide mimics such as LNA (Locked Nucleic Acid), peptide nucleic acid (PNA), phosphorothioate nucleic acid, and morpholino nucleic acid (morpholino oligo)), or a combination thereof. The nucleic acid used in the present invention is not limited to a nucleic acid containing a sequence complementary to at least a part of the base sequence of each target gene, and for example, a nucleic acid containing a sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 95%, 96%, 97%, 98%, or 99% sequence identity to the complementary sequence is also preferably used. In addition, the nucleic acid used in the present invention may be, for example, a nucleic acid (modified form) modified so as to improve the thermal stability of a complex with at least a part of the base sequence of each target gene. Examples of the modified form include a nucleic acid in which all or some of the bases are modified, a nucleic acid in which the sugar moiety of the base, i.e., the sugar moiety of all or some of the bases, and a nucleic acid in which all or some of the phosphodiester bonds of the nucleic acid are modified.

[0043] Nucleic acids that can be used in the present invention can be synthesized by known chemical synthesis methods or enzymatic transcription methods, but are not limited thereto. Examples of chemical synthesis methods include the phosphorothioate method, the phosphoramidite method, and the phosphotriester method, and examples of enzymatic transcription methods include methods using RNA polymerase.

[0044] In the present invention, the compound having inhibitory or suppressive activity against the proteins encoded by the two or more genes, which is inhibitor A, is not limited to, but may be designed or selected from various known or appropriately synthesized compounds that can be used in so-called protein knockdown technology, and examples thereof include compounds that degrade or induce degradation of the protein, and compounds that bind to the protein and inhibit or suppress the activity or function of the protein (e.g., antibodies or antibody fragments against the protein, or decoy nucleic acids, etc.), which may be used in appropriate combination. As the antibody or antibody fragment, the antibodies and antibody fragments described in section 3 above can be similarly used.

[0045] In the present invention, a derivative of the inhibitor A may be used together with or instead of the inhibitor. The derivative may be any derivative of the inhibitor based on the technical common sense of a person skilled in the art, and is not limited thereto. It may also include compounds that undergo metabolism such as oxidation, reduction, hydrolysis, or conjugation in vivo, as well as compounds (so-called prodrugs) that generate the inhibitor or its derivatives by metabolism such as oxidation, reduction, or hydrolysis in vivo. It is preferable that the above derivative has at least the same degree of inhibition or suppression of the expression of each target gene or the same degree of inhibition or suppression activity of the target protein as that of inhibitor A.

[0046] The inhibitor A that can be used in the present invention can also be used in the form of its pharmacologically acceptable salt. Such salts are not limited to, but include, for example, hydrohalides (e.g., hydrochlorides, hydrobromides, and hydroiodides), inorganic acid salts (e.g., sulfates, nitrates, perchlorates, phosphates, carbonates, and bicarbonates), organic carboxylates (e.g., acetates, trifluoroacetates, maleates, tartrates, fumarates, and citrates), organic sulfonates (e.g., methanesulfonates, trifluoromethanesulfonates, ethanesulfonates, benzenesulfonates, toluenesulfonates, and camphorsulfonates), amino acid salts (e.g., aspartates, and glutamates), quaternary amine salts, alkali metal salts (e.g., sodium salts, and potassium salts), and alkaline earth metal salts (e.g., magnesium salts, and calcium salts). Furthermore, inhibitor A may exist in the form of a hydrate or solvate, which may also be used as an active ingredient in the pharmaceutical composition and therapeutic agent of the present invention.

[0047] The content ratio of inhibitor A as an active ingredient in the pharmaceutical composition of the present invention is not limited and can be set appropriately, but can be, for example, within the range of 0.01 to 99% by weight, or may be within the range of 0.01 to 30% by weight, 0.05 to 20% by weight, or 0.1 to 10% by weight, based on the total weight of the pharmaceutical composition. The administration route of the pharmaceutical composition according to the present invention is not limited, and any administration route, such as injection (intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, intra-articular injection), transdermal (external application), intranasal, transmucosal, intranasal, intratracheal, oral, or inhalation, may be adopted as long as inhibitor A, which is an active ingredient, can function in the living body.

[0048] The dosage form of the pharmaceutical composition according to the present invention is not limited, and examples thereof include injections (including drops), inhalants, liquids, emulsions, suspensions, solutions, ointments, nasal drops, tablets, granules, sprays, capsules, syrups, suppositories, tapes, and liposomes. The pharmaceutical composition according to the present invention may contain pharmacologically acceptable carriers, diluents, excipients, binders, disintegrants, lubricants, flavorings, colorants, sweeteners, flavorings, suspending agents, wetting agents, emulsifiers, dispersants, adjuvants, preservatives, buffers, stabilizers, coating agents, etc. that are commonly used in the pharmaceutical field.

[0049] The dosage of the pharmaceutical composition according to the present invention may be any dosage that provides a therapeutically effective amount of inhibitor A as an active ingredient against GCA. The therapeutically effective amount can be appropriately changed depending on the severity of the pathological condition of the patient, sex, age, body weight, etc. For example, about 1 μg to about 2000 mg, more preferably about 10 μg to about 1000 mg, and more preferably about 100 μg to about 500 mg per kg of the patient's body weight can be administered in one administration. The administration can be once to several times a day, for example, divided into 4 to 6 times, or can be administered intermittently once every few days or weeks.

[0050] In the present invention, a kit containing inhibitor A can also be used for preventing or treating GCA. The form of inhibitor A in the kit is not limited, but may be provided in a dissolved state, for example, in consideration of stability (storage) and ease of use. The kit may contain other components as appropriate in addition to inhibitor A. The kit may be one that contains at least the above-mentioned inhibitor A as a component. Therefore, all of the components essential for preventing or treating GCA may be provided together with inhibitor A, or may be provided separately, and is not limited.

[0051] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these. EXAMPLES

[0052] <Summary> In this example, we first revealed the profile of differentially expressed genes (DEGs) in inflammatory TA by cDNA microarray analysis. Signature genes of GCA lesions indicated that macrophage-related cells play a central role in pathogenesis. Notably, several signature genes of osteoclast lineage were detected among the upregulated DEGs. Immunohistochemical analysis showed that MNGCs found in GCA arteries indeed expressed osteoclast markers. Considering the localization of MNGCs in the vicinity of damaged tissues, an osteoclast-like, tissue resorbing function of these MNGCs in GCA is inferred. Finally, comparison with datasets from other inflammatory diseases showed that RA and KD did not share osteoclast markers, but showed a significant overlap of shared DEGs. Taken together, these results suggest that chronic inflammatory diseases, including GCA, have closely related yet distinct molecular pathologies in terms of osteoclast markers.

[0053] <Method> subject In this example, a temporal artery biopsy (TAB) was performed on a patient suspected of GCA for diagnosis by routine pathological examination. A portion of the TAB (<10 mm) was taken and stored in All-protect solution (Qiagen) at 4°C for RNA extraction.

[0054] RNA extraction and microarray analysis Preserved tissues were homogenized in Trizol reagent using a Tissue-Lyzer (Qiagen), and RNA was extracted using the RNeasy kit (Qiagen). RNA quality was assessed with a 2100 Bioanalyzer (Agilent Technologies). Cy3-labeled cRNA was prepared using the Low Input Quick Amp Labeling Kit (Agilent Technologies) according to the manufacturer's instructions. Labeled samples were hybridized to Agilent SurePrint G3 Human GE Ver.3.0 Array (8 × 60K), washed, and scanned using a SureScan Microarray Scanner (Agilent Technologies). Signal intensity was measured using Feature Extraction software (Agilent Technologies) and analyzed using GeneSpring GX software (Agilent Technologies).

[0055] statistical analysis To identify differentially expressed genes (DEGs), we first defined differentially expressed probes (DEPs) between the typical GCA cluster and the non-GCA and atypical GCA clusters. As a result, 3745 probes significantly increased expression and 3706 probes decreased expression (log2fc>1, corrected p-value<0.05). Comparison between GCA and non-GCA based on clinical diagnosis or after removing G2, G4, and N5 by distinct features (PCA, histology, clinical findings) shows that the upregulated DEPs are almost overlapped between the typical GCA and the non-GCA and atypical GCA clusters. Next, the probe-based microarray data (Agilent probe IDs) were converted to gene-based data by a canonical approach to define DEGs. In cases where probe sets for the same gene overlapped, we decided to represent the expression value of the gene by the probe showing the highest value on average of 16 samples, as verified by other researchers. As a result, the volcano plot of DEGs showed 2858 up-regulated and 2744 down-regulated genes in typical GCA clusters. To calculate biological pathway enrichment scores, gene names or gene IDs (if available) of DEGs were uploaded to g:Profiler (https: / / biit.cs.ut.ee / gprofiler).

[0056] Histology and immunohistochemistry (IHC) analysis Formalin-fixed paraffin-embedded (FFPE) samples available from each patient were sectioned at 4 μm thickness for histological examination, hematoxylin and eosin (HE), Elastica van Gieson (EVG) and picro sirius red (PSR) staining. For IHC, FFPE sections were deparaffinized, rehydrated and then subjected to heat-induced epitope retrieval (HIER) in 10 mM citrate buffer (pH 6), 10 mM Tris-EDTA buffer (pH 9) or 1 mM EDTA (pH 8) according to the antibody used (Figure 4). Endogenous peroxidase activity was blocked with 0.5% hydrogen peroxide in 10 mM phosphate-buffered saline (PBS, pH 7.4) for 30 min. Sections were then washed with PBS containing 0.03% Triton X-100 (PBST), blocked for 20 min in PBS containing 5% normal bovine serum (hereafter referred to as NBS+PBS), and incubated with primary antibodies diluted in NBS+PBS overnight at 4°C. Slides were incubated with biotinylated secondary antibodies (1:1000) for 2 h at room temperature, visualized using a standard peroxidase-based method (Vectastain Elite, ABC kit, Vector Laboratories, Burlingame CA, USA) and 3,3'-diaminobenzadine (DAB) as a chromogen, and heavy stained with hematoxylin. Between all incubations, sections were washed three times for 10 min with PBST. Images for HE staining, EVG staining, and IHC were obtained using an inverted Keyence BZ-X810 microscope (Keyence) and a CFI Plan Apo 20× / 0.75 objective lens (Nikon). PSR staining photographs were taken with a polarizing microscope BX53 (Olympus).

[0057] <Result> Histological analysis (Figure 1) Collection of temporal artery biopsies from patients with suspected giant cell arteritis (GCA) Twenty-six patients suspected of GCA and who underwent temporal artery biopsy (TAB) for diagnosis were collected, excluding patients undergoing treatment. Small blocks (<10 mm) were taken from the obtained specimens and stored at 4°C in All-protect solution (Qiagen). Histopathological examination of the specimens was performed using standard procedures. Of the patients from whom specimens were collected, 16 were diagnosed with GCA and 10 with non-GCA. Considering the pathological analysis and clinical findings, 10 GCA specimens and 6 non-GCA specimens were selected as comparison groups and further analyzed in this example. Representative histopathological images stained with HE are shown (Figure 1A, B). In the histopathological images of G5 and G7, which are typical GCA samples, the media was infiltrated with large amounts of macrophages and MNGCs. Elastica van Gieson (EGV) staining further demonstrated fragmentation of elastic lamina and collagen fibers in the medial layer, with proximal localization of MNGCs (Fig. 1C, D). Some of the GCA samples also showed damage to the smooth muscle layer. Furthermore, intimal hyperplasia due to proliferation of fibroblast-like cells, along with ablation of collagen fibers, was evident in most GCA cluster specimens. Similarly, PSR staining and polarized light microscopy demonstrated disappearance of type I collagen fibers in the intima of GCA TAB. Taken together, these studies recapitulated the pathology of chronic granulomatous inflammation and deconstruction of extracellular matrix fibers.

[0058] Gene expression signature in typical GCA vessels (Figure 2) Gene expression profiling was performed using a microarray platform, and principal component analysis (PCA) was performed (Figure 2A). As a result, it was found that the majority of GCA (hereafter referred to as "typical GCA") was clearly separated from other specimens in the space of the first and second principal components. Of note, two of the 10 cases diagnosed as GCA (G2 and G4) belonged to a group consisting mainly of non-GCA specimens. The patient in G2 underwent spontaneous remission, while G4 was diagnosed as an atypical GCA case based on the age at onset and other clinical values. Therefore, in the following, the group consisting of non-GCA and atypical GCA is referred to as the "non-GCA / atypical GCA" cluster. The classification of "typical GCA" versus "non-GCA / atypical GCA" was confirmed by hierarchical clustering. The results suggested that the typical GCA cluster had a gene expression profile that was distinct from the others, consistent with the histological examination. We next performed an analysis of differentially expressed genes (DEGs), which revealed that 2,858 genes were upregulated and 2,744 genes were downregulated. A volcano plot was drawn using protein-coding genes in the DEGs (Figure 2B). We found that immune-related genes that play a central role in adaptive immune responses, such as IGLL5, MZB1, and JCHAIN ​​in B cells and CTLA4 in T cells, were upregulated. However, overall, macrophage signature genes were clearly dominant (MMP7, MMP9, MMP12, CXCL9, CXCL10, CCL18, CHI3L1, MARCO, and FBP1). Conversely, genes related to epithelial and smooth muscle cells were included in the list of downregulated genes. This observation is consistent with established views on GCA. Interestingly, of the 14 osteoclast marker genes, the expression of 9 genes, ACP5, MMP9, DCSTAMP, CA2, VAV3, ATP6V0D2, CLCN7, and TCIRG1, was upregulated (Figure 2C).

[0059] Next, functional enrichment of upregulated genes was evaluated. In the KEGG database, immune cell function and inflammation-related disease pathways were detected, including (1) cytokine-cytokine receptor interactions, (2) rheumatoid arthritis, (3) hematopoietic cell lineage, (4) inflammatory bowel disease, and (5) tuberculosis. Using the WikiPathways database, (1) TYROBP-related networks in microglia, (2) SARS-CoV-2 activation of extrafollicular and follicular B cells, (3) SARS-CoV-2 signaling pathway networks, (4) allograft rejection, and (5) microglial pathogen phagocytosis pathways were detected. As microglia are closely related to macrophages, our results indicate that crosstalk with viral infection responses and the function of macrophage lineage cells play an important role in GCA pathology. In the downregulated genes, only the dilated cardiomyopathy pathway in the KEGG database was detected. Furthermore, the cellular composition of GCA-affected sites was evaluated using the x Cell platform. Consistent with statistical analyses (PCA and hierarchical clustering) using whole transcriptome data, we reproduced the clear classification of typical and non-GCA / atypical GCA clusters. The typical GCA cluster showed high enrichment values ​​for CD4+ T cells, CD8+ T cells, and macrophages, in addition to multipotent immune progenitor cells. In contrast, the non-GCA / atypical GCA cluster suggested enrichment for osteoblasts, fibroblasts, and chondrocytes.

[0060] Osteoclast-like phenotype of multinucleated giant cells (MNGCs) in GCA (Figures 3, 5–7) Comprehensive gene expression analysis showed unexpected upregulation of osteoclast-specific genes such as ACP5, CA2, and ATP6V0D2, in addition to macrophage-related signatures such as FBP1, CHI3L1, and MARCO, which have been reported to be expressed in GCA arteries. MRC1 (CD206) has been established as a marker of immunosuppression and tissue remodeling, i.e., M2 macrophages, and has already been shown to be expressed in macrophages in GCA arteries (references 3 and 4, supra). The inventors confirmed that MNGCs and their surrounding cells were positive for MRC1, indicating the progression of a type II immune response involving M2 macrophages in the affected arteries. Recently, it has been reported that LRRC15 is a marker of myofibroblasts that contribute to immunosuppressive, i.e., type II immune responses, and the inventors have demonstrated that myofibroblasts in the GCA intima express LRRC15. Next, we investigated the expression of ATP6V0D2 and ACP5, which are representative osteoclast markers, and found that both were expressed in MNGCs. ATP6V0D2 positivity was observed in the media and intima of GCA and non-GCA TABs, albeit at low levels. We further investigated the expression of vitamin D receptor (VDR), which has recently been shown to be expressed in osteoclasts and involved in bone homeostasis. VDR signaling was detected in MNGCs and their neighboring cells, probably macrophages and epithelioid cells, in GCA, but not in non-GCA blood vessels. It has been shown that the TYROBP / TREM2 pathway plays an important role in osteoclast differentiation. We then investigated the expression of TREM2, and found that MNGCs were TREM2 positive. It has also been proposed that disease-related osteoclast-like cells differentiate from MHC-II positive dendritic cells and stimulate T cells. In fact, HLA-DRA expression was confirmed in MNGCs in GCA blood vessels. MMP12 is (1) one of the top five genes most highly expressed in typical GCA clusters (log2fc=6.4), (2) an elastin-degrading enzyme whose substrate is extracellular matrix fibers including elastin, and (3) has recently been shown to be involved in granuloma formation and may be a therapeutic target for sarcoidosis granulomas (reference 5 cited above).For these reasons, we investigated the expression of MMP12, and the results showed that MMP12 was expressed in MNGCs of GCA blood vessels.

[0061] Granulomas and crosstalk with other vasculitides (Figure 8) We investigated the gene signature of GCA and other vasculitis and inflammatory diseases, including rheumatoid arthritis (RA) and granulomatosis. We selected four DEG sets derived from vascular inflammatory diseases: large vessel GCA (LV-GCA) derived from inflammatory aortic aneurysm (LV), VZV antigen-negative GCA (VZV-GCA), Kawasaki disease (KD), and Takayasu's arteritis (TAK). A threshold of adjp<0.05 was used to select DEGs from each dataset (only the top 300 and bottom 300 gene lists for TAK).

[0062] Among them, the gene groups whose gene names matched those of our GCA data were used as the common gene set in each comparison. The number of overlapping DEGs (adjp<0.05 log2fc>1) was calculated, and Venn diagrams were created, and the number of overlapping genes was analyzed by Fisher's exact test. Despite the differences in affected tissues, namely aorta (large vessel type GCA (LV-GCA) and TAK), coronary artery (KD), synovial fluid (RA), and lymph node (TB and SA), significant enrichment was detected in all pairwise comparisons examined here (Figures 8A, 8B). In the three GCA-derived datasets (GCA, VZV, LV), 189 common DEGs were upregulated, including two osteoclast marker genes, MMP9 and ACP5 (Figure 8C). Compared with other vasculitides, some of the osteoclast marker genes were shared with TAK and AT (55 hub genes were examined in AT), but not with KD (Figure 8D). Interestingly, six osteoclast marker genes, MMP9, CTSK, DCSTAMP, CLCN7, CA2 and ATP6V0D2, were shared with TB or SA granulomas, but not with RA. Seventy common DEGs were detected among GCA, RA, TB and SA (Fig. 8E). Taken together, DEGs with increased expression in KD and RA were significantly shared with GCA, except for osteoclast maker genes. As for the osteoclast differentiation pathway (KEGG:04380), the enrichment scores of RA and KD were lower than others (Fig. 8F).

[0063] <Consideration> Whole transcriptome from arterial biopsies revealed significant induction of immune-related genes, especially those involved in the functions of macrophages, T cells, and B cells. Similarly, pathway analysis of upregulated DEGs detected the TYROBP phagocytosis pathway, suggesting that the phagocytic activity of macrophages or their relatives may be behind the pathology of GCA. Furthermore, a significant increase in the expression of osteoclast signature genes was observed in typical GCA clusters. Considering that osteoclasts are physiological MNGCs differentiated from macrophages, the involvement of macrophage-derived osteoclast-like MNGCs with phagocytic activity was strongly suggested. Indeed, the inventors showed that MNGCs in inflamed arteries expressed osteoclast markers ACP5 and ATP6V0D2. Furthermore, MNGCs in GCA were positive for TREM2, HLA-DRA, and MMP12, indicating that the characteristics of MNGCs in GCA lesions are similar to those of osteoclasts. Furthermore, when combined with DEG lists published by other researchers, we found overlapping profiles of DEGs derived from granulomas and RA as well as other inflammatory vasculitis. These findings will provide new information on the molecular pathology of GCA and pave the way for developing therapeutic strategies for GCA. Microarray data combined with histological examination obtained from the same specimens showed clear correlations between histological analysis and gene expression profiles. Among the GCA samples, G5 and G7 were plotted close to each other on PCA and showed common pathological features with numerous MNGC infiltrations on histological staining. In contrast, G2 and G4 were similar to non-GCA in histological features as well as gene expression profiles. Non-GCA sample N5 was diagnosed with polyarteritis nodosa, another type of arteritis. Its histopathology did not show clear inflammatory features and was also close to G2 and G4 on PCA. Thus, it was suggested that these three samples may form an intermediate third cluster. Pathway analysis accompanying the identification of DEGs revealed close involvement of macrophage lineage cells.Pathway analysis indicates the close involvement of macrophage lineage cells, consistent with previous reports showing increased expression of macrophage-specific genes such as chemokine family (CXCL9, CXCL10), matrix metalloproteinase family (MMP9), and CHI3L1. Meanwhile, increased expression of the TREM2 gene in GCA is intriguing, considering that TREM2-expressing microglia are associated with amyloid plaque lesions in Alzheimer's disease. Similarities between amyloid plaques and granulomas have been shown, and TREM2-dependent phagocytosis by macrophage lineage cells may play a common role in pathogenesis.

[0064] A detailed examination of the DEGs revealed obvious upregulation of osteoclast signature genes, including ACP5 (log2fc=4.9), CA2 (log2fc=3.5), ATP6V0D2 (log2fc=3.5), DCSTAMP (log2fc=5.3), and MMP9 (log2fc=5.4). Osteoclasts are giant cells with multiple nuclei differentiated from macrophages that resorb mineralized materials such as bone. Despite their morphological similarity to osteoclasts, the expression of osteoclast markers on MNGCs in GCA has not yet been confirmed in detail. We confirmed the expression of ACP5 and ATP6V0D2 in MNGCs of GCA arteries, as well as VDR, TREM2, HLA-DRA, and MMP12. ACP5 and ATP6V0D2 are well-confirmed markers and have been shown to be involved in osteoclast function and bone homeostasis. The VDR pathway plays a pivotal role in calcification and calcium metabolism in the cardiovascular system. Among them, expression of the CYP27B1 gene, which encodes an enzyme essential for synthesizing active vitamin D, was also strongly increased (log2fc=5.5), indicating that both vitamin D production and its receptor-induced signaling were ongoing in the inflamed arteries.

[0065] MNGCs are often localized in close proximity to damaged areas of the elastic lamina and collagen bundles surrounding the smooth muscle layer. Furthermore, MMP12, one of the most efficient elastin-degrading proteases, was positive, suggesting that MNGCs expressing MMP12 damaged elastic fibers composed of elastin. Based on these findings, we hypothesized that MNGCs in GCA lesions may have a hard tissue resorption function similar to osteoclasts, and we named this cell population "myoclasts." Myoclasts strongly express HLA-DRA, but its expression in osteoclasts has not been confirmed, and the two may be different. Finally, TREM2 is also expressed in myoclasts, and it has been shown to associate with TYROBP and constitute a signaling pathway that confers cell fusion ability to macrophages. On the other hand, the phagocytic function of TREM2-expressing microglia has been suggested in relation to Alzheimer's disease. It is interesting to note that both cell fusion ability and phagocytosis have been shown to have a common molecular basis in the fluidity of the cell membrane. Taken together, these findings suggest that myoclasts may play a central role in the pathogenesis of GCA by damaging the extracellular matrix of the smooth muscle layer (MMP12), ingesting foreign substances (phagocytosis), and presenting them to T cells (HLA-DRA). Whole transcriptome data paint a picture of the molecular architecture underlying the disease pathology. Comparison of transcriptomes from various pathologies showed that GCA was closely correlated not only with other vasculitides but also with granulomas in pulmonary lymph nodes and synovial fluid in RA. Interestingly, DEGs regulated in KD and RA were found to overlap in GCA, even though the osteoclast signature was excluded in both diseases. This suggests that the molecular pathology common to GCA may be overlooked in KD and RA. Meanwhile, comparison with tuberculosis and SA granulomas further strengthens the similarity of the molecular pathology of GCA and granulomas. Considering that some osteoclast markers are shared, osteoclast-like MNGCs may have a central role in granuloma-like diseases and represent a common therapeutic target.

Claims

1. A marker for detecting multinucleated giant cells, comprising mRNA of two or more genes selected from the group consisting of MRC1, LRRC15, ACP5, ATP6V0D2, VDR, TREM2, HLA-DRA, and MMP12, or comprising proteins encoded by said two or more genes.

2. The marker according to claim 1, which is used in a method for diagnosing giant cell arteritis.

3. A diagnostic marker for giant cell arteritis, comprising mRNA of two or more genes selected from the group consisting of MRC1, LRRC15, ACP5, ATP6V0D2, VDR, TREM2, HLA-DRA, and MMP12, or comprising a protein encoded by said two or more genes.

4. A detection agent or composition for detecting multinucleated giant cells, comprising a nucleic acid probe capable of binding to the mRNA of two or more genes selected from the group consisting of MRC1, LRRC15, ACP5, ATP6V0D2, VDR, TREM2, HLA-DRA, and MMP12, or comprising a substance capable of detecting proteins encoded by said two or more genes.

5. The detection agent or composition for detection according to claim 4 , wherein the detectable substance is a substance capable of staining the protein encoded by the two or more genes and / or an antibody against the protein encoded by the two or more genes.

6. The detection agent or composition for detection according to claim 4 or 5, which is used in a method for diagnosing giant cell arteritis.

7. A diagnostic agent or composition for diagnosis of giant cell arteritis, comprising a nucleic acid probe capable of binding to the mRNA of two or more genes selected from the group consisting of MRC1, LRRC15, ACP5, ATP6V0D2, VDR, TREM2, HLA-DRA, and MMP12, or comprising a substance capable of detecting a protein encoded by said two or more genes.

8. 8. The diagnostic agent or composition according to claim 7, wherein the detectable substance is a substance capable of staining the proteins encoded by the two or more genes, or an antibody against the proteins encoded by the two or more genes.

9. A microarray for detecting multinucleated giant cells, comprising a nucleic acid probe capable of binding to the mRNA of two or more genes selected from the group consisting of MRC1, LRRC15, ACP5, ATP6V0D2, VDR, TREM2, HLA-DRA, and MMP12.

10. The microarray of claim 9 for use in a method for diagnosing giant cell arteritis.

11. A diagnostic microarray for giant cell arteritis, comprising a nucleic acid probe capable of binding to the mRNA of two or more genes selected from the group consisting of MRC1, LRRC15, ACP5, ATP6V0D2, VDR, TREM2, HLA-DRA, and MMP12.

12. A kit for detecting multinucleated giant cells, comprising a nucleic acid probe capable of binding to the mRNA of two or more genes selected from the group consisting of MRC1, LRRC15, ACP5, ATP6V0D2, VDR, TREM2, HLA-DRA, and MMP12, or comprising the probe immobilized on a carrier, or comprising a substance capable of detecting proteins encoded by the two or more genes.

13. The kit according to claim 12 , wherein the detectable substance is a substance capable of staining the proteins encoded by the two or more genes and / or an antibody against the proteins encoded by the two or more genes.

14. The kit according to claim 12 or 13, which is used in a method for diagnosing giant cell arteritis.

15. A diagnostic kit for giant cell arteritis, comprising a nucleic acid probe capable of binding to the mRNA of two or more genes selected from the group consisting of MRC1, LRRC15, ACP5, ATP6V0D2, VDR, TREM2, HLA-DRA, and MMP12, or comprising the probe immobilized on a carrier, or comprising a substance capable of detecting proteins encoded by the two or more genes.

16. The kit according to claim 15 , wherein the detectable substance is a substance capable of staining the proteins encoded by the two or more genes, or an antibody against the proteins encoded by the two or more genes.

17. Detecting mRNA of two or more genes selected from the group consisting of MRC1, LRRC15, ACP5, ATP6V0D2, VDR, TREM2, HLA-DRA, and MMP12 in a sample collected from a biological tissue, or reacting a sample collected from a biological tissue with a substance capable of detecting a protein encoded by the two or more genes, thereby detecting the protein; A method for detecting the presence or absence of multinucleated giant cells using the detection results obtained as an indicator.

18. The method according to claim 17 , wherein the detectable substance is a substance capable of staining the proteins encoded by the two or more genes and / or an antibody against the proteins encoded by the two or more genes.

19. 19. The method according to claim 17 or 18, used in a method for diagnosing giant cell arteritis.

20. 19. The method according to claim 17 or 18, wherein the sample taken from the biological tissue is a tissue sample derived from an artery.

21. Detecting mRNA of two or more genes selected from the group consisting of MRC1, LRRC15, ACP5, ATP6V0D2, VDR, TREM2, HLA-DRA, and MMP12 in a sample collected from a biological tissue, or reacting a sample collected from a biological tissue with a substance capable of detecting a protein encoded by the two or more genes, thereby detecting the protein; A method for diagnosing giant cell arteritis using the obtained detection results as an indicator.

22. The method according to claim 21 , wherein the detectable substance is a substance capable of staining the proteins encoded by the two or more genes and / or an antibody against the proteins encoded by the two or more genes.

23. 23. The method according to claim 21 or 22, wherein the sample taken from the biological tissue is a tissue sample derived from an artery.

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