KR102181698B1 - Mehtod for screening anti-inflammatory agent - Google Patents

Abstract

The present invention comprises the steps of: (a) treating a candidate material to cells expressing BIG1 or GBF1; (b) measuring the expression of BIG1 or GBF1 on the cells; And (c) selecting a substance that inhibits the expression of BIG1 or GBF1 as compared to the non-treated group of the candidate substance as an inflammation inhibitory substance.

Description

Screening method for anti-inflammatory substances {MEHTOD FOR SCREENING ANTI-INFLAMMATORY AGENT}

The present invention relates to a screening method for selecting an inflammation inhibitory substance.

Interleukin (IL)-1 is a major pro-inflammatory cytokine that is closely related to the progression of many chronic inflammatory or degenerative diseases, including rheumatoid arthritis and Alzheimer's disease. In bone marrow cells, IL-1β is first produced as an inactive precursor form (pro-IL-1β) by binding of Toll-like receptors (TLRs) by microbial or tissue damage-causing ligands. Unlike other cytokines, pro-IL-1β is present in the cytoplasm because it lacks the endoplasmic reticulum (ER)-targeting leader sequence in the Il-1β gene. Most secreted proteins are first transported to the ER through the leader sequence and then follow the existing ER-Golgi secretion pathway. Therefore, it is unclear how IL-1β without a leader sequence is released from the cytoplasm to the extracellular medium.

In the cytoplasm, active caspase-1, called IL-1β converting enzyme, cleaves pro-IL-1β into the mature IL-1β receptor, and active IL-1β is secreted through a pathway independent of the ER-Golgi pathway. The secreted active IL-1β triggers the expression of pro-inflammatory molecules by binding to the IL-1β receptor of the target cell. It has been reported that caspase-1 activity is required for secretion as well as cleavage in the activated form of IL-1β.According to recent studies, active caspase-1 decomposes gasdermin D to decompose gasdemin D pore Formed and can serve as a conduit for IL-1β secretion (US Patent Publication No. 2018-0318414).

The pyrin domain-containing 3 (NLRP3) receptor family, such as the oligomerization domain to which nucleotides are bound, Among the identified inplasmasome sensor molecules, it is one of the best-studied molecules related to the binding of ASC (apoptosis-associated speck-like protein containing a caspase-recruitment domain) and procaspase-1. It provides a variety of stimuli, ranging from microbial infections to endogenous metabolites. There is new evidence that excessive activation of the NLRP3 inplasmosome is closely related to the progression of several chronic inflammatory and metabolic disorders. However, the underlying activation mechanism of NLRP3 inplasmasome by various stimulations is still weak.

It is an object of the present invention to inhibit the expression of BIG1 or GBF1, thereby inhibiting the activation of NLRP3 inplasmasomes, rheumatoid arthritis, Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, multiple sclerosis, amyotrophic lateral sclerosis, and inflammatory spinal injury It is to provide a screening method for determining as an inhibitor of inflammation that can prevent or treat many chronic neuroinflammatory or degenerative diseases, including the like.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems that are not mentioned will be clearly understood by those of ordinary skill in the art from the following description.

Hereinafter, various embodiments described herein are described with reference to the drawings. In the following description, for a thorough understanding of the invention, various specific details, such as specific forms, compositions and processes, etc. are set forth. However, certain embodiments may be practiced without one or more of these specific details, or with other known methods and forms. In other instances, well-known processes and manufacturing techniques have not been described in specific details in order not to unnecessarily obscure the present invention. Reference throughout this specification to “one embodiment” or “embodiment” means that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the present invention. Thus, the context of “in one embodiment” or “embodiment” expressed in various places throughout this specification does not necessarily represent the same embodiment of the invention. Additionally, particular features, shapes, compositions, or properties may be combined in any suitable manner in one or more embodiments.

Unless otherwise defined in the present invention, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

In the present invention, "inhibition of inplasmasome activity" refers to a modification in vivo that causes a decrease in the activation of inplasmasomes of a target cell, etc., and suppresses inflammasome production, suppresses cytokine production due to inflamasome, or ASC ( Adaptor protein apoptosis-associated spec-like protein containing a caspase-recruitment domain) may inhibit the formation of a polymer (oligomer).

In the present invention, inflammasomes are caspase-1-activated complex protein complexes, 1) NOD-like receptor family, pyrin domain-containing 3 (NLRP3), 2) linking protein (sensor protein) ( It is composed of an adaptor protein) ASC (adaptor protein apoptosis-associated spec-like protein containing a caspase-recruitment domain) and 3) an effector protein, procaspase-1. The above-described inplasmasome components are assembled when microbial infection or tissue injury occurs.Inflamasomes activated by assembly in cytosol activate caspase-1 to activate interleukin (IL)-1β or interleukin- 18 is secreted to perform the host's innate immune defense function (Schroder K, Tschopp J, Cell 140:821-832 (2010); Franchi L, Munoz-Planillo R, Nunex, Nat Immunol 13:325-332 (2012)) ). Preferably, the inplasmasome may be an NLRP3 inplasmasome, but is not limited thereto.

In the present invention, NLRP3 inflamasome can induce and/or promote the onset of inflammasome-mediated inflammatory disease when excessively active (Henao-Mejia J, Elinav Em Thaiss CA, Flavell RA (2014) Inflammasomes and metabolic disease.Annu Rev Physiol 76:57-78; Wen H, TingJP, eil LA 2012) A role for the NLRP3 inflammasome in metabolic diseases-did Warburg miss inflammation Nat Immunol 13:352-357.). The inflammasome-mediated inflammatory disease is an abnormal disease that occurs when inplasmasome is abnormally excessively activated, and may preferably be an auto-inflammatory disease or a neuro-inflammatory disease, but is not limited thereto.

In the present invention, auto-inflammatory diseases are Merkle-Wells syndrome (MWS), LADA (adult-type delayed autoimmune diabetes), familial cold auto-inflammatory syndrome (FCAS), cryopyrin-related periodic syndrome (CAPS). , Neonatal-onset multi-organ inflammatory syndrome (NOMID), chronic infantile neurocutaneous joint (CINCA) syndrome, familial Mediterranean fever (FMF), and/or systemic onset Certain forms of pediatric arthritis such as pediatric idiopathic arthritis (SJIA), systemic onset It may be any one or more selected from the group consisting of a specific form of juvenile rheumatoid arthritis, such as juvenile idiopathic rheumatoid arthritis, and/or a specific form of adult rheumatoid arthritis, but is not limited thereto.

In the present invention, the neuroinflammatory disease refers to a disease caused by damage to the nerve tissue by an inflammatory reaction, and preferably Alzheimer's disease, Parkinson's disease, Huntinton's disease, Lou Gehrig's disease, Creutzfeldt-Jakob disease, multiple sclerosis, amyotrophic lateral sclerosis, It may be any one or more diseases selected from the group consisting of diffuse Lewy body disease, leukoencephalitis, temporal lobe epilepsy, and inflammatory spinal cord injury, and more preferably Alzheimer's disease, but is not limited thereto.

In Alzheimer's disease, NLRP3 inflammasome activation of microglia is reported as an important mechanism, and even in an animal model of Alzheimer's disease, caspase-1, a cytokine caused by inplasmasome in the microglia, and The amount of IL-1 is increased (Immunol Rev. 2015 May; 265(1):63-74.).

According to a recent study, compounds that inhibit NLRP3 inflammasome formation, such as MCC950 or ketone metabolite β-hydroxybutyrate, and Glibenclamide, have shown that the auto-inflammatory disease Merkle-Well syndrome. Or it has been reported to exert a therapeutic effect through the process of alleviating the disease in neuro-inflammatory diseases such as Alzheimer's and CNS injury (Nature Medicine Volume: 21, Pages:248-255 Year published: (2015) DOI:doi:10.1038/nm). .3806, Nature Medicine Volume: 21, Pages:263-269 Year published:(2015) DOI:doi:10.1038/nm.3804). Therefore, the material selected by the present invention remarkably inhibits the formation of NLRP3 inflammasomes, so that it can be effectively used for the treatment of the auto-inflammatory disease and neuro-inflammatory disease, which are NLRP3 inflammasome-mediate inflammatory diseases. have.

On the other hand, in the present invention, "prevention" includes, without limitation, any action that blocks the symptoms of obesity or lipid-related metabolic diseases, or inhibits or delays the symptoms by using an inflammation inhibitory substance selected by the screening method according to the present invention. can do.

In addition, in the present invention, "treatment" may include, without limitation, any action that improves or benefits the symptoms of obesity or lipid-related metabolic disease by using an inflammation inhibitory substance selected by the screening method according to the present invention.

The sample of interest in the present invention may contain a biopsy isolated from the subject of interest, or an animal, preferably a tissue, cell, blood, serum, plasma, saliva, or sputum isolated from the subject, and preferably It may be blood, serum or plasma, more preferably monocytes or exosomes isolated from the blood, serum or plasma.

In the present invention, "BIG1" and "GBF1" are proteins belonging to the ADP-ribosylating factor guanine-nucleotide exchange factor (ARFGEF) family, and play an important role in intracellular vesicle transport. ARFGEF protein is involved in the activation of ARF by replacing GDP bound to ARF with GTP. It contains a Sec7 domain capable of causing guanine-nucleotide exchange activity.

The amplification of the genome can be performed by PCR (Polymerase Chain Reaction). PCR may be performed using a PCR reaction mixture containing components known to be necessary for a PCR reaction in the art or using commercially available apples. The PCR reaction mixture may include genomic DNA extracted from apples, SCAR primer set of the present application, an appropriate amount of DNA polymerase, dNTP, PCR buffer solution, and water. The PCR buffer solution may include Tris-HCl, MgCl2, KCl, or the like, but may not be limited thereto. In addition, methods of measuring the mRNA expression level include RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA), Northern Blotting (Northern blotting) and using a DNA chip, but is not limited thereto.

In addition, methods for measuring the protein expression level include Western blot, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immune diffusion method, and locate ( rocket) Immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, Complement Fixation Assay, FACS and protein chips are used, but are not limited thereto.

The present invention provides a composition for screening for inhibition of inflammation comprising a substance for measuring the mRNA level of the BIG1 or GBF1 gene or a substance for measuring the level of the protein encoded by the gene.

In one embodiment of the present invention, when the expression of the BIG1 or GBF1 gene is suppressed, inflammation is suppressed. In another embodiment of the present invention, when the expression of the BIG1 or GBF1 gene is inhibited, the activity of the NLRP3 inplasmasome is inhibited. In another embodiment of the present invention, the composition is used to treat many chronic neuroinflammatory or degenerative diseases including rheumatoid arthritis, Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, multiple sclerosis, amyotrophic lateral sclerosis, and inflammatory spinal injury. It is to predict what can be prevented or cured.

The present invention comprises the steps of: (a) treating a candidate material to cells expressing BIG1 or GBF1; (b) measuring the expression of BIG1 or GBF1 on the cells; And (c) selecting a substance that inhibits the expression of BIG1 or GBF1 as compared to the non-treated group of the candidate substance as an inflammation inhibitory substance.

In one embodiment of the present invention, (d) compared to the untreated group in the cells treated with the candidate substance, when the activity of the NLRP3 inplasmasome is inhibited, the step of selecting as an inflammation inhibitory substance is further included. In another embodiment of the present invention, the anti-inflammatory substance is a number of chronic neuroinflammatory or degenerative diseases including rheumatoid arthritis, Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, multiple sclerosis, amyotrophic lateral sclerosis, and inflammatory spinal injury. It is a substance that can prevent or treat. In another embodiment of the present invention, the cells are dendritic cells, natural killer cells, T cells, B cells, regulatory T cells, Treg cells), natural killer T cells, innate lymphoid cells, macrophages, granulocytes, and chimeric antigen receptor-expressing T cells (CAR-T). -T cell), Lymphokine-activated killer Cell (LAK), Cytokine Induced Killer Cell (CIK), fibroblast, chondrocyte, synovial cell ( synovial cells), skin keratinocytes, adipocytes, osteoblasts, osteoclasts, and peripheral blood mononuclear cells.

The present invention provides an inflammatory substance discrimination kit comprising the above-described composition for suppressing inflammation screening.

The present invention (a) measuring the expression of BIG1 or GBF1 from a sample of interest; (b) measuring the expression of BIG1 or GBF1 from the normal group; (c) Provides a method of comparing the measured value of step (a) with the measured value of step (b), and discriminating that the target sample has suppressed NLRP3 inplasmo when the measured value of step (a) is low. do.

In one embodiment of the present invention, when the NLRP3 inplasma is inhibited, the target sample includes rheumatoid arthritis, Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, multiple sclerosis, amyotrophic lateral sclerosis, and inflammatory spinal injury. It is judged to have low susceptibility to many chronic neuroinflammatory or degenerative diseases.

The screening method for determining as an inflammation inhibitor according to the present invention inhibits the expression of BIG1 or GBF1 in cells by treating the inflammation inhibitor, thereby inhibiting the activation of NLRP3 inplasmasome, and thus, rheumatoid arthritis, Alzheimer's disease, Parkinson's disease, Huntington's disease , Lou Gehrig's disease, multiple sclerosis, amyotrophic lateral sclerosis, and inflammatory spinal injury, and many chronic neuro-inflammatory or degenerative diseases.

However, the effects of the present invention are not limited to the above-mentioned effects, and other effects that are not mentioned will be clearly understood by those skilled in the art from the following description.

1 shows ATP (2mM, 30 min, A in FIG. 1) after treatment with LPS (0.25 μg/ml, 3h) in normal BMDM (Scr) or BIG1 ( Arfgef1 )-specific shRNA (BIG1)-expressing BMDM cells, or It is the result of immunoblot after treatment with alum crystals (500 μg/ml, 6 hours, FIG. 1B).
Figure 2 is a graph quantifying IL-1β in the culture supernatant of shScr or shBIG1 BMDM treated with ATP (2mM, 30 min) and LPS (0.25 μg/ml, 3 hours).
Figure 3 is for DSS-crosslinked pellets (DSS-pel) or cell lysates (Lys) from shScr or shBIG1 BMDM treated with ATP (2mM, 30 min) and LPS (0.25 μg/ml, 3 hours) This is the result of immunoblot.
Figure 4 shows the results of RT-PCR analysis after treatment with LPS (0.25µg/ml, 3h) and ATP (2.5mM, 30 minutes) and without treatment in normal BMDM (Scr) or GBF1-specific shRNA-expressing BMDM. .
5 is a result of immunoblot with culture supernatant (Sup) or cell lysate (Lys).
6A is a representative immunofluorescence image of mouse BMDM stained with untreated mouse BMDM (Unt) or mouse BMDM treated with BFA (2 μg/ml, 1 hour or 3 hours) and anti-GM130 antibody (red). 6B and C show normal BMDM (Scr) or shBig1 BMDM (B) or NLRP3-GFP-expressing BMDM treated with ATP (2mM, 30 minutes) and LPS (0.25 μg/ml, 3 hours) ( C) is a representative immunofluorescence image. For DAPI, nuclear signals are indicated in blue. Scale bar, 10 μm.
7 and 8 are graphs showing that BIG1 knockdown partially impairs LPS-induced mRNA production of pro-inflammatory cytokines.
7 shows immunoblot of cell lysates using the indicated antibodies. Immunoblot was performed with or without treatment with LPS (0.5 μg/ml, 1h or 3h) in the presence or absence of wild type BMDM and BFA (2 μg/ml) (Unt). Normal BMDM (Scr) or BIG1-knocked down BMDM was treated with LPS (0.5 μg/ml, 1, 3 or 6 hours).
8 is a graph quantifying the level of IL-1β or IL-6 mRNA in cell lysates determined by quantitative real-time PCR. * P <0.05; ** P <0.01; *** P <0.001. ns is not significant.
9 is an immunofluorescence image of BIG1-knockdown (shBIG1) mouse BMDM treated with ATP (2mM, 30 min) and LPS (0.25 μg/ml, 3h). It indicates that inhibition of BIG1-mediated vesicle transport does not inhibit ATP-induced mitochondrial spatial rearrangement. Stained with anti-Tom20 (red) and anti-α-tubulin (green) antibodies. Scale bar, 10 μm.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

mouse

C57BL/6 mice were obtained from Orient Bio (Seoul, Korea). Nlrp3 ^-/- mice were obtained from Jackson Laboratory (Bar Harbor, ME, USA) and grown at Yonsei University. Mice were grown under conditions without specific pathogens, and male mice aged 9 to 12 weeks were used in the experiment. The protocol for animal experiments was approved by the Ethics Committee of Yonsei University Medical School Hospital, and all experiments were performed according to the approved guidelines of the institutional ethics committee.

Reagents and antibodies

LPS, ATP, Brefeldin A (BFA; brefeldin A), nigerisin, MCC950 and poly dA:dT were commercially available from Sigma-Aldrich (St. Louis, MO, USA). GolgiPlug was obtained from BD Biosciences (San Jose, CA, USA). Ac-YVAD-chloromethylketone was obtained from Bachem (Torrance, CA, USA). Alum crystals were from InvivoGen (San Diego, CA, USA), Ciliobrevin D from Calbiochem (San Diego, CA, USA), and anti-mouse caspase-1 and anti-NLRP3 antibodies were from AdipoGen (San Diego, CA, USA). In, anti-mouse IL-1β antibody was from R&D Systems (Minneapolis, MN, USA), anti-ASC and anti-phospho-ERK antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-IκB, Anti-phospho-IκB and anti-IL-6 antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA), anti-BFA-inhibited guanine-nucleotide-exchange protein 1 (BIG1; brefeldin A-inhibited guanine nucleotide-exchange). The protein 1) antibody was obtained from Abcam (Cambridge, MA, USA), and the PE-conjugated anti-TLR4 antibody was obtained from BioLegend (San Diego, CA, USA).

Cell culture

Mouse primary bone marrow-derived macrophages (BMDM; bone marrow-derived macrophages) were harvested from the femurs of C57BL/6 or Nlrp3 ^-/- mice. Immortal NLRP3-GFP-expressing and NLPR3-reconstituted (N1-8) BMDMs were described by Dr. It was provided by ES (Thomas Jefferson University, Philadelphia, PA, USA). All BMDM was maintained in L929-conditioned DMEM supplemented with 10% FBS and antibiotics. Human peripheral blood mononuclear cells (PBMCs) were separated from blood by density gradient centrifugation. CD14 ⁺ monocytes were positively isolated from PBMCs using anti CD14 microbeads (Miltenyi Biotec Inc., Auburn, CA, USA). The study protocol was approved by the Institutional Review Board (No.1403-049-564) of Seoul National University Hospital. For knockdown of BIG1 encoded by Arfgef1, lentiviral particles containing untargeted or Arfgef1- specific short-hairpin (sh) RNA (Sigma-Aldrich) were used to infect mouse BMDM. Cells stably expressing shRNA were cloned by puromycin selection.

Immunoblot analysis

Cells were lysed in a buffer containing 20mM HEPES (pH 7.5), 0.5% Nonidet P-40, 50mM KCl, 150mM NaCl, 1.5mM MgCl ₂ , 1mM EGTA and protease inhibitor. The aqueous lysate was analyzed by SDS-PAGE and then transferred to a PVDF membrane. In some experiments, the cell culture supernatant was precipitated with methanol/chloroform and then immunoblot was performed.

mRNA quantification

To measure mRNA production, total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcribed using PrimeScript RT master mix (Takara, Tokyo, Japan). Quantitative real-time PCR was performed using SYBR Premix Ex Taq (Takara). Primers were as follows: 5'-GCC CAT CCT CTG TGA CTC AT-3' and 5'-AGG CCA CAG GTA TTT TGT CG-3' (mouse II-1β ); 5'-AGT TGC CTT GGG ACT GA-3' and 5'-TCC ACG ATT TCC CAG AGA AC-3' (mouse Il-6 ); 5'-CGC GGT TCT ATT TTG TTG GT-3' and 5'-AGT CGG CAT CGT TTA TGG TC-3' (mouse Rn18s ).

Inplasmasome activation assay

In order to stimulate the activation of NLRP3 inplasmasome, BMDM was treated with LPS (0.25 μg/ml, 3 hours), followed by treatment with ATP (2 mM, 40 minutes) or nigericin (5 μM, 40 minutes). Poly dA:dT (1 μg/ml) was transfected with BMDM for 6 hours to induce inflammasome activation (AIM2; absent in melanoma 2). NLR family, CARD domain-containing 4 (NLRC4; NLR family, CARD domain-containing 4) In order to induce inplasmasome activation, Pseudomonas aeruginosa PAO1 was used to infect BMDM as previously described. Activation of inplasmasome was determined by the presence of active caspase-1 p20 and active IL-1β from the culture supernatant in an immunoblot, and determined by quantification of extracellular IL-1β using a Quantikine ELISA kit (R&D Systems).

NLRP3 inplasmasome assembly analysis

To measure the oligomerization of NLRP3, spot-like aggregates of NLRP3-GFP were evaluated by multifocal fluorescence microscopy in NLRP3-GFP-expressing BMDM. To determine ASC oligomerization, disuccinimidyl suberate (DSS, Thermo Fisher Scientific, Waltham, MA, USA)-mediated cross-linking assay was performed. To visualize the molecular interactions of ASC and NLRP3, proximity-ligation using the Duolink in situ red starter kit (Sigma-Aldrich) using anti-ASC or anti-NLRP3 antibodies according to the manufacturer's protocol. Analysis was carried out. Relative proximity linkage signal (PL signal/DAPI signal) was quantified using Image J software (National Institutes of Health, Bethesda, MD, USA) and calculated as relative fold change compared to the untreated control.

Immunofluorescence assay

Cells were grown on coverslips in 12-well plates, and after appropriate treatment the cells were fixed with 4% paraformaldehyde for 30 minutes and permeated with 0.2% Triton X-100 for 15 minutes. After 1 hour blocking with 4% BSA, the cells were incubated with the primary antibody for 1 hour, washed with PBS, and Alexa 488- or FITC-conjugated anti-mouse or anti-rabbit IgG antibody (Jackson ImmunoResearch, West Grove , PA, USA) for 30 minutes. Cells were observed with a multifocal fluorescence microscope (LSM 700, Carl Zeiss, Oberkochen, Germany).

Assay for potassium in cells

In order to measure the level of intracellular potassium, an inductively coupled plasma-optical emission spectrometry was performed using an OPTIMA 8300 ICP spectrometer (Perkin Elmer, Waltham, MA, USA). Cells were lysed with 10% HNO ₃ and intracellular potassium levels were measured.

TLR4 internalization analysis

After appropriate treatment, cells were washed with PBS containing 5% FBS, incubated with anti-TLR4-PE antibody (0.1 μg/ml) for 30 minutes at room temperature, followed by flow cytometry (FACSverse, BD Biosciences). Analyzed.

Statistical analysis

All values are expressed as the mean±standard error (SE) of individual samples. Data were analyzed by comparing the normal group and the BIG-1-knocked down group using a one-way analysis of variance (ANOVA), Dunnett or Bonferroni post-hoc test, or bidirectional ANOVA with Bonferroni post-hoc test. Statistically, the significance level was set to p≤0.05. Analysis was performed using GraphPad Prism (GraphPad Software, San Diego, CA, USA).

BIG1 or GBF1 deficiency inhibits the activation of NLRP3 inplasmasome.

BFA (brefeldin A) treatment clearly reduced NLRP3 inplasmasome assembly and activation in macrophages. BFA inhibited vesicle formed from the corrugated film by disabling ARFGEF (ADP-ribosylation factor guanine nucleotide -exchange factors) , such as an encrypted BIG1 (brefeldin A-inhibited guanine nucleotide -exchange protein 1) by Arfgef1. The present inventors investigated whether impaired BIG1 expression affects NLRP3 inplasmasome activation. BIG1-knocked down bone marrow-derived macrophages (BMDMs) were generated by infecting lentiviral particles expressing Arfgef1-targeted shRNA. After stimulation with LPS/ATP, it was confirmed that the levels of active caspase-1 and IL-1β were significantly reduced compared to normal BMDM as a control in the culture supernatant after stimulation with LPS/ATP (FIGS. 1A and 2). Consistently, it was confirmed that the activation of LPS/alum-stimulated caspase-1 and IL-1β was clearly inhibited in BIG1-knocked down BMDM (Fig. 1B). In addition, due to the deficiency of BIG1, it was confirmed that ASC (apoptosis-associated speck-like protein containing a caspase-recruitment domain) oligomerization induced by LPS/ATP stimulation was significantly suppressed (FIG. 3). These results indicate that BFA-sensitive BIG1 can contribute to the activation of the NLRP3 inplasmasome pathway in BMDM in response to treatment with LPS and NLRP3 agonists.

Since ARFGEF BIG1 acts in the trans-Golgi region, the present inventors further experimented with the influence of cis-Golgi-located ARFGEF GBF1 (Golgi-specific BFA-resistance guanine nucleotide exchange factor 1) on NLRP3 inplasmasome signaling. . GBF1-knocked down BMDM was generated by shRNA delivery (Fig. 4). GBF1-knocked down BMDM confirmed caspase-1 activation and impaired IL-1β secretion significantly impaired by LPS/ATP stimulation (FIG. 5 ). It was confirmed that GBF1-mediated endoplasmic reticulum (ER)-Golgi vesicle transport plays an important role in the activation of NLRP3 inplasmasome.

When BFA is treated but BIG1 is deficient, Golgi organization is destroyed.

Since long-term BFA treatment destroys Golgi structure, the present inventors conducted an experiment on whether NLRP3 inplasmasome signaling impairment in BFA-treated macrophages or BIG1-knocked down macrophages damages Golgi structure. It was confirmed that BFA treatment caused damage to Golgi organization in a time-dependent manner (FIG. 6A). On the other hand, in the BIG1-knockdown BMDM, the intact Golgi structure was confirmed in a stationary state (FIG. 6B). At least in the case of BIG1-knockdown macrophages, it was confirmed that Golgi destruction was not primarily due to the activation of weakened NLRP3 inplasmasome. It was confirmed whether aggregates such as NLRP3 stains were formed around the Golgi. On the other hand, the NLRP3 aggregates formed by LPS/ATP stimulation were not localized with the Golgi apparatus (FIG. 6C), and it was suggested that the Golgi apparatus may not act as a platform for assembly of NLRP3 inplasmasome.

Inhibition of vesicle transport impairs LPS-promoted IκB phosphorylation and subsequent transcription.

In general, NLRP3 inplasmasome activation can be achieved by two signals. Signal 1 for priming and signal 2 for activation. Regarding the inhibition of BFA treatment or BIG1 knockdown-mediated NLRP3 inplasmasome activation, the potential role of BFA in signal 1 and signal 2 induced events was evaluated. The present inventors first confirmed the signal 1-related LPS-induced activation of the NF-κB pathway, which is a major priming for transcription of inplasmasome components including pro-IL-1β. BPS treatment did not change IκB phosphorylation 1 hour after LPS stimulation, but inhibited IκB phosphorylation 3 hours after LPS treatment, whereas LPS generated strong phosphorylation of IκB in BMDM (Fig. 7A). Likewise, BIG1 deficiency slightly impaired IκB phosphorylation at 3 hours after LPS treatment, but not at 1 hour (Fig. 7B).

We measured the mRNA levels of NF-κB target genes including pro-IL-1β and IL-6 after LPS priming. In BIG1-knocked down macrophages, it was shown that the levels of IL-1β and IL-6 mRNA significantly decreased 3 hours or 6 hours after LPS priming (Fig. 8A and B).

LPS can regulate downstream intracellular signaling pathways by inducing TLR4 internalization. Therefore, it was investigated whether inhibition of BIG1 attenuates signal transduction by blocking TLR4 internalization. As a result, LPS priming resulted in time-dependent TLR4 internalization, indicating that TLR4 is implicit upon LPS participation. However, treatment with the inhibitor of BIG1 did not affect LPS-induced TLR4 internalization. These observations indicate that vesicle transport around the Golgi apparatus may be involved in LPS-induced priming events involving the NF-κB mediated pathway, but not TLR4 internalization.

Inhibition of vesicle transport does not inhibit ATP-induced mitochondrial spatial arrangement.

According to a recent study, the ER membrane associated with mitochondria, formed by mitochondrial transport to ER, plays a crucial role as a platform for assembly of NLRP3 inplasmasome. Therefore, the present inventors investigated whether damaged Golgi vesicle transport under NLRP3 activation conditions affects the spatial rearrangement of mitochondria in BMDM. LPS/ATP stimulation caused a clear mitochondrial rearrangement movement around the nucleus even in Nlrp3- deficient macrophages. ATP treatment alone was sufficient to facilitate this mitochondrial rearrangement movement. The rearrangement of mitochondria was blocked by treatment with the dinein inhibitor ciliobrevin D, and it was shown that ATP stimulation moves mitochondria retrogradely along microtubules. On the other hand, BFA treatment had no effect on mitochondrial movement to the nuclear region after LPS/ATP stimulation. Consistent with this finding, ATP-promoted mitochondrial spatial organization was also evident in the BIG1-soluble type BMDM (FIG. 9 ). These results indicate that Golgi vesicle transport damaged due to BIG1 knockdown did not affect ATP-induced mitochondrial movement.

Through the above results, treatment with a substance that inhibits BIG1 or GBF1 inhibits ER-Golgi vesicle transport due to BIG1 or GBF1 deficiency, and effectively inhibits NLRP3 inflammasome activity, which induces auto-inflammatory disease or neuro-inflammatory disease in macrophages. It can be seen that it suppresses.

Although the present invention has been described in detail above, the scope of the present invention is not limited thereto, and various modifications and variations are possible without departing from the technical spirit of the present invention described in the claims. It will be self-evident to those who have the knowledge of.

Claims (11)

A composition for screening a substance that suppresses inflammation caused by NLRP3 inflammasome through inhibition of BIG1 or GBF1 gene expression, including a substance measuring the mRNA level of the BIG1 or GBF1 gene or a substance measuring the protein level encoded by the gene . The method of claim 1,
The substance that inhibits inflammation is to inhibit inflammation through inhibition of BIG1 or GBF1 gene expression. delete The method of claim 1,
The substance screened for suppressing the expression of the BIG1 or GBF1 gene by the composition for screening the substance that suppresses inflammation is capable of preventing or treating auto-inflammatory diseases, neuro-inflammatory diseases, or degenerative diseases,
The auto-inflammatory diseases include Merkle-Wells syndrome (MWS), LADA (adult-type delayed autoimmune diabetes), familial cold auto-inflammatory syndrome (FCAS), cryopyrin-related periodic syndrome (CAPS), neonatal -Onset multi-organ inflammatory syndrome (NOMID), chronic infant neurocutaneous joint (CINCA) syndrome, familial Mediterranean fever (FMF), systemic onset childhood idiopathic arthritis (SJIA), systemic onset childhood idiopathic arthritis, and adult rheumatoid arthritis Is any one disease selected from,
The neuro-inflammatory disease is any one selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, Creutzfeldt-Jakob disease, multiple sclerosis, amyotrophic lateral sclerosis, diffuse Lewy body disease, leukoencephalitis, temporal lobe epilepsy, and inflammatory spinal cord injury. The composition, which is a disease. (a) treating cells isolated from individuals expressing BIG1 or GBF1 with a candidate substance;
(b) measuring the expression of BIG1 or GBF1 on the cells;
(c) selecting a substance that inhibits the expression of BIG1 or GBF1 as an inflammation inhibitor compared to the candidate substance-untreated group; And
(D) compared to the non-treated group in the cells treated with the candidate substance, when the activity of the NLRP3 inplasmasome is inhibited, the method for screening an inflammation inhibitory substance comprising the step of selecting as an inflammation inhibitory substance. delete The method of claim 5,
The inflammation inhibitory substance is capable of preventing or treating auto-inflammatory diseases, neuro-inflammatory diseases, or degenerative diseases,
The auto-inflammatory diseases include Merkle-Wells syndrome (MWS), LADA (adult-type delayed autoimmune diabetes), familial cold auto-inflammatory syndrome (FCAS), cryopyrin-related periodic syndrome (CAPS), neonatal -Onset multi-organ inflammatory syndrome (NOMID), chronic infant neurocutaneous joint (CINCA) syndrome, familial Mediterranean fever (FMF), systemic onset childhood idiopathic arthritis (SJIA), systemic onset childhood idiopathic arthritis, and adult rheumatoid arthritis Is any one disease selected from,
The neuro-inflammatory disease is any one selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, Creutzfeldt-Jakob disease, multiple sclerosis, amyotrophic lateral sclerosis, diffuse Lewy body disease, leukoencephalitis, temporal lobe epilepsy, and inflammatory spinal cord injury. Which is a disease, the method. The method of claim 5,
The cells are dendritic cells, natural killer cells, T cells, B cells, regulatory T cells, Treg cells, and natural killer T cells ( natural killer T cell), innate lymphoid cell, macrophage, granulocyte, chimeric antigen receptor-T cell (CAR-T), lymphokine activity Lymphokine-activated killer Cell (LAK), Cytokine Induced Killer Cell (CIK), fibroblast, chondrocyte, synovial cell, skin keratinocyte ( keratinocyte), adipocytes (adipocyte), osteoblasts (osteoblast), osteoclasts (osteoclast) and peripheral blood mononuclear cells (peripheral blood mononuclear cells) any one selected from the group consisting of. An inflammation inhibitory substance discrimination kit comprising a composition for screening a substance that suppresses inflammation of any one of claims 1, 2 and 4. (a) measuring the expression of BIG1 or GBF1 from the sample of interest isolated from the subject;
(b) measuring the expression of BIG1 or GBF1 from the normal group;
(c) Comparing the measured value in step (a) with the measured value in step (b), and when the measured value in step (a) is low, the method of determining that the target sample has suppressed NLRP3 inplasmasome. The method of claim 10,
In the above method, when NLRP3 inplasmasome is inhibited, it is determined that the individual from which the target sample is derived has low susceptibility to auto-inflammatory disease, neuro-inflammatory disease, or degenerative disease,
The auto-inflammatory diseases include Merkle-Wells syndrome (MWS), LADA (adult-type delayed autoimmune diabetes), familial cold auto-inflammatory syndrome (FCAS), cryopyrin-related periodic syndrome (CAPS), neonatal -Onset multi-organ inflammatory syndrome (NOMID), chronic infant neurocutaneous joint (CINCA) syndrome, familial Mediterranean fever (FMF), systemic onset childhood idiopathic arthritis (SJIA), systemic onset childhood idiopathic arthritis, and adult rheumatoid arthritis Is any one disease selected from,
The neuro-inflammatory disease is any one selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, Creutzfeldt-Jakob disease, multiple sclerosis, amyotrophic lateral sclerosis, diffuse Lewy body disease, leukoencephalitis, temporal lobe epilepsy, and inflammatory spinal cord injury. Which is a disease, the method.

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