Anti-NMDAR encephalitis target spot and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to an anti-NMDAR encephalitis target spot and application thereof. The anti-NMDAR encephalitis target spot comprises an IRF7 gene or / and an expression protein. The application of the target spot comprises but is not limited to preparation of an anti-NMDAR encephalitis drug, a CRISPR / Cas9 system for treating the anti-NMDAR encephalitis drug, an anti-NMDAR encephalitis diagnostic kit and an anti-NMDAR encephalitis disease drug screening model. It is found for the first time that abnormal activation of the IRF7 in peripheral mononuclear cells is positively correlated with the severity of the anti-NMDAR encephalitis disease, and it is proved through a gene knockout (KO) model and in-vitro experiments that IRF7 defects can significantly relieve pathological injuries. A therapeutic drug aiming at the target spot can effectively treat anti-NMDAR encephalitis. The therapeutic drug can relieve BBB damage and disease severity in anti-NMDAR encephalitis by reducing the expression of IRF7.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically to an anti-NMDAR encephalitis target and application thereof. Background Art

[0002] Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is a severe neurological disorder characterized by antibodies targeting NMDARs detected in serum or cerebrospinal fluid (CSF). The antibodies bind to the NMDA receptor, leading to its internalization in the hippocampus. Although patients may require intensive care unit treatment and experience multiple relapses, most patients show remarkable recovery after immunotherapy. Tumors, viral infections, and genetic susceptibility have been implicated in the etiology of the disease.

[0003] The pathophysiology of this disease is complex and involves disruption of the blood-brain barrier (BBB), a tightly regulated interface that maintains the brain microenvironment. BBB disruption allows peripheral immune cells and antibodies to infiltrate the central nervous system (CNS), leading to the progression of autoimmune encephalitis and other CNS diseases. Immune cells infiltrating the CNS exacerbate damage through the secretion of antibodies and proinflammatory cytokines. BBB leakage in patients with anti-NMDAR encephalitis is associated with increased intrathecal IgG synthesis, disease severity, and poor prognosis.

[0004] Current treatment for anti-NMDAR encephalitis relies primarily on immunosuppression, but efficacy is limited in some patients and carries the risk of relapse. Existing technologies have yet to elucidate the role of peripheral immune cells (such as monocytes) and their molecular regulatory mechanisms in disease progression, particularly the key regulatory targets for blood-brain barrier disruption and central inflammatory infiltration. Summary of the Invention

[0005] (1) Technical issues to be resolved

[0006] In view of the above shortcomings and deficiencies of the prior art, the present invention provides a new target for anti-NMDAR encephalitis, which is the IRF7 gene and / or expressed protein;

[0007] Correspondingly, the present invention also provides an application of an anti-NMDAR encephalitis target in the preparation of an anti-NMDAR encephalitis drug and a diagnostic kit.

[0008] (II) Technical Solution In order to achieve the above-mentioned objectives, the main technical solutions adopted by the present invention include:

[0009] In a first aspect, the present invention provides a target for anti-NMDAR encephalitis, which includes the IRF7 gene and / or expressed protein.

[0010] The present invention discovered for the first time that abnormal activation of IRF7 in peripheral monocytes is positively correlated with the severity of anti-NMDAR encephalitis (including BBB destruction and behavioral abnormalities), and demonstrated through gene knockout (KO) models and in vitro experiments that IRF7 deficiency can significantly alleviate pathological damage.

[0011] In a second aspect, the present invention also provides a use of the target described in any of the above schemes in the preparation of anti-NMDAR encephalitis drugs.

[0012] Optionally, the anti-NMDAR encephalitis drug inhibits the expression and / or activity of IRF7.

[0013] The anti-NMDAR encephalitis drugs involved in this program can maintain BBB integrity, reduce central inflammatory infiltration, inhibit the pro-inflammatory phenotype transformation of monocytes / macrophages, and reduce autoantibody-mediated neuronal damage.

[0014] Optionally, the anti-NMDAR encephalitis drug is used to prepare a drug for reducing blood-brain barrier damage and monocyte / macrophage-mediated central inflammatory response.

[0015] Optionally, the anti-NMDAR encephalitis drug is siRNA, shRNA, small molecule inhibitor or / and monoclonal antibody targeting the target.

[0016] In a third aspect, the present invention also provides a CRISPR / Cas9 system for preparing a drug for treating anti-NMDAR encephalitis, comprising: an sgRNA specifically targeting the IRF7 gene;

[0017] Cas9 protein or its coding sequence;

[0018] A vector for delivering the sgRNA and Cas9 protein.

[0019] In a fourth aspect, the present invention also provides a use of the anti-NMDAR encephalitis target described in any of the above schemes in an anti-NMDAR encephalitis diagnostic kit, which includes a specific probe for detecting the expression level of IRF7 in peripheral mononuclear cells.

[0020] In a fifth aspect, the present invention also provides the use of IRF7 in any of the above schemes as a target for anti-NMDAR encephalitis in a disease drug screening model for anti-NMDAR encephalitis. The model is based on an in vitro co-culture system of IRF7-deficient monocytes or / and the blood-brain barrier, and is used to evaluate the effects of drugs on IRF7 expression, monocyte migration ability and / or BBB integrity.

[0021] In a sixth aspect, the present invention also provides the use of IRF7 in any of the above schemes as a target for anti-NMDAR encephalitis and in a diagnostic marker or / and disease severity assessment indicator for anti-NMDAR encephalitis.

[0022] In a seventh aspect, the present invention further provides the use of IRF7 in any of the above schemes as a target for anti-NMDAR encephalitis in a disease drug screening model for anti-NMDAR encephalitis, wherein the model is an IRF7-deficient animal model.

[0023] (3) Beneficial effects

[0024] The beneficial effects of the present invention are:

[0025] The present invention provides a novel anti-NMDAR encephalitis target and application. A therapeutic drug targeting this target can effectively treat anti-NMDAR encephalitis. By reducing IRF7 expression, the therapeutic drug can alleviate BBB disruption and disease severity in anti-NMDAR encephalitis.

[0026] The new anti-NMDAR encephalitis target can be effectively used in drug screening models and assay kits to evaluate the effects on IRF7 expression, monocyte migration ability, or BBB integrity. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a graph showing the BBB permeability assessment results of anti-NMDAR encephalitis patients and a healthy control group (HC) in Example 1;

[0028] Figure 2 This is a graph showing the correlation analysis results between the number of peripheral leukocytes (monocytes, lymphocytes, and neutrophils) and clinical data;

[0029] Figure 3 Figure 2 shows the results of detecting the expression levels of IRF7 and phosphorylated IRF7 in the patient's monocytes;

[0030] Figure 4 This is a flow chart of the experiment in Example 3 for immunizing mice with the GluN1359-378 peptide and monitoring the development of the disease;

[0031] Figure 5 The figure shows the results of Western blotting to determine the expression of GluN1 in the hippocampal membrane of WT wild-type mice and WT model mice and the statistical relative protein expression analysis in Example 3;

[0032] Figure 6 This is a graph showing the experimental results of detecting anti-NMDAR antibodies in mouse serum by cell-based assay (CBA) in Example 3;

[0033] Figure 7 This is a graph showing the results of the open field test for detecting anxiety and depression-like behaviors in mice in Example 3;

[0034] Figure 8This is a diagram showing the results of the Y-maze experiment in Example 3 to detect the spatial memory of mice;

[0035] Figure 9 This is a graph showing the experimental results of flow cytometry analysis of the number of leukocyte populations in mouse peripheral blood in Example 3;

[0036] Figure 10 Graph showing the results of detecting IRF7 expression levels in different leukocyte subsets in Example 3;

[0037] Figure 11 Graph showing the correlation analysis results between monocyte IRF7 expression levels and Y-maze behavioral parameters in Example 3;

[0038] Figure 12 This is a graph showing the results of flow cytometry analysis of changes in the number of infiltrating immune cells in the brain tissue of model mice in Example 3;

[0039] Figure 13 This is a graph showing the results of detecting the expression level of IRF7 in monocytes, B cells, and T cells after infiltration in Example 3;

[0040] Figure 14 Graph showing the correlation analysis results between monocyte IRF7 expression levels and mouse behavioral parameters (O-maze, Y-maze) in Example 3;

[0041] Figure 15 This is a graph showing the experimental results of immunofluorescence detection of autoantibody deposition in the hippocampus of IRF7 knockout mice in Example 3;

[0042] Figure 16 This is a graph showing the expression level changes of GluN1 subunit in mouse hippocampus detected by Western blot in Example 3;

[0043] Figure 17 This is a graph showing the results of the open field test in Example 3 to detect anxiety and depression-like behaviors in each group of mice;

[0044] Figure 18 This is a graph showing the results of the novel object recognition experiment in Example 3 to detect the cognitive functions of mice in each group;

[0045] Figure 19 This is a graph showing the results of the forced swimming test in Example 3 to detect depressive-like behaviors in each group of mice;

[0046] Figure 20 The results of the Y-maze experiment in Example 3 to test the spatial memory of mice in each group;

[0047] Figure 21 This is a graph showing the experimental results of Western blotting to detect the integrity of the blood-brain barrier of each group of mice in Example 3;

[0048] Figure 22 For Example 3 Figure 22 Statistical analysis results diagram;

[0049] Figure 23 The figure shows the results of the assay of related markers in BMDM of IRF7 knockout mice and wild-type mice after BBB damage in Example 3;

[0050] Figure 24 Schematic diagram of the in vitro BBB model in Example 3;

[0051] Figure 25 This is a graph showing the experimental results of immunofluorescence detection of the effect of BMDM on the tight junction protein claudin-5 in Example 3. DETAILED DESCRIPTION

[0052] In order to explain in detail the possible application scenarios, technical principles, specific solutions that can be implemented, and the purpose and effects achieved by this application, the following is a detailed description of the specific embodiments listed. The embodiments described herein are only used to more clearly illustrate the technical solutions of this application and are therefore only examples and are not intended to limit the scope of protection of this application.

[0053] References to "embodiments" herein mean that the specific features, structures, or characteristics described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the word "embodiment" in various places in the specification does not necessarily refer to the same embodiment, nor does it particularly limit its independence or relevance to other embodiments. In principle, in this application, as long as there are no technical contradictions or conflicts, the various technical features mentioned in the embodiments can be combined in any manner to form a corresponding implementable technical solution.

[0054] Unless otherwise defined, the technical terms used herein have the same meanings as those generally understood by those skilled in the art to which this application belongs; the use of relevant terms herein is only for describing specific embodiments and is not intended to limit this application.

[0055] In the description of this application, the term "and / or" is used to describe a logical relationship between objects, indicating that three relationships can exist. For example, A and / or B means: A exists, B exists, and both A and B exist. In addition, the character " / " in this document generally indicates that the objects before and after are in a logical "or" relationship.

[0056] In this application, terms such as "first" and "second" are merely used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual quantity, priority or sequence relationship between these entities or operations.

[0057] Without further limitations, in this application, the words "include", "comprise", "have" or other similar expressions used in the sentences are intended to cover non-exclusive inclusion. These expressions do not exclude the presence of additional elements in the process, method or product including the elements, so that the process, method or product including a series of elements may include not only those defined elements, but also other elements not explicitly listed, or elements inherent to such process, method or product.

[0058] Consistent with the understanding in the Examination Guidelines, in this application, expressions such as "greater than," "less than," and "exceed" are understood to exclude the number itself; expressions such as "above," "below," and "within" are understood to include the number itself. Furthermore, in the description of the embodiments of this application, "multiple" means more than two (including two), and similar expressions related to "multiple" are also understood in this manner, such as "multiple groups," "multiple times," etc., unless otherwise specifically defined.

[0059] In the description of the embodiments of the present application, the space-related expressions used, such as "center", "longitudinal", "lateral", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "vertical", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise", "axial", "radial", "circumferential", etc., indicate the orientation or position relationship based on the orientation or position relationship shown in the specific embodiments or drawings, and are only for the convenience of describing the specific embodiments of the present application or facilitating the reader's understanding, and do not indicate or imply that the device or component referred to must have a specific position, a specific orientation, or be constructed or operated in a specific orientation. Therefore, it should not be understood as a limitation on the embodiments of the present application.

[0060] Unless otherwise expressly specified or limited, in the description of the embodiments of the present application, the terms "installed", "connected", "connected", "fixed", "set", etc. used should be understood in a broad sense. For example, the "connection" can be a fixed connection, a detachable connection, or an integrated setting; it can be a mechanical connection, an electrical connection, or a communication connection; it can be a direct connection or an indirect connection through an intermediate medium; it can be the internal connection of two elements or the interaction relationship between two elements. For those skilled in the art of the present application, the specific meanings of the above terms in the embodiments of the present application can be understood according to the specific circumstances.

[0061] Example 1

[0062] This example provides a method for verifying the correlation between peripheral monocytes and BBB permeability in patients with anti-NMDAR encephalitis and healthy patients.

[0063] This example recruited 31 patients with NMDAR encephalitis and 30 healthy controls from the Department of Neurology, The Third Affiliated Hospital of Sun Yat-sen University. The included patients met the clinical diagnostic criteria according to the guidelines. The study protocol adhered to the ethical principles outlined in the 1975 Declaration of Helsinki and was approved by the local ethics committee of the Third Affiliated Hospital of Sun Yat-sen University ([(2019)2-637]). Informed written consent was obtained from all patients or their representatives.

[0064] In this method, 18 patients with anti-NMDAR encephalitis and 17 sex- and age-matched healthy controls (HC) were examined for BBB permeability assessment using 99mTc-DTPA SPECT / CT. Figure 1 Figure 2. Representative 99mTc-DTPA SPECT / CT images of healthy controls (HCs) and patients with anti-NMDAR encephalitis (cases). BBB leakage analysis results were performed. Bave (referring to BBB permeability) was analyzed for HCs and patients with anti-NMDAR encephalitis. N = 17 per group. ***P < 0.001; by Welcht test (mean ± SEM). Figure 1 It can be seen that the average BBB value (BBB permeability index) was significantly increased in patients compared with the healthy control group, indicating that the BBB in patients was damaged.

[0065] Figure 2 Correlations between various peripheral blood counts (monocytes, lymphocytes, and neutrophils) and clinical data (Bave, mRS, CSF total protein concentration, and CSF leukocyte count) were analyzed. Correlation coefficients were determined using Spearman correlation analysis (n = 30-35). *P < 0.05, **P < 0.01. Figure 2 It can be seen that the patient's peripheral monocyte count showed a positive correlation with Bave, mRS and CSF total protein concentration, but had no statistical correlation with CSF white blood cell (WBC) count. This correlation indicates that the peripheral monocyte count is positively correlated with the severity of BBB destruction in patients with anti-NMDAR encephalitis, emphasizing the key role of peripheral monocytes in BBB destruction. In addition, the peripheral lymphocyte count was negatively correlated with mRS, but had no significant correlation with Bave, CSF WBC count or CSF total protein concentration. Neutrophil count had no statistical correlation with the above clinical data.

[0066] Flow cytometric analysis was performed to assess IRF7 activation in circulating monocytes from recently recruited healthy controls and patients with anti-NMDAR encephalitis.

[0067] Figure 3Figure 2 shows flow cytometric analysis of IRF7 and phosphorylated IRF7 expression in peripheral blood mononuclear cells (CD14+) from healthy controls (HCs) and patients. Mean fluorescence intensity (MFI) of IRF7 (PE-conjugated) and phosphorylated IRF7 (Alexa Fluor 488-conjugated) in circulating monocytes was measured. N = 10 per group. *P < 0.05, **P < 0.01; Student's t-test (mean ± SEM). The results show elevated expression of IRF7 and phosphorylated IRF7 in monocytes from patients, suggesting activation of IRF7 in monocytes associated with anti-NMDAR encephalitis.

[0068] Figure 1-3 Middle: mRS is modified Rankin Scale; CSF is cerebrospinal fluid; TP is total protein; WBC is cells; p-IRF7 is phosphorylated IRF7; HCs are healthy controls.

[0069] Example 2

[0070] A method for constructing an anti-NMDAR encephalitis mouse model, comprising the following steps:

[0071] S1 Animals: Female C57BL / 6N mice and IRF7 knockout mice on a C57BL / 6N background (C57BL / 6N-Irf7 em3yagen) were housed in the animal facility of South China Agricultural University under designated pathogen-free conditions with a 12-h light-dark cycle. All animal studies were reviewed and approved by the Animal Research Ethics Committee of South China Agricultural University.

[0072] S2 peptide design: The 359-378 peptide of GluN1 can successfully induce anti-NMDAR encephalitis in mice. This example uses this peptide with the sequence RKLVQVGIYNGTHVIPNDRK, purchased from China Peptide Company (Shanghai, China) with a purity of 99%;

[0073] S3 Active Immunity:

[0074] The anti-NMDAR encephalitis mouse model was established using 6-8 week old mice. The GluN1359-378 peptide was dissolved in normal saline, while Mycobacterium tuberculosis H37 Ra (231141, BD) was dissolved in an equal volume of complete Freund's adjuvant (F5881, Sigma-Aldrich). These solutions were mixed to form the immunogen. The immunogen was subcutaneously injected into the dorsal region of the mice, with each animal receiving a total of 800 μg of GluN1359-378 peptide and 600 μg of Mycobacterium tuberculosis H37 Ra in a volume of 200 μL. 50 μL of the immunogen was subcutaneously injected into four different sites on the back of the mouse. At the same time, on the day of the first immunization, 450 ng of pertussis toxin (PTX, P7208, Sigma-Aldrich) was intraperitoneally injected into each mouse. 48 hours after the first immunization, 400 ng of PTX was intraperitoneally injected into each mouse. 7 days after the first immunization, the mice underwent a second immunization, and the procedures were the same as the first.

[0075] In other specific embodiments, in order to determine whether there are anti-NMDAR antibodies in mouse serum, serum was collected 14 days after the first immunization, and immunofluorescence assay was performed on HEK-293 T cells transfected with GluN1. Polyplus) transfected these cells with 500 ng M68CT-GluN1 plasmid. Subsequently, the cells were fixed with 4% paraformaldehyde on ice for 1 hour and then blocked with 3% bovine serum albumin (BSA, SigmaAldrich) for 1 hour at room temperature. The cells were incubated with mouse serum (diluted 1:1 in PBS) at 4°C overnight. A rabbit anti-GluN1 antibody (21287, 1:100, SignalwayAntibody) was used as a positive control. The next day, the cells were washed and incubated with an anti-rabbit secondary antibody conjugated to AlexaFluor488 (1:1000, 711-545-152, Jackson) and an anti-mouse secondary antibody conjugated to Cy3 (1:1000, 115-165-003, Jackson) at room temperature for 1 hour.

[0076] This model can be used to perform behavioral assessments, open field tests, novel object recognition tests, zero maze, Y maze, forced swim test, human peripheral blood mononuclear cell isolation, immunofluorescence staining, Western blotting, and flow cytometry.

[0077] Behavioral assessments began 4 weeks after the first immunization. Mice were acclimated to the experimenter and experimental apparatus before these assessments. To mitigate the effects of odor cues from the previous mouse, the apparatus was cleaned before each trial. Locomotor activity of the mice was monitored and analyzed using DigBehv software (Shanghai Metrology Software Technology Co., Ltd.).

[0078] Open field test (OFT) method: The open field test is used to evaluate the motor ability and anxiety and depression behaviors of mice. In this experiment, each mouse is placed in a 40x40x40cm 3 Each mouse was placed in a box and allowed to explore freely for 10 minutes. During this process, key parameters were recorded, including total distance traveled, total activity time, center distance, and center time.

[0079] Novel Object Recognition Test (NORT) Method: It is designed to assess the cognitive ability of mice. Mice usually show a preference for novel objects rather than familiar objects. The day before the test, an open field test was performed to acclimate the mice to a 40x40x40cm 3 . In this experiment, two identical objects were placed at the 1 / 3 and 2 / 3 points on the diagonal of the box. During the training phase, each mouse was placed in the center of the box and allowed to freely explore the two objects for 10 minutes. Subsequently, after an interval of 2 hours, one of the objects was replaced with a new object that was different in shape and color from the familiar object. The mice were then allowed to explore the familiar and new objects for 10 minutes. The exploration time for the familiar (a) and new objects (b) was recorded. If the total exploration time for the two objects was less than 20 seconds, the data was excluded. The recognition index was calculated using the following formula: 100% × time spent exploring b / (time spent exploring a + time spent exploring b).

[0080] The Zero Maze (Zero Maze): The Zero Maze is a validated instrument for assessing anxiety-like behavior in mice. Anxious mice prefer the safety of enclosed spaces to open areas. This experiment captures the conflict between the desire to seek refuge in an enclosed arm and the curiosity to explore the open arms. The maze consists of a PVC circular corridor with a diameter of 40 cm, a track width of 5 cm, and a height of 50 cm above the ground. The circular track is symmetrically divided into four quadrants, with two open arms and two enclosed arms facing each other. The walls of the enclosed arms are 12 cm high. Each mouse is placed at the entrance of the same enclosed arm and allowed to freely explore the maze for 10 minutes.

[0081] Y-maze: A maze is an instrument used to assess spatial memory. It is made of plastic and has three arms with identical features. The experiment consists of a training and a testing phase. Initially, during the training phase, access to one arm is blocked by a barrier. Each mouse is placed in the center of the maze and allowed to freely explore both accessible arms for 8 minutes. After a 2-hour interval, the testing phase begins. The barrier is removed, and the mouse is placed in the center and allowed to freely explore all three arms for another 8 minutes.

[0082] Forced Swim Test (FST): This test assesses depressive-like behavior in mice, characterized by immobility time in water. Each mouse is placed in a transparent cylindrical container with water at 24 ± 1°C. A video camera placed in front of the apparatus records the mouse's movements in the water. Immobility is defined as the cessation of active movement and a floating position. The total recording time is 6 minutes, with immobility time measured from the second minute.

[0083] Human peripheral blood mononuclear cell isolation: Human monocytes were isolated by density gradient centrifugation using a commercial human peripheral blood mononuclear cell isolation kit (P8680, Solarbio). The separation solution consisted of reagent A and reagent D (3:2 volume ratio). The separation process strictly adhered to the manufacturer's protocol. Briefly, the blood was carefully layered on the separation solution and centrifuged at 800g for 30 minutes. Subsequently, the cells in the second layer were collected and washed with PBS. The cells were then suspended in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS). The suspended cells were seeded into tissue culture dishes at a density of 1 million / ml. After a 4-hour incubation period, the supernatant was discarded and the adhered monocytes were harvested for subsequent experiments.

[0084] Immunofluorescence staining method: In the in vivo experiments, mice were sacrificed after behavioral assessment and perfused with PBS and 4% paraformaldehyde. The brain tissue was removed and embedded in optimal cutting temperature compound and cut into 25 μm sections. The brain sections were then washed with PBS and blocked with PBS containing 0.03% Triton-X100 and 3% BSA for 1 hour at room temperature, followed by incubation with primary antibodies in PBS containing 0.03% Triton-X100 and 3% BSA at 4°C overnight. After washing, the sections were incubated with secondary antibodies for 1 hour at room temperature. The sections were then mounted with mounting medium containing DAPI (ab104139, Abcam). The primary antibodies used were rabbit anti-GluN1 antibody (1:100, Ab-897, Signalway Antibody), rabbit anti-NeuN antibody (1:200, ab177487, abcam), rat anti-Laminine antibody (1:200, MA106100, Invitrogen), and rabbit anti-Fibrinogen antibody (1:100, NBP2-80414, Novus). Secondary antibodies used were anti-rabbit secondary antibody conjugated to Alexa Fluor 488 (1:1000, 711-545-152, Jackson), anti-mouse secondary antibody conjugated to Cy3 (1:1000, 115-165-003, Jackson), and anti-rat secondary antibody conjugated to Cy3 (1:1000, 112-165-003, Jackson). To assess antibody deposition in brain tissue, brain sections were incubated with an anti-mouse secondary antibody (1:500, 115-165-003, Jackson) in PBS containing 0.03% Triton-X100 and 3% BSA for 2 days at 4°C. To investigate IRF7 activation in human peripheral blood mononuclear cells, isolated monocytes were plated on coverslips coated with poly-L-lysine (Sigma). Monocytes were then fixed with 4% paraformaldehyde and permeabilized with PBS containing 0.25% Triton-X100. Cells were then blocked with PBS containing 0.03% Triton-X100 and 3% BSA for 1 hour at room temperature and incubated with the primary antibody against phospho-IRF7 (1:100, AF3885, Affinity Biosciences) in PBS containing 0.03% Triton-X100 and 3% BSA overnight at 4°C. After washing, the mononuclear cells were incubated with anti-rabbit secondary antibodies conjugated to AlexaFluor 647 (1:1000, ab150075, Abcam) and Phalloidin-iFluor 488 (1:1000, AAT-23115, AATB Bioquest) at room temperature for 1 h. The sections and cells were mounted with mounting medium containing DAPI (ab104139, Abcam).

[0085] Western Blotting: Mouse cortical proteins were extracted using RIPA lysis buffer (P0013, Beyotime). In this experiment, 40 μg of cortical proteins were used for each sample. In this example, membrane proteins were extracted from hippocampus using a membrane protein extraction kit (BB-3103, Bestbio) according to the manufacturer's protocol. In this example, the extracted membrane proteins were mixed with a loading buffer containing 20% sodium dodecyl sulfate (SDS) and boiled at 65°C for 30 minutes. The proteins were separated using 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. The following primary antibodies were used: rabbit anti-GluN1 antibody (1:1000, 27676-1-AP, Proteintech), rabbit anti-Na,K-ATPase antibody (1:1000, 3010S, Cell Signaling Technology), rabbit anti-ZO-1 (1 μg / mL, 61-7300, Invitrogen), rabbit anti-occludin antibody (1:1000, 91131, Cell Signaling Technology), rabbit anti-Claudin-5 antibody (1:900, 34-1600, Invitrogen), rabbit anti-Claudin-1 antibody (1:1000, 13050-1-AP, Proteintech), and mouse anti-GAPDH (1:50000, 60004-1-Ig, Proteintech). Band intensities were semiquantitatively analyzed using ImageJ (NIH) software.

[0086] Flow cytometry method: Flow cytometry was used to quantify leukocytes in human blood, mouse blood, and cerebral hemispheres. In the in vivo experiments, mice were sacrificed after behavioral assessment. In this example, mouse blood was collected and perfused with 20 ml of PBS. The hemispheres were then separated and digested with 0.25% trypsin-EDTA (ThermoFisher) at 37°C for 25 minutes. The brain tissue was homogenized and then filtered through a 70 μm cell strainer. The homogenate was gently placed in a percoll solution and myelin was removed by density gradient centrifugation in solutions containing 30% and 70% percoll. In this example, brain cells were collected and washed with PBS. Human and mouse blood was treated with ACK lysis buffer to lyse red blood cells and then washed with PBS. After fixation and permeabilization (Invitrogen, Intracellular Fixation and Permeabilization Buffer Kit), brain cells and blood cells were stained with antibodies. The following antibodies were used: Bv421 anti-mouse CD45 (1:400, 103134, Biolegend), PE / Cy7 anti-mouse CD12 (1:400, 101216, Biolegend), APC anti-mouse Ly24 (1:400, 128016, Biolegend), AF488 anti-mouse F4 / 80 (1:400, 123120, Biolegend), Percp / Cy5.5 anti-mouse CD3 (1:400, 100218, Biolegend). The cells were stained with antibodies against phospho-IRF7 (1:200, AF3885, Affinity Biosciences). Anti-mouse 488 (1:1000, ab150109, Abcam) was then used as a secondary antibody in combination with the primary antibody against phospho-IRF7. Data were analyzed using FlowJo software.

[0087] Example 3

[0088] This example provides an experimental and measurement result using the anti-NMDAR encephalitis mouse model and related methods in Example 2:

[0089] The anti-NMDAR encephalitis mouse model in Example 2 and wild-type (WT) model mice were used as controls. 359-378Mice were immunized with a peptide containing the N368 / G369 region of the GluN1 subunit for two rounds of immunization, and the development of the disease was monitored by various experiments, such as Figure 4 Flow chart of the experiment for immunizing mice with GluN1359-378 peptide and monitoring disease development;

[0090] The pathophysiology of anti-NMDAR encephalitis is characterized by the internalization of NMDA receptors in the hippocampus due to the presence of pathogenic autoantibodies. Figure 5 Figure 1 shows Western blot analysis of GluN1 expression in hippocampal membranes of wild-type and WT model mice, as well as statistical relative protein expression analysis. Data are fold changes compared with the WT-naive group, normalized to Na,K-ATPase expression levels. N = 3 per group. *P < 0.05; Student's t-test (mean ± SEM). Western blot analysis shows decreased expression of the GluN1 subunit in hippocampal membranes of wild-type (WT) model mice compared with wild-type mice, indicating receptor internalization.

[0091] Figure 6 Figure 1 shows the results of an experiment to detect anti-NMDAR antibodies in mouse serum for obtaining serum samples and performing cell-based assays (CBA), a tool commonly used in clinical settings. The figure shows immunofluorescence of HEK-293T cells transfected with GluN1. The cells were stained with mouse serum (red) extracted 14 days after the first immunization. A commercially available anti-GluN1 antibody (green) was used as a positive control. The results show the presence of anti-NMDAR antibodies in the serum of wild-type model mice compared to wild-type mice.

[0092] To assess whether the model mice exhibited psychiatric symptoms similar to those seen in patients with anti-NMDAR encephalitis, a series of behavioral assessments were performed. Schematic diagram of the open field test repeated three times and representative images of the mouse movement trajectory. Figure 7 Figure 2 shows the results of an open field test to assess anxiety and depression-like behaviors in mice. Total distance traveled, total activity time, center distance, and center time in the WT-naive (i.e., wild-type) mice (n=14) group compared with WT model mice (n=12). *P<0.05, **P<0.01, ***P<0.001; Student's t-test (mean ± SEM). The open field test showed that wild-type model mice exhibited reduced total activity, center distance, and center time compared with wild-type mice, indicating anxiety and depression-like behaviors.

[0093] In this example, mice were subjected to a Y-maze to assess spatial memory. Figure 8Shown is a graph showing the results of the Y-maze experiment to detect spatial memory in mice, which shows the time WT wild-type mice and WT model group mice stayed in the new arm and the time they entered the new arm. N = 14 mice per group. Although there was no difference in the frequency of entering the new arm, the wild-type model mice spent less time exploring the area, indicating that spatial memory was impaired. In this example, *P < 0.05; by t-test (mean ± SEM). In the figure, PTX, pertussis toxin; CBA, cell-based assay; WT, wild type.

[0094] 2. Determination of the Correlation between Increased IRF7 Expression in Peripheral Monocytes and Behavioral Abnormalities in Model Mice

[0095] In this example, flow cytometry was used to quantify the leukocyte population in the peripheral blood of mice. Figure 9 Figure 1 shows the results of flow cytometric analysis of circulating leukocytes in mouse peripheral blood. Flow cytometric gating strategy for total leukocytes (CD45+), myeloid cells (CD45+CD12+), lymphocytes (CD45+CD12-), monocytes (CD45hiCD12+LY24hi), T cells (CD45+CD12-CD3+), and B cells (CD45+CD12-B220+) are shown, with the percentage of cells within singlets recorded. N = 6-8. *P < 0.05, ***P < 0.001; Student's t-test (mean ± SEM). Figure 9 The results showed that the number of total leukocytes, total myeloid cells, and monocytes in wild-type model mice was significantly increased compared with wild-type mice, while there was no significant difference in the total lymphocyte, B cell, and T cell populations. Further examination of the expression of IRF7 in these subpopulations revealed that Figure 10 Figure 2 shows the results of flow cytometric analysis of IRF7 MFI in circulating monocytes, T cells, and B cells. N = 6-10. *P < 0.05, ***P < 0.001; t-test (mean ± SEM). Figure 10 The results showed that the expression level of IRF7 in monocytes of wild-type model mice was higher than that in other leukocyte subsets, and the expression was significantly increased compared with T cells, while the expression of IRF7 in T cells was reduced. In addition, the expression level of IRF7 in B cells of wild-type mice and wild-type model mice remained unchanged. Correlation studies found that there was a negative correlation between the percentage of circulating monocytes and the total activity time in the open field test, which suggested that peripheral monocytes may be involved in the pathogenesis. The correlation results between the Y maze behavioral assessment data are shown in Figure 2. Figure 11Figure 2 shows IRF7-MFI in circulating monocytes from WT model mice. N = 11 model mice. Correlation coefficients (r) and P values were determined using Helmholtz-Schmidt correlation analysis. FSC, forward scatter; SSC, side scatter; A, plot; H, depth; W, width; WT, wild type; MFI, mean fluorescence intensity. Among the behavioral tests performed, the Y-maze best reflects hippocampal function, particularly in assessing spatial and working memory. Figure 11 showed a negative correlation between IRF7 expression in monocytes and the time spent in the new arm of the Y-maze. There was no statistically significant correlation between IRF7 expression in monocytes and behavioral assessment data from the open field test or the 0 maze. These correlations indicate that in wild-type model mice, the number of peripheral monocytes and their IRF7 expression levels are associated with disease severity. However, the number of circulating B cells and T cells and the expression level of IRF7 in these cells did not show a statistically significant correlation with behavioral assessment data. This suggests that monocytes may be involved in the pathogenesis of anti-NMDAR encephalitis, and that it is the specific IRF7 expression in monocytes (rather than B cells or T cells) that leads to disease progression.

[0096] 2. Determination of the Correlation between Increased IRF7 Expression in Brain-Infiltrating Monocytes and Behavioral Abnormalities in Model Mice

[0097] In this example, flow cytometry was used to evaluate the immune cells infiltrating into the mouse brain. Figure 12 This figure shows the results of flow cytometric analysis of changes in the number of infiltrating immune cells in mouse brain tissue. The gating strategy for total infiltrating leukocytes (CD45hi), the percentages of myeloid cells (CD45hiCD12+), lymphocytes (CD45hiCD12-), monocytes (CD45hiCD12+LY24hi), T cells (CD45hiCD12-CD3+) and B cells (CD45hiCD12-B220+) in single cells were recorded. N=6-8. ***P<0.001; by t-test (mean ± SEM), the results showed that compared with wild-type mice, the presence of various leukocytes (including monocytes, B cells and T cells) in the brains of wild-type model mice increased, indicating the presence of leukocyte infiltration after immunization. It is worth noting that Figure 13 Figure 2 shows the results of flow cytometric analysis of IRF7 expression levels in monocytes, T cells, and B cells after infiltration. N = 10-12. *P < 0.05; by Student's test or Welcht's test (mean ± SEM). Figure 13showed that IRF7 expression was increased in the monocyte population of wild-type model mice, while IRF7 levels in B cells and T cells were not significantly changed. Correlation analysis showed that the percentage of infiltrating monocytes was negatively correlated with the center distance and center time in the open field test. There was no significant correlation between the percentage of infiltrating monocytes and behavioral assessment data in the 0 maze or Y maze. In addition, Figure 14 Correlation between Y-maze and IRF7-MFI behavioral assessment data, with permeabilized monocytes shown. N = 10. Correlation was determined using Pearson correlation analysis. FSC, forward scatter; SSC, side scatter; A, area; H, height; W, width; hi, height; WT, wild type; MFI, mean fluorescence intensity. Figure 14 The results showed that the expression level of IRF7 in monocytes was positively correlated with the time spent in the closed arm of the 0-maze and negatively correlated with the number of entries into the new arm of the Y-maze. Although the percentage of infiltrating B cells was negatively correlated with the center time and the percentage of infiltrating T cells was positively correlated with the total distance traveled in the open field test, the expression level of IRF7 in B cells and T cells did not show a statistically significant correlation with the behavioral assessment data. These observations suggest that infiltrating monocytes and B cells may be involved in the pathogenesis of anti-NMDAR encephalitis. However, IRF7 in infiltrating monocytes, rather than IRF7 in B cells or T cells, appears to have a key influence on disease progression. The different expression patterns of IRF7 among immune cell subsets emphasize the complexity of the neuroinflammatory response.

[0098] The test results showed that the expression model of IRF7 in mouse infiltrating monocytes was positively correlated with behavioral abnormalities

[0099] 3. To elucidate the pathogenic role of IRF7 in anti-NMDAR encephalitis, this example immunized WT mice and IRF7 knockout (KO) mice, and evaluated the disease severity of the two groups of mice through a series of experiments.

[0100] Figure 15 Figure 1 shows immunofluorescence analysis of the hippocampus of IRF7 knockout mice, with autoantibody deposits marked in red and NeuN in green. The experiment was repeated three times, and representative images are shown. Immunofluorescence analysis revealed reduced autoantibody deposition in the hippocampus of IRF7 knockout mice compared to wild-type mice. Figure 16 Representative results from immunofluorescence experiments in the mouse hippocampus show reduced GluN1 expression in the hippocampus of wild-type and IRF7-knockout mice, with the latter showing greater retention of this subunit. Behavioral assessments were also performed to assess disease severity. Figure 17Figure 1 shows the results of the open field test for WT-naive, WT-model, KO-naive, and KO model mice. The figure shows the total distance traveled, total activity time, center distance traveled, and center time traveled for each group of mice, with N = 7-9; *P < 0.05, **P < 0.01, ***P < 0.001; analyzed by one-way analysis of difference (mean ± SEM). In the open field test, IRF7 knockout mice traveled greater total distance, total activity time, center distance traveled, and center time traveled, indicating reduced depressive and anxiety-like behaviors compared to wild-type mice. Figure 18 Schematic diagram of the novel object recognition test results. The experimental mice were: WT-naive, WT-model, KO-naive mice and KO model mice. The novel object recognition test showed that there was no significant difference in the degree of cognitive impairment between wild-type model mice and IRF7 knockout model mice. Figure 19 Figure 1 shows the forced swim test results for T-naive, WT, KO, and KO mice. The figure shows the immobility time measured for each group of mice. N = 7-9; *P < 0.05, **P < 0.01. One-way analysis of difference (mean ± SEM) showed that both wild-type and IRF7 knockout mice exhibited prolonged immobility time in the forced swim test (an indicator of depressive-like behavior), suggesting a depressive state. The immobility time in wild-type mice was even longer than that in IRF7 knockout mice. Figure 20 Figure 2 shows the movement trajectories of WT-naive, WT-model, KO-naive, and KO-model mice in a Y-shaped maze. The figure shows the rate of change, dwell time, and number of entries into the novel arm for each group of mice. N = 7-9; *P < 0.05; one-way analysis of variance (mean ± SEM). WT, wild type; KO, knockout. The test results showed that the rate of change was reduced in both wild-type and IRF7 knockout mice, suggesting that both groups of mice had impaired spatial memory. However, compared with wild-type mice, IRF7 knockout mice made more entries into the novel arm, suggesting that this group of mice had milder spatial memory impairment. There was no significant difference in the time spent in the novel arm between wild-type and IRF7 knockout mice. Essentially, these findings indicate that IRF7 deficiency ameliorates the symptoms of anti-NMDAR encephalitis in mice, providing compelling evidence for the role of this gene in disease pathogenesis.

[0101] The results of this test demonstrated that knocking out IRF7 could reduce the severity of the disease in the mouse model.

[0102] 4. To explore the effect of IRF7 deficiency on monocyte / macrophage-mediated BBB disruption, this example also conducted in vivo and in vitro experiments.

[0103] First, to elucidate the mechanism by which IRF7-deficient mice exhibit reduced severity of anti-NMDAR encephalitis, in vivo experiments were performed. This example focused on the integrity of the blood-brain barrier by isolating cortical tissue and performing Western blot analysis to assess the integrity of the vascular basement membrane. Immunofluorescence staining of laminin and fibrinogen was then used to further assess the integrity of the vascular basement membrane. Figure 21 The figure shows the Western blot results of the expression of ZO-1, occludin, claudin-5 and claudin-1 in the mouse cortex. Figure 22 For Figure 21 Statistical analysis results. N = 5 per group. *P < 0.05; univariate analysis of differences (mean ± SEM). The figure shows that both wild-type and IRF7 knockout mice have BBB damage, but the extent of damage in the latter is less severe than that in the former. Claudin-5 expression in wild-type mice is lower than that in wild-type mice, suggesting BBB damage. Conversely, claudin-5 expression in IRF7 knockout mice is higher than that in wild-type mice, indicating that BBB integrity is preserved.

[0104] To explore the effects of IRF7 deficiency on monocyte / macrophage-mediated BBB disruption, in vitro experiments were also performed. MMPs are involved in the degradation of proteins in the extracellular matrix, leading to BBB disruption and the progression of neuroinflammation and brain damage. BMDMs from wild-type and IRF7 knockout mice were cultured, and MMPs, tissue inhibitors of metalloproteinases (TIMPs), and macrophage polarization markers were assessed by qPCR. Figure 23 Figure 3: Markers of BBB damage expressed in BMDM from wild-type and IRF7-KO mice, including MMPs, Timps, and macrophage polarization, were analyzed by qPCR. N = 3 per group. *P < 0.05; BMDM from IRF7-knockout mice exhibited reduced expression of Mmp8 and the M1 macrophage marker Il2, while increased expression of Timp1, compared to wild-type BMDM. This pattern suggests that IRF7 deficiency impairs the ability of BMDM to degrade the BBB and favors a shift toward an M2 macrophage phenotype, characterized by anti-inflammatory and reparative properties. To directly assess the effects of IRF7 deficiency on macrophage-mediated tight junction disruption, wild-type and IRF7-knockout BMDM were co-cultured with the endothelial cell line bEnd.3. TER and NaF permeability were measured. Figure 24 Schematic diagram of the in vitro BBB model. bEnd.3 cells were treated with mock, WT-BMDM, and KO-BMDM. MFI of NaF in the bottom chamber of the three groups. N = 3-4 per group. *P < 0.05; by one-way ANOVA (mean ± SEM). Figure 24Figure 3 shows that wild-type BMDMs significantly reduced TER and increased NaF permeability, whereas IRF7-knockout BMDMs did not exhibit this effect. Figure 25 Immunofluorescence staining of claudin-5 (green) in bEnd.3 cells to detect tight junction disruption. The experiment was repeated three times, and representative images are shown. MMPs, matrix metalloproteinases; TIMPS, tissue inhibitor of metalloproteinases; BMDM, myeloid-derived macrophages; TER, trans-internal resistance; MFI, mean fluorescence intensity. WT, wild type; KO, knockout; Il1b,

[0105] Interleukin 1b; Il6, interleukin 6; Arg1, arginase-1; Mrc1, mannose receptor type C 1. Figure 25 Immunofluorescence analysis revealed that wild-type BMDMs were more efficient at disrupting the tight junction protein claudin-5 compared to IRF7-knockout BMDMs. Together, these in vitro findings suggest that IRF7 deficiency in monocytes / macrophages reduces their ability to disrupt BBB integrity, likely by regulating the expression of MMPs and TIMPs and influencing macrophage polarization. These results highlight the critical role of IRF7 in monocyte / macrophage-mediated neuroinflammation and suggest that targeting IRF7 may be a promising therapeutic strategy for treating anti-NMDAR encephalitis.

[0106] This result demonstrates that IRF7 deficiency leads to a weakened ability of monocytes / macrophages to disrupt the blood-brain barrier.

[0107] Example 4

[0108] This example provides a method for constructing an IRF7 knockout (KO) mouse cell culture model. Bone marrow cells were extracted from the femurs and tibias of 6-12 week-old C57BL / 6N wild-type (WT) and IRF7 knockout (KO) mice. These cells were then cultured and differentiated into bone marrow-derived macrophages (BMDMs) for 6 days. The culture medium was CSF1-conditioned RPMI-1640 medium containing 10% FBS and 1% PS. Simultaneously, the bEnd.3 endothelial cell line was cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS and 1% PS.

[0109] This example uses the above-mentioned mouse cell culture model to perform quantitative real-time polymerase chain reaction (qPCR): to verify the differential expression of genes. According to the manufacturer's protocol, total RNA was extracted from BMDM using the RNA-Quick purification kit (RN001, ESscience). Subsequently, complementary DNA (cDNA) was synthesized using the Rapid Reverse Transcription Kit (RT001, ESscience). Quantitative real-time polymerase chain reaction (qPCR) was performed on a 480II (Roche). Gene expression levels were normalized to GAPDH. Primers were synthesized by Sangon Biotech (Guangzhou, China) and are listed in Table S4. Fold changes were scaled by group and displayed in a heat map.

[0110] In this example, the above mouse cell culture was used to construct an in vitro blood-brain barrier (BBB) model.

[0111] bEnd.3 cells at a concentration of 1 million cells / ml were seeded onto a 12-well transwell membrane with a pore size of 0.4 μm (Corning Costar). These cells were allowed to establish tight junctions (TJ) for at least 2 days. Subsequently, BMDM was resuspended and added to the bEnd.3 monolayer at a density of 0.5 million cells / well. After treatment, the culture medium in the upper chamber was replaced with 250 μM sodium fluoride (NaF), and the culture plate was incubated for 30 minutes. After incubation, the fluorescence intensity of the culture medium in the lower chamber was measured. The transendothelial resistance (TER) of the in vitro BBB model was determined using a Millicell-ERS-2 epithelial voltammeter (Millipore MERS00002). The culture medium in the upper and lower chambers was replaced with PBS, and the membrane area (cm 2 ) multiplied by the voltmeter reading to determine TER. The relative change in TER relative to baseline was calculated. For immunofluorescence analysis, bEnd.3 cells were seeded on poly-L-lysine-coated coverslips in 48-well culture plates at a density of 0.2 million cells / well. After allowing TJs to form for at least 2 days, BMDMs were resuspended and added to bEnd.3 cells at a density of 0.12 million cells / well. The cells were fixed with 4% paraformaldehyde and blocked with 3% BSA. The cells were then incubated overnight at 4°C with the primary antibody rabbit anti-claudin-5 (1:200, 34-1600, Invitrogen). After washing, an anti-rabbit secondary antibody conjugated to AlexaFluor488 (1:1000, 711-545-152, Jackson) was added. The cells were then sealed with culture medium containing DAPI (ab104139, Abcam) for visualization of the nuclei.

[0112] Statistical data in the embodiments of the present invention were analyzed using GraphPad Prism 8.3.0 software. All data are expressed as mean ± standard error mean (SEM). Single factor analysis was used to analyze the differences in the mean between the groups using analysis of variance (ANOVA). Student's t test or Welch's t test was used for two-group comparisons. Pearson or Spearman correlation analysis was used to analyze the correlation between the data. In all analyses, P < 0.05 was considered to be statistically significant.

[0113] The results of this example demonstrate that IRF7 in peripheral monocytes is closely linked to the pathogenesis of anti-NMDAR encephalitis, particularly in regulating BBB integrity. The results of this example demonstrate that the number of peripheral monocytes is positively correlated with disease severity (e.g., blood-brain barrier disruption) in patients. Furthermore, this example also demonstrates that IRF7 is activated in circulating monocytes in patients. The anti-NMDAR encephalitis mouse model presented in this example demonstrates that an increase in peripheral monocytes enhances IRF7 expression, and both are positively correlated with adverse behavioral outcomes. Increased numbers of CNS-infiltrating monocytes and IRF7 expression in these cells are positively correlated with adverse behavioral outcomes. In IRF7-KO mice, this example demonstrates that IRF7 deficiency mitigates disease and reduces BBB disruption. Furthermore, in vitro experiments in this example demonstrate that IRF7-deficient BMDM exhibit reduced BBB compromise compared to WT BMDM. In summary, this example demonstrates the critical role of IRF7 in the pathogenesis of anti-NMDAR encephalitis by regulating BBB disruption and the inflammatory response of monocytes / macrophages.

[0114] Peripheral monocyte counts in patients with anti-NMDAR encephalitis positively correlated with BBB permeability, highlighting a potential role for monocytes in BBB disruption. This is consistent with previous findings showing elevated circulating monocyte levels in patients with central nervous system diseases characterized by BBB leakiness and expands our understanding of immune cell trafficking to the central nervous system during neuroinflammatory conditions. Previous studies have implicated leukocyte infiltration in BBB disruption, and the findings presented here specifically highlight monocytes as key players in this process. The increased number of peripheral monocytes in these patients suggests a potential role in disease pathogenesis, potentially through the secretion of proinflammatory cytokines and matrix metalloproteinases that degrade the BBB. The activation of IRF7 in these patients' monocytes, as evidenced by elevated levels of both IRF7 and phosphorylated IRF7, provides new insights into the immune mechanisms underlying anti-NMDAR encephalitis. IRF7 is known for its role in promoting the type I interferon response and has been implicated in a variety of autoimmune diseases. The findings presented here suggest that IRF7 may be a driver of the inflammatory process that leads to the neurological symptoms of anti-NMDAR encephalitis.

[0115] The results of the assays in Examples 1-4 of the present invention show that anti-NMDAR encephalitis induced in mice resulted in an increase in the number of peripheral monocytes and a significant upregulation of IRF7 in monocytes. The positive correlation between the number of peripheral monocytes, the expression of IRF7 in these cells, and adverse behavioral outcomes suggests that IRF7 in monocytes is involved in the pathophysiology of anti-NMDAR encephalitis. In the peripheral blood of the mouse model, IRF7 was found to be primarily expressed in monocytes rather than B cells or T cells, and IRF7 expression was specifically increased in monocytes, indicating a unique role for monocytes in the context of this disease. This is consistent with the current understanding of the specialized role of monocytes in immune responses. In addition, the number of circulating B cells or T cells or their IRF7 expression levels were not associated with behavioral outcomes, suggesting that monocytes play a more specialized role in disease pathology. This finding warrants further study to elucidate the different mechanisms by which monocytes and lymphocytes cause neuroinflammation. In addition, the present invention found that the number of infiltrating monocytes and the increase in IRF7 expression in these cells were associated with behavioral abnormalities. In addition, the present invention also found that the number of B cells and T cells was correlated with behavioral data, but the level of IRF7 expression in these cells was not statistically correlated with behavioral abnormalities. The results of the present invention indicate that infiltrating monocytes, B cells, and T cells may act as mediators of the neuroinflammatory process, and emphasize that IRF7 in monocytes rather than lymphocytes plays a pathogenic role. The present invention used IRF7 gene knockout mice in the study, and the results showed that the severity of the disease and the degree of BBB damage were reduced, providing strong evidence for the role of IRF7 in the pathogenesis of the disease. This finding is consistent with the results of studies that have shown that IRF7 can regulate immune responses and affect the disease outcomes of autoimmune diseases.

[0116] Using an in vitro BBB model, this example demonstrates that IRF7 deficiency in BMDMs impairs BBB disruption. This finding, consistent with the reduced disease severity and BBB disruption seen in IRF7-KO mice, suggests that IRF7 is a key mediator of monocyte / macrophage-induced neuroinflammation. As suggested by the qPCR data in this example, the precise molecular mechanisms by which IRF7 regulates BBB integrity and monocyte and macrophage function likely involve the regulation of matrix metalloproteinases, tissue inhibitors of metalloproteinases, and inflammatory cytokines. This is consistent with previous studies implicating IRF7 in regulating inflammatory responses and macrophage polarization. The results of this example also demonstrate that IRF7 deficiency is associated with a shift in macrophage polarization toward M2 macrophages. This type of macrophage is associated with anti-inflammatory and tissue repair processes, participating in wound healing, tissue remodeling, and resolution of inflammation. This finding suggests that IRF7 not only influences the ability of monocytes to disrupt the BBB but also modulates their inflammatory potential. These results are consistent with studies implicating IRF7 in regulating proinflammatory monocytes and modulating immune cell migration.

[0117] Example 5

[0118] This example provides the use of IRF7 as a target in the preparation of anti-NMDAR encephalitis drugs.

[0119] In some other specific embodiments, the related drugs may be but are not limited to:

[0120] 1. siRNA / shRNA drugs, IRF7-specific siRNA or shRNA

[0121] By silencing IRF7 mRNA through RNA interference technology, IRF7 protein expression is reduced, thereby inhibiting the pro-inflammatory phenotype transformation of monocytes / macrophages, reducing blood-brain barrier destruction and central inflammatory response.

[0122] siRNA: 5'-GGAUCAAGUUCCAGAUCUATT-3' (sense strand)

[0123] shRNA: Targets a specific sequence of IRF7 mRNA and is stably expressed via a vector (such as lentivirus).

[0124] More specifically, the siRNA / shRNA pharmaceutical composition is composed of the following weight ratios:

[0125] siRNA: 0.1-5 mg / kg body weight (preferably 1 mg / kg)

[0126] Liposomes (DOTAP / DOPE molar ratio 1:1): total lipid content 10-50 mg / mL

[0127] Normal saline: Make up to the injection volume (intravenous dose is 0.5-2 mL / kg)

[0128] Preparation: Mix siRNA and liposomes at a 1:10 (w / w) ratio, vortex, and incubate for 30 minutes to form a complex. Dissolve the complex in saline, adjust the pH to 7.4, and filter sterilize. Aliquot into single-use aliquots (1 mL containing 1 mg of siRNA) and store at -80°C.

[0129] Usage: Intravenous injection once a week for 4 weeks to inhibit monocyte IRF7 expression and reduce BBB leakage.

[0130] The siRNA / shRNA drugs or their combination in this embodiment can effectively reduce the expression of IRF7 protein, thereby inhibiting the pro-inflammatory phenotype transformation of monocytes / macrophages, reducing blood-brain barrier damage and central inflammatory response.

[0131] 2. Small molecule inhibitors, IRF7 activity inhibitors, inhibit IRF7 transcriptional activity by blocking IRF7 phosphorylation or nuclear translocation, reducing the release of pro-inflammatory cytokines (such as IL-6 and TNF-α). They are used for oral or intravenous administration to improve BBB integrity and behavioral symptoms.

[0132] IRF7 activity inhibitors were obtained through high-throughput screening of IRF7-specific small molecule inhibitors with an IC50 value of X nM. IRF7 pathway-related inhibitors: TBK1 / IKKε inhibitors (Amlexanox) indirectly regulate IRF7 activity.

[0133] More specifically, the pharmaceutical composition is composed of the following weight ratios:

[0134] The oral tablet formulation (each tablet) contains:

[0135] IRF7-specific small molecule inhibitor (IC50=50nM): 10-100mg (preferably 50mg);

[0136] Microcrystalline cellulose: 30 mg;

[0137] Lactose: 20 mg;

[0138] Magnesium stearate: 1mg.

[0139] The preparation method is as follows: uniformly mix an IRF7-specific small molecule inhibitor (IC50=50 nM), microcrystalline cellulose, lactose and magnesium stearate, press into tablets, and coat (hydroxypropyl methylcellulose layer) to mask the bitter taste.

[0140] Dosage: Take 1-2 tablets orally once daily for 8 weeks. Combining with the TBK1 inhibitor Amlexanox (25 mg / time) may enhance efficacy.

[0141] 3. Anti-IRF7 monoclonal antibodies specifically bind to the IRF7 protein, blocking its function or promoting its degradation, thereby inhibiting the inflammatory response of monocytes and macrophages. They are administered intravenously or intrathecally to directly target inflammation in the central nervous system.

[0142] Humanized anti-IRF7 monoclonal antibodies (eg, mAb-IRF7) are prepared by hybridoma technology or phage display technology.

[0143] More specifically, the anti-IRF7 monoclonal antibody drug is an anti-IRF7 monoclonal antibody injection.

[0144] The drug composition includes: humanized anti-IRF7 monoclonal antibody (mAb-IRF7) and buffer (10 mM phosphate, 150 mM NaCl, pH 6.0).

[0145] Ratio (per injection): mAb-IRF7: 10-200 mg / mL (preferably 50 mg / mL);

[0146] Buffer: make up to 5mL;

[0147] Preparation method: mAb-IRF7 was expressed and purified by CHO cells (purity > 95%), dialyzed into buffer, filtered and sterilized, and then packaged.

[0148] Usage: Intravenous injection, once a week (5 mg / kg), or intrathecal injection (1 mg / time), directly targeting central inflammation.

[0149] 4. IRF7 gene knockout CRISPR / Cas9 system. Mechanism of action: Cas9 protein targets the IRF7 gene via sgRNA, achieving gene knockout or mutation, permanently inhibiting IRF7 expression. This system can be used for in vitro monocyte editing or in vivo targeted therapy.

[0150] sgRNA sequence: 5'-GGCUCAAGUUCCAGAUCUAG-3' (targeting the exon region of the IRF7 gene).

[0151] Delivery vehicle: AAV or liposome encapsulation for in vivo delivery.

[0152] More specifically, in the CRISPR / Cas9 system for IRF7 gene knockout, the ratio (per dosage unit) is: sgRNA: 1×10^13 vg (viral genome) / kg;

[0153] Cas9 mRNA: 0.5 mg / kg;

[0154] AAV9 vector: total titer 1×10^14 vg / mL.

[0155] The preparation method is:

[0156] sgRNA and Cas9 mRNA were cloned into AAV9 vector, packaged into HEK293 cells, and purified and dissolved in PBS.

[0157] Usage: Single intravenous injection to edit the IRF7 gene in peripheral monocytes and suppress inflammation in the long term.

[0158] 5. IRF7 inhibitors combined with immunotherapy: IRF7 inhibitors (such as siRNA or small molecule compounds) are combined with immunotherapy (such as IVIG or rituximab) to synergistically suppress inflammatory responses and autoantibody production. This approach is used for patients with refractory or recurrent anti-NMDAR encephalitis.

[0159] The combination of RF7 siRNA and intravenous immunoglobulin (IVIG); the combination of IRF7 small molecule inhibitor and rituximab.

[0160] More specifically, the IRF7 inhibitor combined with the immune drug is:

[0161] IRF7 siRNA: 1 mg / kg;

[0162] Intravenous immunoglobulin (IVIG): 0.4 g / kg;

[0163] Normal saline: dilute to a total dose of 200 mL;

[0164] Dosage regimen: siRNA and IVIG are infused simultaneously once a month for 6 months.

[0165] IRF7 inhibitors combined with immune drugs can also:

[0166] Small molecule inhibitor + rituximab:

[0167] Small molecule inhibitors: 50 mg / time (oral, once a day)

[0168] Rituximab: 375 mg / m 2 (Intravenous drip, once a week × 4 weeks)

[0169] Rituximab depletes B cells, and small molecule inhibitors inhibit monocyte IRF7, synergistically reducing autoantibody production.

[0170] Experimental siRNA efficiency verification:

[0171] In vitro, IRF7 mRNA was significantly reduced after 24 hours of 1 μM siRNA treatment in monocytes (qPCR).

[0172] The pharmaceutical compositions provided in this embodiment can effectively treat anti-NMDAR encephalitis.

[0173] Example 6

[0174] This example provides an anti-NMDAR encephalitis diagnosis qPCR detection kit based on IRF7 expression level.

[0175] qPCR detection reagent composition:

[0176] IRF7 primer pairs:

[0177] Forward primer: 5′-CAGCAGCCTTTCCAGAAGAC-3′;

[0178] Reverse primer: 5′-GTCTTGGTCTTGGAGGCTGA-3′;

[0179] IRF7 TaqMan probe: 5′-FAM-ATGCCTGGAGGCCGTCAA-BHQ1-3′;

[0180] Internal reference gene (GAPDH) primers and probes:

[0181] Forward primer: 5′-GAAGGT GAA GGT CGGAGT C-3′;

[0182] Reverse primer: 5′-GAAGAT GGT GAT GGGATT TC-3′;

[0183] Probe: 5′-HEX-CAAGCT TCC CGT TCT CAG CC-BHQ1-3′;

[0184] Master mix: 2× qPCR master mix containing dNTPs, Taq DNA polymerase, and MgCl2;

[0185] More specifically, in the detection kit,

[0186] 2× premix: 10 μL;

[0187] 0.5 μL each of the forward primer (10 μM) and reverse primer (10 μM) of the IRF7 primer pair;

[0188] IRF7 TaqMan probe (5 μM): 0.4 μL;

[0189] 0.5 μL each of the forward primer (10 μM) and reverse primer (10 μM) of the GAPDH primer pair;

[0190] GAPDH probe (5 μM): 0.4 μL;

[0191] cDNA template: 2 μL; nuclease-free water: make up to 20 μL;

[0192] qPCR kit preparation method: Synthesize IRF7 and GAPDH primers and probes, verify amplification efficiency (efficiency must reach 90-110%). Aliquot the premix, primers, and probe into lyophilized tubes and store at -20°C.

[0193] The detection method used in the detection kit of this embodiment is:

[0194] Peripheral blood (2 mL) was collected from the patient, and mononuclear cells were isolated by density gradient centrifugation. Total RNA was extracted using an RNA extraction reagent, and the concentration and purity were determined (A260 / A280 ≥ 1.8). 1 μg of RNA was used to synthesize cDNA according to the reverse transcription kit instructions.

[0195] qPCR amplification: Prepare the reaction system according to the ratio and set the amplification program: pre-denaturation: 95°C for 3 minutes, cycle: 95°C for 10 seconds → 60°C for 30 seconds (40 cycles).

[0196] Result analysis: The ΔΔCt method was used to calculate the relative expression of IRF7 (GAPDH was used as the internal reference).

[0197] Diagnostic threshold: IRF7 expression ≥ 2 times the mean of healthy controls was considered positive.

[0198] Example 7

[0199] This embodiment provides a qPCR flow cytometry detection kit for diagnosing anti-NMDAR encephalitis based on IRF7 expression levels, which comprises:

[0200] Fluorescently labeled antibodies: anti-human CD14-FITC (monocyte marker), anti-human IRF7-PE (target protein detection), isotype control antibody (IgG-PE), cell lysis buffer: lysis buffer containing Triton X-100 (pH 7.4).

[0201] Fixative: 4% paraformaldehyde solution.

[0202] Positive control: lysate of monocytic cell lines that highly express IRF7 (such as THP-1 cells).

[0203] Negative control: IRF7 knockout THP-1 cell lysate or healthy human peripheral blood mononuclear cell samples.

[0204] Standard curve sample: serial dilution of IRF7 recombinant protein (0.1-100 ng / mL).

[0205] Auxiliary reagents:

[0206] RNA extraction reagents (such as TRIzol or magnetic bead kit).

[0207] cDNA synthesis reagents (including reverse transcriptase, random primers, and dNTPs).

[0208] Flow cytometry wash buffer (PBS containing 1% BSA).

[0209] More specifically, in the detection kit,

[0210] Anti-CD14-FITC: 1 μL (1:100 dilution);

[0211] Anti-IRF7-PE or isotype control: 1 μL (1:50 dilution);

[0212] Cell suspension: 100 μL (1×10^6 cells);

[0213] Preparation method of flow cytometry kit: Pre-dilute the fluorescent antibody in proportion and then dispense into light-proof tubes (store at 4°C).

[0214] The lysate and fixative were aliquoted into 1 mL tubes.

[0215] Control sample preparation method:

[0216] Positive control: THP-1 cells were cultured, induced to overexpress IRF7, and then lysed and quantitatively aliquoted.

[0217] Negative control: THP-1 cells in which IRF7 was knocked out using CRISPR / Cas9.

[0218] The detection method used in the detection kit of this embodiment is:

[0219] Take 100 μL of anticoagulated blood, add lysis buffer to remove red blood cells, and collect mononuclear cells by centrifugation.

[0220] Antibody staining: Resuspend cells in wash buffer, add fluorescent antibodies, and incubate in the dark for 30 minutes.

[0221] The cells were fixed with 4% paraformaldehyde for 10 minutes, and the IRF7-PE signal in CD14+ cells was detected by flow cytometry.

[0222] Interpretation of results:

[0223] Positive standard: IRF7 mean fluorescence intensity (MFI) ≥ 1.5 times that of healthy controls.

[0224] Clinical verification: The IRF7 positive detection rate is above 60%.

[0225] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A target for anti-NMDAR encephalitis, characterized in that: It includes IRF7 gene and / or expressed protein.

2. Use of the target as claimed in claim 1 in the preparation of anti-NMDAR encephalitis drugs.

3. The use according to claim 2, characterized in that: The anti-NMDAR encephalitis drug inhibits the expression and / or activity of IRF7.

4. The use according to claim 3, characterized in that: The anti-NMDAR encephalitis drug is used in the preparation of a drug for reducing blood-brain barrier damage and central inflammatory response mediated by monocytes / macrophages.

5. The use according to claim 3, characterized in that: The anti-NMDAR encephalitis drug is siRNA, shRNA, small molecule inhibitor or / and monoclonal antibody targeting the target.

6. A CRISPR / Cas9 system for preparing a drug for treating anti-NMDAR encephalitis, characterized in that: It includes: sgRNA that specifically targets the IRF7 gene; Cas9 protein and / or its coding sequence; A vector for delivering the sgRNA and Cas9 protein.

7. The use of the anti-NMDAR encephalitis target in an anti-NMDAR encephalitis diagnostic kit according to claim 1, characterized in that: It includes a specific probe for detecting the expression level of IRF7 in peripheral mononuclear cells.

8. A disease drug screening model for anti-NMDAR encephalitis, characterized by: The model is based on an in vitro co-culture system of IRF7-deficient monocytes or / and the blood-brain barrier, and is used to evaluate the effects of drugs on IRF7 expression, monocyte migration ability, or BBB integrity.

9. A disease drug screening model for anti-NMDAR encephalitis, characterized by: The model is an IRF7-deficient animal model.

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