Anti-nmdar encephalitis drugs, efficacy analysis and mouse model establishment method

Abstract

The present application relates to the field of biological medicine, and more particularly to a kind of anti-NMDAR encephalitis drug, curative effect analysis and mouse model establishment method.Mouse model has anti-GluN1 autoantibody, blood-brain barrier damage, IL-1β produced by endothelial cell;Whether mouse blood-brain barrier damage can be improved, the activity of endothelial cell IL-1β receptor is determined to determine the therapeutic effect by mouse model.Anakinra (Anakinra) is the drug for treating anti-NMDAR encephalitis.The present application can more accurately analyze and evaluate the cause and treatment effect of encephalitis by establishing humanized mouse model with anti-GluN1 (GluN1 is one subunit of NMDAR) antibody, blood-brain barrier damage and IL-1β produced by endothelial cell.

Description

[0001] The present application relates to the field of biological medicine, and particularly relates to an anti-NMDAR encephalitis drug, efficacy analysis and mouse model establishment method. BACKGROUND

[0002] Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is a rare and severe autoimmune neurological disease mediated by antibodies, and its clinical features include mental and behavioral changes, movement disorders, memory disorders, and epilepsy. Its possible pathogenesis is that the selective binding of anti-NMDAR antibodies (NMDAR-Abs) to NMDAR on the surface of neurons reduces the density of NMDAR and causes the function of neurons to decline. Although viral infection and tumor carrying may be important causes of anti-NMDAR encephalitis, the etiology and pathogenesis of the disease are largely unknown.

[0003] Because the etiology and pathogenesis of anti-NMDAR encephalitis are unclear, the evaluation of the therapeutic efficacy of anti-NMDAR encephalitis is limited, and some patients have poor efficacy and a high relapse rate after treatment.

[0004] Mouse models are powerful research tools for promoting the understanding of the pathogenesis of diseases. So far, several types of anti-NMDAR encephalitis mouse models have been established, including antibody passive transfer mouse models and active immunization mouse models. However, these mouse models are limited by genetic or species differences. For example, although the passive transfer of anti-NMDAR antibodies to mice can cause the function of NMDAR-mediated synaptic transmission in the brain of mice to be low, this model fails to generalize the immune factors and lacks the characteristics of immune cell infiltration and neuroinflammation during the disease process. While the NMDAR peptide-induced active immunization mouse model can trigger a strong immune response, produce anti-NMDAR antibodies, and initiate brain infiltration of immune cells in mice. However, these models ignore the etiology and immune pathogenic mechanisms related to the etiology of human autoimmune reactions.

[0005] The pathogenesis of anti-NMDAR encephalitis requires two main components. First, B cells are abnormally activated and differentiated into effector B cells, which then produce and secrete autoantibodies in the serum. Then the autoantibodies enter the brain, and anti-GluN1 autoantibodies cause selective and reversible reduction of NMDARs in synapses, leading to cognitive decline and neuropsychiatric symptoms in patients. There are several pathways for antibodies to enter the central nervous system, including the blood-brain barrier, the olfactory pathway, etc. The blood-brain barrier is an important pathway for anti-NMDAR autoantibodies to enter, because blood-brain barrier damage is associated with disease severity and intrathecal IgG synthesis in patients with anti-NMDAR encephalitis. Bypassing the damaged blood-brain barrier is crucial for the development of previous animal models of anti-NDMAR encephalitis. In the passive transfer model, patient-derived anti-NMDAR antibodies are directly delivered to the brain by intracerebroventricular injection, and administration of pertussis toxin to disrupt the blood-brain barrier is a prerequisite for active immunization models. Other models also ignore the pathogenic role of blood-brain barrier damage in this autoimmune encephalitis. Using these models also cannot determine the cause of blood-brain barrier damage.

[0006] Transferring patient-derived peripheral blood cells into mice with severe combined immunodeficiency (SCID) is a widely accepted humanized mouse model that has been used for a long time to study autoimmune diseases such as myasthenia gravis, nephrotic syndrome, systemic lupus, systemic sclerosis, etc. However, the establishment of a humanized mouse model needs to overcome the adaptability of the mouse immune system to have better model stability. SUMMARY

[0007] (I) Technical problems to be solved

[0008] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present application provides an anti-NMDAR encephalitis therapeutic effect analysis method, which can more accurately analyze and evaluate the pathology and treatment efficacy of encephalitis by establishing a humanized mouse model with anti-GluN1 (GluN1 is a subunit of NMDAR) antibodies, blood-brain barrier damage, and IL-1β produced by endothelial cells.

[0009] Correspondingly, the present application also provides a stable and new anti-NMDAR encephalitis humanized mouse model that can produce blood-brain barrier damage without the need for additional drugs, overcoming technical bias.

[0010] Correspondingly, the present application also provides a drug for treating anti-NMDAR encephalitis, which can improve blood-brain barrier damage, thereby achieving a better effect of treating anti-NMDAR encephalitis.

[0011] (II) Technical solutions

[0012] In order to achieve the above-mentioned purposes, the main technical solutions adopted by the present application include:

[0013] In a first aspect, the present application provides a method for analyzing the therapeutic effect of anti-NMDAR encephalitis, comprising the following steps:

[0014] S1: Establishing a humanized mouse model, which has anti-GluN1 autoantibodies, blood-brain barrier damage, and IL-1β produced by endothelial cells.

[0015] S2: Determining the therapeutic effect by analyzing whether the blood-brain barrier damage of the mouse can be improved and the IL-1β receptor activity of the endothelial cells can be inhibited.

[0016] The present application shows through experiments that the production of anti-GluN1 autoantibodies, blood-brain barrier damage, and IL-1β produced by endothelial cells is the pathogenesis of anti-NMDAR encephalitis, and in particular, it is clear that IL-1β is produced by endothelial cells; thus, the use of the IL-1 receptor antagonist Anakinra can improve blood-brain barrier damage and neuropsychiatric behavior, thereby alleviating the disease condition.

[0017] The present application has better accuracy by specifically establishing a mouse model with anti-GluN1 autoantibodies, blood-brain barrier damage, and IL-1β produced by endothelial cells, and thereby evaluating the therapeutic effect of drugs and other methods.

[0018] The mouse model in the present application can be established by the following methods, but is not limited thereto. Other mouse models can also be established by antibody transfer passive models, peptide segment-induced active models, and humanized mouse models.

[0019] In a second aspect, the present application further provides a method for constructing a humanized mouse model of anti-NMDAR encephalitis, comprising the following steps: injecting peripheral blood mononuclear cells (PBMCs) of an anti-NMDAR encephalitis patient into BRGSF mice through intraperitoneal injection (i.p.), and continuing to culture for more than 8 weeks.

[0020] The method for constructing a humanized mouse model provided by the present application can specifically cause the mouse to produce anti-GluN1 autoantibodies, blood-brain barrier damage, and IL-1β produced by endothelial cells; in particular, it causes the endothelial cells to produce IL-1β and damage the blood-brain barrier.

[0021] The humanized model of the present application can be used to study the pathogenic effect of blood-brain barrier damage in anti-NMDAR encephalitis patients.

[0022] In the humanized mouse model established in the present application, the production of human anti-NMDAR antibodies was detected in the serum of BRGSF mice, which indicated that the patient-derived B cell subpopulation was proliferated and functionally normal, which was a necessary condition for successful modeling. Human anti-NMDAR antibodies were also significantly elevated in the CSF of mice, while hippocampal NMDAR protein was also reduced in model mice. These results indicate that anti-NMDAR autoantibodies effectively enter the central nervous system, rather than artificially damaging the blood-brain barrier, and that these autoantibodies detected in humanized mice have function, similar to patient-derived anti-NMDAR antibodies. The model provides an in vivo experimental platform for studying blood-brain barrier damage, and also suggests the pathogenic role of PBMCs in anti-NMDAR encephalitis. Unlike previous models that used pertussis toxin or genetic knockout operations (Apoe knockout) to damage the blood-brain barrier, PBMC transfer reflects the situation where patients exhibit donor-dependent blood-brain barrier permeability, which means that patient-derived lymphocytes can be harmful to blood-brain barrier integrity.

[0023] In the stroke model, it has been demonstrated that infiltrating T lymphocytes (such as Th17 and Th1 cells) can modulate cytokines (such as INFγ and IL17), chemokines (Ccl2 and Cxcl1) or ROS to degrade tight junctions in the blood-brain barrier. It should be noted that these T lymphocytes can not directly secrete pro-inflammatory mediators that damage the blood-brain barrier, but rather amplify the inflammatory cascade by remodeling gene expression in cells within the blood-brain barrier. Using the humanized model of the present application, it can be observed that only in mice transferred with patient PBMCs, there is a significant infiltration of CD4 + and CD8 + T lymphocytes into brain tissue, which has a potential link to blood-brain barrier dysfunction. This is further supported by in vitro experiments, which found that after patient PBMC intervention, the trans-endothelial electrical resistance (TEER) value decreased, the fluorescein isothiocyanate-dextran (FITC-Dextran) permeability increased, and the tight junction proteins decreased in the endothelial cell line in the BBB model experiment. Therefore, in addition to autoantibody secretion, the use of the humanized model of the present application can reveal new pathogenic effects of lymphocyte subpopulations in damaging the integrity of the blood-brain barrier.

[0024] Alternatively, in order to improve the stability of the humanized mouse model, the anti-NMDAR encephalitis patient should be diagnosed as being in the acute phase and not treated with immunotherapy.

[0025] The humanized mouse carrying part or all of human pathophysiological systems provided by the application is a valuable supplement for developing a new type of anti-NMDAR encephalitis animal model, and improves the in-depth research and understanding of the pathology and treatment effect of anti-NMDAR encephalitis.

[0026] Autoimmune encephalitis is a special neuroimmunological disease, and the immune response thereof is mainly directed against specific antigens such as central NMDAR. It has been verified that the mouse model of the application only produces an immune response against NMDAR, and it is determined that the produced antibody is specifically combined with NMDAR protein, and no immune response is produced against other brain antigens.

[0027] The construction method of the anti-NMDAR encephalitis humanized model of the application does not need additional substances, and the cell barrier can be successfully destroyed by directly transferring human peripheral PBMC. In particular, the mouse of the application is a BALB / c Rag2 - / - Il2rg - / - Sirpα NOD Flk2 - / - The mouse (also referred to as BRGSF mouse) is a severe combined immunodeficiency (SCID) mouse, which lacks functional B, T and NK cells, and is highly permissive to human PBMC implantation due to the expression of SirpαNOD.

[0028] The PBMC of the anti-NMDAR encephalitis patient implanted into the BRGSF mouse of the application can produce a core clinical phenotype, and closely reflect the characteristics of the human disease in the mouse, and the pathogenic role of the changed endothelial cells in the blood-brain barrier (BBB) is determined, and endothelial-derived interleukin 1β (IL-1β) is determined as a therapeutic target of anti-NMDAR encephalitis.

[0029] Alternatively, the extracted PBMC can be directly transferred into the mouse, or can be frozen in liquid nitrogen for use. Specifically, the freezing method is as follows: the PBMC is resuspended in a freezing preservation solution containing 10% dimethyl sulfoxide, then transferred to a 2mL freezing vial, and frozen in a NALGENE TM Cryo 1℃ freezing container (NALGENE, Waltham, MA, USA) at-80℃ overnight. Then, the frozen PBMC is stored in liquid nitrogen.

[0030] Alternatively, in order to prevent the death caused by graft-versus-host disease (GVHD) rejection, the number of cells of the peripheral blood PBMC transplanted into each mouse is 0.5×10 6 ~1.9×10 6

[0031] ​In order to further improve the stability of the humanized mouse model, the extraction method of peripheral blood mononuclear cells of the anti-NMDAR encephalitis patient is as follows: after collecting the peripheral blood sample of the anti-NMDAR encephalitis patient, the peripheral blood mononuclear cells are separated by using the density gradient centrifugation method, and then are transferred into different mice by intraperitoneal injection.

[0032] Optionally, in step S2, whole blood, cerebrospinal fluid, bone marrow, spleen and brain of the model mouse are collected for analysis.

[0033] In a third aspect, the present application further provides a humanized mouse model prepared by the construction method in the above-mentioned scheme.

[0034] In a fourth aspect, the present application further provides a drug for treating anti-NMDAR encephalitis, which comprises the following component: Anakinra.

[0035] (III) Beneficial Effects

[0036] The beneficial effects of the present application are as follows: the analysis method of the anti-NMDAR encephalitis therapeutic effect of the present application can more accurately analyze and evaluate the pathology and therapeutic effect of encephalitis by establishing a humanized mouse model with anti-GluN1 autoantibody, blood-brain barrier damage and IL-1β produced by endothelial cells;

[0037] The humanized mouse model of the present application can realize the pathogenesis of encephalitis caused by promoting IL-1β-dependent blood-brain barrier dysfunction.

[0038] The drug for treating anti-NMDAR encephalitis of the present application can achieve good therapeutic effect by improving blood-brain barrier damage and neuropsychiatric behavior, thereby reducing recurrence. Anakinra as an IL1 receptor antagonist can improve blood-brain barrier damage and neuropsychiatric behavior. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 The experimental design and verification schematic diagram for PBMC implantation of BRGSF mice;

[0040] Figure 2 The test result graph for the production of anti-GluN1 autoantibody in the model group mice and the reduction of GluN1 expression in hippocampal tissue;

[0041] Figure 3 The test result graph of abnormal animal behavior, structure and function changes of different brain regions of the model group mice;

[0042] Figure 4 The test result graph of blood-brain barrier leakage of the model group mice;

[0043] Figure 5Figure showing the results of the test for the endothelial cell IL-1β damage to the blood-brain barrier for the model group of mice;

[0044] Figure 6 Figure showing the results of the test for the improvement of the disease disability and tight junction protein expression of the humanized mouse model by Anakinra (Anakinra, ank). DETAILED DESCRIPTION

[0045] In order to better explain the present application, so as to be understood, the present application is described in detail below through specific embodiments.

[0046] Example 1

[0047] The present embodiment provides a method for establishing a humanized mouse model, the steps of which are as follows: the peripheral blood mononuclear cells (PBMCs) of anti-NMDAR encephalitis patients are injected intraperitoneally into BRGSF mice, and the mice are continuously cultured for more than 8 weeks until the model is successfully established. The number of cells of the peripheral blood PBMCs transplanted into each mouse is 0.5×10 6 ~ 1.9×10 6 During the process of transplanting the peripheral blood PBMCs into the mice, the PBMCs are mixed in RPMI 1640 culture medium before being transplanted.

[0048] The number of cells of the peripheral blood PBMCs transplanted into each mouse can be, but is not limited to, 0.5×10 6 , 1.9×10 6 , 1.5×10 6 , 1×10 6 , 1.2×10 6 .

[0049] The present embodiment can implant the PBMCs of anti-NMDAR encephalitis patients into BRGSF mice to produce core clinical phenotypes and closely reflect the characteristics of human diseases in mice. Using this humanized mouse

[0050] model to determine the pathogenic role of the changed endothelial cells in the blood-brain barrier (BBB) and the endothelial-derived interleukin 1β (IL1-β) as a therapeutic target for anti-NMDAR encephalitis. The humanized mouse model obtained in the present embodiment is specifically applied, and the analysis method is as follows:

[0051] S1 PBMCs of anti-NMDAR encephalitis patients and healthy controls HC: 13 anti-NMDAR encephalitis patients and 13 healthy controls (HC) similar in age and gender were selected from the Department of Neurology, the Third Affiliated Hospital of Sun Yat-sen University in Guangzhou, China. The demographic and clinical characteristics of the subjects are shown in Table 1.

[0052] Table 1 Demographic and clinical characteristics of the subjects

[0053] Demographics and clinical characteristics of healthy donors and anti-NMDAR encephalitis patients

[0054]

[0055] PBMCs from patients and healthy controls were used for in vivo experiments.

[0056] PBMCs from patients and healthy controls were used for in vitro experiments.

[0057] F: female, M: male; MRI: magnetic resonance imaging;

[0058] mRS: modified Rankin Scale; NR: not relevant.

[0059] PBMCs were collected from all patients and HC by using PBMC isolation kit (LTS1077, TBD Science, China); whole blood (5 mL) in EDTA anticoagulation tube was mixed with 5 mL PBS and slowly added to a 50 mL centrifuge tube containing 10 mL separation solution. PBMCs were obtained by density gradient centrifugation at 400 x g for 30 min at 20 °C.

[0060] A schematic diagram of S2 design is shown in Figure 1 A. Female BRGSF mice aged 8-10 weeks were purchased from SJA Biotech Co., Ltd. (Suzhou, Jiangsu, China) and housed in the animal facility of South China Agricultural University under the designated pathogen-free conditions with a 12-hour light / dark cycle. All animal studies were reviewed and approved by the Animal Research Ethics Committee of South China Agricultural University (2020b102); mice were randomly divided into three groups and injected with cell culture medium (NC group), PBMCs of HC (HC group), and PBMCs of anti-NDMAR encephalitis patients (model group), respectively;

[0061] wherein, Figure 1 A: Experimental design and verification diagram of PBMC-transferred BRGSF mice.

[0062] B: Representative images of flow cytometry of mouse (m) CD45 + , human (h) CD44 + , hCD3 + , hCD4 + , hCD8 + , and hCD19 + cells in the blood, bone marrow, and spleen of BRGSF mice. HC and Patient refer to healthy controls and patients, respectively (n = 3 / group).

[0063] PBMCs from healthy subjects and anti-NMDAR encephalitis patients (1 x 10 6 cells in 100 μL RPMI 1640 medium) were injected intraperitoneally into each BRGSF mouse in the HC and model groups, respectively. Each mouse only received one injection of PBMCs. The negative control group (NC group) mice were injected intraperitoneally with the same amount of RPMI 1640 medium. Eighteen of the 26 PBMC samples (n = 9 in each group) were transferred to BRGSF mice (Table 1), and the detailed number of recipient mice for each PBMC donor is shown in Table 2. Ten of the 26 PBMC samples (n = 5 in each group) were used for in vitro experiments (Table 1). Among them, the PBMCs from patient 4 and healthy subject (HC) 7 in Table 1 were used in both in vivo and in vitro experiments (Table 1). This method was performed in accordance with the Declaration of Helsinki in 1964 and was approved by the Institutional Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University (

[2019] 02-637-01). All participants agreed and signed the written informed consent form; after 8 weeks of transplantation according to the above method, the whole blood, cerebrospinal fluid (CSF), bone marrow, spleen and brain of each mouse can be collected for analysis. The pathogenesis of the model can be successfully explored by flow cytometry, immunostaining, dot blotting, Western blotting, behavioral tests, functional magnetic resonance imaging and RNA transcriptome sequencing methods.

[0064] Table 2 Mice transplanted with PBMCs

[0065]

[0066] The present application also designs an in vitro BBB model experiment, the method is as follows: insertable cell culture insert with 3 μm pores and 0.3 cm 2 surface area (353096, Falcon, Kennett Square, PA, USA) is coated with 0.2% gelatin (G0040, solarbio) on both sides and placed in a 24-well cell culture plate (3533504, Falco). Human astrocyte cell line SVGp12 (1.2 x 10 5 cells; CRL-8621 TM) were obtained from Hunan Fenghui Biotechnology Co., Ltd. (Changsha, Hunan, China) and inoculated on the bottom of cell culture inserts and cultured in Dulbecco's Modified Eagle Medium (DMEM; C1995500BT, Gibco, Franklin, TN, USA) supplemented with 10% fetal bovine serum (FBS; ST30-3302, PAN, Cologne, Germany), 100 IU / mL penicillin (15140122, Gibco) and 100 pg / mL streptomycin (15140122, Gibco). Human cerebral endothelial cells hCMEC / D3 (1.2 x 105cells; SCC066) were purchased from EK Bioscience (Shanghai, China) and inoculated on the top of cell culture inserts and cultured in Endothelial Cell Medium (ECM; 1001, Sciencell, Carlsbad, CA, USA). The medium was changed every 2-3 days. Cultures were maintained at 37 °C in 5% CO2. 5 + + + +

[0067] The Shapiro-Wilk test was used to test the normality of the data. To compare the means of two groups, the Student t-test was used if the data were normally distributed, otherwise the Mann-Whitney U test was performed. To compare more than two groups of patients, one-way analysis of variance (ANOVA) and post-hoc tests were used. A p-value or adjusted p-value less than 0.05 was considered statistically significant. For the analysis of structural MRI, Mann-Whitney U test was performed using sklearn to compare the size of brain regions between different groups and highlight the brain regions with significantly different volumes in the standard template. For the analysis of ALFF, comparisons were made using paired t-tests under the null hypothesis (p < 0.05).

[0068] In the above specific application of the mouse model of the present embodiment, the following tests were performed by setting injection of cell culture medium (NC group), PBMC of healthy subjects HC (HC group), and PBMC of anti-NDMAR encephalitis patients (model group) as controls.

[0069] Test result I: The model group of mice PBMC transferred to BRGSF mice induced the production of autoantibodies and down-regulation of GluN1 expression in hippocampal tissue.

[0070] Eight weeks after injection, the transplanted lymphocytes were detected in the peripheral blood, bone marrow (BM) and spleen of the PBMC humanized mice, as shown by the presence of CD45 + , CD4 + , CD8 + and CD19 + human cells on flow cytometry Figure 1B). Histologically, CD4 + T cells, CD20 + B cells and CD138 + plasma cells were observed in the spleens of all PBMC humanized mice, but not in the NC group of mice Figure 2 A). Transfer of PBMCs led to the formation of white pulp in the spleens of immunodeficient mice, indicating that model mice formed germinal center-like structures Figure 2 A).

[0071] Figure 2 The production of anti-GluNl autoantibodies in model mice and the loss of GluNl in the hippocampus, Figure 2 A: Representative micrographs of H&E staining and immunohistochemical staining for hCD4, hCD20 and hCD138 in BRGSF mouse spleen sections injected with medium (NC) or PBMCs (HC or patient) (n=3 / group). B: Immunofluorescence of M68CT-GluNl in HEK-293T cells (n=3 / group). Red, signal detected by commercial anti-GluNl antibody. Green, signal detected by sera from mice from different groups. Nuclei were counterstained with DAPI (blue). C: Dot blot using sera from mice (n=10 / group) to detect the extracellular region of GluNl (N21-Q559). Commercial GluNl antibody (AB) and mouse IgG were used as positive and negative controls, respectively (n=3). D: Dot blot using CSF from mice (pooled from 10 mice / group) to detect cerebrospinal fluid GluNl expression. E: Western blot of hippocampal GluNl and PSD95 from NC, HC and patient group mice (n=3 / group). F: Statistical analysis of relative protein expression in E. **p<0.01, one-way ANOVA and post-hoc test.

[0072] that the recipient mice were able to produce anti-NMDAR antibodies. To this end, the present application used immunostaining and dot blot of HEK-293T cells transfected with GluNl (NMDAR subunit). As shown in Figure 2 Figure 2, antibodies against GluNl in sera from model mice were positive in immunofluorescence Figure 2 B) and dot blot analysis Figure 2 C and 2D, whereas no GluNl antibodies were present in sera from NC or HC groups Figure 2 C). Since cerebrospinal fluid from mice is limited, the present application pooled CSF from all mice in each group and used dot blot to detect antibodies. In line with sera, autoantibodies against GluNl were positive in model group CSF, but not in NC and HC groups Figure 2D). In addition, Western blotting showed that the expression level of GluN1 in the hippocampus of the model group mice was lower than that of the NC group mice and HC-PBMC transferred mice (n = 3, Figure 3 E and 2F). There was no statistically significant difference in the expression of PSD95 protein between the groups. These results suggest that the anti-NMDAR antibodies produced by the humanized mice may act by selectively reducing GluN1 expression.

[0073] Test results II. PBMC transfer of the model group mice changed the behavior of the recipient mice and induced changes in their structure and function

[0074] To determine whether the PBMC transfer of the above model group mice changed the behavior of the recipient mice and induced changes in their structure and function, the following tests were performed:

[0075] Anti-NMDAR encephalitis patients can exhibit psychiatric symptoms, cognitive deficits, and motor abnormalities. To assess whether PBMC implantation can reproduce the neuropsychiatric disorders of patients, the present application performed a series of standardized tests. The BRGSF mice of the above three groups of cell culture medium (NC group), PBMC of HC (HC group), and PBMC of anti-NDMAR encephalitis patients (model group) were examined for behavioral changes 8 weeks after PBMC transplantation.

[0076] In the elevated plus maze test, the model group mice carrying patient PBMC transfer spent significantly more time in the open arm and less time in the closed arm than the NC group mice and the mice carrying PBMC of healthy controls (HC group), indicating an agoraphobia-like behavior Figure 3 A). At the same time, the recognition ability of mice to new objects was used to measure cognition. The discrimination index of the model group mice carrying patient PBMC was lower in the new object recognition test than that of the NC group and HC group mice Figure 3 B), indicating memory and learning deficits. In addition, in the forced swimming test, the mice of the model group also exhibited a depression-like behavior, showing a longer immobility time than the NC group mice and the HC-PBMC treated mice Figure 3 C). While the open field test confirmed the hyperlocomotor phenotype of the model group mice, with a significant increase in total travel distance compared to the NC group mice and the HC group mice Figure 3 D). There was no difference in any behavior between untreated mice and mice transferred with PBMC of healthy controls. In summary, the neuropsychiatric behavior of humanized PBMC mice closely depends on the source of PBMC, and transferring PBMC from anti-NMDAR encephalitis patients can summarize the neuropsychiatric abnormalities observed in patients.

[0077] Figure 1 Abnormal animal behavior, structural and functional changes in different brain regions of the model group mice:

[0078] The following parameters are considered: A: Representative movement trajectories of mice in the elevated maze (EPM) group (n=9), HC group (n=9), and patient group (n=10) to examine claustrophobic-like behaviors; B: Representative movement trajectories of mice in the NC group (n=7), HC group (n=9), and patient group (n=10) in the Novel Object Recognition Test (NORT) to examine memory and learning deficits; C: Inactivity time of animals in the NC group (n=9), HC group (n=9), and patient group (n=10) during the forced swimming test to assess depressive-like behaviors; D: Representative movement trajectories of animals in the NC group (n=9), HC group (n=9), and patient group (n=10) during the mine test to examine hyperactive movement and anxiety behaviors; E: Comparison of different brain region volumes among the groups; F: Comparison of ALFF values ​​between the NC group and the patient group, and between the HC group and the patient group (n=6 / group) **p<0.01, one-way ANOVA and post-hoc tests.

[0079] This invention also explores whether changes in neuropsychiatric behavior are related to changes in the volumetric structure and intrinsic brain function of different brain regions. Eight weeks after PBMC injection, mice underwent MR imaging (MRI). Figure 3 A); This reveals that the volume of most brain regions, including different hippocampal areas, did not differ significantly between the two groups. Figure 3 E). However, compared with the NC group, the model group had significantly lower lateral entorhinal area (p = 0.026) and postsubiculum (p = 0.045) volumes. There was no significant difference in brain volume between the HC and model groups (lateral entorhinal area: p = 0.211; postsubiculum: p = 0.297). Figure 3 E).

[0080] On the other hand, the model group mice exhibited altered functional activity in multiple brain regions. Compared to the NC and HC groups, the model group mice showed significantly increased ALFF values ​​in the hippocampus, thalamus, prefrontal cortex, cingulate cortex, area 2 (Cg2), and DS (dorsal subiculum). Figure 3 F, intergroup adjusted p<0.05), but ALFF values ​​were decreased in several other regions, such as BMP (basolateral amygdala, posterior part) and CE (cerebellum) (F, intergroup adjusted p<0.05), but ALFF values ​​were decreased in several other regions, such as BMP (basomedial amygdala, posterior part) and CE (cerebellum) (F, intergroup adjusted p<0.05), but ALFF values ​​were decreased in Figure 4 F, p<0.05 after intergroup correction. This invention proposes that the model group significantly altered brain function, but not brain structure, and that the abnormal psychological processing may be attributed to neuropsychiatric disorders in mice with patient-associated immune systems.

[0081] Test result three: humanized model group mice showed blood brain barrier leakage

[0082] Anti-NMDAR encephalitis patients are characterized by cytokine elevation and immune cell infiltration in CSF, which is associated with blood brain barrier damage in patients. In the present humanized PBMC mouse model, fibrinogen extravasation was observed in the brains of mice in the model group, but not in mice in the NC and HC groups, indicating the presence of blood brain barrier leakage in mice in the model group Figure 4 A). Tight junction proteins such as Occludin, Claudin-1, Claudin-5 and ZO-1 are important components that connect brain microvascular endothelial cells (BMVECs) and seal the intracellular space between BMVECs to maintain the integrity of the blood brain barrier. Consistent with fibrinogen extravasation, the expression levels of Claudin-1, Occludin, ZO-1 and Claudin-5 were significantly reduced in mice injected with patient-derived PBMCs compared to mice injected with healthy control-derived PBMCs Figure 4 B and 4C). In addition, in mice injected with the model group, this was accompanied by significant infiltration of CD4+ T cells, CD8+ T cells and B220+ B cells in the meninges Figure 4 D) and CD8+ T cells in the hippocampus Figure 4 E) in the hippocampus. No immune cell infiltration was observed in mice from the NC and HC groups. These results indicate that patient-derived PBMCs are involved in blood brain barrier leakage, causing immune cells to enter the central nervous system by suppressing tight junction protein expression.

[0083] Figure 5 Figure for blood brain barrier leakage in model group mice: A: Immunofluorescence of laminin (Lam; blood vessels, red) and fibrinogen (Fib; blood brain barrier leakage index, green) in the brain of mice (n=3 / group). Nuclei were counterstained with DAPI (blue). B: Western blot of tight junction proteins (Claudin-1, Occludin, ZO-1 and Claudin-5) on total tissue lysates of whole brain (n=3 / group). C: Statistical analysis of laminin relative protein expression: Immunofluorescence of laminin (Lam; blood vessels, red) and human CD4 (CD4+ lymphocytes, green), or human CD8 (CD8+ lymphocytes, green), or B220 (B lymphocytes, red) in the meninges of mice (n=3 / group). Nuclei were counterstained with DAPI (blue). E: Immunofluorescence of human CD4 (green), human CD8 (red) or B220 (red) in the hippocampus (n=3 / group). Nuclei were counterstained with DAPI (blue). In the present figures, the specific description of red, blue and other colors is that after conversion to black and white, it is specifically represented as different shades of black.

[0084] Test results IV. Blood-brain barrier of mice in the humanized model group of endothelial cell IL-1β injury

[0085] To explore the potential molecular mechanism of blood-brain barrier leakage, the present application performed transcriptome analysis. As described above, mice were randomly divided into three groups (n = 3 for each group) and treated accordingly. At 8 weeks after culture medium or PBMC injection, the brain was collected for bulk RNA-seq analysis. Deconvolution analysis showed that the brain transcriptome of model group mice was associated with loss of neuronal fraction compared to NC group mice and healthy control PBMC-transplanted mice (A and 5B). The present application did not interpret this result as neuronal loss in model group mice, on the contrary, the expression of neuronal-related markers can be widely affected by model group. Further DEG analysis showed that there were 417 common DEGs between model group and NC group (490 DEGs, Figure 5 C) and model group and HC group (1268 DEGs). The differential expression of 22 common DEGs was verified by quantitative RT-PCR (D). Based on DEG (E), the present application constructed a protein-protein interaction (PPI) network of 417 common DEGs retrieved from STRING database, and then performed molecular complex detection (MCODE) analysis to identify highly interconnected regions. Notably, IL-1β is the most highly connected gene in the complete network and is identified as a hub gene of important MCODE modules (F-5G). Figure 5 C) and model group and HC group (1268 DEGs). The differential expression of 22 common DEGs was verified by quantitative RT-PCR (D). Based on DEG (E), the present application constructed a protein-protein interaction (PPI) network of 417 common DEGs retrieved from STRING database, and then performed molecular complex detection (MCODE) analysis to identify highly interconnected regions. Notably, IL-1β is the most highly connected gene in the complete network and is identified as a hub gene of important MCODE modules (F-5G). Figure 5 C) and model group and HC group (1268 DEGs). The differential expression of 22 common DEGs was verified by quantitative RT-PCR (D). Based on DEG (E), the present application constructed a protein-protein interaction (PPI) network of 417 common DEGs retrieved from STRING database, and then performed molecular complex detection (MCODE) analysis to identify highly interconnected regions. Notably, IL-1β is the most highly connected gene in the complete network and is identified as a hub gene of important MCODE modules (F-5G). Figure 5 C) and model group and HC group (1268 DEGs). The differential expression of 22 common DEGs was verified by quantitative RT-PCR (D). Based on DEG (E), the present application constructed a protein-protein interaction (PPI) network of 417 common DEGs retrieved from STRING database, and then performed molecular complex detection (MCODE) analysis to identify highly interconnected regions. Notably, IL-1β is the most highly connected gene in the complete network and is identified as a hub gene of important MCODE modules (F-5G). Figure 3 C) and model group and HC group (1268 DEGs). The differential expression of 22 common DEGs was verified by quantitative RT-PCR (D). Based on DEG (E), the present application constructed a protein-protein interaction (PPI) network of 417 common DEGs retrieved from STRING database, and then performed molecular complex detection (MCODE) analysis to identify highly interconnected regions. Notably, IL-1β is the most highly connected gene in the complete network and is identified as a hub gene of important MCODE modules (F-5G). Figure 5 )。

[0086] C) and model group and HC group (1268 DEGs). The differential expression of 22 common DEGs was verified by quantitative RT-PCR (D). Based on DEG (E), the present application constructed a protein-protein interaction (PPI) network of 417 common DEGs retrieved from STRING database, and then performed molecular complex detection (MCODE) analysis to identify highly interconnected regions. Notably, IL-1β is the most highly connected gene in the complete network and is identified as a hub gene of important MCODE modules (F-5G). Figure 5 C) and model group and HC group (1268 DEGs). The differential expression of 22 common DEGs was verified by quantitative RT-PCR (D). Based on DEG (E), the present application constructed a protein-protein interaction (PPI) network of 417 common DEGs retrieved from STRING database, and then performed molecular complex detection (MCODE) analysis to identify highly interconnected regions. Notably, IL-1β is the most highly connected gene in the complete network and is identified as a hub gene of important MCODE modules (F-5G).

[0087] C) and model group and HC group (1268 DEGs). The differential expression of 22 common DEGs was verified by quantitative RT-PCR (D). Based on DEG (E), the present application constructed a protein-protein interaction (PPI) network of 417 common DEGs retrieved from STRING database, and then performed molecular complex detection (MCODE) analysis to identify highly interconnected regions. Notably, IL-1β is the most highly connected gene in the complete network and is identified as a hub gene of important MCODE modules (F-5G). Figure 5I). Then, the present application examined the source of IL-1 β elevation by immunofluorescence. P-glycoprotein-labeled microvascular endothelial cells Figure 5 J) but not astrocytes (Gfap+, Figure 5 K) co-stained with anti-IL-1 β specific antibodies, suggesting that patient-derived PBMCs upregulate endothelial IL-1 β to damage the blood brain barrier. This was also supported by in vitro blood brain barrier model studies. Compared to HC controls, patient-derived PBMCs decreased TEER and increased permeability to dextran in the hCMEC / D3 microvascular endothelial cell line Figure 5 L and 5M). Moreover, patient-derived PBMCs also directly upregulated IL-1 β protein levels in hCMEC / D3 cells, while decreasing Claudin-1, Occludin, ZO-1 and Claudin-5 Figure 5 N). The present application found that the disrupted tight junctions in endothelial cells were IL-1 β dependent. Overexpression of IL-1 β in hCMEC / D3 cells significantly decreased Claudin-1, Occludin, ZO-1 and Claudin-5 protein levels Figure 5 O), while knockdown of IL-1 β showed the opposite effect in in vitro experiments Figure 6 P). Therefore, patient PBMCs can directly upregulate endothelial IL-1 β, thereby decreasing tight junctions in brain microvascular endothelial cells, leading to blood brain barrier leakage.

[0088] Figure 6Endothelial IL-1β damages the blood-brain barrier in model mice; wherein, A: UMAP plot of 16028 cells from normal mouse brain. Neurons; ASC, astrocytes; Immune, immune cells; EP, endothelial progenitor cells; OLG, oligodendrocytes and vascular endothelial cells. B: Statistical analysis of the proportion of neurons in mouse brain by MuSic deconvolution analysis of bulk RNA-Seq data (n=3 / group). C and D: Volcano plots showing DEGs between each group (n=3 / group). E: RT-qPCR validation of DEGs in C and D: Bar plots summarizing 20 GOBP terms (adjusted p<0.05) based on commonly (between patients and HC and patients and NC) downregulated DEGs and interaction networks for all relevant GOBP terms. Nodes refer to GOBP terms whose semantics are similar to the summary terms of the same color. G: Bar plots summarizing 16 GOBP terms (adjusted p<0.05) based on commonly upregulated DEGs and interaction networks for all relevant GOBP terms. H: PPI network of common DEGs. I: Western blotting (n=3 / group) and statistical analysis of IL-1β on whole brain total tissue lysates. J: Immunofluorescence of P-glycoprotein (brain microvascular endothelial cells, red) and IL-1β (green) in meninges (n=3 / group). Nuclei were counterstained with DAPI (blue). K: Immunofluorescence of GFAP (astrocytes, red) and IL-1β (green) in meninges (n=3 / group). Nuclei were counterstained with DAPI (blue). L: Relative changes in transendothelial electrical resistance (TEER) of different treated in vitro blood-brain barrier models (n=5 / group). M: FITC-dextran recovery in different treated in vitro blood-brain barrier models (n=5 / group). N: Western blotting of IL-1β, Occludin, Claudin-1, ZO-1, and Claudin-5 on hCMEC / D3 cell lysates treated with different treatments (N=5 / group). O: Western blotting of IL-1β, Occludin, Claudin-1, ZO-1, and Claudin-5 on hCMEC / D3 cell lysates transfected with empty vector or IL-1β overexpression plasmid (n=3). P: Western blotting of IL-1β, Occludin, Claudin-1, ZO-1, and Claudin-5 on hCMEC / D3 cell lysates transfected with medium, siRNA control (siNC), or Il1b siRNA (si1 and si2) (n=3) **p<0.01, one-way ANOVA with post-hoc test.

[0089] Test results five, blocking IL-1β signaling pathway improves the phenotype of humanized anti-NMDAR encephalitis model

[0090] The therapeutic value of IL-1β blockade was investigated in a humanized mouse model of NMDAR encephalitis. Anakinra, an Il-1 receptor antagonist approved for the treatment of rheumatoid arthritis, blocks IL-1β signaling. A schematic diagram of the experimental design is shown below. Figure 6 As shown in Table A. The sources of PBMCs used in the in vitro blood-brain barrier (BBB) ​​model are shown in Table 1. Based on the in vitro BBB model experiments, Anakinra reversed the inhibition of Occludin, ZO-1, and Claudin-5 expression induced by patient-derived PBMCs in hCMEC / D3 cells. Figure 6 B). Therefore, compared with the vector control, Anakinra treatment significantly enhanced TEER in hCMEC / D3 cells in the in vitro BBB model group of patient-derived PBMCs, while reducing FITC-glucan permeability (B). Figure 6 (C and 6D). Anakinra also demonstrated its efficacy in in vivo model experiments. Eight weeks after PBMC injection, mice treated with Anakinra showed significantly improved hyperactive motor behavior, claustrophobia-like behavior, and depressive-like behavior compared to the control group. Figure 6 E-6G). At the molecular level, Anakinra reversed the inhibition of Claudin-1, Occludin, and Claudin-5 expression in the mouse brain (E-6G). Figure 6 H-6I) and improved the protein expression level of GluN1 in the hippocampus. Figures 1-6 These results indicate that Anakinra improves autoimmune encephalitis by restoring blood-brain barrier permeability.

[0091] ​Figure. Among them, A: schematic diagram of experimental design for drug efficacy analysis. B: Western blotting and statistical analysis of relative protein expression of Claudin-1, Occludin, ZO-1 and Claudin-5 on cell lysates from hCMEC / D3 cells (n = 5 / group). C: Relative changes in transendothelial electrical resistance (TEER) of the in vitro blood-brain barrier model (n = 5 / group). D: FITC-dextran recovery rate of the in vitro blood-brain barrier (n = 5 / group). E: Representative movement trajectories of model group mice (n = 6) and ank-treated mice (n = 6) in the open field test. F: Representative movement trajectories of mice from different groups (n = 6) in the elevated plus maze (EPM). G: Immobility time of mice from different groups (n = 6) in the forced swimming test. H: Western blotting of tight junction proteins (Claudin-1, Occludin, ZO-1 and Claudin-5) on total tissue lysates of whole brain from mice of different groups (n = 3 / group). Western blotting expression of hippocampal GluN1 (n = 3 / group). I: Statistical analysis of relative protein expression in H. J: Statistical analysis of relative protein expression in H. *p < 0.05, **p < 0.01, Student t test.

[0092] In summary, the present application transfers the PBMCs of patients to BRGSF mice to establish a humanized mouse model of anti-NMDAR encephalitis. The engraftment of patient lymphocytes induces the production and entry of anti-GluN1 autoantibodies into the brain, promotes lymphocyte infiltration, and leads to abnormal neuropsychiatric behavior and functional changes in multiple brain regions related to neuropsychiatric behavior. It is emphasized that blood-brain barrier leakage is a key pathological change in this humanized mouse model, and the IL-1β pathway is involved in the BBB damage process. In addition, the present application proves that, in the anti-NMDA encephalitis humanized mouse model of the present application, blocking IL-1β receptors by Anakinra can reverse blood-brain barrier leakage, immune cell infiltration and neuropsychiatric disorders. The present application provides a clinically relevant model of anti-NMDAR encephalitis and emphasizes that blocking IL-1β signaling is a promising strategy for improving this autoimmune encephalitis.

[0093] In the mouse model of the present embodiment, anti-NMDAR encephalitis patient-derived PBMCs can trigger the pathogenesis of encephalitis by causing IL-1β-dependent blood-brain barrier dysfunction, and IL1 receptor antagonist Anakinra is a therapeutic drug for anti-NMDAR encephalitis. The new animal model expands the potential of the present application to directly evaluate the effects of cellular and humoral immune functions on the progression of this autoimmune encephalitis. The mouse model of the present embodiment is simple to establish, can realize the establishment of an anti-NMDAR encephalitis patient-specific mouse model, and can be used to evaluate various potential therapies or develop precision medicine.

[0094] The present application is described in detail in the accompanying drawings ​ The specific description of the colors such as red, blue, etc. is specifically manifested as the difference in the depth of black and white after being converted into black and white drawings.

[0095] Embodiment 3

[0096] The present embodiment provides a drug for treating anti-NMDAR encephalitis, Anakinra;

[0097] In some other specific embodiments, hormones, mycophenolate mofetil can also be included.

[0098] Specifically, the hormones include methylprednisolone, prednisone acetate, methylprednisolone.

[0099] Anakinra is a biological agent, which is an artificially synthesized protein. It is an interferon antagonist used to treat some autoimmune diseases, especially rheumatoid arthritis. Anakinra reduces inflammation and related symptoms by preventing the process of interferon signaling; in the present embodiment, Anakinra has a significant therapeutic effect on the drug for treating anti-NMDAR encephalitis; wherein Anakinra and hormones, mycophenolate mofetil and other drugs have a synergistic effect on the therapeutic effect.

[0100] The dosages of Anakinra and hormones, mycophenolate mofetil are determined according to the actual height and weight of the patient, and the conventional medication is used.

[0101] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for establishing a mouse model of NMDAR encephalitis, characterized in that: It includes the following steps: Peripheral blood mononuclear cells from patients with anti-NMDAR encephalitis were injected intraperitoneally into BRGSF mice and cultured for more than 8 weeks to induce endothelial IL-1β expression; the number of peripheral blood mononuclear cells transplanted into each mouse was 0.5 × 10⁻⁶. 6 ~1.9×10 6 indivual; The method for extracting peripheral blood mononuclear cells from patients with anti-NMDAR encephalitis is as follows: after collecting peripheral blood samples from patients with anti-NMDAR encephalitis, the peripheral blood mononuclear cells are separated using density gradient centrifugation and then transferred into different mice via intraperitoneal injection.

2. The method for establishing an anti-NMDAR encephalitis mouse model as described in claim 1, characterized in that: The patients with anti-NMDAR encephalitis should be diagnosed in the acute phase and not have received immunotherapy.

3. The method for establishing an anti-NMDAR encephalitis mouse model as described in claim 1, characterized in that: The analysis was performed by collecting whole blood, cerebrospinal fluid, bone marrow, spleen, and brain from model mice.

4. An anti-NMDAR encephalitis mouse model obtained by the method for establishing an anti-NMDAR encephalitis mouse model according to any one of claims 1-3.

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