Use of miR-423-5p in enhancing b-lymphocyte immune response in eae models, methods and kits

Abstract

The application relates to the field of immunology, and particularly relates to application, a method and a kit of miR-423-5p in enhancing B lymphocyte immune response in an EAE model. In the process of modeling of a standard EAE active immunization model, miR-423-5p is injected intravenously (30 ug per mouse) on the 1st, 5th, 9th, 13th and 17th days to stimulate B cell proliferation and promote antibody production. A novel EAE animal model with significant B cell contribution is successfully constructed, the disease phenotype of which is that the onset is earlier, the progress is faster, the disease condition is more serious, the score is higher, and the weight loss is more significant; meanwhile, the lymphocyte infiltration of the thoracic and lumbar segments of the spinal cord of the model mice is obviously increased, the expression of the disease-related cytokine IFN-gamma in serum is up-regulated, and the expressions of B cell antibodies IgG and IgM are also significantly up-regulated.

Description

Technical Field

[0001] This invention relates to the field of immunology, specifically to the application, method, and kit of miR-423-5p in enhancing B lymphocyte immune responses in an EAE model. Background Technology

[0002] However, existing standard EAE models have significant limitations in reproducing the immune mechanisms. In these models, the use of potent T-cell adjuvants (CFAs) and exogenous toxins (PTXs) induces an excessive and dominant T-cell (especially Th1 and Th17)-mediated inflammatory response. This artificially enhanced T-cell response produces a “masking effect”: although B cells play a crucial role in the pathology of real MS patients (such as generating oligoclonal bands, participating in antigen presentation, and forming ectopic lymphoid follicles), in the standard EAE model, the B-cell immune response is masked by the extremely active T-cell response.

[0003] Specifically, this "T cell-dominated" microenvironment makes B cell function insignificant and difficult to detect. On the one hand, the function of B cells as professional antigen-presenting cells (APCs) to present antigens to T cells appears negligible in the context of strong adjuvant nonspecific activation of T cells; on the other hand, the regulatory functions of B cells (such as regulatory B cells secreting IL-10) or pathogenic functions (such as producing autoantibodies) are often regarded as secondary factors or difficult to separate from background noise by conventional means in the face of severe T cell-mediated tissue damage.

[0004] Therefore, existing EAE models fail to accurately reflect the complex network of T-cell and B-cell synergistic pathogenesis in MS, resulting in drugs screened based on these models often being effective only against the T-cell pathway, while showing poor clinical efficacy against B-cell-related MS subtypes. Currently, there is an urgent need for an improved model or detection method that can overcome the "masking effect" of T-cell responses on B-cell responses, in order to accurately assess the specific contributions of B cells and potential therapeutic targets in multiple sclerosis. Summary of the Invention

[0005] To address the shortcomings of existing standard EAE active immunization models where T cell responses are overly dominant and B cell immune responses are masked and difficult to detect and evaluate, this paper provides the application, methods, and kits for miR-423-5p in enhancing B lymphocyte immune responses in EAE models.

[0006] To achieve the above objectives, the technical solution specifically adopted by the present invention is as follows: A method for enhancing B lymphocyte immune responses in a standard experimental autoimmune encephalomyelitis (EAE) active immunization model, comprising introducing exogenous miR-423-5p as an immunomodulator in the context of conventional EAE modeling. Specifically, the method includes administering miR-423-5p to the model animals during the establishment of the EAE active immunization model. miR-423-5p can specifically act on B cell signaling pathways (or regulate antigen presentation processes), thereby further amplifying B cell activation signals in the context of antigen stimulation, achieving the purpose of stimulating B cell proliferation and promoting antibody production.

[0007] To ensure the stability and significance of the aforementioned enhancement effects, this invention has screened the application method, timing, and dosage: Administration method (intravenous injection): Compared to subcutaneous or intraperitoneal injection, this invention uses intravenous injection. Intravenous injection allows miR-423-5p to rapidly enter the bloodstream, bypassing degradation in local tissues and more efficiently accumulating in peripheral immune organs such as the spleen and lymph nodes, thereby directly acting on antigen-activated B cells.

[0008] Timing of administration (days 1, 5, 9, 13, and 17): This is a validated specific time window. Day 1 coordinates with antigen-induced immune response initiation; days 5 to 9 cover the latency period, continuously stimulating B cell proliferation in lymphoid tissue; days 13 to 17 correspond to the antibody maturation phase before the disease peak. Intervention at these specific time points synergizes with the natural course of the EAE model, maximizing the enhanced effect.

[0009] Dosage (30 μg / animal): 30 micrograms (30 μg) was determined as the single-dose dose through dose-response experiments. This dose achieves a significant biological effect while avoiding non-specific immune responses that may be caused by excessively high doses.

[0010] Furthermore, the method described in this invention is applicable to standard EAE active immunization models. Specifically, the model is induced by subcutaneous immunization with an emulsion mixture of myelin antigen peptides (preferably MOG35-55) and complete Freund's adjuvant (CFA), followed by intraperitoneal injection of pertussis toxin (PTX) at a specific time. This invention is an improvement on this classic model, without altering the original model's construction basis, and has good compatibility.

[0011] Furthermore, based on the above findings, this invention also proposes the application of miR-423-5p in the preparation of reagents for enhancing B lymphocyte immune responses in a standard EAE active immunization model. The reagent may be a drug, adjuvant, or research reagent, and its core function is to upregulate B cell responses.

[0012] Furthermore, this invention also provides a kit for constructing an EAE animal model with enhanced B cell immune response. The kit comprises: Core component: Effective amount of miR-423-5p; Guidance information: The instruction manual clearly describes the specific steps (i.e., the aforementioned method) for using this component for intervention.

[0013] This kit commercializes immune modulation protocols, making it easier for researchers to standardize their procedures.

[0014] This invention has the following characteristics and beneficial effects: A novel animal model of acute exacerbation of immune encephalopathy (EAE) with significant B-cell contribution was successfully constructed. The disease phenotype was characterized by earlier onset, faster progression, more severe illness, and more significant weight loss. Simultaneously, the model mice showed significantly increased lymphocyte infiltration in the thoracic and lumbar spinal cord segments, upregulated expression of the disease-associated cytokine IFN-γ in serum, upregulated miR-423-5p expression in splenic B cells, and significantly upregulated expression of B-cell antibodies IgG and IgM. These results, from clinical scoring, histopathology, cytokine levels, and humoral immunity perspectives, confirmed a significant enhancement of the B-cell immune response. Attached Figure Description

[0015] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 For model flowchart; Figure 2 Clinical scoring results; Figure 3 The model shows the lymphocyte infiltration in the spinal cord of the thoracic (left) and lumbar (right) vertebral segments; Figure 4 shows the experimental results. In the figure: (a) shows the expression of miR-423-5p in the spleen B lymphocytes of normal mouse EAE model and EAE+423 model mouse (qPCR results); (b) shows the IFN-γ IgG IgM content in the serum of the three model mice (ELISA results). Detailed Implementation

[0016] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0017] Experimental data: 1. Laboratory animals SPF-grade female C57BL / 6 mice, 6-8 weeks old and weighing 18-22g, were selected. Animals were housed in a temperature- and humidity-controlled environment with 12-hour light-dark cycles and free access to food and water. They underwent a one-week acclimatization period before the experiment.

[0018] 2. EAE active immune induction Day 0: Move MOG35 55% of the peptide was dissolved in sterile PBS to a concentration of 2 mg / mL. An equal volume of MOG35 was then added... The 55% solution was mixed with complete Freund's adjuvant (CFA, containing 4 mg / mL inactivated Mycobacterium tuberculosis) and emulsified repeatedly using a glass syringe until a stable water-in-oil emulsion was formed (it did not spread when dropped onto water). Each mouse received a subcutaneous injection of the emulsion at four points along both sides of the spine on its back, with a total injection volume of 100 μL per mouse, containing MOG35. 55200μg.

[0019] Day 0 and Day 2: Each mouse was given a booster immunization by intraperitoneal injection of 200 ng of pertussis toxin (PTX) (dissolved in 200 μL of sterile PBS).

[0020] 3. Intravenous injection of miR-423-5p Injection timing: Injections were administered on days 1, 5, 9, 13, and 17 post-immunization.

[0021] Injection method: Fix the mouse in a mouse tube and immerse its tail in warm water (37-40℃) for 1-2 minutes to dilate the tail vein.

[0022] The miR-423-5p mimic was diluted to a final concentration of 50 μM with sterile saline.

[0023] Each mouse was injected with 100 μL (containing 30 μg miR-423-5p) via the tail vein each time.

[0024] Control group mice were injected with an equal volume of negative control miRNA (scramble miRNA).

[0025] 4. Postoperative care and observation After the injection, gently press the injection site with a sterile dry cotton ball to stop the bleeding, then put the mouse back into its original cage and continue to feed it, taking care to keep it warm.

[0026] 5. Clinical score Starting from day 0 of immunization, mice were clinically scored daily at the same time point. The internationally recognized EAE five-point scoring system was used. 0 points: No signs of disease 1 point: Reduced tail tension (tail drag) or slightly clumsy gait 2 points: Loss of tail tension (complete tail drag) and / or moderately clumsy gait and / or poor righting ability. 3 points: Limb weakness 4 points: Limb paralysis 5 points: Near-death state or death Mouse weight changes were recorded daily, and the percentage of weight loss was calculated with the weight on the day of immunization as the baseline.

[0027] 6. Experimental endpoint and sample collection Animals were euthanized on day 20 post-immunization (peak disease period). The following samples were collected: Serum: Blood was collected from the heart, centrifuged to separate the serum, and stored at -80℃ for future testing.

[0028] Spinal cord: After perfusion and fixation with 4% paraformaldehyde, the spinal cord (especially the thoracic and lumbar segments) was harvested for pathological sections and HE staining to observe lymphocyte infiltration.

[0029] Spleen: The spleen was aseptically harvested and a single-cell suspension was prepared for B cell isolation and qPCR detection.

[0030] 7. Experimental Results like Figure 2 As shown, mice in the negative control group (scramble miRNA) began to develop symptoms on day 9 post-immunization, exhibiting tail weakness and gait abnormalities. The symptoms subsequently worsened, reaching a peak between days 18 and 20 post-immunization, with an average clinical score approaching 3. Some mice showed severe neurological damage, including limb weakness and limited mobility. In contrast, mice in the miR-423-5p intervention group showed a significantly delayed onset of disease and a substantial reduction in overall severity. Their average clinical scores at all time points were significantly lower than those in the negative control group, and no severe neurological damage was observed. The trend in mouse body weight change was highly consistent with the clinical score results. Mice in the negative control group experienced continuous weight loss with the onset of EAE, indicating significant bodily depletion. Mice in the miR-423-5p intervention group showed a significantly smaller rate of weight loss compared to the control group, with more stable overall body weight, indicating effective improvement in systemic inflammation and bodily damage. In conclusion, miR-423-5p can effectively delay the onset of EAE in mice, reduce disease severity, and alleviate bodily depletion during the course of the disease, demonstrating a significant protective effect against experimental autoimmune encephalomyelitis.

[0031] Figure 3HE-stained pathological sections of spinal cord tissue from the thoracic (left) and lumbar (right) vertebrae of mice provide a direct visual representation of the degree of lymphocyte infiltration and inflammatory damage in the spinal cord tissue of each group. In the negative control group, mice showed extensive lymphocyte aggregation and infiltration in both the thoracic and lumbar vertebrae, with wide and deep inflammatory cell infiltration, disrupting the normal structure of the spinal cord. The inflammatory infiltration in the lumbar vertebrae was more pronounced than in the thoracic vertebrae, consistent with the typical pathological characteristics of the EAE model, which is characterized by predominantly lumbosacral spinal cord inflammation. In contrast, the miR-423-5p intervention group showed a significant reduction in the number of lymphocytes infiltrating the thoracic and lumbar vertebrae, a significantly smaller area of ​​inflammatory infiltration, significantly better spinal cord tissue structure integrity than the control group, and a substantial reduction in the degree of inflammatory damage. The above pathological results were highly consistent with the clinical scores and weight change phenotypes, further confirming from a histological perspective that miR-423-5p can significantly inhibit inflammatory cell infiltration in the spinal cord tissue of EAE mice, reduce central nervous system inflammatory damage, improve the pathological damage phenotype of the spinal cord, and exert a neuroimmunoprotective effect.

[0032] Figure 4 shows the regulatory effects of miR-423-5p on humoral immunity and inflammatory factor expression in EAE mice, verified by qPCR and ELISA. Figure 4(a) shows the qPCR results of spleen B lymphocytes, indicating that compared with the normal group, the basal expression level of miR-423-5p in spleen B cells of EAE model mice was significantly downregulated; however, after intervention with miR-423-5p mimics, the expression level of miR-423-5p in spleen B lymphocytes of EAE+miR-423-5p group mice significantly rebounded, successfully achieving effective overexpression intervention of the target gene.

[0033] In summary, molecular biological assays showed that miR-423-5p effectively upregulated the expression of miR-423-5p in splenic B lymphocytes of EAE mice, significantly reduced the secretion of pro-inflammatory factors and autoimmune antibodies in vivo, and inhibited the over-activated autoimmune inflammatory response in EAE mice. This elucidated the protective mechanism of miR-423-5p against EAE at the molecular immune level.

[0034] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely preferred examples and are not intended to limit the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for enhancing B lymphocyte immune responses in a standard experimental autoimmune encephalomyelitis active immunization model, characterized in that, The method includes: During the establishment of the EAE active immunization model, miR-423-5p was administered to the model animals to stimulate B cell proliferation and promote antibody production.

2. The method according to claim 1, characterized in that, The method of administration is intravenous injection.

3. The method according to claim 2, characterized in that, The timing of administration is as follows: intravenous injections are administered on days 1, 5, 9, 13, and 17 after the start of modeling.

4. The method according to claim 3, characterized in that, The dosage of miR-423-5p administered each time was 30 μg per 20g model animal.

5. The method according to claim 1, characterized in that, The standard EAE active immunization model was induced by subcutaneous injection of a mixture of immune myelin antigen peptides and complete Freund's adjuvant, supplemented by intraperitoneal injection of pertussis toxin.

6. The method according to claim 5, characterized in that, The myelin antigen peptide is MOG. 35-55 .

7. Application of miR-423-5p in the preparation of reagents for enhancing B lymphocyte immune responses in an EAE active immunization model.

8. The application according to claim 7, characterized in that, The enhanced B lymphocyte immune response includes stimulating B cell proliferation and promoting antibody production.

9. A kit for constructing an EAE animal model with enhanced B cell immune response, characterized in that, The kit comprises: an effective amount of miR-423-5p, and instructions for use, which instruct the application of the miR-423-5p to a model animal according to the method of any one of claims 1-6.

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