Composition for predicting permeability of blood-brain barrier

Abstract

When the composition of the present invention is used, it is possible to provide accurate basic information on the regulation of blood-brain barrier permeability, which limits future drug delivery, by predicting the permeability of the blood-brain barrier. In addition, when the pharmaceutical composition of the present invention is used, it is possible to effectively prevent, ameliorate or treat neuroinflammation by reducing blood-brain barrier permeability.

Description

Composition for predicting blood-brain barrier permeability

[0001] The present invention relates to a composition for controlling the permeability of the blood-brain barrier and the use thereof.

[0002] The blood-brain barrier (BBB) ​​refers to a specialized vascular structure in the brain composed of multiple layers of endothelial cells, perivascular cells, and astrocytes. It plays a role in physically separating the brain parenchyma from circulating blood, thereby preventing the influx of blood-mediated toxins and circulating immune cells. Additionally, the BBB contributes to brain homeostasis by selectively transporting nutrients, ions, and water to the brain. BBB dysfunction is associated with vascular disorders such as stroke (including ischemic stroke) and multiple neurological disorders such as Parkinson's disease, Alzheimer's disease, epilepsy, and brain tumors; therefore, it is considered a consequence of various brain pathological conditions. It is known that damage to the BBB leads to a noticeable influx of circulating immune cells into the brain parenchyma, causing significant inflammation. Consequently, BBB disruption can exacerbate the pathological state of neurological diseases and is recognized as a potential risk factor for various neurodegenerative diseases. Maintaining BBB integrity is critical for preserving brain functional homeostasis.

[0003] Furthermore, recent studies have demonstrated that systemic or peripheral inflammation caused by COVID-19 or sepsis can impair BBB integrity, leading to subsequent neuroinflammation. However, the precise mechanisms underlying the regulation of BBB permeability in response to various pathological conditions, including peripheral inflammation, are not yet fully understood.

[0004] The inventors confirmed that microglia NLRP3 activation is critical for peripheral inflammation-induced BBB disruption and that BBB permeability can be predicted by identifying direct signaling mediator markers associated with neutrophils. Furthermore, they identified NLRP3-driven chemokines directly associated with BBB permeability as potential therapeutic targets to alleviate neuroinflammation, thereby completing the present invention.

[0005] One objective of the present invention is to provide a composition for predicting the permeability of the blood-brain barrier (BBB).

[0006] Another objective of the present invention is to provide a kit for predicting the permeability of the blood-brain barrier (BBB).

[0007] Another objective of the present invention is to provide a method for providing information for predicting the permeability of the blood-brain barrier (BBB).

[0008] Another objective of the present invention is to provide a composition that controls the permeability of the blood-brain barrier (BBB).

[0009] Another objective of the present invention is to provide a composition for treating neuroinflammation-related diseases.

[0010] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below.

[0011] Various embodiments described herein are described with reference to the drawings. In the following description, for a complete understanding of the invention, various specific details, such as specific forms, compositions, and processes, are described. However, specific embodiments may be practiced without one or more of these specific details, or in combination with other known methods and forms. In other examples, known processes and manufacturing techniques are not described as specific details so as not to unnecessarily obscure the invention. Reference throughout this specification to "one embodiment" or "an embodiment" means that the particular features, forms, compositions, or characteristics described in association with the embodiment are included in one or more embodiments of the invention. Accordingly, the context of "in one embodiment" or "an embodiment" expressed at various places throughout this specification does not necessarily represent the same embodiment of the invention. Additionally, particular features, forms, compositions, or characteristics may be combined in any suitable way in one or more embodiments.

[0012] Unless otherwise specifically defined in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains.

[0013]

[0014] 1. Applications of predicting Blood-Brain Barrier (BBB) ​​permeability

[0015] According to one embodiment of the present invention, the invention relates to a composition for predicting the permeability of the blood-brain barrier (BBB) ​​of an individual.

[0016] In the present invention, the prediction composition may include a preparation for measuring the expression level of at least one protein or gene selected from the group consisting of chemokines, interferon (IFN)-stimulated genes (ISGs), and matrix metalloproteinases (MMPs).

[0017] The preparation for measuring the level of the protein present according to the present invention may include, but is not limited to, at least one selected from the group consisting of an antibody, an oligopeptide, a ligand, a PNA (peptide nucleic acid), and an aptamer that specifically binds to at least one protein selected from the group consisting of chemokines, interferon (IFN)-stimulated genes (ISG), and matrix metalloproteinases (MMPs).

[0018] The "antibody" of the present invention refers to a protein molecule capable of specifically binding to at least one protein selected from the group consisting of chemokines, interferon (IFN)-stimulated genes (ISG), and matrix metalloproteinases (MMPs). The form of the antibody is not particularly limited, but may include polyclonal antibodies, monoclonal antibodies, or any part of an antibody that possesses antigen-binding ability, and may include all types of immunoglobulin antibodies. Additionally, special antibodies such as humanized antibodies may be included, and the antibody includes not only a complete form having two full-length light chains and two full-length heavy chains, but also functional fragments of the antibody molecule. A functional fragment of an antibody molecule refers to a fragment possessing at least an antigen-binding function, and may include Fab, F(ab'), F(ab') 2, Fv, etc., but is not limited thereto.

[0019] In the present invention, the "oligopeptide" is a peptide composed of 2 to 20 amino acids and may include dipeptides, tripeptides, tetrapeptides, and pentapeptides, but is not limited thereto.

[0020] In the present invention, the "PNA (Peptide Nucleic Acid)" refers to an artificially synthesized polymer similar to DNA or RNA, which was first introduced in 1991 by Professors Nielsen, Egholm, Berg, and Buchardt of the University of Copenhagen, Denmark. While DNA has a phosphate-ribose sugar backbone, PNA has a repeating N-(2-aminoethyl)-glycine backbone connected by peptide bonds, which significantly increases its binding affinity and stability to DNA or RNA, and is therefore used in molecular biology, diagnostic analysis, and antisense therapy. PNA is disclosed in detail in the literature [Nielsen PE, Egholm M, Berg RH, Buchardt O (December 1991). "Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide". Science 254 (5037): 1497-1500].

[0021] The "aptamer" of the present invention refers to a single-stranded oligonucleotide and is a nucleic acid molecule having binding activity to the protein. The aptamer may have various three-dimensional structures depending on its base sequence and may have high affinity for a specific substance, such as in an antigen-antibody reaction. The aptamer may inhibit the activity of a specific target molecule by binding to the target molecule. The aptamer may be RNA, DNA, modified nucleic acid, or a mixture thereof, and may be in a linear or cyclic form.

[0022] The preparation for measuring the expression level of a gene encoding the protein of the present invention may include, but is not limited to, one or more selected from the group consisting of primers, probes, and antisense nucleotides that specifically bind to a gene encoding at least one protein selected from the group consisting of chemokines, interferon (IFN)-stimulated genes (ISGs), and matrix metalloproteinases (MMPs).

[0023] In the present invention, the "primer" is a fragment that recognizes a target gene sequence and includes a forward and reverse primer pair, but preferably is a primer pair that provides analysis results having specificity and sensitivity. High specificity can be conferred when the nucleic acid sequence of the primer is a sequence that is inconsistent with the non-target sequence present in the sample, so that it amplifies only the target gene sequence containing the complementary primer binding site and does not induce non-specific amplification.

[0024] In the present invention, the term "probe" refers to a substance capable of specifically binding to a target substance to be detected within a sample, and means a substance capable of specifically confirming the presence of the target substance within the sample through said binding. The type of probe is not limited to substances commonly used in the industry, but preferably may be PNA (peptide nucleic acid), LNA (locked nucleic acid), peptide, polypeptide, protein, RNA, or DNA, and most preferably PNA. More specifically, the probe may be a biomaterial derived from an organism or similar, or manufactured in vitro, and may be, for example, enzymes, proteins, antibodies, microorganisms, animal and plant cells and organs, nerve cells, DNA, and RNA; DNA may include cDNA, genomic DNA, and oligonucleotides; RNA may include genomic RNA, mRNA, and oligonucleotides; and examples of proteins may include antibodies, antigens, enzymes, peptides, etc.

[0025] In the present invention, "LNA (Locked nucleic acids)" refers to nucleic acid analogs containing a 2'-O, 4'-C methylene bridge [J Weiler, J Hunziker and J Hall Gene Therapy (2006) 13, 496.502]. LNA nucleosides contain common nucleic acid bases of DNA and RNA and can form base pairs according to the Watson-Crick base pairing rule. However, due to the 'locking' of the molecule caused by the methylene bridge, LNAs are unable to form an ideal shape in Watson-Crick bonding. When LNAs are included in DNA or RNA oligonucleotides, LNAs can pair more quickly with complementary nucleotide chains, thereby increasing the stability of the double helix.

[0026] In the present invention, "antisense" refers to an oligomer having a backbone between nucleotide base sequences and subunits, wherein the antisense oligomer hybridizes with a target sequence within RNA by Watson-Crick base pairing, thereby allowing the formation of a heterodimer of mRNA and RNA oligomers within the target sequence. The oligomer may have exact sequence complementarity or approximate complementarity with respect to the target sequence.

[0027] Since information on at least one protein selected from the group consisting of chemokines, interferon (IFN)-stimulated genes (ISGs), and matrix metalloproteinases (MMPs) according to the present invention, or the gene encoding them, is known, a person skilled in the art can easily design a primer, probe, or antisense nucleotide that specifically binds to the gene encoding the protein based on this.

[0028] The diagnostic composition of the present invention can predict the permeability of the blood-brain barrier (BBB) ​​of an individual by comparing the expression level of at least one protein or gene encoding it, selected from the group consisting of chemokines, interferon (IFN)-stimulated genes (ISG), and matrix metalloproteinases (MMPs) measured in a biological sample isolated from a target individual, with a control group to determine whether the expression level has increased or decreased.

[0029] The chemokines of the present invention are small proteins that serve to guide immune cells to specific locations and are well known to play an important role in immune responses. They primarily function to attract immune cells to sites of inflammation or to regulate the distribution of immune cells within tissues. Chemokines are associated with various diseases, particularly inflammatory diseases, autoimmune diseases, infections, and cancer.

[0030] 본 발명에서 상기 케모카인 단백질은 CXCL1(NCBI GenBank accession No.: NP_001502.1), CXCL2(NCBI GenBank accession No.: NP_002080.1), CXCL3(NCBI GenBank accession No.: NP_002081.2), CXCL5(NCBI GenBank accession No.: NP_002985.1), CXCL8(NCBI GenBank accession No.: NP_000575.1), CXCL9(NCBI GenBank accession No.: NP_002407.1), CXCL10(NCBI GenBank accession No.: NP_001556.2), CXCL11(NCBI GenBank accession No.: NP_005400.1), CXCL12(NCBI GenBank accession No.: NP_954637.1), CXCL13(NCBI GenBank accession No.: NP_006410.1), CCL2(NCBI GenBank accession No.: NP_002973.1), CCL3(NCBI GenBank accession No.: NP_002974.1), CCL4(NCBI GenBank accession No.: NP_002975.1), CCL5(NCBI GenBank accession No.: NP_002976.2), CCL7(NCBI GenBank accession No.: NP_006264.2), CCL11(NCBI GenBank accession No.: NP_002977.1), CCL13(NCBI GenBank accession No.: NP_005399.1), CCL20(NCBI GenBank accession No.: NP_004582.1), CCL24(NCBI GenBank accession No.: NP_002982.2) 및 CCL26(NCBI GenBank accession No.: NP_006063.It may include one or more selected from the group consisting of 1), specifically one or more selected from the group consisting of CXCL1, CXCL2, CXCL3, CXCL5, and CXCL10, but is not limited thereto.

[0031] The interferon (IFN)-stimulated genes (ISG) of the present invention refer to a set of genes induced by interferon signals. It is well known that when interferon binds to receptors on the cell surface, signals are transmitted through the JAK and STAT pathways, and in this process, transcription factors are activated to promote ISG expression. They primarily perform various antiviral and immunomodulatory functions, such as inhibiting viral replication, enhancing immune responses, and inducing apoptosis.

[0032] In the present invention, the interferon stimulating gene-related proteins are RSAD2 (NCBI GenBank accession No.: NP_542388.2), OASL (NCBI GenBank accession No.: NP_003724.1), IFIT1 (NCBI GenBank accession No.: NP_001539.3), ISG15 (NCBI GenBank accession No.: NP_005092.1), TRIM5 (NCBI GenBank accession No.: NP_149023.2), IFIT3 (NCBI GenBank accession No.: NP_001540.2), USP18 (NCBI GenBank accession No.:NP_059110.2), IRF7 (NCBI GenBank accession No.: NP_001563.2), SAMD9l (NCBI GenBank accession No.: NP_689916.2), SAMHD1 (NCBI GenBank accession No.: NP_056289.2), IRGM(NCBI GenBank accession No.: NP_001333486.1), DDX60(NCBI GenBank accession No.: NP_060101.3), SLFN5(NCBI GenBank accession No.: NP_659412.3), DDX58(NCBI GenBank accession No.: NP_055129.2), PARP12(NCBI GenBank accession No.: NP_073587.1), SP100(NCBI GenBank accession No.: NP_001073860.1), IFIH1(NCBI GenBank accession No.: NP_071451.2), GBP5(NCBI GenBank accession No.: NP_443174.1), SLFN11(NCBI GenBank accession No.: NP_001362936.1) and GBP3(NCBI GenBank accession No.: NP_060754.It may include one or more selected from the group consisting of 2), but is not limited thereto.

[0033] The matrix metalloproteinases (MMPs) of the present invention refer to a group of enzymes that degrade proteins associated with the extracellular matrix (ECM), and are known to perform functions primarily in processes such as tissue remodeling, inflammatory responses, wound healing, and cancer metastasis. In particular, they act as important pathological factors in cancer, inflammatory diseases, and cardiovascular diseases.

[0034] In the present invention, the substrate metalloproteinase may include one or more selected from the group consisting of MMP8 (NCBI GenBank accession No.: NP_002415.1), MMP9 (NCBI GenBank accession No.: NP_004985.2) and MMP25 (NCBI GenBank accession No.: NP_071913.1), but is not limited thereto.

[0035] In the present invention, the "Blood-Brain Barrier (BBB)" refers to a physical and biochemical barrier existing between the blood and the brain to protect the brain. The blood-brain barrier prevents harmful substances or pathogens in the blood from entering the brain, while simultaneously allowing nutrients, ions, or water necessary for brain function to pass through selectively. It acts as a key defense mechanism that protects the brain's nerve cells and important neural functions. The blood-brain barrier is a special vascular structure of the brain composed of multiple layers of endothelial cells, perivascular cells, and astrocytes. Since these cells form a highly tightly packed structure, they prevent substances in the blood from moving freely between the cells. Although the blood-brain barrier is an important defense mechanism for protecting the brain, it often makes the treatment of brain diseases difficult, and various studies are being conducted to address this issue.

[0036] In the present invention, the term "blood-brain barrier permeability" refers to the degree of destruction of the blood-brain barrier; an increase in blood-brain barrier permeability can be interpreted as a high degree of destruction of the blood-brain barrier, and a decrease in blood-brain barrier permeability can be interpreted as a low degree of destruction of the blood-brain barrier. The degree of blood-brain barrier permeability includes all means capable of quantifying and comparing the level of blood-brain barrier permeability of an individual with a control group in which neuroinflammation-related diseases have not developed.

[0037] In the present invention, the "control group" may be a normal control group in which no neuroinflammation-related disease has occurred, or may be the average to median value of the expression level of at least one protein or gene encoding it selected from a group consisting of chemokines, interferon (IFN)-stimulated genes (ISGs), and matrix metalloproteinases (MMPs) in brain immune cells. The expression level of the marker protein or the nucleic acid molecule encoding it in the control group can be compared with the expression level of the marker protein or the nucleic acid molecule encoding it in a biological sample derived from a patient with a neuroinflammation-related disease to be analyzed, and the level of blood-brain barrier permeability of the individual can be predicted and diagnosed by determining whether there is a significant change in the expression level.

[0038] In the present invention, the term "prediction" means confirming the existence or characteristics of a pathological condition, and more specifically, determining the criteria for the blood-brain barrier (BBB) ​​permeability of an individual, and in particular, determining an increase or decrease in the blood-brain barrier (BBB) ​​permeability of an individual.

[0039] In one embodiment of the present invention, when one or more chemokines selected from the group consisting of CXCL1, CXCL2, CXCL3, CXCL5, and CXCL10 are expressed at a higher level compared to the control group, it can be predicted that the blood-brain barrier (BBB) ​​permeability of the individual will increase.

[0040] In another embodiment of the present invention, when one or more interferon-stimulating genes selected from the group consisting of RSAD2, OASL, IFIT1, ISG15, TRIM5, IFIT3, USP18, IRF7, SAMD9l, SAMHD1, IRGM, DDX60, SLFN5, DDX58, PARP12, SP100, IFIH1, GBP5, SLFN11, and GBP3 are expressed at a higher level compared to the control group, it can be predicted that the blood-brain barrier (BBB) ​​permeability of the individual will increase.

[0041] In another embodiment of the present invention, when at least one substrate metalloproteinase selected from the group consisting of MMP8, MMP9, and MMP25 is expressed at a higher level compared to the control group, it can be predicted that the blood-brain barrier (BBB) ​​permeability of the individual will increase.

[0042] In the present invention, the destruction or damage of the blood-brain barrier (BBB) ​​is considered a significant cause of neuroinflammation, and dysfunction of the blood-brain barrier is regarded as a result of various neuropathological conditions, including ischemic stroke. Furthermore, when the BBB is damaged, circulating immune cells noticeably infiltrate the brain parenchyma, causing significant inflammation. Such damage (destruction) of the BBB can exacerbate the pathological condition of neurological diseases and is a potential risk factor for various neurodegenerative diseases. The purpose of the present invention is to select patients for whom increased blood-brain barrier (BBB) ​​permeability is predicted and for whom the pathological condition of neurological diseases may worsen, and to apply customized treatment accordingly.

[0043]

[0044] According to another embodiment of the present invention, the invention relates to a kit for predicting the blood-brain barrier (BBB) ​​permeability of an individual comprising the composition of the present invention.

[0045] The kit of the present invention, when using the composition of the present invention, can predict that blood-brain barrier (BBB) ​​permeability will increase in a target individual when at least one protein or gene selected from the group consisting of chemokines, interferon (IFN)-stimulated genes (ISG), and matrix metalloproteinases (MMPs) is present at a high level compared to a control group. Preferably, it can be predicted that the pathological state of a neurological disease may worsen when the expression level of at least one protein or gene selected from the group consisting of chemokines, interferon (IFN)-stimulated genes (ISG), and matrix metalloproteinases (MMPs) is present at a high level compared to a control group.

[0046] The kit of the present invention can predict that when one or more chemokines selected from the group consisting of CXCL1, CXCL2, CXCL3, CXCL5, and CXCL10 are expressed at a higher level than in the control group in the target individual using the composition of the present invention, the blood-brain barrier (BBB) ​​permeability of the target individual will increase.

[0047] The kit of the present invention can predict that when one or more interferon-stimulating genes selected from the group consisting of RSAD2, OASL, IFIT1, ISG15, TRIM5, IFIT3, USP18, IRF7, SAMD9l, SAMHD1, IRGM, DDX60, SLFN5, DDX58, PARP12, SP100, IFIH1, GBP5, SLFN11, and GBP3 are expressed at a higher level than in the control group in the target individual, the blood-brain barrier (BBB) ​​permeability of the target individual will increase.

[0048] The kit of the present invention can predict that when at least one substrate metalloproteinase selected from the group consisting of MMP8, MMP9, and MMP25 is expressed at a higher level than in the control group in the target individual using the composition of the present invention, the blood-brain barrier (BBB) ​​permeability of the target individual will increase.

[0049] In the above-described prediction kit of the present invention, the descriptions regarding proteins of chemokines, interferon (IFN)-stimulated genes (ISG) and matrix metalloproteinases (MMPs) or genes encoding them, the blood-brain barrier (BBB), the permeability of the blood-brain barrier, etc., are the same as those described in the prediction composition, and are omitted to avoid excessive complexity in this specification.

[0050] The kit of the present invention may be an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a Rapid kit, or an MRM (Multiple reaction monitoring) kit, but is not limited thereto.

[0051] The kit of the present invention may further include one or more other component compositions, solutions, or devices suitable for the analysis method. For example, the kit of the present invention may further include essential elements necessary to perform a reverse transcription polymerase chain reaction. The reverse transcription polymerase chain reaction kit includes a primer pair specific to a gene encoding a marker protein. The primer is a nucleotide having a sequence specific to the nucleic acid sequence of the gene and may have a length of about 7 bp to 50 bp, more preferably about 10 bp to 30 bp. It may also include a primer specific to the nucleic acid sequence of a control gene. Furthermore, the reverse transcription polymerase chain reaction kit may include a test tube or other suitable container, a reaction buffer (with varying pH and magnesium concentration), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitor DEPC-water, sterile water, etc.

[0052] In addition, the diagnostic kit of the present invention may include essential elements necessary for performing DNA chip operations. The DNA chip kit may include a substrate to which cDNA or oligonucleotides corresponding to a gene or a fragment thereof are attached, and reagents, preparations, enzymes, etc., for producing fluorescently labeled probes. Additionally, the substrate may include cDNA or oligonucleotides corresponding to a control gene or a fragment thereof.

[0053] In addition, the diagnostic kit of the present invention may include essential elements necessary for performing ELISA. The ELISA kit includes an antibody specific to the protein. The antibody is an antibody that has high specificity and affinity for the marker protein and has little cross-reactivity with other proteins, and is a monoclonal antibody, a polyclonal antibody, or a recombinant antibody. In addition, the ELISA kit may include an antibody specific to a control protein. Furthermore, the ELISA kit may include reagents capable of detecting the bound antibody, such as a labeled secondary antibody, chromophores, an enzyme (e.g., conjugated with the antibody) and its substrate, or other substances capable of binding to the antibody.

[0054] In the diagnostic kit of the present invention, a nitrocellulose membrane, a PVDF membrane, a well plate synthesized from polyvinyl resin or polystyrene resin, a glass slide glass, etc., may be used as a fixative for the antigen-antibody binding reaction, but is not limited thereto.

[0055] In addition, the label of the secondary antibody in the diagnostic kit of the present invention is preferably a conventional chromogenic agent that produces a color reaction, and labels such as fluorescein and dyes such as HRP (horseradish peroxidase), alkaline phosphatase, colloid gold, FITC (poly L-lysine-fluorescein isothiocyanate), and RITC (rhodamine-B-isothiocyanate) may be used, but are not limited thereto.

[0056] In addition, for the diagnostic kit of the present invention, it is preferable to use a chromogenic substrate to induce color development depending on the label that performs the color reaction, and TMB (3,3',5,5'-tetramethylbezidine), ABTS [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)], OPD (o-phenylenediamine), etc. may be used. At this time, it is more preferable that the chromogenic substrate be provided in a state dissolved in a buffer solution (0.1 M NaAc, pH 5.5). A chromogenic substrate such as TMB is degraded by HRP used as a label for the secondary antibody conjugate to produce a chromogenic precipitate, and the presence or absence of the marker proteins is detected by visually confirming the degree of precipitation of this chromogenic precipitate.

[0057] In the diagnostic kit of the present invention, the washing solution preferably comprises phosphate buffer solution, NaCl, and Tween 20, and a buffer solution (PBST) composed of 0.02 M phosphate buffer solution, 0.13 M NaCl, and 0.05% Tween 20 is more preferably used. After the antigen-antibody binding reaction, a secondary antibody is reacted with the antigen-antibody conjugate, and then an appropriate amount of the washing solution is added to the immobilizer to wash 3 to 6 times. A sulfuric acid solution (H2SO4) may preferably be used as the reaction stopping solution.

[0058]

[0059] According to another embodiment of the present invention, the invention relates to a method for providing information for predicting the permeability of the blood-brain barrier (BBB) ​​of an individual.

[0060] The method of the present invention comprises the step of measuring the expression level of at least one protein or a gene encoding the same selected from the group consisting of chemokines, interferon (IFN)-stimulated genes (ISGs), and matrix metalloproteinases (MMPs) in a biological sample isolated from a target individual.

[0061] The above method of the present invention may be used to predict the permeability of the blood-brain barrier (BBB) ​​in biological samples separated from a target individual, thereby selecting patients for whom the pathological condition of a neurological disease may worsen.

[0062] In the present invention, the "desired individual" refers to a mammal including a human, and may be selected from the group consisting of, for example, humans, rats, mice, guinea pigs, hamsters, rabbits, monkeys, dogs, cats, cattle, horses, pigs, sheep, and goats, and preferably may be a human, but is not limited thereto.

[0063] In the present invention, the term "human" may refer to a person who has developed or is suspected of having a neuroinflammation-related disease, and who requires or is expected to receive appropriate treatment for a neuroinflammation-related disease due to abnormal blood-brain barrier function, but is not limited thereto.

[0064] The "biological sample" of the present invention refers to any substance, biological fluid, tissue, or cell obtained from or derived from a patient who has developed a neuroinflammation-related disease or an individual suspected of having a neuroinflammation-related disease and thus suspected of having blood-brain barrier dysfunction, such as, for example, blood including whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, and serum; sputum; tears; mucus; nasal washes; nasal aspirate; breath; urine; semen; saliva; peritoneal washings; pelvic fluids; cystic fluid; meningeal fluid; amniotic fluid; glandular fluid; and pancreatic fluid. It may include, but is not limited to, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, organ secretions, cell, cell extract, or cerebrospinal fluid.

[0065] The step of measuring the level of the protein present in the present invention may be performed using the composition of the present invention by protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radioimmunodiffusion, Ouchteroni immunodiffusion, Rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoresis analysis, liquid chromatography-mass spectrometry (LC-MS), LC-MS / MS (liquid chromatography-mass spectrometry / mass spectrometry), Western blotting, or ELISA (enzyme-linked immunosorbent assay), but is not limited thereto.

[0066] The step of measuring the expression level of the gene encoding the protein of the present invention may be performed using the composition of the present invention by reverse transcription polymerase chain reaction (RT-PCR), competitive reverse transcription polymerase chain reaction (Competitive RT-PCR), real-time reverse transcription polymerase chain reaction (Real-time RT-PCR), RNase protection assay (RPA), Northern blotting, or a DNA chip, but is not limited thereto.

[0067] In the method for providing information for the above prediction according to the present invention, descriptions regarding chemokines, interferon (IFN)-stimulated genes (ISG) and matrix metalloproteinases (MMPs) proteins or genes encoding them, agents for measuring expression levels, blood-brain barrier (BBB), permeability of the blood-brain barrier, control groups, neuroinflammation-related diseases, etc. are the same as those described in the diagnostic composition, and are therefore omitted to avoid excessive complexity in this specification.

[0068] The method of the present invention predicts that the permeability of the blood-brain barrier (BBB) ​​of the target individual will increase if the expression level of at least one protein selected from the group consisting of chemokines, interferon (IFN)-stimulated genes (ISGs), and matrix metalloproteinases (MMPs) measured in the biological sample, or the gene encoding such protein, is higher than that of a normal control. More specifically, if one or more chemokines selected from the group consisting of CXCL1, CXCL2, CXCL3, CXCL5, and CXCL10 are expressed at a higher level than that of the control; It can be predicted that blood-brain barrier (BBB) ​​permeability will increase when one or more interferon-stimulating genes selected from the group consisting of RSAD2, OASL, IFIT1, ISG15, TRIM5, IFIT3, USP18, IRF7, SAMD91, SAMHD1, IRGM, DDX60, SLFN5, DDX58, PARP12, SP100, IFIH1, GBP5, SLFN11, and GBP3 are expressed at a higher level compared to the control group; or when at least one substrate metalloproteinase selected from the group consisting of MMP8, MMP9, and MMP25 is expressed at a higher level compared to the control group.

[0069]

[0070] 2. Use for the prevention or treatment of diseases related to nerve inflammation

[0071] According to another embodiment of the present invention, the invention relates to a pharmaceutical composition for reducing the permeability of the blood-brain barrier (BBB).

[0072] The above composition of the present invention may include, as an active ingredient, a preparation that reduces the expression level or activity of the CXCR2 protein or the gene encoding it.

[0073] The agent of the present invention that reduces the expression level or activity may be a protein activity inhibitor or a gene expression inhibitor.

[0074] The above composition of the present invention may include a preparation that inhibits the activity of the CXCR2 protein or a preparation that reduces the expression level of the gene encoding it.

[0075] In the present invention, the "CXCR2" is one of the chemokine receptors that induces the migration of immune cells and is known to play an important role in various inflammatory and immune responses. It is composed of a structure having seven helical domains that penetrate the cell membrane, and when CXCR2 binds to a ligand, the G protein is activated, thereby triggering a series of signaling pathways within the cell. This signaling is known to primarily alter intracellular calcium concentration and activate various signaling pathways (e.g., MAPK, PI3K / AKT pathways) to induce responses such as chemotaxis, survival, and proliferation. CXCR2 primarily guides immune cells to the site of inflammation and also plays an important role in various physiological and pathological processes.

[0076] The significance of the present invention lies in confirming that the destruction of the blood-brain barrier (BBB) ​​caused by peripheral inflammation is significantly prevented by inhibiting the activity of the CXCR2 protein, in particular among various chemokine receptors, thereby inhibiting the migration of neutrophils.

[0077] The agent for inhibiting the activity of the CXCR2 protein according to the present invention may comprise one or more selected from the group consisting of compounds, peptides, peptide mimetics, aptamers, antibodies, and natural products that specifically bind to CXCR2, and the agent for reducing the expression of the CXCR2 gene may comprise one or more selected from the group consisting of antisense nucleotides that bind complementarily to the CXCR2 gene, preferably the mRNA of the gene, short interfering RNA (siRNA), short hairpin RNA, and ribozymes, but is not limited thereto and may include all of the means that can be easily derived by known techniques in the art as a means of acting directly or indirectly on the target protein or gene CXCR2 to produce an effect of inhibiting its activity or expression.

[0078] In the present invention, the "peptide mimetics" are peptides or non-peptides that inhibit the binding domain of a protein that leads to the inhibition of the activity of the CXCR2 protein. Major residues of non-hydrolyzable peptide analogs include β-turned dipeptide cores (Nagai et al. Tetrahedron Lett 26:647, 1985), keto-methylene pseudopeptides (Ewenson et al. J Med chem 29:295, 1986; and Ewenson et al. in Peptides: Structure and Function (Proceedings of the 9th AmeriCan Peptide Symposium) Pierce chemiCal co. Rockland, IL, 1985), azepines (Huffman et al. in Peptides: Chemistry and Biology, GR Marshall ed., EScOM Publisher: Leiden, Netherlands, 1988), benzodiazepines (Freidinger et al. in Peptides: Chemistry and Biology, GR Marshall ed., EScOM Publisher: Leiden, Netherlands, 1988), and β-aminoalcohols (Gordon et It can be produced using a substituted gamma-lactam ring (Garvey et al. in Peptides: Chemistry and Biology, GR Marshell ed., EScOM Publisher: Leiden, Netherlands, 1988).

[0079] In the present invention, the "aptamer" is a single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) characterized by having a stable tertiary structure and the ability to bind to a target molecule with high affinity and specificity. Since the first development of the aptamer discovery technology called SELEX (Systematic Evolution of Ligands by EXponential enrichment) (Ellington, AD and Szostak, JW., Nature 346:818-822, 1990), many aptamers capable of binding to various target molecules, ranging from small organic molecules to peptides and membrane proteins, have been continuously discovered. Aptamers are compared to monoclonal antibodies due to their inherent high affinity (usually at the pM level) and specificity, and they have high potential as alternative antibodies, particularly to the extent that they are referred to as "chemical antibodies."

[0080] In the present invention, the "antibody" may be either manufactured through protein injection or purchased commercially. Additionally, the antibody includes polyclonal antibodies, monoclonal antibodies, and fragments capable of binding to epitopes.

[0081] Here, the polyclonal antibody can be produced by a conventional method of injecting the CXCR2 protein into an animal and collecting blood from the animal to obtain serum containing the antibody. Such polyclonal antibodies can be purified by any method known in the art and can be produced from any animal species host such as goat, rabbit, sheep, monkey, horse, pig, cattle, dog, etc.

[0082] In addition, the monoclonal antibody can be produced using any technology that provides for the generation of antibody molecules through the culture of a continuous cell line. Such technologies include, but are not limited to, hybridoma technology, human B-cell line hybridoma technology, and EBV-hybridoma technology.

[0083] In addition, antibody fragments containing specific binding sites for the CXCR2 protein can be prepared. For example, although not limited to these, an F(ab')2 fragment can be prepared by digesting an antibody molecule with pepsin, and a Fab fragment can be prepared by reducing the disulfide bridge of an F(ab')2 fragment. As another method, a Fab expression library can be made small so that a monoclonal Fab fragment with desired specificity can be identified quickly and easily.

[0084] In the present invention, the antibody may be bound to a solid substrate to facilitate subsequent steps, such as washing or separation of the complex. Examples of solid substrates include synthetic resins, nitrocellulose, glass substrates, metal substrates, glass fibers, microspheres, and microbeads. Additionally, the synthetic resins include polyester, polyvinyl chloride, polystyrene, polypropylene, PVDF, and nylon.

[0085] In the present invention, the "antisense nucleotide" is defined in the Watson-Click base pair as a molecule that binds (hybridizes) to the complementary base sequence of DNA, immature mRNA, or mature mRNA, thereby interfering with the flow of genetic information from DNA to protein. The property of antisense nucleotides to be specific to target sequences makes them exceptionally multifunctional. Since antisense nucleotides are long chains of monomer units, they can be easily synthesized against target RNA sequences. Many recent studies have demonstrated the utility of antisense nucleotides as a biochemical means for studying target proteins. Given the recent significant advances in oligonucleotide chemistry and the synthesis of nucleotides exhibiting enhanced cell line adsorption, target binding affinity, and nuclease resistance, the use of antisense nucleotides can be considered as a new type of inhibitor.

[0086] In the present invention, the "siRNA" and "shRNA" are nucleic acid molecules capable of mediating RNA interference or gene silencing, and are used as efficient gene knockdown methods or gene therapy methods because they can suppress the expression of target genes. shRNA forms a hairpin structure by binding between complementary sequences within a single-stranded oligonucleotide, and in vivo, the shRNA is cleaved by a dicer to become siRNA, a double-stranded oligonucleotide, which is a small RNA fragment of 21 to 25 nucleotides in size, and can specifically bind to mRNA having a complementary sequence to suppress expression. Therefore, the choice of which means to use between shRNA and siRNA can be determined by a person skilled in the art, and if the mRNA sequences they target are the same, a similar expression reduction effect can be expected. For the purposes of the present invention, the expression of CXCR2 can be suppressed by specifically acting on the gene encoding CXCR2 to cleave the CXCR2 gene (e.g., mRNA molecule) and inducing RNA interference (RNAi). siRNA can be synthesized chemically or enzymatically. The method of preparing siRNA is not particularly limited and methods known in the art may be used. Examples include, but are not limited to, a method of directly chemically synthesizing siRNA, a method of synthesizing siRNA using in vitro transcription, a method of cleaving long double-stranded RNA synthesized by in vitro transcription using an enzyme, a method of expression through intracellular delivery of an shRNA expression plasmid or viral vector, and a method of expression through intracellular delivery of a PCR (polymerase chain reaction)-induced siRNA expression cassette.

[0087] In the present invention, the term "ribozyme" refers to an RNA molecule having catalytic activity. Ribozymes having various activities are known, and the ribozyme of the CXCR2 gene includes known or artificially produced ribozymes, and a ribozyme having selectively target-specific RNA cleavage activity can be prepared by known standard techniques.

[0088] The composition of the present invention can be applied to the prevention or treatment of neuroinflammation-related diseases in which abnormalities in blood-brain barrier function are anticipated by promoting a reduction in blood-brain barrier (BBB) ​​permeability.

[0089] The pharmaceutical composition of the present invention may be characterized in that it is in the form of a capsule, tablet, granule, injection, ointment, powder, or beverage, and the pharmaceutical composition may be characterized in that it is intended for humans.

[0090] The pharmaceutical composition of the present invention is not limited to these, but may be formulated and used in the form of oral formulations such as powders, granules, capsules, tablets, and aqueous suspensions, as well as topical preparations, suppositories, and sterile injectable solutions, according to conventional methods. The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. For oral administration, the pharmaceutically acceptable carrier may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavorings, etc.; for injectable preparations, it may include buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc., mixed with other agents; and for topical administration, it may include bases, excipients, lubricants, preservatives, etc. The formulations of the pharmaceutical composition of the present invention may be prepared in various ways by mixing with the pharmaceutically acceptable carriers described above. For example, for oral administration, it can be manufactured in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injectables, it can be manufactured in the form of unit ampoules or multiple doses. In addition, it can be formulated into solutions, suspensions, tablets, capsules, sustained-release formulations, etc.

[0091] Meanwhile, examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil. Additionally, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc. may be additionally included.

[0092] The routes of administration of the pharmaceutical composition of the present invention are not limited to but include oral, intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal. Oral or parenteral administration is preferred.

[0093] The term "parenteral" in the present invention includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intradural, intralesional, and intracranial injection or infusion techniques. Preferably, the pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration, but is not limited thereto.

[0094] The pharmaceutical composition of the present invention may vary depending on several factors including the activity of the specific compound used, age, body weight, general health, gender, diet, time of administration, route of administration, elimination rate, drug combination, and the severity of the specific disease to be prevented or treated, and the dosage of the pharmaceutical composition may be appropriately selected by a person skilled in the art, depending on the patient's condition, body weight, degree of disease, drug form, route of administration, and duration, and may be administered at a dose of 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg per day. Administration may be administered once a day or divided into several doses. The dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated as a pill, coated tablet, capsule, liquid, gel, syrup, slurry, or suspension.

[0095]

[0096] According to another embodiment of the present invention, the invention relates to a pharmaceutical composition for the prevention or treatment of neuroinflammation-related diseases.

[0097] The above composition of the present invention may include, as an active ingredient, a preparation that reduces the expression level or activity of the CXCR2 protein or the gene encoding it.

[0098] The agent of the present invention that reduces the expression level or activity may be a protein activity inhibitor or a gene expression inhibitor.

[0099] The above pharmaceutical composition of the present invention may include a preparation that inhibits the activity of the CXCR2 protein or a preparation that reduces the expression level of the gene encoding it.

[0100] In the present invention, the pharmaceutical composition can treat or prevent neuroinflammation-related diseases by inducing inhibition of the permeability of the blood-brain barrier (BBB) ​​by administering to a patient a preparation that inhibits the activity of the CXCR2 protein or a preparation that reduces the expression level of the gene encoding it.

[0101] The pharmaceutical composition of the present invention has the function of preventing, improving, or treating neuroinflammation-related diseases by inhibiting damage (destruction) of the blood-brain barrier, and the neuroinflammation-related diseases may be one or more selected from the group consisting of Alzheimer's disease, Parkinson's disease, ischemic stroke, encephalitis, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), and Huntington's disease.

[0102] In addition, the above pharmaceutical composition can be applied to the prevention or treatment of neuroinflammation-related diseases in which abnormalities in blood-brain barrier function are ultimately predicted by controlling the permeability of the blood-brain barrier (BBB).

[0103] In the present invention, the term "prevention" may be included without limitation as long as it is any act that can block symptoms caused by a neuroinflammation-related disease or inhibit or delay the progression to a neuroinflammation-related disease using the composition of the present invention.

[0104] In the present invention, the term "treatment" refers to a series of activities performed to alleviate or / and improve a desired disease. For the purposes of the present invention, treatment includes activities that suppress or delay neuroinflammation-related diseases, and may include, without limitation, any act that improves or benefits symptoms caused by neuroinflammation-related diseases.

[0105] The description of the agent that inhibits the activity of the CXCR2 protein or the agent that reduces the expression level of the gene encoding it in the pharmaceutical composition for treating neuroinflammation-related diseases of the present invention, the pharmaceutical composition, etc., is the same as described above, so it is omitted to avoid excessive complexity in this specification.

[0106]

[0107] According to another embodiment of the present invention, the invention relates to a pharmaceutical composition for reducing the permeability of the blood-brain barrier (BBB) ​​or a pharmaceutical composition for the prevention or treatment of neuroinflammation-related diseases, comprising a matrix metalloproteinase (MMP) inhibitor as an active ingredient.

[0108] In the present invention, the MMP inhibitor may be one or more selected from the group consisting of ilomastat, batimastat, marimastat, doxycycline, prinostat, rebimastat, SB-3CT, TAPI-2, bryostatin-1, and PD166793, and specifically may be ilomastat, but is not limited thereto as long as it corresponds to a substance that inhibits the activity of MMP enzymes.

[0109] In the present invention, the ilomastat is known as a broad-spectrum matrix metalloproteinase (MMP) inhibitor and inhibits enzymatic activity by binding to zinc ions located at the active site of MMPs. Through this, it is known to prevent the degradation of the extracellular matrix (ECM), maintain tissue structure, or alleviate pathological changes (e.g., cancer metastasis, inflammation). The MMPs targeted for inhibition include various types such as MMP-2 and MMP-9, and are known to be involved in the progression of cancer, inflammatory diseases, and fibrosis.

[0110] In the pharmaceutical composition for reducing the permeability of the blood-brain barrier (BBB) ​​or the pharmaceutical composition for treating neuroinflammation-related diseases according to the present invention, the descriptions regarding the blood-brain barrier, permeability, neuroinflammation-related diseases, pharmaceutical composition, etc. are the same as those described above, and are therefore omitted to avoid excessive complexity in this specification.

[0111]

[0112] According to another embodiment of the present invention, the invention relates to a method for reducing blood-brain barrier permeability comprising the step of administering an effective amount of a composition for reducing blood-brain barrier (BBB) ​​permeability, which includes as an active ingredient a preparation that reduces the expression level or activity of the CXCR2 protein or the gene encoding it, to a subject requiring administration.

[0113] According to another embodiment of the present invention, the invention relates to a method for preventing or treating a neuroinflammation-related disease comprising the step of administering an effective amount of a composition for preventing or treating a neuroinflammation-related disease, comprising as an active ingredient a preparation that reduces the expression level or activity of the CXCR2 protein or the gene encoding it, to a subject requiring administration.

[0114] According to another embodiment of the present invention, the invention relates to a method for reducing blood-brain barrier permeability comprising the step of administering an effective amount of a composition for reducing blood-brain barrier (BBB) ​​permeability, which includes a matrix metalloproteinase (MMP) inhibitor as an active ingredient, to a subject requiring administration.

[0115] According to another embodiment of the present invention, the invention relates to a method for preventing or treating a neuroinflammation-related disease, comprising the step of administering an effective amount of a composition for preventing or treating a neuroinflammation-related disease, comprising a matrix metalloproteinase (MMP) inhibitor as an active ingredient, to a subject requiring administration.

[0116] In the present invention, "administration" means providing a predetermined composition of the present invention to a subject by any appropriate method.

[0117] The "subject" requiring administration according to the present invention may include both mammals and non-mammalians. Here, examples of mammals may include, but are not limited to, humans, non-human primates, such as chimpanzees, other ape or monkey species; domesticated animals, such as cattle, horses, sheep, goats, pigs; domesticated animals, such as rabbits, dogs, or cats; and laboratory animals, such as rodents, such as rats, mice, or guinea pigs. Additionally, examples of non-mammalians in the present invention may include, but are not limited to, birds or fish.

[0118] In the present invention, the formulation of the composition administered as described above is not particularly limited and may be administered as a solid formulation, a liquid formulation, or an aerosol formulation for inhalation, and may be administered as a solid formulation intended to be converted into a liquid formulation for oral or parenteral administration immediately before use, and may be administered in the form of, for example, oral formulations such as powders, granules, capsules, tablets, and aqueous suspensions, external formulations, suppositories, and sterile injectable solutions, but is not limited thereto.

[0119] In addition, in the present invention, a pharmaceutically acceptable carrier may be additionally administered together with the composition of the present invention during administration. Here, the pharmaceutically acceptable carrier may use a binder, lubricant, disintegrant, excipient, solubilizer, dispersant, stabilizer, suspending agent, colorant, flavoring agent, etc. for oral administration; may use a mixture of a buffer, preservative, analgesic, solubilizer, isotonic agent, stabilizer, etc. for injectables; and may use a base, excipient, lubricant, preservative, etc. for topical administration. The formulation of the composition of the present invention can be prepared in various ways by mixing it with the pharmaceutically acceptable carrier described above. For example, for oral administration, it may be prepared in the form of a tablet, troche, capsule, elixir, suspension, syrup, wafer, etc.; and for injectables, it may be prepared in the form of a unit dosing ampoule or a multi-dose formulation. Other formulations may be prepared as a solution, suspension, tablet, capsule, sustained-release formulation, etc.

[0120] Meanwhile, examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil. Additionally, fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc. may be additionally included.

[0121] The routes of administration of the composition according to the present invention include, but are not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal. Oral or parenteral administration is preferred.

[0122] In the present invention, "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intradural, intralesional, and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration.

[0123] In the present invention, "pharmaceuticalally effective amount" refers to a sufficient amount of agent to provide a desirable biological result. Such result may be a reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desirable change in the biological system. For example, "effective amount" for therapeutic use is the amount of the composition disclosed in the present invention required to provide a clinically significant reduction in the disease. In any individual case, an appropriate "effective" amount may be determined by a person skilled in the art using routine experiments. Accordingly, the expression "effective amount" generally refers to an amount of the active substance that has a therapeutic effect. In the case of the present invention, the active substance is a agent that inhibits the expression or activity of CXCR2 or MMP targets.

[0124] The composition of the present invention may vary depending on several factors including the activity of the specific agent used, age, body weight, general health, gender, diet, time of administration, route of administration, release rate, drug combination, and the severity of the specific disease to be prevented or treated, and the dosage of the composition may be appropriately selected by a person skilled in the art, depending on the patient's condition, body weight, degree of disease, drug form, route of administration, and duration, and may be administered at a dose of 0.0001 to 100 mg / kg or 0.001 to 100 mg / kg per day. The administration may be given once a day or divided into several doses. The dosage does not limit the scope of the present invention in any way. The composition according to the present invention may be formulated into pills, coated tablets, capsules, liquids, gels, syrups, slurries, suspensions, etc.

[0125] By using the composition of the present invention, the permeability of the blood-brain barrier can be predicted, thereby providing accurate basic information on controlling blood-brain barrier permeability that limits future drug delivery. Furthermore, by using the pharmaceutical composition of the present invention, the permeability of the blood-brain barrier can be reduced, thereby effectively preventing, improving, or treating neuroinflammation.

[0126] Figure 1 shows the results of repeated peripheral LPS stimulation inducing NLRP3-dependent blood-brain barrier disruption according to one embodiment of the present invention, Figures 1a and 1b show the results of quantifying IL-1β (a) and IL-6 (b) in serum and brain extracts of mice administered (ip) of PBS or LPS (0.8 mg / kg), Figure 1c shows a representative organ image (c) of a mouse injected with PBS or LPS, and Figure 1d shows the results of quantifying Evans blue of the brain (d). Figure 1e shows the results of quantifying Evans Blue in brain tissue 3 to 24 hours after the second LPS injection, Figure 1f shows the in vitro imaging results of active Caspase-1 in brain, spleen, and lung tissues of mice injected with PBS or LPS and a Caspase-1 activation probe 6 hours after the last LPS injection, and Figure 1g shows WT and Nlrp3 mice injected with PBS or LPS 6 hours after the last injection. - / - These are representative immunoblot results obtained from mouse brain tissue extracts, and Figures 1h and 1i show WT and Nlrp3 injected with PBS or repeated LPS 6 hours after the last injection. - / - These are the results of quantifying IL-1β(h) and IL-6(i) in mouse brain extracts. Figures 1j and 1k show WT and Nlrp3 mice injected with PBS or repeated LPS 6 hours after the last injection. - / -Figure 11 shows the levels of Il1b(j) and Il6(k) mRNA in mouse brain tissue extracts. WT and Nlrp3 mice injected with PBS or repeatedly injected with LPS. - / - These are the results of quantifying Evans blue in mouse brain tissue, and Figure 1m shows two-photon in vivo imaging of mice injected with PBS or repeated LPS 24 hours after the last injection. Texas Red-dextran (10 kD) was intravenously injected prior to sacrifice, and white arrows indicate intravenous dextran leakage; a rainbow intensity scale was used to indicate dextran leakage (scale bar, 50 μm). Figure 1n shows the results of quantifying vascular leakage by calculating the intensity of extravenous dextran every minute, and data are expressed as mean ± SEM (**P < 0.01, ***P < 0.001, ****P < 0.0001). Figure 1o shows the results of analyzing the extravascular leakage of NaF. Figure 1p shows the results of comparing the degree of increase in BBB permeability caused by intravenous injection (Intravenous, IV) of a cytokine cocktail (IL-1β 10 μg / kg + IL-6 35 μg / kg + TNFα 35 μg / kg) to confirm NLRP3-dependent BBB permeability.

[0127] Figure 2 shows the results of repeated peripheral LPS stimulation inducing the infiltration of NLRP3-dependent bone marrow cells into the brain and gliosis. Figure 2a shows total immune cells (CD45 + ), microglia (CD45 int , CD11b int ), brain-infiltrating bone marrow cells (CD45 hi , CD11b hi ), neutrophils (CD45 hi , CD11b hi , Ly6C int , Ly6G + ), monocyte (CD45 hi , CD11b hi , Ly6C + , Ly6G- Figures 2b and 2c show the flow cytometry-based quantification results of immune cells (b) and brain-infiltrating bone marrow cells (c) in brain tissue of WT mice injected with PBS or LPS 6 hours after the last injection, and Figures 2d to 2f and 2h show PBS or repeated LPS-injected WT and Nlrp3 - / - Figure 2 shows the quantification results of immune cells (d), brain-infiltrating cells (e), neutrophils (f), and microglia (h) in mouse brain tissue via flow cytometry 6 hours after the last injection, and Figure 2g shows the control group (LysM-GFP) and NLRP3 KO (LysM-GFP; NLRP3 24 hours after the last LPS injection, injected with PBS or repeated LPS) 24 hours after the last LPS injection. - / - This shows a representative image of extravascular neutrophils injected with 70 kDa Texas Red-dextran prior to the sacrifice of mice (scale bar, 50 μm). Figures 2i and 2j show the quantification results of brain-infiltrating cells (i) and neutrophils (j) in the brains of PBS- or repeated LPS-injected WT mice 6 hours after the second LPS injection by flow cytometry, and Figure 2k shows PBS- or repeated LPS-injected WT and Nlrp3 mice 6 hours after the last injection. - / - Representative immunofluorescence images of the mouse hippocampus; coronal brain sections were stained with anti-GFAP (green) and anti-Iba1 (red), and DAPI represents the nuclear signal (blue) (scale bar, 50 μm). Figures 2l and 2m show WT and Nlrp3 upon LPS injection. - / - These are the quantification results of the mean fluorescence intensity of GFAP(l) standardized by DAPI and the number of GFAP+ astrocytes (m) in mice. Figures 2n and 2o show WT and Nlrp3 upon LPS injection. - / -These are the results of the quantification of the mean fluorescence intensity of Iba1(n) standardized by DAPI in mice and the quantification of the number of Iba1+ microglia(o). Data are expressed as mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001).

[0128] Figure 3 illustrates the results confirming that IL-1R signaling is not required for BBB disruption and brain infiltration of bone marrow cells caused by peripheral inflammation, and Figure 3a shows the experimental plan for pretreatment with MCC950 (10 mg / kg) or IL-1RA (10 mg / kg) 30 minutes prior to PBS or LPS injection. Figure 3b shows the quantification results of Evans Blue extravascular efflux into the brain in response to repeated LPS injections in mice pretreated with MCC950 or IL-1RA. Figure 3c is a representative flow cytometry result depicting the immune cell (CD45+) population in the stimulated mouse brain as in Figure 3b, and Figures 3d and 3e are the flow cytometry-based quantification results of brain-infiltrating bone marrow cells (d) and neutrophils (e) in PBS or LPS-treated brains. Figures 3f and 3g show WT and IL-1RA upon LPS injection - / - The results show the quantification (f) and representative image (g) of Evans blue extravasation in the mouse brain, and Fig. 3h shows WT and Il1r1 upon repeated LPS injection. - / - This is a representative flow cytometry result of the brain showing a population of immune cells (CD45+) in mice. Figures 3i and 3j show WT and Il1r1 upon repeated LPS injections. - / - These are the results of flow cytometry-based evaluation of brain-infiltrating cells (i) and neutrophils (j) in the mouse brain, and Fig. 3k shows WT and Nlrp3 upon repeated LPS stimulation. - / - and Il1r1 - / -This is a representative immunofluorescence image of the hippocampus region of the mouse brain (scale bar, 100 μm). WT, Nlrp3 treated as shown in Figs. 3l and 3k - / - and Il1r1 - / - These are the results of quantification of dextran fluorescence standardized by DAPI in the mouse hippocampus, and Fig. 3m shows WT and Nlrp3 upon repeated LPS injection. - / - This is the result of quantifying IL-18 in mouse brain extracts. Figure 3n shows WT (n = 3, PBS; n = 6, LPS x2) and Nlrp3 under repeated LPS. - / - This shows the results of the quantification of Evans blue deposition in the brains of mice (n = 7, PBS; n = 12, LPS x2). Data are expressed as mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001).

[0129] Figure 4 illustrates the contribution of microglia NLRP3 inflammasome activation to peripheral LPS-induced BBB disruption. Figure 4a shows the results of displaying a comprehensive single-cell map of the mouse brain using a UMAP plot of single-cell RNA sequencing data containing 105,838 genes; the merged dataset includes samples from PBS- or LPS-infused WT and NLRP3 KO groups. The UMAP visualization represented the complex cellular environment as clusters distinguished by 17 distinct colors. Figure 4b illustrates the cell cluster ratios for each group, and Figure 4c shows a bar graph representing the log2 folding change (log2FC) of each cell type when comparing the WT-LPS x2 group and the KO-LPS x2 group. The right bar indicates an increase in the WT-LPS x2 group, and the left bar indicates an increase in the KO-LPS x2 group. Figure 4d shows a UMAP plot (bottom panel) for neutrophil clusters in WT and NLRP3 KO mice injected with PBS or LPS. Figure 4d shows a heatmap illustrating the expression of inflammasome marker genes in each cluster, with color intensity adjusted for average expression levels. Figure 4e shows the VISION scoring of inflammasome genes for all cell clusters within all samples. The violin plot shows the inflammasome scores using the genes derived from Figure 4d. Figures 4f and 4g show the results of NLRP3 expression in Cx3cr1 cells (f) or Aldh1l1 cells (g) and other cell types in controls and NLRP3 mutant (D301N) mice upon tamoxifen treatment (left panel), and the results of quantifying Evans Blue extravascular efflux in the brains of NLRP3 mutant mice upon tamoxifen-treated controls and repeated PBS or LPS injection (right panel).Figure 4h shows the results of NLRP3 expression in Tmem119 cells and other cell types in control and NLRP3 mutant mice upon tamoxifen treatment (top panel), and representative images of the brain in tamoxifen-treated control and mutant mice upon PBS or repeated LPS injection (bottom left panel) and quantified results of Evans Blue extravasation (bottom right panel). Figure 4i shows representative flow cytometry results of the brain showing microglia and brain-infiltrating bone marrow populations, and Figures 4j and 4i show the quantified results of brain-infiltrating bone marrow cells or neutrophils. Figure 4k shows tamoxifen-treated control mice (Gsdmd. flox / flox ) and microglia (MG) specific GSDMD - / - Mouse(Tmem119 creER / + ;Gsdmd flox / flox Figure 4 shows the quantitative results of Evans Blue deposition upon repeated LPS injection, and Figure 41 shows the control group and MG-GSdmd upon repeated LPS injection as measured by flow cytometry. - / - Results of quantification of neutrophils in mouse brains are shown. Data are expressed as mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001).

[0130] Figure 5 illustrates the results showing that the NLRP3 inflammasome reprograms BBB constituent cells in the direction of chemotaxis and cell migration; Figure 5a shows a Venn diagram illustrating the number of genes specifically upregulated in brain endothelial cells after LPS x2 treatment versus PBS treatment groups in WT mice, and the number of genes upregulated in WT-LPS x2 versus WT-LPS x2; Figure 5b shows the heatmap results indicating 20 genes notably upregulated in the WT LPS x2 group of Figure 5a, excluding unannotated genes; Figure 5c shows the results of gene ontology (GO) analysis for the 33 genes identified in Figure 5a; Figures 5d and 5e are heatmaps showing the expression of major adhesion genes (d) or adhesion molecules (e) in brain endothelial cells of WT and Nlrp3-deficient mice after PBS or LPS stimulation; and Figures 5f to 5h show the WT LPS x2 identified in the gene ontology The heatmap results showing specific terms are perivascular cells of GO BP (f), astrocytes of GO molecular function (MF) (g), and astrocytes of Sigma DB (h). This analysis utilized genes that were particularly increased in the WT LPS x2 group compared to the WT PBS and KO LPS x2 groups. Figure 5i is a UMAP plot showing the astrocyte and astrocyte 2 clusters of the WT-PBS and WT-LPS x2 groups, and Figure 5j is a violin plot showing the expression of the top 10 upregulated genes in astrocyte 2 compared to the astrocyte cluster, where bold text indicates IFN-related or stimulated genes.Figure 5k is a UMAP plot showing 13 subclusters of microglia, Figure 5l is a heatmap result showing categories representing specific functional terms of microglia subclusters, and Figures 5m and 5n are trajectory analyses showing the direction of microglia fate between distant cluster lineages. The UMAP visualization is color-coded to represent subclusters of microglia (m) or to explain the trajectories from homeostatic microglia to individual subtypes of ISG and DAM1. Figure 5o is a UMAP visualization result depicting the density of microglia for each experimental group with density values ​​between 0 and 1; Figure 5p is a Venn diagram result showing the number of genes specifically increased in all clusters of the WT LPS x2 group compared to the WT PBS group (fold change > 1.5, p < 0.05) and the KO LPS x2 group (fold change > 1.5, p < 0.05); Figure 5q is a heatmap indicating NLRP3 specificity, showing an increase in 30 genes derived from Figure 5p, with unannotated genes excluded. Figures 5r and 5s are the results of quantification of brain-infiltrating myeloid cells (r) or neutrophils (s) in the brains of WT and Rsad2-deficient mice upon repeated LPS injection by flow cytometry. Data are expressed as mean ± SEM.

[0131] Figure 6 illustrates the results showing that microglia NLRP3-GSDMD activation induces the production of Cxcr2-specific chemokines through the release of GDF-15, and Figures 6a and 6b are heatmaps showing the increased expression of NLRP3-dependent chemokine genes across all groups of clusters (a) or endothelial cells (b), where color intensity represents the converted average expression level. Figures 6c to 6f show WT, NLRP3 - / - , Gsdmd - / - and Il1r1 - / -Figure 6g shows the results of quantifying the mRNA levels of Cxcl1(c), Cxcl2(d), Cxcl3(e), and Cxcl5(f) in whole brain extracts; Figure 6g shows the results of quantifying the brain mRNA levels of Cxcl1 in control and Tmem119-specific NLRP3 mutant (D301N) expressing mice 6 hours after the second LPS injection; and Figure 6h shows the control (Gsdmd) in PBS or repeated cases by flow cytometry. flox / flox , n=6) and MG-Gsdmd - / - Mouse(Tmem119 creER / + , Gsdmd flox / flox Figure 6i shows the results of quantifying mRNA levels of Cxcl1 and Cxcl2 in the brains of individuals (n = 4), Figure 6i illustrates the experimental plan for isolating secretions from microglia treated with LPS (0.25 μg / ml, 3 hours) followed by ATP treatment (2.5 mM, 20 min) or ATP alone, and Figure 6j shows the WT or Gsdmd at appropriate treatment determined by mass spectrometry analysis. - / - (KO) A heatmap showing the relative concentration of target proteins in the culture supernatant of microglia, where L+A represents LPS + ATP treatment as in Fig. 6i. Fig. 6k is a heatmap result showing the expression level of Gdf-15 in specific cell clusters based on scRNA-seq data, and Fig. 6l is the result of quantifying the mRNA levels of multiple chemokines in mixed glial cultures that were not or were treated with GDF-15 (100 ng / ml, 6 hours). Data are expressed as mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Fig. 6m shows the results confirming whether the production of GDF-15, discovered by secretome analysis, is an NLRP3 and GSDMD-dependent phenomenon, and Fig. 6n shows the results confirming the changes in the expression levels of CXCL1 and CXCL2 in brain tissue after intravenous injection of GDF-15 into mice.

[0132] Figure 7 illustrates the results showing that neutrophil recruitment contributes to BBB disruption upon peripheral inflammatory stimulation; Figure 7a shows the immunoblot results detecting pro-IL-1β, IL-1β, and NLRP3 in brain lysates of WT mice at different time points (0, 1–6 hours) after the second LPS injection; Figures 7b and 7c show the quantification results of brain-infiltrating bone marrow cells (b) or neutrophils (c) in the brains of WT mice when LPS injection was repeated at different time points using PBS or flow cytometry. Figure 7d shows the violin plot results illustrating chemokine receptor gene expression in neutrophils of all groups, and Figure 7e shows a one plot visualizing intercellular communication in the CXCL signaling pathway for each experimental group. The plots indicate interactions between two cell types in the PBS group and five cell types in the LPS x2 group. The width and color of the arrows indicate the potential for interaction. Figure 7f shows the quantification results of Evans Blue deposition in the brains of WT mice pretreated with IgG (n = 4) or anti-Ly6G (n = 6) antibodies 1 hour prior to the first LPS injection and subjected to repeated LPS injections. Figure 7g shows the quantification results of Evans Blue deposition in the brains of WT mice pretreated with vehicle (n = 6) or anti-Cxcr2 antagonists (n = 8) 1 hour prior to the first LPS injection and subjected to repeated LPS injections. Figure 7h shows WT (blue) and Ccr2 - / -This is the result of quantifying Evans Blue extravasation into the brain following LPS injection in mice (yellow). Figure 7i is a heatmap showing various matrix metalloproteinase (MMP) genes in the brains of all clusters of WT and NLRP3 KO mice upon PBS or LPS injection. Figure 7j shows a dot plot visualizing MMP signaling markers for each cell cluster in all groups, Figure 7k shows the result of quantifying brain Mmp9 mRNA levels in WT mice 1 to 6 hours after the second LPS injection, and Figures 7l and 7m show WT and NLRP3 at 3 or 6 hours. - / - These are the results of quantifying brain Mmp9 (l, n = 4) or Mmp8 (m, n = 3-8) mRNA levels in mice, and Figure 7n shows WT (n = 5) or Il1r 6 hours after the second LPS injection. - / - Figure 70 shows the results of quantifying brain Mmp9 mRNA levels in mice (n = 6 or 7), and Figure 70 shows the results of quantifying brain mRNA levels of Mmp9 in control (n = 2) and Tmem119 mutant mice (n = 4 or 5) upon PBS or single / repeated LPS injection 6 hours after the last injection (*P < 0.05, **P < 0.01, ***P < 0.001). Figure 7p shows the results of LPS administration on BBB permeability after administering ilomastat, a broad-spectrum MMP inhibitor, to mice.

[0133] The present invention will be described in more detail below through examples. These examples are intended solely to explain the present invention more specifically, and it will be obvious to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the invention.

[0134]

[0135] Experimental method

[0136] Mouse preparation

[0137] C57BL / 6, Nlrp3 D301NneoR ,Cx3cr1 creERT ,Tmem119 creERT , Aldh1l1 cre / ERT2 , Il1r - / - , Gsdmd - / - and Ccr2 - / - The mice were purchased from Jackson Laboratory, and Gsdmd flox / flox Mice were obtained from the RIKEN BioResource Research Center. All experiments were performed under specific pathogen-free conditions, and male or female mice aged 8 to 12 weeks were used. For cell-specific NLRP3 (D301N) mutation expression, loxP flanked neomycin-resistant cassette (reverse) on intron 2 and Nlrp3 with a point mutation on exon 3 were used. D301NneoR Cx3cr1 mouse creERT , Tmem119 creERT and Aldh1l1 cre / ERT Mice were crossbred. To induce active Cre recombinase expression, mice were treated with tamoxifen (75 mg / kg) for 5 days to excise the neomycin-resistant cassette, and NLRP3 (D301N) was expressed in Cx3cr1-, Tmem119-, and Aldh1l1- specific cells. Gsdmd flox / flox Tmem119 mouse creERT Microglia-specific Gsdmd-deficient mice were produced by crossbreeding with mice. The animal experiment protocol was approved by the Ethics Committee, and all experiments were performed in accordance with the guidelines approved by the Ethics Committee.

[0138]

[0139] Mouse handling

[0140] To induce peripheral inflammation, mice were injected intraperitoneally with lipopolysaccharide (LPS, Sigma-Aldrich, L3012, 0.8 mg / kg) 1 to 2 times at 24-hour intervals, and to block NLRP3 inflammasome-mediated signaling, mice were administered MCC950 (Sigma-Aldrich, PZ0280, 10 mg / kg) or an IL-1 receptor antagonist (Prospec, cyt-203, 10 mg / kg) 30 minutes before LPS injection.

[0141]

[0142] BBB Permeability Analysis

[0143] To evaluate BBB integrity, Evans blue (Sigma, E2129) (0.2 mg / kg) was intravenously injected into mice via the tail vein. After 1 hour, the mice were perfused with PBS under deep anesthesia, and the brains were harvested and homogenized with saline. Subsequently, Evans blue was extracted using trichloroacetic acid and quantified using a Varioskan Flash 3001 microplate fluorescence meter (Thermo Fisher) at excitation 620 nm / emission 680 nm. Additionally, BBB permeability was determined by measuring the leakage of Texas red-conjugated dextran (70 kDa) (Thermo Fisher, D1830). Dextran (1 mg) was administered via the tail vein one hour before euthanasia, and brain tissue was isolated and immunohistochemical analysis was performed to quantify dextran leakage.

[0144] Flow cytometry

[0145] Mouse brain tissue was obtained after removing the olfactory regions, brain stem, and meninges. The obtained brain was homogenized in RPMI-1640 medium containing DNase I and collagenase IV using a Dounce homogenizer. The homogenate was filtered through a cell filter (70 µm) and myelin debris was removed by applying a 30% Percoll gradient, and the single-cell suspension was used for flow cytometry analysis. Cells were stained with fluorescent dye-conjugated monoclonal antibodies against CD45 (BioLegend, 103116), CD11b (Invitrogen, 12-0112-82), Ly6C (Invitrogen, 53-5932-82), and Ly6G (Invitrogen, 17-9668-82), and dead cells were excluded by counterstaining with 4',6-diamidino-2-phenylindol (4',6-diamidino-2-phenylindol, DAPI) (Invitrogen, D1306). Analysis was performed using FACSVerse (BD Biosciences) and FlowJo software (TreeStar).

[0146]

[0147] Immunohistochemistry

[0148] Mice were anesthetized and perfused with PBS and 4% paraformaldehyde. Brain tissue was post-fixed in 4% PFA overnight at 4°C, then placed in 30% sucrose until the samples sank to the bottom and became dehydrated. The brain tissue was coated with an OCT compound and sliced ​​into 30 μm thick slices using a Leica CM1860 cryostat.

[0149] Brain sections were permeated with 0.3% Triton X-100 for 30 minutes and blocked with 4% BSA solution for 1 hour at room temperature (RT). Sections were incubated overnight at 4°C with anti-GFAP (Invitrogen, PA1-10004) and anti-Iba1 (Wako, 019-19741) antibodies dissolved in 1% BSA, followed by secondary antibody staining for 3 hours at room temperature. Sections were labeled with DAPI to visualize nuclei, mounted using mounting solution (Invitrogen, P36934), and covered with coverslips. Images were acquired using a confocal microscope (LSM980, Carl Zeiss) and processed using Zen Blue software; mean fluorescence intensity (MFI), cell number, and microglia morphology were quantified using ImageJ software and Imaris v.9.7.

[0150]

[0151] Two-photon intravital microscopy

[0152] Cranial window implantation surgery was performed as previously described for in vivo imaging of the mouse brain using a two-photon microscope. Briefly, mice were anesthetized and positioned on a custom stereotactic stage on a 37°C heated pad (Live Cell Instruments). Hair was removed from the frontal and parietal skull regions. A cranial window was created using a microdrill to form a circle of approximately 4 mm in the parietal bone of the skull. The exposed cerebral cortex was covered with a 5 mm circular cover glass using tissue adhesive, and 500 μg of 10 kDa or 70 kDa Texas Red-dextran (Thermo Fisher) was injected intravenously (iv) into the mouse, placed in 200 μl of PBS, and placed on a custom microscope platform for imaging. To track and quantify vascular permeability, the post-capillary vein of the mouse's right parietal cortex was imaged in 1 μm optical sections for over 20 minutes. Images were analyzed using Volocity software (PerkinElmer), and vascular permeability was quantified by the intensity of 10 kDa Texas Red-tagged dextran observed outside the vascular surface as previously described.

[0153]

[0154] In vitro detection of active caspase-1

[0155] Cy5.5- and BHQ-3 conjugated active caspase-1 probes were obtained, and to detect active caspase-1 activity within the trachea, a caspase-1 specific probe (100 μg / mouse) was administered via the tail vein 1 hour prior to IV euthanasia. Each organ was obtained after transcardiac perfusion with PBS for in vitro imaging, and Cy5.5 fluorescence, which is generated only when active caspase-1 is present in the isolated organs, was analyzed using an IVIS Spectrum In Vivo imaging system (PerkinElmer).

[0156]

[0157] cell culture

[0158] Mouse brain mixed glial cells were prepared from the whole brains of mice aged 1 to 3 days and cultured for 3 weeks. The mixed glial cells were cultured in Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 (DMEM / F12, 1:1) supplemented with 10% fetal bovine serum (FBS) and antibiotics. Mouse bone marrow cells were isolated from the femur and tibia and differentiated into bone marrow-derived macrophages (BMDM). BMDM were maintained in L929-condition DMEM supplemented with 10% FBS and antibiotics.

[0159]

[0160] Immunoblot analysis

[0161] Brain tissue was lysed in a buffer containing 20 mM HEPES (pH 7.5), 0.5% Nonidet P-40, 50 mM KCl, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, and a protease inhibitor. The soluble lysate was fractionated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene fluoride (PVDF) membrane, and then stained with appropriate primary and horseradish peroxidase (HRP)-conjugated secondary antibodies. Anti-NLRP3 (AG-20B-0014) and anti-caspase-1 (AG-20B-0042) antibodies were purchased from AdipoGen. The anti-mouse IL-1B antibody was purchased from R&D Systems (AF-401-NA), the anti-GSDMD antibody from Abcam (209845), and the anti-B-actin antibody from Santa Cruz Biotechnology (sc-47778). All blots were obtained from at least three independent experiments.

[0162]

[0163] Enzyme-linked immunosorbent assay (ELISA)

[0164] Whole blood samples were obtained and centrifuged for serum separation, and serum samples were diluted for ELISA. For brain tissue lysates, brain was homogenized using a Dounce tissue grinder in a buffer containing 10 mM Tris-Cl (pH 7.4), 2 mM EDTA, 150 mM NaCl, and a protease inhibitor (TEN buffer). After centrifugation, the supernatant was collected and Bradford assay was performed for protein quantification. The concentrations of IL-1B, IL-6, IL-18, CXCL1, and CXCL2 were measured using the ELISA DuoSets kit (R&D Systems) according to the manufacturer's instructions.

[0165]

[0166] Secretome Analysis via Mass Spectrometry

[0167] To identify NLRP3-GSMD-specific secreted proteins, WT and Gsdmd-deficient microglia were isolated from the primary mixed glial cell culture by shaking (200 rpm, 4 hours). The microglia were then treated with LPS (0.25 μg / ml, 3 hours) followed by ATP treatment (2.5 mM, 20 min) or ATP alone. The cell-free supernatants of the WT and Gsdmd-deficient microglia were then analyzed by mass spectrometry. To prepare proteins for digestion, each cell supernatant (140 μl) was mixed with 100 mM triethylammonium bicarbonate (TEAB) buffer (60 μl, pH 8.5). Subsequently, the mixture was reduced with 200 mM Tris-2-carboxyethylphosphine (TCEP) at 55 °C for 1 hour, followed by alkylation with 375 mM iodoacetamide (IAA) for 30 minutes in the absence of light. A clear protein pellet was obtained via acetone precipitation, reconstituted with 100 mM TEAB buffer, and trypsinized at 37 °C for 16 hours. The resulting peptide mixture was then labeled using the TMT 10plex® isobaric mass tagging kit reagent (Thermo Fisher Scientific) according to the manufacturer's instructions for accurate relative quantification. After vacuum drying, the samples were desalted using a peptide desalination spin column (Thermo Fisher Scientific), re-dried, and suspended in 0.1% formic acid. Analysis was performed using a Dionex Ultimate 3000 RSLC nano-HPLC system coupled with an Orbitrap Q Exactive mass spectrometer (Thermo Fisher Scientific). Quantification and normalization were performed using Proteome Discoverer 2.3 software (Thermo Fisher Scientific).

[0168]

[0169] Neutrophil depletion

[0170] To induce neutrophil depletion, anti-mouse Ly6G (Bio X Cell, BE0075-1) or control IgG (Bio X Cell, BE0089) antibodies (50 μg / mouse) were injected intraperitoneally into mice 1 hour prior to the first LPS injection. Mouse brain tissue was obtained 6 hours after the second LPS injection, and to confirm neutrophil depletion, blood samples were collected 6 hours after the last LPS injection (LPS x2), and neutrophil (CD45+, CD11b+, Ly6G+) and monocyte (CD45+, CD11b+, Ly6C+, Ly6G-) populations were analyzed using a flow cytometer.

[0171]

[0172] Single-cell isolation for scRNA sequencing analysis

[0173] Mouse brain tissue was enzymatically separated into single-cell suspensions using the Adult Brain Dissociation Kit (130-107-677, Miltenyi Biotec) according to the manufacturer's instructions. Dissociated samples were collected and filtered through a 70 μm cell filter; myelin debris was removed using a debris remover, and red blood cell (RBC) lysis was performed using an RBC remover. Hemispheres of three mice (two males and one female) were WT and Nlrp3 - / - All mice were collected into a single replica for each group (PBS, LPS x1, LPS x2).

[0174]

[0175] Library preparation and single-cell transcriptome analysis

[0176] The library was prepared using a Chrome controller according to the 10X Chrome Next GEM Single Cell 3' v3.1 protocol (CG000315). Briefly, a single-cell suspension was diluted in nuclease-free water to obtain a sample with a cell count of 10,000. The same calculated cell suspension was loaded onto a Chrome Next GEM chip, and the RNA transcripts of the single cells were barcoded and reverse transcribed. After processing, the product was purified and concentrated using Polymerase Chain Reaction (PCR) to generate the final cDNA library. The resulting library was quantified using qPCR according to the qPCR Quantification Protocol Guide and verified using an Agilent Technologies 4200 TapeStation. The library was finally sequenced using the HiSeq platform (Illumina) according to the read lengths specified in the user guide.

[0177] Single-cell transcriptomics was analyzed using Cell Ranger v6.1.2 (10x Genomics). Briefly, FASTQ files were generated by demultiplexing raw BCL files on an Illumina sequencing instrument using the 'cellranger mkfastq' tool. Subsequently, the raw FASTQ files underwent further processing using the 'cellranger count' tool, which included mapping against the mouse reference genome (mm10-2020-A), quantifying gene expression using Unique Molecular Identifiers (UMIs), identifying cell clusters, and performing Differentially Expressed Genes (DEG) analysis. The final dataset was generated by aggregating multiple independent samples using "cellranger aggr". Analysis was performed using the Python package Scanpy, and cell clustering and UMAP analysis were conducted based on statistically significant principal components. DEGs between the control and experimental groups were determined using the Wilcoxon rank sum test with Scanpy's '-FindMarkers' function.

[0178] The false discovery rate (FDR) was controlled using adjusted p-values ​​and Bonferroni corrections. Gene enrichment and functional annotation analysis for the list of important probes were performed using g:Profiler (https: / biit.cs.ut.ee / gprofiler / ).

[0179]

[0180] Single-cell RNA sequencing of the whole brain transcriptome

[0181] Quality Management and Data Analysis

[0182] A double elimination process was performed on the dataset acquired using Scrublet software. Cells with fewer than 500 unique transcripts were removed, and cells exceeding 20% ​​of transcripts mapped to mitochondrial genes were filtered out. Cells displaying more than 8,500 unique genes were considered outliers and discarded, resulting in the removal of 19,243 cells. The count data was log-normalized using the pp.normalize function and processed as pp.highly_variable_gene using the "seurat_v3" flavor containing 2,000 genes. For the tl.umap function, the number of principal components was set to 40, and batch correction was performed on each sample using the Harmony algorithm to reduce batch effects between samples.

[0183]

[0184] Marker gene identification and cluster identity evaluation

[0185] To characterize the clusters obtained from the harmony-corrected subjects, differential expression analysis between clusters was performed, and they were classified based on estimated marker gene expression. Gene set scoring was performed using VISION scoring with inflammasome gene sets as shown in Figure 4d. The inventors generated ranks for the DEGs of each cluster using the tl.rank_genes_groups function and then compared them with the remaining cells using the Wilcoxon Rank-sum test modified with Benjamini-Hochberg correction. Visualization of specific marker genes was derived using pl.matrix plots.

[0186]

[0187] Gene-set enrichment analysis (GSEA)

[0188] Gene set enrichment analysis was performed using Gprofiler formula version 1.0.0 and GSEApy. Differential gene analysis was performed using the tl.rank_genes_groups Wilcoxon Rank-sum test and tie correction, and results were expressed as percentages. Reference datasets included WikiPathways_2019_Mouse, MSigDB_Hallmark_2020, Gene Ontology: Biological Processes_2023, and Molecular Function_2023. Filtered DEGs for endothelial cells, pericytes, astrocytes, and microglia were obtained by comparing WT PBS with LPS Х2 (Log2FC = 2) with a p-value less than 0.05 and a log fold change greater than 1. When comparing WT LPS X2 and NLRP3 KO type LPS X2, DEG was analyzed in astrocytes and microglia using the same criteria. However, for endothelial cells, DEG was calculated with a p-value cutoff of 0.05 and a minimum log2FC of 0.5. For perivascular cells, DEG was calculated using a minimum log2FC of 0.5. For the DEG analysis of the entire cell population comparing WT-PBS versus LPS X2 and WT-LPS X2 versus KO-LPS X2, parameters including a p-value cutoff of 0.05, a non-zero proportion <50% from reference, a non-zero group proportion > 25%, and a log fold change > 0.58 were utilized.

[0189]

[0190] Analysis of intercellular communication

[0191] CellChat, a tool equipped with a ligand-receptor interaction database, was used to explore intercellular communication networks. This analysis requires two inputs: cell gene expression data and assigned cell labels. CellChat uses this data to automatically organize cells by constructing a shared neighbor graph based on inter-cell distances in low-dimensional space or pseudotemporal trajectory space. Unique pathways for each group were identified using triple mean-based communication probability calculations.

[0192]

[0193] scFates for trajectory analysis

[0194] To analyze trajectories within microglia, these cell types were initially subsetted, and the principal component analysis representation of the data matrix was calculated using the sc.pp.neighbors function. Subsequently, a neighborhood graph was inserted using UMAP via the tl.umap function following the use of the sc.tl.diffmap function. By identifying connecting nodes between clusters, trajectories were established within the microglia subclusters, and three important pathways were defined. DEG analysis was performed to identify specific marker genes for each trajectory pathway. The generated marker genes were visually displayed using a pl. matrix plot. Through this comprehensive approach, distinct pathways within microglia can be described, and unique molecular features associated with each trajectory can be characterized, thereby enhancing the understanding of the transcriptional dynamics of the microglia subclusters.

[0195]

[0196] Statistical analysis

[0197] All values ​​are expressed as the mean and the standard error of the mean (SEM). The number of animals in each experimental group is indicated in the legend of each figure. Data were analyzed using the two-tailed Student's t-test, one-way analysis of variance (ANOVA), Dunnett's post hoc test, or two-way ANOVA with the Bonferroni post hoc test. The statistical significance level was set at P ≤ 0.05, and all statistical analyses were performed using GraphPad Prism 8.0.

[0198]

[0199] Experimental results

[0200]

[0201] Peripheral LPS stimulation induces BBB disruption in an NLRP3-dependent manner.

[0202] To determine whether peripheral inflammation could alter the inflammatory state in the brain, mice were injected intraperitoneally with a mild dose of lipopolysaccharide (LPS, 0.8 mg / kg) daily. A single LPS administration (LPS x1 at 6 hours after injection) strongly increased the levels of pro-inflammatory cytokines such as IL-1B and IL-6 in the circulating blood, but not in the brain (see Figures 1a and 1b). In contrast, repeated LPS administration at 24-hour intervals (LPS x2) did not stimulate the production of pro-inflammatory cytokines in the blood, indicating LPS tolerance, but it was confirmed to induce significant upregulation of IL-1B and IL-6 levels in the brain.

[0203] Additionally, to evaluate BBB permeability, extravascular leakage of Evans blue administered intravenously one hour prior to euthanasia was measured. Similar to cytokine production, repeated LPS stimulation significantly increased brain leakage of Evans blue compared to a single injection (see Figures 1c and 1d), whereas intravenous Evans blue was found to freely penetrate other organs regardless of LPS stimulation. The increase in BBB permeability caused by LPS persisted for 24 hours (see Figure 1e) but decreased starting 48 hours after LPS stimulation. This suggests that repeated peripheral inflammation can induce transient BBB disruption.

[0204] To determine whether peripheral LPS stimulation promotes the activation of the inflammasome complex in the brain, we used a probe specific to active caspase-1 that emits fluorescence only when active caspase-1 is cleaved. In particular, repeated LPS stimulation, rather than a single administration, induced potent caspase-1 activation in the brain, whereas caspase-1 activation was detected in the spleen or lungs even after a single LPS injection (see Fig. 1f). Supporting these findings, repeated LPS injections were confirmed to induce IL-1β cleavage in the brain in an NLRP3-dependent manner (see Fig. 1g). Additionally, it was confirmed that NLRP3-deficient mice showed reduced production of pro-inflammatory cytokines in the brain in response to peripheral LPS stimulation at the protein (see Figs. 1h and 1i) and mRNA (see Figs. 1j and 1k) levels.

[0205] Along with the production of these cytokines, it was confirmed that brain leakage of Evans blue due to peripheral inflammation in Nlrp3-deficient mice was significantly reduced (see Fig. 1l). To further confirm the aforementioned NLRP3-dependent BBB disruption, the inventors performed two-photon in vivo imaging to confirm the leakage of Texas Red-labeled dextran from blood vessels into the brain parenchyma. Repeated LPS stimulation caused a strong increase in dextran fluorescence in the extravascular space (see Fig. 1m), which was significantly reduced in Nlrp3-deficient mice (see Fig. 1n). These results demonstrate that repeated peripheral LPS stimulation impairs BBB integrity in an NLRP3-dependent manner. Further verification was sought using a different method to validate the existing results regarding the extravascular leakage of Evans blue measured by BBB permeability. We conducted an extravascular leakage experiment of NaF (Fluorescein Sodium), which is one of the experimental techniques used to evaluate vascular permeability and the degree of damage to the vascular barrier, and as shown in Figure 10, we were able to confirm that NLRP3-dependent BBB permeability was observed.

[0206] Although the results of analyzing BBB permeability under conditions of repeated LPS intraperitoneal administration were confirmed above, we intended to further verify whether the same effect occurs in other systemic inflammation models. To confirm NLRP3-dependent BBB permeability, we examined the effect of increased BBB permeability after intravenous injection (IV) of a cytokine cocktail (IL-1β 10 μg / kg + IL-6 35 μg / kg + TNFα 35 μg / kg). The experimental results confirmed that the increase in BBB permeability was an NLRP3-dependent phenomenon even in the systemic inflammation model (see Figure 1p).

[0207]

[0208] Peripheral LPS stimulation induces circulating immune cell infiltration into the brain and activation of brain-resident glial cells in an NLRP3-dependent manner.

[0209] Considering that BBB disruption can lead to the subsequent infiltration of circulating immune cells into the brain parenchyma, brain immune cell populations were analyzed using flow cytometry. It was confirmed that repeated LPS stimulation, unlike a single injection, significantly increased CD45+ total immune cells, including neutrophils and monocytes, as well as CD45hiCD11bhi brain-infiltrating bone marrow cells (see Figures 2a to 2c).

[0210] In contrast, the number of brain-resident microglia was not affected by single or repeated LPS stimulation. Consistent with BBB permeability, NLRP3 deficiency significantly reduced the increase in whole-brain immunity and brain-infiltrating myeloid cells induced by peripheral LPS (see Figs. 2d to 2f). Additionally, two-photon in vivo imaging showed that repeated LPS stimulation promoted NLRP3-dependent descent of LysM+ neutrophils from blood vessels into brain parenchymal regions (see Fig. 2g). However, it was confirmed that peripheral LPS stimulation did not alter the number of microglia regardless of the presence of NLRP3 (see Fig. 2h). The time interval effect between LPS priming and secondary LPS stimulation was evaluated. Notably, unlike the 24-hour interval, secondary LPS stimulation after 48 hours of LPS priming did not induce myeloid cell infiltration into the brain (see Figs. 2i and 2j), suggesting that the effect of LPS priming may be transient in terms of BBB permeability.

[0211] The inventors further evaluated changes in brain-resident glial cells in the hippocampal region of wild-type (WT) and NLRP3-deficient mice using immunofluorescence staining with antibodies targeting astrocyte-specific GFAP and microglia-specific Iba1 (see Fig. 2k). Repeated LPS stimulation resulted in a significant increase in the intensity and number of astrocytes in WT mice, which was significantly reduced in the absence of NLRP3 (see Figs. 2l and 2m). In contrast, peripheral LPS stimulation enhanced the intensity of microglia in the WT mouse brain in an NLRP3-dependent manner without affecting the number of microglia (see Figs. 2n and 2o). Repeated LPS stimulation induced significant morphological changes in the active form of microglia, as determined by an increase in soma volume and a decrease in total length, which were largely reversed by MCC950, a well-known NLRP3-specific inhibitor. These observations suggest that the NLRP3 inflammasome is important for the peripheral inflammation-inducing activation of brain-resident glial cells.

[0212]

[0213] BBB disruption induced by peripheral LPS stimulation is independent of IL-1 receptor signaling.

[0214] Next, we investigated whether inhibition of NLRP3 inflammasome signaling could reduce BBB destruction induced by peripheral LPS (see Fig. 3a). MCC950 was used to inhibit NLRP3 activation, and an IL-1 receptor antagonist (IL-1RA) was used to inhibit IL-1β signaling, a major component of the inflammasome response. In particular, MCC950 was found to effectively reduce recurrent Evans Blue leakage caused by LPS, whereas IL-1RA failed to inhibit BBB destruction (see Fig. 3b). Consistently, IL-1RA failed to block the brain infiltration of bone marrow cells induced by peripheral LPS, whereas MCC950 was found to exhibit a notable protective effect (see Figs. 3c to 3e). These results suggest that although IL-1β is a major product of NLRP3 inflammasome activation, IL-1β is not required for BBB destruction induced by recurrent peripheral inflammation. To further confirm IL-1β-independent BBB disruption, BBB permeability analysis was performed in IL-1 receptor-deficient mice. Consistent with the results for IL-1RA, IL-1 receptor deficiency did not affect peripheral LPS-induced Evans blue leakage (see Figs. 3f and 3g) or the infiltration of bone marrow cells into the brain (see Figs. 3h to 3j). To further support these results, BBB permeability was evaluated to observe the leakage of Texas red-labeled dextran (70 kDa) from the circulation into the brain parenchyma (see Fig. 3k). Repeated LPS stimulation resulted in a significant increase in dextran fluorescence in the hippocampal region of the mouse brain, which was significantly reduced in Nlrp3-deficient mice (see Fig. 3l). However, IL-1 receptor deficiency did not block peripheral LPS-induced dextran leakage (see Fig. 3l).In addition to IL-1β, IL-18 is another inflammasome-specific effector cytokine, but IL-18 levels in the brain were low and remained unchanged by peripheral LPS stimulation regardless of the presence of NLRP3 (see Fig. 3m).

[0215] To elucidate the underlying mechanism of NLRP3 inflammasome-dependent BBB impairment other than IL-1β or IL-18, we analyzed the role of gasdermin D (GSDMD), which is cleaved into an N-GSDMD fragment by active caspase-1. The N-GSDMD fragment formed GSDMD pores in the plasma membrane, promoting the secretion of IL-1β and the thermal degradation of inflammasome-activated cells; indeed, bone marrow-derived macrophages deficient in GSDMD failed to secrete IL-1β despite intracellular caspase-1 activation. In particular, GSDMD deficiency significantly attenuated Evans Blue leakage induced by peripheral LPS (see Fig. 3n), suggesting that the release of inflammatory mediators or subsequent pyroptosis by GSDMD is necessary for peripheral inflammation-induced BBB disruption.

[0216]

[0217] Microglia NLRP3-GSDMD activation is important for peripheral inflammation-induced BBB disruption.

[0218] To gain insight into the underlying mechanisms of NLRP3-mediated BBB impairment, single-cell RNA sequencing (RNA-seq) analysis was performed on mouse brains to compare WT and NLRP3-deficient conditions. In the control group, WT and NLRP3-deficient mice were injected with PBS, while in the peripheral inflammation group, WT mice were administered single or repeated LPS injections, whereas NLRP3 knockout (KO) mice were administered repeated LPS injections. Clustering analysis was performed on approximately 110,000 quality-controlled cells, and 16 clusters were identified in the Uniform Manifold Approximation and Projection (UMAP) after batch effect correction, as shown in Figure 4a (see Figure 4a).

[0219] Repeated LPS stimulation substantially increased the populations of neutrophil and astrocyte clusters within the brain (see Fig. 4b). Importantly, as shown in Fig. 4c, this increase was dependent on NLRP3. In contrast, repeated LPS injections reduced the number of endothelial cells, which are found specifically in WT brains (see Figs. 4b and 4c). Subsequently, the expression of inflammasome constituent proteins was analyzed based on clusters, and specifically, brain-resident microglia and brain-infiltrating neutrophil or monocyte-derived macrophages (Mo-Mac) showed relatively high expression of inflammasome components such as NLRP3, Asc (Pycard), and Caspase-1, along with inflammasome-associated effector molecules such as IL-1B and GSDMD (see Figs. 4d and 4e).

[0220] To analyze which specific cell type's NLRP3 inflammasome activity is important for peripheral LPS-induced BBB disruption, we prepared mice expressing a tamoxifen-induced hyperactive NLRP3 mutant (D301N). This mutant was originally identified in patients with autoinflammatory diseases and is considered a structural active form of NLRP3.

[0221] Using the Cre-Lox system, mice with hyperactive NLRP3 in Cx3cr1-expressing cells, such as monocytes and microglia, were first prepared via tamoxifen injection. Interestingly, Evans blue leakage induced by peripheral LPS was significantly increased in mice expressing the Cx3cr1-specific NLRP3 mutation (see Fig. 4f), which indicates the importance of NLRP3 in microglia and monocytes. However, it was confirmed that the astrocyte-specific expression of the NLRP3 hyperactive mutation did not affect BBB disruption induced by peripheral LPS (see Fig. 4g).

[0222] To further investigate the role of microglia NLRP3 activation in BBB integrity, Tmem119-specific NLRP3 (D301N) expressing mice were prepared in which only the NLRP3 mutation was expressed in microglia upon tamoxifen injection. In particular, it was confirmed that NLRP3 (D301N) expression in microglia led to a significant increase in Evans blue leakage upon repeated LPS stimulation (see Fig. 4h). Supporting these results, it was confirmed that subsequent infiltration of bone marrow cells into the brain significantly increased in microglia-specific NLRP3 mutation-expressing mice in response to repeated LPS injections (Figs. 4i and 4j).

[0223] To analyze the potential role of microglia GSDMD-mediated pyroptosis in NLRP3-dependent BBB impairment, microglia-specific (Tmem119-creER) GSDMD-deficient mice were prepared. In particular, tamoxifen-induced GSDMD depletion in microglia significantly reduced peripheral LPS-induced Evans Blue leakage (see Fig. 4k) and neutrophil infiltration into the brain (see Fig. 4l). These results demonstrate that repeated peripheral inflammation promotes BBB disruption through the activation of NLRP3-GSDMD signaling in microglia.

[0224]

[0225] Peripheral inflammation induces NLRP3-dependent reprogramming of BBB-composing cells toward chemotaxis and immune cell migration.

[0226] To analyze changes induced by the inflammasome in BBB-containing cells, differentially expressed genes (DEG) analysis was performed on endothelial cells of the brains of WT and Nlrp3-deficient mice. Comparing the WT-PBS group and the WT-LPS x2 group, it was observed that the expression of 1,644 genes significantly increased by more than twofold in the LPS-stimulated group (see Fig. 5a). Furthermore, comparing the WT-LPS x2 group with the NLRP3 KO-LPS x2 group, 314 genes were identified in which expression increased by more than 1.4fold in the WT-LPS x2 group (see Fig. 5a). Surprisingly, 33 genes were found to overlap between the two gene lists, indicating distinct and significant expression in the WT-LPS x2 group (see Fig. 5b). Gene ontology (GO) analysis was performed on genes specifically upregulated by NLRP3 in endothelial cells, and an association with myeloid cell migration and chemotaxis was confirmed (see Fig. 5c). In addition, the inventors confirmed changes in the expression of major tight junction proteins and adhesion molecules in endothelial cells. Transcriptomic analysis showed that repeated LPS stimulation led to the upregulation and downregulation of tight junction proteins (see Fig. 5d), but no significant difference was observed between WT and NLRP3-deficient mice.

[0227] Furthermore, peripheral LPS stimulation induced the upregulation of most adhesion molecules in endothelial cells. However, it was confirmed that this upregulation was independent of the presence of NLRP3 (see Fig. 5e). Similarly, NLRP3-specific DEGs in perivascular cells were analyzed, and 69 genes exhibiting higher expression levels in the WT-LPS x2 group were identified. GO analysis of NLRP3-specific DEGs in perivascular cells also showed a strong association with the chemotaxis and migration of granulocytes, such as neutrophils (see Fig. 5f). Based on these results, GO analysis of 148 genes with significantly increased expression in WT-LPS x2 astrocytes identified significant changes related to chemotaxis and cell migration (see Fig. 5g). Interestingly, NLRP3-specific DEGs in astrocytes showed notable changes in chemokine signaling and interferon (IFN) responses (see Figs. 5g and 5h). Repeated LPS experiments induced expansion of astrocyte 2 subsets (see Fig. 5i). Many genes with increased expression specific to the astrocyte 2 cluster were associated with a type 1 IFN response (see Fig. 5j).

[0228] In addition to the cells constituting the BBB, we analyzed the recurrent changes induced by LPS in microglia, which are the brain's major inflammasome-activating cells. Considering that microglia plasticity varies depending on the situation, we performed sub-clustering of microglia and annotated them from mg1 to mg13 (see Fig. 5k). In particular, the mg8 and mg9 clusters showed specific increases in the LPS x2 group of WT mice, and DEG analysis confirmed that these were primarily associated with the IFN response (see Fig. 5l). To further elucidate the differences between WT and NLRP3 KO in microglia, trajectory analysis was performed (see Figs. 5m and 5n). The analysis results showed that the 'homeostasis' cluster was the starting point and the 'phagocytosis' cluster was the endpoint for each group (see Fig. 5n, bottom left). However, LPS x2 stimulation generated two additional endpoints: an interferon-stimulated genes (ISG)-high cluster (mg8, see Fig. 5n, middle bottom) and a damage-associated microglia1 (DAM1) cluster (mg10, see Fig. 5n, right bottom). Density analysis using UMAP indicated a noticeable difference in density between WT and NLRP3 KO at the LPS x2 level (see Fig. 5o). While microglia reached the final endpoint (both ISGs-high and DAM1) in the WT-LPS x2 group, the trajectory stopped midway through the trajectory path and did not reach the endpoint in the KO-LPS x2 group (see Fig. 5o). These results suggest that NLRP3 inflammasome activation promotes the reprogramming of microglia into ISG and DAM1 phenotypes.

[0229] Next, NLRP3-specific DEGs were analyzed across the entire cluster, and 30 genes specifically upregulated in the WT-LPS x2 group were identified (see Fig. 5p). It was confirmed that most of the NLRP3-dependent upregulated genes in all clusters were IFN-related genes or ISGs (see Fig. 5q).

[0230] GO analysis of NLRP3-specific genes also revealed a significant increase in type 1 IFN-related genes. We analyzed the expression of IFN-B in brain extracts and observed NLRP3-specific upregulation of Ifnb1 mRNA in mouse brains 3 hours after secondary LPS stimulation. Among NLRP3-specific ISGs, the expression of the Rsad2 gene, which encodes viperin, was upregulated in the brains of WT mice. However, Rsad2 deficiency did not significantly attenuate brain infiltration of bone marrow cells induced by peripheral LPS (see Figs. 5r and 5s). These results suggest that type 1 IFN signaling may be involved downstream of the NLRP3 inflammasome and contribute to BBB disruption.

[0231]

[0232] The microglial NLRP3-GSDMD axis promotes the production of neutrophil-specific chemokines in BBB-composing cells.

[0233] NLRP3-specific DEG and GO analysis confirmed that inflammasome activation significantly alters the expression of genes associated with BBB chemotaxis or chemokine signaling. Furthermore, chemokine transcriptome levels in all clusters were analyzed in response to LPS stimulation. It was confirmed that several chemokines, such as Cxcl5, Ccl20, and Cxcl3, were upregulated by peripheral LPS stimulation only in NLRP3-expressing WT mice (see Fig. 6a). The inventors also analyzed chemokine levels in BBB-containing cells, microglia, and endothelial cells (see Fig. 6b) and confirmed that some chemokines (Cxcl1, Cxcl2, Cxcl3, Cxcl5, and Cxcl10) were generally upregulated in an NLRP3-dependent manner. Based on these transcriptome analyses, it can be seen that repeated LPS stimulation induces the upregulation of Cxcl1 and Cxcl2 in whole mouse brain extracts. Interestingly, the production of these chemokines was significantly reduced by NLRP3 or GSDMD deficiency but not by IL-1 receptor deficiency (see Figs. 6c to 6f). Consistent with these results, it was confirmed that Cxcl1 mRNA expression increased in mouse brains expressing microglia-specific hyperactivated NLRP3 mutants after repeated LPS stimulation (see Fig. 6g). In contrast, microglia-specific deletion of GSDMD significantly reduced the production of Cxcl1 and Cxcl2 mRNA induced by repeated LPS in the brain (see Fig. 6h), and these results collectively confirm that NLRP3-GSDMD signaling in microglia is important for the production of these CXCL chemokines induced by peripheral inflammation in the brain.

[0234] To gain insight into how NLRP3-GSDMD activation induces CXCL chemokine production in microglia, mass spectrometry-based protein analysis was performed on the culture supernatants of WT and GSDMD-deficient microglia (see Fig. 6i). When comparing the LPS / ATP-treated group with the PBS-treated or ATP-treated groups in WT and GSDMD-KO microglia, 11 proteins specifically released by NLRP3-GSDMD were identified in the microglia (see Fig. 6j). Among these, Growth Differentiation Factor-15 (GDF-15) is considered a potential NLRP3-specific secreted protein and has been reported as a characteristic of many pathological diseases, including chronic inflammation. Transcriptomic analysis also confirmed significant expression of GDF-15 in microglia (see Fig. 6k). Furthermore, treatment with recombinant GDF-15 potently induces the production of Cxcl1, Cxcl2, and Cxcl3 mRNAs in mouse mixed glial cultures, including astrocytes (see Fig. 6l). These results indicate that microglia NLRP3-GSDMD activation can promote the production of CXCL chemokines in the BBB region through the induction of GDF-15 secretion. Additionally, secretome analysis confirmed that the production of GDF-15 is NLRP3 and GSDMD-dependent (see Fig. 6m), and direct intravenous injection of growth differentiation factor-15 (GDF-15) into mice increased the production of Cxcl1 and Cxcl2 in brain tissue (see Fig. 6n). This suggests a process in which GDF-15 is produced by NLRP3-GSDMD, leading to neutrophil recruiting (CXCR2) through an increase in CXCL1 / 2, ultimately resulting in BBB damage.

[0235]

[0236] Neutrophil recruitment contributes to peripheral inflammation-induced BBB disruption.

[0237] Notably, CXCL chemokine production was higher at 3 hours after secondary LPS stimulation than at 6 hours (see Figs. 6c to 6e). Additionally, time-dependent experiments showed that activation of the brain inflammasome complex was observed starting 3 hours after secondary LPS treatment (see Fig. 7a). Conversely, myeloid cell infiltration significantly increased at 5 hours after secondary LPS stimulation (see Figs. 7b and 7c). These results suggest that inflammasome-dependent chemokine production may trigger the recruitment of circulating neutrophils. An investigation into the expression of chemokine receptors in neutrophils infiltrated the brain revealed higher expression of Cxcr2 and Ccr1 (see Fig. 7d). Cxcr2 was specific to NLRP3-dependent upregulated chemokines such as Cxcl1, Cxcl2, Cxcl3, and Cxcl5. Analysis of intercellular communication of CXCL signaling using CellChat showed that the general activation of the CXCL signaling pathway in the WT-LPS x2 group was clearly distinct from the activation in other groups (see Fig. 7e). In particular, Cxcl1- and Cxcl2-Cxcr2 signaling pathways were prominent in the WT-LPS x2 group but appeared much less frequently in NLRP3-deficient cells. These pathways affect the interactions between neutrophils and endothelial cells, microglia, or perivascular cells in the WT-LPS x2 group. These results suggest that interactions between neutrophils and BBB cells mediated by CXCL signaling are dependent on NLRP3, and reveal the role of the NLRP3 inflammasome in regulating neutrophil recruitment and BBB integrity during inflammation.

[0238] To determine whether recruited neutrophils could contribute to BBB breakdown, neutrophils were depleted using an anti-Ly6G antibody prior to LPS stimulation. In particular, neutrophil depletion significantly reduced BBB breakdown caused by peripheral inflammation (see Fig. 7f). Thus, blocking neutrophil recruitment by a Cxcr2 antagonist significantly attenuated LPS-promoted BBB permeability (see Fig. 7g). In contrast, a deficiency of Ccr2, which is essential for monocyte recruitment, did not affect BBB integrity in response to repeated LPS stimulation (see Fig. 7h). These results imply that the influx of inflammasome-dependent neutrophils into cerebral blood vessels contributes to BBB breakdown.

[0239] To investigate how resident microglia and recruited neutrophils promote BBB degradation, the expression of matrix metalloproteinases (MMPs), which are critical for BBB disruption, was examined in brain extracts. Specifically, repeated LPS stimulation was found to increase the expression of certain MMPs, such as MMP8, MMP9, and MMP25, in an NLRP3-dependent manner (see Fig. 7i). These NLRP3-dependent MMPs were highly expressed in neutrophils (see Fig. 7j). As brain MMP production increased starting 4 hours after secondary LPS stimulation (see Fig. 7k), cells other than neutrophils can also produce MMPs in response to NLRP3 activation. Consistent with scRNA-seq data, MMP9 production was impaired in brain extracts from NLRP3-deficient mice but was not impaired in Il1r-derived mice (see Figs. 7l to 7n). Furthermore, mice expressing a microglia-specific acquired function NLRP3 mutation showed increased MMP9 expression in brain extracts upon repeated LPS stimulation compared to control mice (see Fig. 7o). These results demonstrate that the microglia NLRP3 inflammasome promotes the production of specific MMPs capable of mediating BBB disruption. Conversely, we sought to investigate the effects of LPS administration on BBB permeability by injecting the pan-MMP inhibitor ilomastat into mouse blood vessels. The results showed that increased BBB permeability in the ilomastat-administered group was significantly inhibited (see Fig. 7p).

[0240]

[0241] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

A composition for predicting the permeability of the blood-brain barrier (BBB) ​​of an individual, comprising a preparation for measuring the expression level of at least one protein or gene selected from the group consisting of chemokines, interferon (IFN)-stimulated genes (ISG), and matrix metalloproteinases (MMPs). In Article 1, A composition comprising one or more selected from the group consisting of antibodies, oligopeptides, ligands, PNA (peptide nucleic acid), and aptamers that specifically bind to the protein, for measuring the expression level of the above protein. In Article 1, A composition comprising one or more selected from the group consisting of primers, probes, and antisense nucleotides that specifically bind to the gene, for measuring the expression level of the gene. In Article 1, A composition wherein the above chemokine is one or more selected from the group consisting of CXCL1, CXCL2, CXCL3, CXCL5, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CCL2, CCL3, CCL4, CCL5, CCL7, CCL11, CCL13, CCL20, CCL24, and CCL26. In Article 4, A composition in which the above chemokine is one or more selected from the group consisting of CXCL1, CXCL2, CXCL3, CXCL5, and CXCL10. In Article 1, A composition wherein the interferon-stimulating gene (ISG) is one or more selected from the group consisting of RSAD2, OASL, IFIT1, ISG15, TRIM5, IFIT3, USP18, IRF7, SAMD9l, SAMHD1, IRGM, DDX60, SLFN5, DDX58, PARP12, SP100, IFIH1, GBP5, SLFN11, and GBP3. In Article 1, A composition in which the above-mentioned substrate metalloproteinase is one or more selected from the group consisting of MMP8, MMP9, and MMP25. In Article 5, A composition in which the blood-brain barrier (BBB) ​​permeability of an individual is predicted to increase when one or more chemokines selected from the group consisting of CXCL1, CXCL2, CXCL3, CXCL5, and CXCL10 are expressed at a higher level compared to a control group. In Article 6, A composition in which the blood-brain barrier (BBB) ​​permeability of an individual is predicted to increase when one or more interferon-stimulating genes selected from the group consisting of RSAD2, OASL, IFIT1, ISG15, TRIM5, IFIT3, USP18, IRF7, SAMD9l, SAMHD1, IRGM, DDX60, SLFN5, DDX58, PARP12, SP100, IFIH1, GBP5, SLFN11, and GBP3 are expressed at a higher level compared to a control group. In Article 7, A composition in which the blood-brain barrier (BBB) ​​permeability of an individual is predicted to increase when at least one substrate metalloproteinase selected from the group consisting of MMP8, MMP9, and MMP25 is expressed at a higher level compared to the control group. A kit for predicting blood-brain barrier (BBB) ​​permeability comprising a composition of any one of claims 1 to 10. A method for providing information for predicting the permeability of the blood-brain barrier (BBB) ​​of an individual, comprising the step of measuring the expression level of at least one protein or gene selected from the group consisting of chemokines, interferon (IFN)-stimulated genes (ISGs), and matrix metalloproteinases (MMPs) in a biological sample isolated from a target individual. In Article 12, The above chemokine is one or more selected from the group consisting of CXCL1, CXCL2, CXCL3, CXCL5, and CXCL10, and The above interferon-stimulating gene (ISG) is one or more selected from the group consisting of RSAD2, OASL, IFIT1, ISG15, TRIM5, IFIT3, USP18, IRF7, SAMD9l, SAMHD1, IRGM, DDX60, SLFN5, DDX58, PARP12, SP100, IFIH1, GBP5, SLFN11, and GBP3, and A method in which the substrate metalloproteinase is one or more selected from the group consisting of MMP8, MMP9, and MMP25. In Article 13, A method for predicting that the blood-brain barrier (BBB) ​​permeability of an individual will increase when one or more chemokines selected from the group consisting of CXCL1, CXCL2, CXCL3, CXCL5, and CXCL10 are expressed at a higher level compared to the control group. In Article 13, A method for predicting that the blood-brain barrier (BBB) ​​permeability of an individual will increase when one or more interferon-stimulating genes selected from the group consisting of RSAD2, OASL, IFIT1, ISG15, TRIM5, IFIT3, USP18, IRF7, SAMD91, SAMHD1, IRGM, DDX60, SLFN5, DDX58, PARP12, SP100, IFIH1, GBP5, SLFN11, and GBP3 are expressed at a higher level compared to a control group. In Article 13, A method for predicting that the blood-brain barrier (BBB) ​​permeability of an individual will increase when at least one substrate metalloproteinase selected from the group consisting of MMP8, MMP9, and MMP25 is expressed at a higher level compared to the control group. A pharmaceutical composition for reducing the permeability of the blood-brain barrier (BBB), comprising as an active ingredient a preparation that reduces the expression level or activity of the CXCR2 protein or the gene encoding it. In Article 17, A composition in which the agent that reduces the expression level or activity is a protein activity inhibitor or a gene expression inhibitor. In Article 18, The above protein activity inhibitor is a composition comprising one or more selected from the group consisting of compounds, peptides, peptide mimetics, aptamers, antibodies, and natural products that specifically bind to the CXCR2 protein. In Article 18, The above gene expression inhibitor is a composition comprising one or more selected from the group consisting of an antisense nucleotide that binds complementarily to the CXCR2 gene or a part thereof, short interfering RNA (siRNA), short hairpin RNA, and ribozyme. A pharmaceutical composition for the prevention or treatment of neuroinflammation-related diseases, comprising as an active ingredient a preparation that reduces the expression level or activity of the CXCR2 protein or the gene encoding it. In Article 21, A composition in which the agent that reduces the expression level or activity is a protein activity inhibitor or a gene expression inhibitor. In Article 22, The above protein activity inhibitor is a composition comprising one or more selected from the group consisting of compounds, peptides, peptide mimetics, aptamers, antibodies, and natural products that specifically bind to the CXCR2 protein. In Article 22, The above gene expression inhibitor is a composition comprising one or more selected from the group consisting of an antisense nucleotide that binds complementarily to the CXCR2 gene or a part thereof, short interfering RNA (siRNA), short hairpin RNA, and ribozyme. In Article 21, A composition wherein the above-mentioned neuroinflammation-related disease is one or more selected from the group consisting of Alzheimer's disease, Parkinson's disease, ischemic stroke, encephalitis, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), and Huntington's disease. A pharmaceutical composition for reducing the permeability of the blood-brain barrier (BBB), comprising a matrix metalloproteinase (MMP) inhibitor as an active ingredient. In Article 26, A composition wherein the above-mentioned MMP inhibitor is one or more selected from the group consisting of ilomastat, batimastat, marimastat, doxycycline, prinostat, rebimastat, SB-3CT, TAPI-2, bryostatin-1, and PD166793. A pharmaceutical composition for the prevention or treatment of neuroinflammation-related diseases, comprising a matrix metalloproteinase (MMP) inhibitor as an active ingredient.

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