Patent Application
20230295280
The present application contains a Sequence Listing, which is being submitted via EFS-Web on even date herewith. The Sequence Listing is submitted in a file entitled “Sequence_Listing.txt,” which was created on Apr. 30, 2021, and is approximately 17 kb in size. This Sequence Listing is hereby incorporated by reference.
The present invention relates to a dendritic cell maturation-suppressing agent, a dendritic cell maturation-suppressing method, a pharmaceutical composition, and a method for screening a dendritic cell maturation-suppressing agent or a terapeutic ingredient for a dendritic cell-mediated disease.
Antigen-presenting cells such as dendritic cells and macrophages express a diverse array of costimulatory receptors. The costimulatory receptors transmit activation signals to T cells cooperatively with interactions of MHC class I molecules and MHC class II molecules with T cell receptors to thereby activate acquired immune responses. Furthermore, the costimulatory receptors also transmit inhibitory signals for T cell anergy. These positive and negative signals are delicately controlled by interactions of receptors and ligands. Therefore, appropriate signaling by receptor-ligand axes of the antigen-presenting cells is extremely important for regulating immune response via T cells. For example, autoimmune diseases such as rheumatoid arthritis are closely related to a decrease in costimulation signals.
A leukocyte immunoglobulin-like receptor (LILR) family includes a number of immunoglobulin-like receptors. Some of the immunoglobulin-like receptors are expressed in myeloid cells such as dendritic cells and macrophages and mediate costimulation to immune cells. Some reports in the past suggested that genes in the LILR family are closely related to autoimmune diseases.
Recently, TARM1 (T cell-interacting, activating receptor on myeloid cells-1) was identified as an immunoglobulin-like receptor coded by a leukocyte-receptor complex region of the LILR family (see Non-Patent Document 1). The TARM1 is composed of two extracellular immunoglobulin-like domains, a transmembrane domain, and a short cytoplasmic domain having no typical signal motif and specifically associates with an FCRγ chain, an adapter molecule having ITAM (Immunoreceptor tyrosine-based activation motif). As a result of an amino acid sequence analysis, the TARM1 has been found to be highly homologous with human monocyte-derived dendritic cells or OSCAR (Osteoclast-associated receptor) mediating costimulation of mouse osteoclasts. The TARM1 promotes production of TNF-a and IL-6 from macrophages and neutrophils by cross-linking with anti-TARM1 monoclonal antibodies in the presence of Toll-like receptor (TLR) ligands. Furthermore, inflammatory stimuli such as LPSs (lipopolysaccharides) facilitate an expression of a TARM1 gene.
Patent Document 1 describes, for example, that dendritic cells express TARM (e.g., TARM1), that inflammatory stimuli facilitate an expression of the TARM, that the dendritic cells adhere to T cells via the TARM, that anti-TARM antibodies suppress the dendritic cells from adhering to the T cells, and that anti-TARM antibodies have a therapeutic effect for collagen-induced arthritis (CIA). However, a relationship between the TARM1 and maturation of the dendritic cells has still not be known.
Patent Document 1: PCT International Publication No. WO2007/037430
Non-Patent Document 1: V. Radjabova et al., J. Immunol., 195, 3149-3159, 2015
An object of the present invention is to provide a novel, dendritic cell maturation-suppressing agent capable of suppressing dendritic cell maturation; a dendritic cell maturation-suppressing method using such a maturation-suppressing agent; a pharmaceutical composition containing such a maturation-suppressing agent as an active ingredient; a method for screening a dendritic cell maturation-suppressing agent; and a method for screening a therapeutic ingredient for a dendritic cell-mediated disease.
Specific means for solving the above-mentioned object include the following embodiments.
<1> A dendritic cell maturation-suppressing agent being capable of inhibiting binding between a TARM1 protein and a collagen protein to suppress dendritic cell maturation.
<2> The dendritic cell maturation-suppressing agent according to <1>, the agent being capable of binding to a collagen-binding domain in the TARM1 protein or the collagen protein.
<3> The dendritic cell maturation-suppressing agent according to <2>, the agent being an antibody capable of binding to the collagen-binding domain in the TARM1 protein or the collagen protein, or a functional fragment of the antibody.
<4> The dendritic cell maturation-suppressing agent according to <2>, the agent being the TARM1 protein or the collagen protein, or a functional equivalent thereof.
<5> The dendritic cell maturation-suppressing agent according to <4>, the agent being any one of polypeptides (a) to (d) below or a fusion protein of the polypeptide with an IgG Fc region:
<6> The dendritic cell maturation-suppressing agent according to <4>, the agent being a collagen polypeptide, a polypeptide including a collagen-like domain, or a fusion protein of the collagen polypeptide or the polypeptide including a collagen-like domain with an IgG Fc region.
<7> A method for suppressing dendritic cell maturation, the method including:
bringing the dendritic cell maturation-suppressing agent according to any one of <1> to <6> into contact with an immature dendritic cell.
<8> A pharmaceutical composition for use in treating a dendritic cell-mediated disease, including the dendritic cell maturation-suppressing agent according to any one of <1> to <6> as an active ingredient.
<9> The pharmaceutical composition according to <8> for use in treating rheumatoid arthritis.
<10> A screening method, including:
<11> A screening method, including:
The present invention makes it possible to provide a novel, dendritic cell maturation-suppressing agent capable of suppressing dendritic cell maturation; a dendritic cell maturation-suppressing method using such a maturation-suppressing agent; a pharmaceutical composition containing such a maturation-suppressing agent as an active ingredient; a method for screening a dendritic cell maturation-suppressing agent; and a method for screening a therapeutic ingredient for a dendritic cell-mediated disease.
A dendritic cell maturation-suppressing agent according to the present embodiment inhibits a binding of a TARM1 protein to a collagen protein to thereby suppress maturation of the dendritic cell.
A TARM1 protein is a protein expressed on a cell membrane of a dendritic cell and is known to exhibit enhanced expression upon inflammatory stimulation. Nucleotide sequence information on a gene coding for the TARM1 protein and amino acid sequence information on the TARM1 protein are available from the GenBank database at the National Center for Biotechnology Information (NCBI). By way of example, nucleotide and amino acid sequences of human TARM1 are shown in SEQ ID NOs: 1 and 2, and nucleotide and amino acid sequences of mouse TARM1 are shown in SEQ ID NOs: 3 and 4.
The present inventors have found that the TARM1 protein expressed on the cell membrane of the dendritic cell binds to a type II collagen protein also expressed on the cell membrane of the dendritic cell, in other words, the type II collagen protein is a ligand for the TARM1 protein; that the type II collagen protein regulates maturation of the dendritic cell in a TARM1-dependent manner; that the maturation of the dendritic cell is suppressed by inhibiting the binding of the TARM1 protein to the type II collagen protein using a fusion protein (TARM1-Fc) of an extracellular domain of the TARM1 protein with an IgG Fc region, and the like.
Furthermore, the present inventors also have found that the TARM1 protein binds to a type I collagen protein, in other words, the type I collagen is a ligand for the TARM1 protein. It is known that the maturation of the dendritic cell is promoted upon binding to the type I collagen protein (U. Brand et al., Eur. J. Immunol., 28, 1673-1680, 1998). Therefore, the maturation of the dendritic cell can be suppressed by inhibiting the binding of the TARM1 protein to the type I collagen protein.
The dendritic cell maturation-suppressing agent according to the present embodiment can suppress a dendritic cell-mediated immune response by suppressing the maturation of the dendritic cell. Therefore, the dendritic cell maturation-suppressing agent according to the present embodiment can be used as, for example, a therapeutic ingredient for a dendritic cell-mediated disease. In fact, the present inventors have confirmed that TARM1-Fc has a therapeutic effect in a collagen-induced arthritis (CIA) model. Note that, the collagen-induced arthritis (CIA) model is a model of rheumatoid arthritis which is one of autoimmune diseases.
The dendritic cell maturation-suppressing agent according to the present embodiment is not particularly limited as long as the agent has an effect of inhibiting the binding of the TARM1 protein to the collagen protein. Examples of the dendritic cell maturation-suppressing agent include a molecule inhibiting the binding of the TARM1 protein to the collagen protein by binding to a collagen-binding domain in the TARM1 protein, a molecule inhibiting the binding of the TARM1 protein to the collagen protein by binding to the collagen protein, and the like.
The dendritic cell maturation-suppressing agent according to the present embodiment may be an antibody binding to the collagen-binding domain in the TARM1 protein or the collagen protein, or a functional fragment thereof.
Examples of the antibody binding to the collagen-binding domain in the TARM1 protein include those recognizing a part or whole of the collagen-binding domain in the TARM1 protein as an epitope. As mentioned below, two immunoglobulin-like domains in the TARM1 protein have a binding capability for the collagen protein. Therefore, for example, those recognizing a part or whole of at least one immunoglobulin-like domain as an epitope can be used as the antibody binding to the TARM1 protein.
Examples of the antibody binding to the collagen protein include those recognizing at least part of the type I collagen protein or the type II collagen protein as an epitope.
An isotype of the antibody is not particularly limited and may be any of IgG, IgM, IgA, IgD, and IgE. Furthermore, the antibody may be a polyclonal antibody or a monoclonal antibody. Examples of the monoclonal antibody include a monoclonal antibody obtained via immunization of a non-human animal, a chimeric antibody, a humanized antibody, and a fully human antibody.
Examples of the functional fragment of the antibody include Fab, Fab′, F(ab′)2, Fv, and scFv.
Furthermore, the dendritic cell maturation-suppressing agent according to the present embodiment may also be a small molecule compound, a polypeptide, a protein, a polysaccharide, and a nucleic acid, in addition to the antibodies.
As mentioned above, the TARM1 protein binds to the collagen protein. Therefore, the TARM1 protein or a functional equivalent thereof may also be used as the dendritic cell maturation-suppressing agent. Examples of the functional equivalent of the TARM1 protein include any of polypeptides (a) to (d) below or a fusion protein of the polypeptide with an IgG Fc region:
The polypeptide (a) includes at least amino acid residues at positions 27 to 120 or 124 to 219 from an N terminus in an amino acid sequence of a human TARM1 protein set forth in SEQ ID NO: 2. Regions at positions 27 to 120 and 124 to 219 from the N terminus correspond to two immunoglobulin-like domains in the human TARM1 protein. The polypeptide (a) including at least one immunoglobulin-like domain in the human TARM1 protein has the binding capability for the collagen protein.
Note that, it is known that an OSCAR protein highly homologous with the TARM1 protein also has two immunoglobulin-like domains and each of the immunoglobulin-like domains has the binding capability for the collagen protein (A. D. Barrow et al., J. Clin. Invest., 121, 3505-3516, 2011; L. Zhou et al., Blood, 127, 529-537, 2016; J. Haywood et al., Proc. Natl. Acad. Sci. USA, 113, 1038-1043, 2016).
The polypeptide (b) includes at least amino acid residues at positions 27 to 120 or 124 to 218 from an N terminus in an amino acid sequence of a mouse TARM1 protein set forth in SEQ ID NO: 4. Regions at positions 27 to 120 and 124 to 218 from the N terminus correspond to two immunoglobulin-like domains in the mouse TARM1 protein. The polypeptide (b) including at least one immunoglobulin domain in the mouse TARM1 protein has the binding capability for the collagen protein.
The polypeptide (c) includes an amino acid sequence in which one or several amino acid residues are substituted for, deleted from, or added to an amino acid sequence of the polypeptide (a) or (b) and has the binding capability for the collagen protein. The number of amino acid residues to be substituted for, deleted from, or added to is not particularly limited as long as the polypeptide retains the binding capability for the collagen protein. The number of amino acid residues to be substituted for, deleted from, or added to may be, for example, 1 to 30, 1 to 25, 1 to 20, or 1 to 15.
When one amino acid is substituted with another amino acid, usually, side chains of the amino acids preferably retain their property before and after the substitution. When amino acids are classified in accordance with properties of side chains of the amino acids, for example, the amino acids may be classified into hydrophilic amino acids (D, E, K, R, H, S, T, N, and Q); hydrophobic amino acids (A, G, V, I, L, F, Y, W, M, C, and P); acidic amino acids (D and E); basic amino acids (K, R, and H); amino acids having aliphatic side chains (A, G, V, I, and L); amino acids having aromatic-containing side chains (F, Y, and W); amino acids having sulfur atom-containing side chains (M and C); and the like (alphabets within parentheses denotes one letter codes of amino acids).
The polypeptide (d) includes an amino acid sequence having 90% or more sequence identity to an amino acid sequence of the polypeptide (a) or (b) and has the binding capability for the collagen protein. The sequence identity to the polypeptide (a) or (b) may be 92% or more, 95% or more, 98% or more, or 99% or more.
Note that, the phrase “sequence identity” means consistency between sequences when two amino acid sequences are aligned and may be calculated using, for example, the BLAST program (www.ncbi.nlm.nih.gov/Blast/cgi).
Furthermore, as mentioned above, the collagen protein binds to the TARM1 protein. Therefore, the collagen protein or a functional equivalent thereof may be used as the dendritic cell maturation-suppressing agent. Examples of the functional equivalent of the collagen protein include a collagen polypeptide, a polypeptide including a collagen-like domain, and a fusion protein of the collagen polypeptide or the polypeptide including a collagen-like domain with an IgG Fc region.
A method for suppressing maturation of a dendritic cell according to the present embodiment includes bringing the above-mentioned dendritic cell maturation-suppressing agent according to the present embodiment into contact with an immature dendritic cell. The dendritic cell maturation-suppressing agent is as mentioned above and a detailed description thereof is omitted.
A method for bringing the dendritic cell maturation-suppressing agent into contact with an immature dendritic cell is not particularly limited. Examples thereof include a method of culturing the immature dendritic cell in a medium containing the dendritic cell maturation-suppressing agent.
Note that, whether the dendritic cell is an immature dendritic cell or a mature dendritic cell may be determined by, for example, analyzing maturation markers of the dendritic cell such as CD40, CD80, CD83, CD86, and I-A/I-E with flow cytometry.
A pharmaceutical composition according to the present embodiment includes the above-mentioned dendritic cell maturation-suppressing agent according to the present embodiment as an active ingredient. The dendritic cell maturation-suppressing agent is as mentioned above and a detailed description thereof is omitted.
The pharmaceutical composition according to the present embodiment can suppress the maturation of the dendritic cell, and therefore, can be suitably used for treating a dendritic cell-mediated disease. In other words, the dendritic cell-mediated disease can be treated by administering the pharmaceutical composition according to the present embodiment to a patient with the disease. Examples of the dendritic cell-mediated disease include an autoimmune disease, an allergic disease, an inflammatory disease, and the like. Examples of the autoimmune disease include rheumatoid arthritis, systemic lupus erythematosus, Sjogren’s syndrome, connective tissue disease, Guillain-Barre syndrome, myasthenia gravis, multiple sclerosis, Goodpasture’s syndrome, Basedow’s disease, and type I diabetes. Examples of the allergic disease include airway hyperreactivity (asthma), atopic dermatitis, and the like. Examples of the inflammatory disease include ulcerative colitis, Crohn’s disease, psoriasis, psoriatic arthritis, nephritis, and the like.
Note that, the term “treating” includes suppression of acuteness of symptoms of the disease, in addition to elimination or alleviation of the symptoms.
The pharmaceutical composition according to the present embodiment may include ingredients other than the dendritic cell maturation-suppressing agent. For example, the pharmaceutical composition may include an organic or inorganic carrier conventionally used as formulation materials. This carrier is formulated as an excipient, a lubricant, a binder, a disintegrant, or the like in the case of a solid formulation; or a solvent, a solubilizing agent, a suspending agent, a tonicity adjusting agent, or a buffering agent, or the like in the case of a liquid formulation. Furthermore, a therapeutic agent for the autoimmune disease may include a formulation additive such as a preservative, an antioxidant, a surfactant, a pH adjusting agent, a coloring agent, a sweetener, or the like.
A dosage form of the pharmaceutical composition is not particularly limited. Examples of the dosage form of the pharmaceutical composition include an oral preparation such as a tablet, a capsule, a granule, a powder, a troche, a syrup, an emulsion, a suspension, a film, and the like; and a parenteral preparation such as an injection, an infusion, a topical agent, a suppository, a pellet, a transnasal agent, a transpulmonary agent (inhalant), an eye drop, and the like.
A dose of the pharmaceutical composition is appropriately determined depending on a subject (age, sex, body weight, etc.), an administration route, a target disease, a symptom, and the like. Usually, the dose of 1 to 5000 mg/day, preferably 5 to 2000 mg/day, more preferably 50 to 2000 mg/day per adult (body weight: 60 kg) may be administered in one or several divided doses. However, an actual dose may be widely varied based on a physician’s discretion and may be outside of the above-mentioned range.
A first aspect of a screening method according to the present embodiment includes measuring a binding activity of any of polypeptides (a) to (d) below with a collagen protein in a presence and absence of a test substance; and selecting the test substance as a candidate for a dendritic cell maturation-suppressing agent when the binding activity in the presence of the test substance is lower than the binding activity in the absence of the test substance:
Furthermore, a second aspect of a screening method according to the present embodiment includes measuring a binding activity of any of the polypeptides (a) to (d) above with a collagen protein in a presence and absence of a test substance; and selecting the test substance as a candidate for a therapeutic ingredient for a dendritic cell-mediated disease when the binding activity in the presence of the test substance is lower than the binding activity in the absence of the test substance.
The polypeptides (a) to (d) are as mentioned above and a detailed description thereof is omitted.
The binding activity of any of the polypeptides (a) to (d) with the collagen protein may be measured according to known methods. For example, the binding activity may be measured by adding any of the polypeptides (a) to (d) to a plate on which the collagen protein is immobilized, leaving to stand for a predetermined time, removing an unbound polypeptide by, for example, washing, and measuring an amount of a bound polypeptide. When any of the polypeptides (a) to (d) is added, the polypeptide may be appropriately labeled with a label substance such as an enzyme, a radioisotope, a fluorescent dye, or the like.
The test substance is selected as a candidate for the dendritic cell maturation-suppressing agent or the therapeutic ingredient for a dendritic cell-mediated disease when the binding activity in the presence of the test substance is lower than the binding activity in the absence of the test substance. By way of example, the test substance may be selected as a candidate for the dendritic cell maturation-suppressing agent or the therapeutic ingredient for a dendritic cell-mediated disease when the binding activity in the presence of the test substance is 50% or lower relative to the binding activity in the absence of the test substance.
Note that, examples of the test substance include, but not limited to, a small molecule compound, a protein, a polypeptide, a polysaccharide, and a nucleic acid. The test substance may be a novel substance or a known substance.
Hereinafter, the present invention will be more specifically described with reference to Examples, but the present invention is not limited to the following Examples.
CD57BL/6J mice, BALBc/A mice, and DBA/1J mice were purchased from Japan SLC, Inc. or CLEA Japan, Inc. Tarm1 heterozygous deficient mice (Tarm1egfp/+ mice) and Tarm1 homozygous deficient mice (Tarm1egfp/egfp mice) were generated by homologous recombination of an exon 1 of a Tarm1 gene in the C57BL/6J mice with an eGFP gene and a neomycin-resistant gene. The Tarm1egfp/egfp mice were born at an expected Mendelian ratio and exhibited no clear abnormality. OT-II TCR transgenic mice (OT-II Tg mouse) were kindly provided by Dr. H. Kishimoto, the Research Institute for Biomedical Sciences, the Tokyo University of Science.
All mice were bred under SPF conditions in a clean room at the Research Institute for Biomedical Sciences, the Tokyo University of Science. For experiments, age (6- to 12-week-old) - and sex-matched mice were used. Animal experiments were approved by Committee at the Tokyo University of Science and conducted in accordance with the ethical guideline for animal experimentation and the guideline for safety of genetic engineering techniques.
CIA was induced as follows according to the previous report (D. D. Brand et al., Nature protocols, 2, 1269-1275, 2007; J. J. Inglis et al., Nature protocols, 3, 612-618, 2008). Complete Freund’s adjuvant (CFA; 1 mg/mL) was prepared by adding heat-killed M. tuberculosis (H37Ra; Difco) to Incomplete Freund’s adjuvant (IFA; Thermo Fisher Scientific). An emulsion was prepared by mixing 4 mg/mL of type II collagen (SIGMA-ALDRICH) with CFA at a volume ratio of 1:1. A total of 200 µL of the emulsion was intradermally administered to 3 positions near the base of the tail of each of the mice (8- to 10-week-old). Fourteen days after the priming, the same volume of the emulsion was administered as a booster. Swelling of each paw was scored and evaluated as follows: 0: No change, 1: Swelling or focal redness of the finger joint, 2: Mild swelling of the ankle joint, 3: Severe swelling of the entire paw. Scores of four paws were added, so that the maximum score was 12.
For verification of a therapeutic effect of Tarm1-Fc for autoimmune arthritis, Tarm1-Fc (1 µg per ankle joint) or IgG Fc (1 µg per ankle joint) were administered to the joint cavities of the left or right ankle joints of the DBA/1J mice in which CIA (mild score) had been induced. On day 0, male DBA⅟J mice (6- to 8-week-old) were immunized with 100 µL of emulsion composed of 2 mg/mL type II collagen and 1.65 mg/mL CFA (M. A. Murayama et al., Nature communications, 6, 8483, 2015). On day 21, the same volume of the emulsion was further administered as a booster. Progression of arthritis was scored and evaluated as follows: 0: No change, 1: Erythema and mild swelling of the tarsal joint, 2: Erythema and mild swelling extending from the tarsal joint to the digit, 3: Erythema and moderate swelling extending from the metatarsal joint, 4: Erythema and severe swelling extending to the ankle, the paw, and the digit, or ankylosis of the paw. Scores of four paws were added, so that the maximum score was 16.
The mice were sacrificed under anesthesia followed by amputating the paws. The paws were fixed in 10% neutral buffered formalin and decalcified in 10% EDTA. Preparation of paraffin-embedded sections and immunohistochemical staining were delegated to the Institute of Medical Science, the University of Tokyo. The procedures will be briefly described. Serial tissue sections (5 µm) were stained with hematoxylin and eosin (HE) and safranin O, and scored and evaluated as follows (J. J. Inglis et al., Nature protocols, 3, 612-618, 2008): 0: No inflammatory sign, 1: Mild inflammation with synovial surface hyperplasia but without cartilage erosion, 2: Change of Grade 1 and granulomatous lesion in the synovial sublining, 3: Change of Grade 2, and pannus formation and cartilage/bone erosion. Indexes of arthritis in the ankle joints were estimated from grades of the bones including the talus, the tibia, and the calcaneus.
Chicken type II collagen-specific IgG was measured for antibody titer by ELISA. Sera were collected on day 42 after immunization. 96-well plates were coated with chicken type II collagen (20 µg/mL), added with serially diluted sera, and incubated at room temperature for 1 hour. The plates were washed with PBS/0.05% Tween 20, added with an alkaline phosphatase-labeled rabbit anti-mouse IgG antibody (Zymed), and incubated at room temperature for 1 hour. Then, SIGMA 104 phosphatase substrate (SIGMA-ALDRICH) was added thereto and absorbance at a wavelength of 415 nm was measured with a microplate reader (MTP-300; Hitachi).
Flow cytometry was performed according to the previous report (R. Yabe et al., FEBS Journal, 277, pp. 4010-4026, 2010). The procedures will be briefly described. Cells were blocked with a 2.4G2 antibody and then stained by treating with a fluorescence-labeled antibody at 4° C. for 30 minutes. Then, the cells were analyzed with a flow cytometer (FACS Canto II or FACSVerse; Becton Dickinson) and the resultant data were analyzed with FlowJo (Tree Star). Dead cells were stained with 7AAD (SIGMA-ALDRICH) and PI (SIGMA-ALDRICH).
For staining a dendritic cell subset, the lymph nodes were subjected to enzymolysis with 200 U/mL of type VIII collagenase (SIGMA-ALDRICH).
For cell sorting, a single cell suspension was incubated with a biotinylated antibody and then with anti-biotin beads (Miltenyi Biotec). The thus-labeled cells were isolated with a magnetic cell sorting system (autoMACS; Miltenyi Biotec) in a negative selection mode. Negative cells were stained with a fluorescence-labeled antibody, and CD11c+ cells and CD4+CD3+ cells were highly purified with a flow cytometer (FACS Aria II (Becton Dickinson) or MoFlo XDP (Beckman Coulter)).
For screening of a Tarm1 ligand, cells were blocked with a 2.4G2 antibody and then incubated with Tarm1-Fc on ice for 1 hour. Then, the cells were washed and further incubated with a PE-labeled anti-human IgG-Fc antibody (HP6017; BioLegend).
Antibodies used for the cytometry analysis are presented in Table 1.
The inguinal lymph nodes were isolated from mice sacrificed on day 10 after priming. Single-cell suspensions were prepared from the draining lymph nodes and cultured in 96-well plates in the presence of heat-denatured chicken type II collagen (2 x 105/well) at predetermined concentrations. After incubation for 66 hours, the cells were labeled by treating with [3H] thymidine (0.25 µCi/mL; PerkinElmer) for 6 hours. The cells were harvested with Micro 96 cell harvester (Skatron) and [3H] radioactivity was measured with Micro Beta Systems (Pharmacia Biotech). Culture supernatants were collected 66 hours after stimulation.
Production of cytokines was measured using Mouse TNF ELISA MAX (BioLegend), Duo set IFN-γ (R&D systems), Duo set IL-17 (R&D systems), Cytometric Bead Array (CBA) mouse TNF Flex Set (BD Biosciences), CBA mouse IL-6 Flex Set (BD Biosciences), and CBA mouse IL-10 Flex Set (BD Biosciences).
RNA was extracted from tissues and cells and purified with Sepasol-RNA I Super (Nacalai Tesque) and GenElute mammalian total RNA miniprep kit (SIGMA-ALDRICH). The resultant RNA was reverse-transcribed with high capacity cDNA reverse transcription kit (Applied Biosystems). For quantitative-PCR, SYBR Premix Ex Taq I kit (TaKaRa) or SYBRP Premix Ex Taq II kit (TaKaRa) was used along with primer sets presented in Table 2 below.
Bone marrow was extracted from the tibia and the femur of C57BL/6J mice and bone marrow cells were collected by erythrocyte destruction with a hemolysis buffer (140 mM NH4Cl, 17 mM Tris-HCl, pH 7.2).
For preparation of GM-CSF-induced dendritic cells (GM-DCs), the bone marrow cells were cultured in an RPMI 1640 medium containing 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 0.1% 2-mercaptoethanol, and recombinant mouse GM-CSF (2 to 20 ng/mL; Peprotech) at a cell density of 2 x 105/mL. On day 3 of culture, the same volume of the medium was further added to wells. On days 6 and 8 of culture, low-adherent cells were collected and cultured in a fresh medium containing GM-CSF (1 to 10 ng/mL). Non-adherent cells were used as GM-DCs.
For preparation of Flt3L-induced dendritic cells (FL-DCs), the bone marrow cells were cultured in an RPMI 1640 medium containing 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 0.1% 2-mercaptoethanol, and recombinant mouse Flt3L (100 ng/mL; Peprotech) at a cell density of 2 x 106/mL. Adherent cells were collected by pipetting with 2.5 mM EDTA/PBS and used as FL-DCs.
For preparation of M-CSF-induced bone marrow-derived macrophages (BMMs), the bone marrow cells were cultured in an RPMI 1640 medium containing 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 0.1% 2-mercaptoethanol, and recombinant human M-CSF (20 ng/mL; R&D systems) at a cell density of 2 x 106/mL. On day 3 of culture, a half volume of the medium was further added to wells. Adherent cells were collected by detaching the cells with 2.5 mM EDTA/PBS and used as BMMs.
GM-DCs were cultured at 37° C. for predetermined periods of time in the presence of LPS (100 ng/mL; SIGMA-ALDRICH) of E. coli O111:B4, CpG (1 µM; Eurofins Genomics), poly(I:C) (100 µg/mL; InvivoGen), TNF (10 ng/mL; Peprotech), IL-1β (10 ng/mL; Peprotech), or type II collagen (1 µg/mL, 10 µg/mL, or 100 µg/mL; SIGMA-ALDRICH).
DNA microarray analysis was delegated to The Chemicals Evaluation and Research Institute, Japan. The procedures will be briefly described. The extent of degradation and quality of RNA were checked with 2100 Bioanalyzer (Agilent Technologies). The RNA was reverse-transcribed to cDNA and then transcribed to Cy3-labeled cRNA, a transcript, using Low Input Quick Amp Labeling Kit (Agilent). The resultant sample was hybridized to SurePrint G3 Mouse GE Microarray (Ver.2.0) in an oven and the microarray was scanned with DNA Microarray Scanner (Agilent Technologies).
Genes of which expression was down-regulated to half or less in GM-DCs of Tarm1egfp/egfp mice were subjected to a gene ontology analysis with PANTHER Overrepresentation Test and modified with Bonferroni correction.
Cells isolated from the spleens and the lymph nodes of OT-II Tg mice were blocked with a 2.4G2 antibody, treated with a biotinylated anti-CD11c antibody (BioLegend), a biotinylated anti-CD11b antibody (BioLegend), or a biotinylated anti-B220 antibody (BioLegend), and treated with anti-biotin beads (Miltenyi Biotec). The thus-labeled cells were removed with a magnetic cell sorting system (autoMACS; Miltenyi Biotec). A negative fraction was further treated with anti-CD4 beads (Miltenyi Biotec) and the thus-labeled cells (CD4+ T cells) were collected with a magnetic cell sorting system. The CD4+ T cells were incubated with 5 µM CFSE (Invitrogen) at 37° C. for 5 minutes. Then, the thus-CFSE-labelled CD4+ T cells (2 x 105/well) were co-cultured with GM-DCs (2 x 104/well) at 37° C. for 3 days in the presence of OVA (1 µg/mL, 10 µg/mL, or 100 µg/mL). Cell proliferation was analyzed with flow cytometry.
IgG Fc fusion proteins were synthesized according to the previous report (R. Yabe et al., FEBS Journal, 277, 4010-4026, 2010). Lipofectamine LTX (Invitrogen) was used to transfect 293T cells with plasmids. Twenty-four hours after the transfection, the medium was exchanged with an OPTI-MEM medium (Invitrogen) containing 2% low-IgG FBS (Invitrogen). Then, fusion proteins secreted into the medium were purified by affinity chromatography with a Protein A Sepharose carrier.
Cells were treated with a trypsin-EDTA solution (0.05% or 0.02%; SIGMA-ALDRICH) or 200 U/mL collagenase (Wako chemicals) at 37° C. for 30 minutes.
Maxisorp 96-well plates (ThermoFisher SCIENTIFIC) were coated with type I collagen, type II collagen, or BSA (0 to 10 µg/mL), washed with a washing buffer (TBS containing 0.05% Tween 20 and 2 mM CaCl2), and then blocked with a blocking buffer (TBS containing 0.05% Tween 20 and 5% BSA). A binding buffer (TBS containing 0.05% Tween 20, 2 mM CaCl2, and 0.5% BSA) containing the IgG Fc fusion protein (5 µg/mL) was incubated at room temperature for 2 hours. An HRP-labeled goat anti-human IgG antibody (Fcγ fragment-specific; 5000-fold diluted; Jackson ImmunoResearch) was incubated at room temperature for 1 hour. Then, SureBlue TMB Microwell Peroxidase Substrate (SeraCare) was added as a color development reagent to each well and the reaction was quenched with 1N HCl. Color development was monitored at a wavelength of 450 nm using iMark microplate reader (BIO-RAD).
Cells were lysed with TBS containing 1% NP-40 and a protease inhibitor cocktail (for mammal cell/tissue extracts) (Nacalai tesque). The resultant cell lysate was pre-cleared and then incubated at 4° C. overnight with a Protein A Sepharose carrier on which Tarm1-Fc or IgG Fc had been immobilized. After washing, the resultant slurry was heated at 95° C. for 5 minutes with an SDS sample buffer. The thus-heated sample was centrifuged, the supernatant thereof was subjected to SDS-PAGE and then electroblotting onto a PVDF membrane. Then, the PVDF membrane was blocked with 4% Block Ace (DS Pharma Biomedical Co.), incubated with biotinylated anti-type II collagen antibodies (A2-10, F10-21, D8-6, and D1-2G; Chondrex), and further incubated with HRP-labeled streptavidin (BioLegend). Thereafter, the PVDF membrane was incubated with ECL Prime Western Blotting Detection System (GE Healthcare) and chemiluminescence was detected with a digital imager (LAS 4000 system; Fuji Film Life Science).
For statistical processing, the two-tailed Student’s t-test, the chi-square test, and the Mann-Whitney U-test were used and a p-value less than 0.05 was determined as statistically significant (*p < 0.05; **p < 0.01; ***p < 0.001). Data were presented as mean ± SD.
It has been conventionally suggested by a gene expression analysis that an immunoglobulin-like gene in a leukocyte immunoglobulin-like receptor (LILR) family is involved in progression of arthritis (N. Fujikado et al., Arthritis research & therapy, 8, R100, 2006). Therefore, firstly, expression of a Tarm1 gene in the ankles of IL-1 receptor antagonist gene (Il1rn) deficient mice (Il1rn-/- mice) and human T-cell leukemia virus type I (HTLV-I) transgenic mice (HTLV-I Tg mice) (R. Horai et al., The Journal of experimental medicine, 191, 313-320, 2000; Y. Iwakura et al., Science, 253, 1026-1028, 1991) was verified. Both the Il1rn-/- mice and the HTLV-I Tg mice spontaneously develop arthritis similar to rheumatoid arthritis.
Then, a correlation between the expression of the Tarm1 gene and expression of inflammatory cytokine (TNF, IL-6, and IL-1β) genes was examined.
Then, Tarm1 homozygous deficient mice (Tarm1egfp/egfp mice) were examined for sensitivity against collagen-induced arthritis (CIA).
Then, immune cell (CD11c+, I-A/I-E+CD11c+, CD44+CD4+, and CD19+) populations in the Tarm1egfp/egfp mice and the WT mice on day 42 after immunization were analyzed by flow cytometry.
Then, antibody titers of serum IgGs specific for type II collagen in the Tarm1egfp/egfp mice and the WT mice were measured with ELISA.
It was suggested from the above results that the Tarm1egfp/egfp mice were more resistant to CIA compared to the WT mice.
Firstly, the Tarm1egfp/egfp mice and the WT mice on day 10 after CIA induction were verified for proliferating recall ability of the draining lymph nodes (dLNs). On day 10 after CIA induction, the lymph nodes were collected from the Tarm1egfp/egfp mice and the WT mice, re-stimulated with type II collagen at various concentrations in vitro for 66 hours, and then measured for [3H]-thymidine uptake into an acid precipitable fraction. The results are shown in
Then, amounts of proinflammatory cytokines contained in culture supernatants after the dLN cells were re-stimulated with the type II collagen at various concentrations in vitro for 66 hours were measured with ELISA.
Then, the Tarm1egfp/egfp mice and the WT mice on day 10 after CIA induction were verified for activation of dendritic cells in the draining lymph nodes (dLNs).
Then, the Tarm1egfp/egfp mice and the WT mice on day 10 after CIA induction were verified for expression of I-A/I-E in CD11c+ cells in the regional lymph nodes (dLNs).
Then, a priming capability of dendritic cells for type II collagen was verified when dendritic cells and T cells isolated from the Tarm1egfp/egfp mice and the WT mice were co-cultured. On day 10 after CIA induction, the dendritic cells and the T cells were isolated from the draining lymph nodes (dLNs) from the Tarm1egfp/egfp mice and the WT mice and co-cultured in the presence of the type II collagen.
It was suggested from the above results that T cell priming was attenuated due to a decreased function of the dendritic cells from the Tarm1egfp/egfp mice.
A function of Tarm1 in an immune system was examined. Firstly, an expression amount of the Tarm1 gene in GM-CSF-induced dendritic cells (GM-DCs), M-CSF-induced bone marrow-derived macrophages (BMMs), osteoclasts (OCs), T cells (Thy1.2+ cells), and B cell (B220+ cells) was analyzed by quantitative RT-PCR.
Then, an expression of the Tarm1 gene in a dendritic cell subset was verified using the Tarm1egfp/+ mice. Dendritic cells were isolated from the Tarm1egfp/+ mice and the WT mice, the expression of the Tarm1 gene was verified by analyzing an expression of an eGFP gene with flow cytometry.
Then, a mixed emulsion of type II collagen and complete Freund’s adjuvant (CFA) was administered to the Tarm1egfp/egfp mice and the WT mice to thereby induce CIA, dendritic cells were isolated on day 7 after CIA induction, and the expression of the Tarm1 gene was verified by analyzing an expression of the eGFP gene with flow cytometry.
Then, an expression of the Tarm1 gene was examined in dendritic cells induced from bone marrow cells from the Tarm1egfp/+ mice and the WT mice in vitro.
Then, whether the expression of Tarm1 gene is induced by stimulating GM-DCs was verified. GM-DCs were treated with inflammatory cytokines (TNF and IL-1β) and pathogen-associated molecular patterns (LPS, CpG, and poly(I:C)) and then an expression amount of the Tarm1 gene was analyzed with quantitative RT-PCR.
Then, percentages of CD11c+ cells and I-A/I-EhiCD11c+ cells on predetermined days after a GM-CSF treatment in Tarm1egfp/egfp mouse-derived and WT mouse-derived GM-DCs were analyzed with flow cytometry.
Then, expression of dendritic cell activation markers (I-A/I-E, CD86, and CD80) on predetermined days after a GM-CSF treatment in the Tarm1egfp/egfp mouse-derived and WT mouse-derived GM-DCs was analyzed with flow cytometry. The results are shown in
Then, expression of the genes in the Tarm1egfp/egfp mouse-derived and WT mouse-derived GM-DCs was analyzed with a DNA microarray.
Then, an effect of deficiency in the Tarm1 gene on an antigen-presenting capability of GM-DCs was examined. OT-II Tg mouse-derived CFSE-labeled CD4+ T cells were co-cultured with the Tarm1egfp/egfp mouse-derived and WT mouse-derived GM-DCs in the presence of OVA at predetermined concentrations. A CFSE intensity was measured with flow cytometry on day 3 of culture. The results are shown in
It was suggested from the above results that maturation of GM-DCs was suppressed in the Tarm1egfp/egfp mice.
It is thought from the results of Test Examples 1 to 3 that the Tarm1 may regulate maturation of GM-DCs through self-recognition. Therefore, ligands for the Tarm1 on the GM-DCs were analyzed with flow cytometry using Tarm1-Fc which is a fusion of an extracellular domain of the Tarm1 with an IgG Fc region. For a negative control, IgG Fc was used instead of the Tarm1-Fc.
An immunoglobulin-like member is known to act in cis on itself (A. D. Barrow and J. Trowsdale, Immunological reviews, 224, 98-123, 2008; J. Trowsdale et al., Immunological reviews, 267, 117-136, 2015) . Therefore, the Tarm1egfp/egfp mouse-derived GM-DCs were also analyzed with flow cytometry for binding to the Tarm1-Fc in the same manner, and the Tarm1-Fc was found to bind to the Tarm1egfp/egfp mouse-derived CD11c+ cells (
Then, a binding of trypsinized or untreated GM-DCs to Tarm1-Fc was analyzed with flow cytometry. For a negative control, IgG Fc was used instead of the Tarm1-Fc. The results are shown in
It has been conventionally reported that collagen is a functional ligand for OSCAR (A. D. Barrow et al., J. Immunol., 194, 3317-3326, 2015; A. D. Barrow et al., J. Clin. Invest., 121, 3505-3516, 2011; J. Haywood et al., Proc. Natl. Acad. Sci. USA, 113, 1038-1043, 2016; L. Zhou et al., Blood, 127, 529-537, 2016). Furthermore, it has also been reported that an interaction between collagen and OSCAR is involved in activation and maturation of dendritic cells (H. S, Schultz et al., J. Immunol., 194, 3169-3179, 2015). Moreover, it has also been reported that other immunoglobulin-like leukocyte receptor complexes such as leukocyte-associated immunoglobulin-like receptor 1 (LAIR1) and glycoprotein VI (GPVI) recognize collagen-like molecules (R. J. Lebbink et al., The Journal of experimental medicine, 203, 1419-1425, 2006; R. J. Lebbink et al., Biology, 28, 202-210, 2009; Y. Miura et al., The Journal of biological chemistry, 277, 46197-46204, 2002). Therefore, the possibility that the collagen-like molecules are endogenous ligands for the Tarm1 was examined as follows.
Firstly, a solid phase-binding assay was performed to examine Tarm1-Fc for its ability to bind to type I collagen, type II collagen, or BSA immobilized on plates. For a negative control, IgG Fc was used instead of the Tarm1-Fc. The results are shown in
Furthermore,
Then, the collagen-like molecules on GM-DCs were analyzed with flow cytometry.
Then, a binding of collagen-treated or untreated GM-DCs to Tarm1-Fc was analyzed with flow cytometry. For a negative control, IgG Fc was used instead of the Tarm1-Fc. The results are shown in
Then, immunoprecipitation and immunoblotting (western blotting) were performed using whole cell lysates (WCLs)of GM-DCs. The whole cell lysates (WCLs) of GM-DCs were subjected to immunoprecipitation using Tarm1-Fc or IgG Fc and the resultant precipitates were subjected to immunoblotting using an anti-type II collagen antibody. Furthermore, the whole cell lysates were subjected to immunoblotting using the anti-type II collagen antibody and an anti-β-actin antibody. The results are shown in
Then, the Tarm1egfp/egfp mouse-derived and WT mouse-derived GM-DCs were stimulated with the type II collagen and expression of dendritic cell activation markers (CD86 and I-A/I-E) was analyzed with flow cytometry. The results are shown in
Furthermore,
It was suggested from the above results that the type II collagen is a ligand for the Tarm1 on the GM-DCs and regulates maturation of the GM-DCs in a Tarm1-dependent manner.
For verification of a physiological activity of the Tarm1-Fc on autoimmune arthritis, Tarm1-Fc and IgG Fc were administered to the joint cavities in the left and right ankle joints of DBA/1J mice in which CIA had been induced. A severity of arthritis is shown in
Then, expression of cytokine (TNF, IL-6, IL-1β, IL-10, and IL-17a) genes in the joints of the DBA/1J mice that received the Tarm1-Fc and the IgG Fc was analyzed with quantitative PCR. The results are shown in
It was suggested from the above results that administration of the Tarm1-Fc suppress progression of autoimmune arthritis.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2018-194614 | Oct 2018 | JP | national |
This application is the U.S. National Phase under 35 U.S.C. § 371 of International application PCT/JP2019/040206, filed Oct. 11, 2019, designating the U.S., and published in Japanese as WO 2020/080298 on Apr. 23, 2020, which claims priority to Japanese Patent Application No. 2018-194614, filed Oct. 15, 2018, the entire contents of which are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2019/040246 | 10/11/2019 | WO |
