Preparation method and application of dendritic cell-derived exosome

Abstract

The invention discloses a preparation method and application of a dendritic cell-derived exosome, and the preparation method comprises the following steps: (1) inducing in vitro differentiation of bone marrow-derived dendritic cells (DCs) to tolerant dendritic cells (tolDCs) through interleukin-10, and ensuring that the tolDCs highly express key antioxidant and anti-inflammatory proteins, namely, heme oxygenase-1 (HO-1); (2) separating and purifying an exosome HO-1 high-tolDex from the cell culture supernatant of the tolDCs (HO-1 high-tolDCs) with high expression of the HO-1 by virtue of methods such as ultracentrifugation and the like; (3) carrying out comprehensive physicochemical property and biological function identification on the prepared HO-1high-tolDex, and (4) verifying that the HO-1high-tolDex can effectively inhibit excessive activation and inflammatory response of target cells through an in-vitro cell experiment and an in-vivo experiment of a systemic lupus erythematosus (SLE) animal model, alleviate immune pathological injury of model animals, inflammation of kidneys and oxidative stress injury, and improve the bioavailability of the model animals. The immune tolerance is induced; the cell-free immunomodulator is a new generation of efficient and safe cell-free immunomodulator.

Description

Technical Field

[0001] The present invention relates to the technical field of engineered exosomes, and particularly to a preparation method and application of dendritic cell-derived exosomes. Background Art

[0002] Systemic lupus erythematosus (SLE) is a classic autoimmune disease characterized by a decrease or loss of immune tolerance, resulting in the production of numerous autoantibodies against the body's own tissues and organs, leading to widespread chronic inflammation and tissue and organ damage, which can affect multiple organs, including the skin, joints, and kidneys. The pathogenesis of SLE is complex. Uncontrolled chronic inflammation is a core driver of SLE disease progression and tissue damage, leading to multi-organ dysfunction. Furthermore, a persistent inflammatory microenvironment is accompanied by severe oxidative stress, which is not only a consequence of inflammation but can also exacerbate inflammation and cell damage, creating a vicious cycle. For example, infiltration and activation of inflammatory cells release large amounts of reactive oxygen species (ROS) and reactive nitrogen species, depleting endogenous antioxidant systems. Oxidative stress products, in turn, activate inflammatory signaling pathways such as NF-κB, promoting the release of inflammatory factors. This causal interaction between persistent inflammation and oxidative stress is a major obstacle to SLE recurrence, exacerbated organ damage, and treatment difficulties. Therefore, developing therapeutic strategies that effectively inhibit inflammation and reduce oxidative stress is crucial for controlling SLE disease progression.

[0003] Dendritic cells (DCs) are the most powerful professional antigen-presenting cells in the body, playing a central role in bridging innate and adaptive immune responses. In autoimmune diseases such as systemic lupus erythematosus (SLE), DC dysfunction and overactivation are crucial components of disease progression. Abnormally activated or dysfunctional DCs can aberrantly activate autoreactive T and B cells, disrupting immune tolerance and promoting the production of autoantibodies and the inflammatory cascade, thereby inducing and exacerbating tissue damage. Therefore, modulating DC function and inducing their differentiation toward tolerance, i.e., the formation of tolerogenic dendritic cells (tolDCs), has emerged as a potential therapeutic strategy for autoimmune diseases such as SLE. TolDCs typically express low levels of costimulatory molecules and high levels of immunosuppressive molecules (such as HO-1, PD-L1, IL-10, and TGF-β), suppressing the activation of autoreactive T cells and inducing the production of regulatory T cells, thereby restoring immune tolerance. However, there are obvious limitations in directly using in vitro induced tolDCs for cell therapy. For example, in vitro induced tolDCs have poor stability in the complex in vivo microenvironment and are easily reversed or reactivated by stimulation of inflammation or danger signals, converting into immune-activating mDCs. This not only fails to achieve the purpose of immunosuppression, but may also aggravate inflammatory responses and immune attacks. There are also challenges in the preparation, storage, transportation and in vivo distribution of cell therapy itself.

[0004] Exosomes are membrane-bound nanovesicles secreted by various cells. They are approximately 30-200 nm in diameter and have a lipid bilayer structure. They are richly encapsulated with bioactive molecules such as proteins, lipids, and nucleic acids. They serve as important vehicles for intercellular communication and exert diverse biological functions in physiological and pathological processes. Exosomes possess excellent biocompatibility, low immunogenicity, and relative stability in body fluids, making them a promising tool for cell-free therapeutics or drug delivery vehicles. Dendritic cell-derived exosomes (Dex) are also secreted by DCs. Dex (mDex) secreted by mature DCs (mDCs) highly express MHC molecules and co-stimulatory molecules on their surface, allowing them to present antigens and effectively activate T cells, showing potential as adjuvants for tumor vaccines. Dex (im-Dex) secreted by immature DCs (imDCs) inherit the tolerance-inducing properties of imDCs and are thought to suppress T cell activation by carrying tolerance-associated molecules or miRNAs. Compared with cell therapy, exosomes have many advantages: such as better stability, easy storage (can be frozen), low immunogenicity, strong ability to penetrate biological barriers, and can be administered through simple injection, which avoids the safety and operational issues brought about by cell transplantation.

[0005] However, natural exosomes still have limitations as therapeutic tools. First, natural exosomes lack targeting and are difficult to deliver accurately to the lesion site, resulting in low therapeutic efficiency and potential off-target effects. For example, in SLE, if exosomes cannot be effectively enriched in the affected organs or lymph nodes, their therapeutic effect will be greatly reduced. Secondly, the composition and function of natural exosomes depend on the physiological state of their parent cells. It is difficult to customize the design according to the needs of complex diseases (such as SLE with simultaneous inflammation, oxidative stress and immune disorders), and it is difficult to achieve multifunctional synergistic treatment. For example, natural imDC-Dex may have a certain tolerance induction ability, but its anti-inflammatory and antioxidant functions may not be sufficient to cope with the severe pathological microenvironment of SLE. In order to overcome these shortcomings, engineered or recombinant exosomes came into being. By modifying the parent cells or directly modifying the exosomes, specific functions can be given to exosomes. For example, exosomes can be modified with targeting ligands to enhance their accumulation at lesions; specific therapeutic drugs (such as anti-inflammatory small molecules, siRNAs, and proteins) can be loaded into exosomes to enhance their therapeutic efficacy; and the membrane protein composition of exosomes can be modified to regulate their interactions with target cells. For example, some studies have enhanced their ability to target inflammatory sites by modifying the surface of mesenchymal stem cell-derived exosomes with specific peptides; others have enhanced their anti-inflammatory effects by loading exosomes with anti-inflammatory drugs. These examples demonstrate the enormous potential of engineered exosomes to improve therapeutic efficiency and functionality.

[0006] Despite the rapid development of engineered exosome technology, there is currently a lack of engineered exosomes that simultaneously integrate the triple functions of immune tolerance, potent anti-inflammatory, and antioxidant properties, and that are derived from nanoscale vesicles with well-defined immunomodulatory functions. Single-functional therapies often struggle to address the chronic inflammation, oxidative stress, and immune imbalance that are at the core of SLE pathology. Therefore, the development and preparation of novel exosomes that can simultaneously address these challenges is urgently needed. Summary of the Invention

[0007] In response to the shortcomings of the existing technology, the purpose of the present invention is to provide a method for preparing and applying dendritic cell-derived exosomes. The present invention utilizes DCs with natural immune regulatory capabilities as the parent cells of the exosomes and endows them with additional anti-inflammatory and antioxidant functions through engineering methods. This is a new approach to overcome existing treatment bottlenecks and achieve multi-dimensional intervention for SLE.

[0008] Therefore, the preparation of a new type of engineered exosomes derived from modified dendritic cells that has immune tolerance induction, anti-inflammatory and antioxidant functions is expected to become a new generation of efficient and safe cell-free immune modulators targeting autoimmune diseases such as SLE.

[0009] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing dendritic cell-derived exosomes, comprising the following steps:

[0010] (1) Inducing bone marrow-derived dendritic cells (DCs) to differentiate into tolerogenic dendritic cells (tolDCs) in vitro through interleukin-10 (IL-10) and ensuring that these tolDCs highly express the key antioxidant and anti-inflammatory protein: heme oxygenase-1 (HO-1);

[0011] (2) From the above-mentioned HO-1 highly expressed tolDCs (HO-1 high -tolDCs) were isolated and purified from the cell culture supernatant by ultracentrifugation: HO-1 high -tolDex;

[0012] (3) The prepared HO-1 high -tolDex conducts comprehensive physicochemical characterization and biological function identification, including its morphology, particle size, marker expression, HO-1 enrichment, and anti-inflammatory and antioxidant functions;

[0013] (4) Through in vitro cell experiments and in vivo experiments in systemic lupus erythematosus (SLE) animal models, it was demonstrated that HO-1 high -tolDex can effectively inhibit the overactivation and inflammatory response of target cells, reduce immunopathological damage, renal inflammation and oxidative stress damage in model animals, and induce immune tolerance.

[0014] As a preferred method, HO-1-highly expressed tolerogenic dendritic cells (HO-1 high Preparation method of dendritic cells (-tolDCs): 6-8 week old C57BL / 6 mice were taken and killed by cervical dislocation. The femur and tibia were separated under sterile conditions. The bone marrow cavity was repeatedly flushed with RPMI-1640 complete medium using a syringe to collect the bone marrow cell suspension. The red blood cell lysis buffer (ACK lysis buffer) was added to lyse the red blood cells for 5 minutes, and the cells were washed with phosphate buffered saline (PBS) and resuspended. The cells were cultured at 1×10 6 The cells were seeded at a density of 100 cells / mL in RPMI-1640 complete medium containing 20 ng / mL recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and 10 ng / mL recombinant mouse interleukin-4 (rmIL-4) and cultured in a 37°C, 5% CO2 incubator; on the 3rd and 5th days, half of the medium was replaced and an equal amount of cytokines was supplemented; on the 6th-7th day, the suspended and semi-suspended cells were collected as immature dendritic cells (imDCs).

[0015] The obtained imDCs were adjusted to a cell density of 1×10 6 / mL, and grouped for processing:

[0016] ①Control (Ctrl) group (imDCs): immature DCs without any treatment;

[0017] ② Lipopolysaccharide (LPS) group (mDCs): imDCs were stimulated and cultured with 100 ng / mL LPS for 24 hours to induce them to transform into mature dendritic cells (mDCs);

[0018] ③IL-10 group (HO-1 high -tolDCs): imDCs were treated with 40 ng / mL recombinant mouse IL-10 (rmIL-10) for 48 hours to induce their differentiation into HO-1 high -tolDCs; ④IL-10+LPS group: imDCs were first treated with 40 ng / mL rmIL-10 for 48 hours, and then 100 ng / mL LPS was added for co-culture for 24 hours to detect the stability of IL-10-induced tolDCs under LPS stimulation.

[0019] Preferably, dendritic cell-derived exosomes: HO-1 high Preparation of tolDex, inducing HO-1 high -tolDex:

[0020] Collect cell culture supernatant, culture in serum-free medium or exosome-depleted serum medium for at least 24 hours, and isolate exosomes by differential ultracentrifugation:

[0021] ① Centrifuge the collected supernatant at 2000g for 30 minutes at 4°C to remove cells and large cell debris, and collect the supernatant;

[0022] ② Centrifuge the supernatant from step ① at 10,000 g for 20-30 minutes at 4°C to remove larger vesicles and apoptotic bodies, and collect the supernatant;

[0023] ③ Filter the supernatant from step ② through a 0.22 μm filter to further remove residual larger particles;

[0024] ④ Ultracentrifuge the supernatant from step ② or ③ at 100,000 g for 70-90 minutes at 4°C, discard the supernatant, and collect the precipitate to extract crude exosomes;

[0025] ⑤ Resuspend the pellet with pre-chilled PBS and wash again by ultracentrifugation at 100,000 g for 70-90 minutes at 4°C;

[0026] ⑥ Discard the supernatant and precipitate the purified exosomes. Resuspend the exosomes with 100-200 μL sterile PBS and determine the exosome protein concentration using a BCA protein concentration assay kit. Aliquot and store at -80°C for later use.

[0027] As a preferred, HO-1 high -tolDex identification:

[0028] (1) Transmission electron microscopy (TEM) morphology observation;

[0029] (2) Nanoparticle tracking analysis (NTA) to detect particle size distribution and concentration;

[0030] (3) Western blot was used to detect the content of exosome marker proteins and HO-1.

[0031] The present invention also provides the use of exosomes prepared by the method for preparing dendritic cell-derived exosomes as described in the above technical solution in autoimmune diseases.

[0032] The beneficial effects of the present invention are: compared with the existing technology, the present invention uses DCs with natural immune regulation capabilities as the parent cells of exosomes, and endows them with additional anti-inflammatory and antioxidant functions through engineering means. It is a new idea to overcome the existing treatment bottleneck and achieve multi-dimensional intervention in SLE disease.

[0033] HO-1 of the present invention high The tolDex technology solution demonstrates significant synergistic effects in immune tolerance induction, anti-inflammatory, and antioxidant capabilities, overcoming the shortcomings of traditional therapies and natural exosomes. It provides a new, efficient, and safe cell-free solution for drug design and development of autoimmune diseases such as SLE, with important scientific significance and huge potential for clinical translation.

[0034] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of IL-10-induced high expression of HMOX1 and HO-1 proteins in tolerance DCs;

[0036] Figure 2 HO-1 high -Schematic diagram of the mean fluorescence intensity of the co-stimulatory molecule CD40 on the surface of tolDCs;

[0037] Figure 3 HO-1 high -Schematic diagram of changes in inflammatory factors and oxidative stress levels produced by tolDCs;

[0038] Figure 4 HO-1 high -Schematic diagram of the chemotactic ability of tolDCs;

[0039] Figure 5 HO-1 high -tolDCs-derived exosomes (HO-1 high -tolDex) preparation process and identification diagram;

[0040] Figure 6 In vitro experiments have shown that HO-1 high Schematic diagram of how tolDex effectively inhibits the overactivation of DCs;

[0041] Figure 7 HO-1 high -Schematic diagram of tolDex inhibiting the overactivation of DCs in the spleen and lymph nodes of SLE model mice;

[0042] Figure 8 HO-1 high -Schematic diagram of tolDex inhibiting the levels of inflammatory factors in the kidneys of SLE model mice;

[0043] Figure 9 HO-1 high -Schematic diagram of tolDex upregulating the content of HMOX1 / HO-1 in the kidneys of SLE model mice. DETAILED DESCRIPTION

[0044] See also Figures 1 to 9 The present invention discloses a method for preparing dendritic cell-derived exosomes, comprising the following steps:

[0045] (1) Inducing bone marrow-derived dendritic cells (DCs) to differentiate into tolerogenic DCs (tolDCs) in vitro through interleukin-10 (IL-10) and ensuring that these tolDCs highly express the key antioxidant and anti-inflammatory protein: heme oxygenase-1 (HO-1);

[0046] (2) From the above-mentioned HO-1 highly expressed tolDCs (HO-1 high -tolDCs) were isolated and purified from the cell culture supernatant by ultracentrifugation, namely HO-1 high -tolDex;

[0047] Preferably, in addition to the above-mentioned ultracentrifugation method for separating and purifying exosomes, other methods can also be used.

[0048] (3) The prepared HO-1 high-tolDex conducts comprehensive physicochemical characterization and biological function identification, including its morphology, particle size, marker expression, HO-1 enrichment, and anti-inflammatory and antioxidant functions;

[0049] (4) Through in vitro cell experiments and in vivo experiments in systemic lupus erythematosus (SLE) animal models, it was demonstrated that HO-1 high -tolDex can effectively inhibit the overactivation and inflammatory response of target cells, reduce immunopathological damage, renal inflammation and oxidative stress damage in model animals, and induce immune tolerance.

[0050] As a preferred method, HO-1-highly expressed tolerogenic dendritic cells (HO-1 high Preparation method of dendritic cells (-tolDCs): 6-8 week old C57BL / 6 mice were taken and killed by cervical dislocation. The femur and tibia were separated under sterile conditions. The bone marrow cavity was repeatedly flushed with RPMI-1640 complete medium using a syringe to collect the bone marrow cell suspension. The red blood cell lysis buffer (ACK lysis buffer) was added to lyse the red blood cells for 5 minutes, and the cells were washed with phosphate buffered saline (PBS) and resuspended. The cells were cultured at 1×10 6 The cells were seeded at a density of 100 cells / mL in RPMI-1640 complete medium containing 20 ng / mL recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and 10 ng / mL recombinant mouse interleukin-4 (rmIL-4) and cultured in a 37°C, 5% CO2 incubator; on the 3rd and 5th days, half of the medium was replaced and an equal amount of cytokines was supplemented; on the 6th-7th day, the suspended and semi-suspended cells were collected as immature dendritic cells (imDCs).

[0051] The obtained imDCs were adjusted to a cell density of 1×10 6 / mL, and grouped for processing:

[0052] ①Control (Ctrl) group (imDCs): immature DCs without any treatment;

[0053] ② Lipopolysaccharide (LPS) group (mDCs): imDCs were stimulated and cultured with 100 ng / mL LPS for 24 hours to induce them to transform into mature dendritic cells (mDCs);

[0054] ③IL-10 group (HO-1 high -tolDCs): imDCs were treated with 40 ng / mL recombinant mouse IL-10 (rmIL-10) for 48 hours to induce their differentiation into HO-1 high-tolDCs; ④IL-10+LPS group: imDCs were first treated with 40 ng / mL rmIL-10 for 48 hours, and then 100 ng / mL LPS was added for co-culture for 24 hours to detect the stability of IL-10-induced tolDCs under LPS stimulation.

[0055] Prepared HO-1 high -tolDex not only inherits the immune tolerance-inducing potential of its parent tolDCs, but also possesses powerful anti-inflammatory and antioxidant capabilities. As a "cell-free" therapeutic tool, HO-1 high Compared to cell therapy, tolDex offers advantages such as greater safety, stability, and ease of standardization. Compared to natural exosomes, the engineered exosomes of the present invention significantly enhance their therapeutic efficacy by ensuring efficient loading of HO-1. These carefully designed and modified DC-derived engineered exosomes are expected to become a new generation of multifunctional immunomodulators, synergistically exerting multiple therapeutic effects, including immune tolerance reestablishment, inflammatory environment improvement, and oxidative stress clearance. They offer a safer, more efficient, and clinically transformative treatment option for autoimmune diseases such as SLE, and have important theoretical significance and practical application value for promoting the development and targeted delivery of new SLE drugs.

[0056] Preferred: Dendritic cell-derived exosomes: HO-1 high Preparation of tolDex, inducing HO-1 high -tolDex:

[0057] Collect cell culture supernatant, culture in serum-free medium or exosome-depleted serum medium for at least 24 hours, and isolate exosomes by differential ultracentrifugation:

[0058] ① Centrifuge the collected supernatant at 2000g for 30 minutes at 4°C to remove cells and large cell debris, and collect the supernatant;

[0059] ② Centrifuge the supernatant from step ① at 10,000 g for 20-30 minutes at 4°C to remove larger vesicles and apoptotic bodies, and collect the supernatant;

[0060] ③ Filter the supernatant from step ② through a 0.22 μm filter to further remove residual larger particles;

[0061] ④ Ultracentrifuge the supernatant from step ② or ③ at 100,000 g for 70-90 minutes at 4°C, discard the supernatant, and collect the precipitate to extract crude exosomes;

[0062] 5. Resuspend the pellet in pre-chilled PBS and wash again by ultracentrifugation at 100,000 g for 70-90 minutes at 4°C.

[0063] 6. Discard the supernatant and precipitate the purified exosomes. Resuspend the exosomes with 100-200 μL sterile PBS and determine the exosome protein concentration using a BCA protein concentration assay kit. Aliquot and store at -80°C for later use.

[0064] The present invention describes the technical solution in more detail as follows:

[0065] 1. Tolerogenic dendritic cells (HO-1 high Induction and culture of β-tolDCs

[0066] 1.1 Induction and culture of mouse bone marrow-derived dendritic cells (BM-DCs)

[0067] 6-8 week old C57BL / 6 mice were killed by cervical dislocation, and the femur and tibia were isolated under sterile conditions. RPMI-1640 complete medium (containing 10% fetal bovine serum (FBS, preferably exosome-depleted FBS) and 1% penicillin / streptomycin) was drawn with a syringe to repeatedly flush the bone marrow cavity and collect the bone marrow cell suspension. Red blood cell lysis buffer (ACK lysis buffer) was added to lyse the red blood cells for 5 minutes, and the cells were washed with PBS and resuspended. The cells were plated at 1×10 6 The cells were seeded at a density of 100 cells / mL in RPMI-1640 complete medium containing 20 ng / mL recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and 10 ng / mL recombinant mouse interleukin-4 (rmIL-4) and cultured in a 37°C, 5% CO2 incubator. On the 3rd and 5th days, half of the medium was replaced and an equal amount of cytokines were supplemented. On the 6th and 7th days, suspended and semi-suspended cells were collected, which were immature dendritic cells (imDCs). Animal Ethics Statement: The animal experiments were reviewed and approved by the Animal Ethics Committee of this unit, Animal Ethics Number: xmsq2022-0792, and the execution process strictly complied with the ethical requirements of experimental animal welfare.

[0068] 1.2 Induction of HO-1 high -tolDCs

[0069] The obtained imDCs were adjusted to a cell density of 1×10 6 The cells were divided into groups and treated as follows: ① control (Ctrl) group (imDCs): immature DCs without any treatment; ② lipopolysaccharide (LPS) group (mDCs): imDCs were stimulated with 100 ng / mL LPS for 24 hours to induce them to transform into mature dendritic cells (mDCs); ③ IL-10 group (HO-1 high-tolDCs): imDCs were treated with 40 ng / mL recombinant mouse IL-10 (rmIL-10) for 48 hours to induce their differentiation into HO-1 high -tolDCs; ④IL-10+LPS group: imDCs were first treated with 40 ng / mL rmIL-10 for 48 hours, and then 100 ng / mL LPS was added for co-culture for 24 hours to detect the stability of IL-10-induced tolDCs under LPS stimulation.

[0070] 1.3HO-1 high Phenotypic and functional characterization of tolDCs

[0071] (1) HO-1 high -tolDCs highly express HMOX1 and HO-1 proteins

[0072] 1) RT-qPCR detection of HMOX1 mRNA expression: DCs from each group were collected and total RNA was extracted using TRIzol reagent. 1 μg of total RNA was taken and reverse transcription kit, such as PrimeScript TM RT Master Mix was used to reverse transcribe it into cDNA. Using cDNA as template, SYBR Green qPCR Master Mix and specific primers were used for amplification on a real-time fluorescence quantitative PCR instrument. Reaction conditions: 95℃ pre-denaturation for 30 seconds, then 95℃ denaturation for 5 seconds, 60℃ annealing and extension for 30 seconds, for a total of 40 cycles. -ΔΔCt The relative expression level of HMOX1 mRNA was calculated by the primer sequence as follows:

[0073] HMOX1: forward 5'-CCTCTGACGAAGTGACGCC-3',

[0074] reverse 5′-CAGCCCCACCAAGTTCAAA-3′;

[0075] β-actin: forward 5'-ACCTTCTACAATGAGCTGCG-3',

[0076] Reverse 5′-CTGGATGGCTACGTACATGG-3′.

[0077] Experimental results showed that IL-10 could induce the increase of HMOX1 transcription level in DCs, such as Figure 1 As shown in A.

[0078] 2) Western blot analysis of HO-1 protein expression: Cells from each group were collected and lysed on ice for 30 minutes in RIPA lysis buffer containing protease and phosphatase inhibitors. The cells were centrifuged at 12,000 rpm at 4°C for 15 minutes, and the supernatant was collected. Protein concentration was determined and quantified using a BCA protein concentration assay kit. Equal amounts of protein (e.g., 20-30 μg) were subjected to sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gel electrophoresis and then transferred to a PVDF membrane. Blocked with 5% skim milk or BSA for 1 hour at room temperature. Primary antibodies (e.g., rabbit anti-mouse HO-1 antibody, 1:1000 dilution; mouse anti-mouse β-actin antibody, 1:5000 dilution) were added and incubated overnight at 4°C. After washing the membrane with Tris-buffered saline containing Tween 20 (TBST), the corresponding horseradish peroxidase-conjugated secondary antibody (1:5000 dilution) was added and incubated for 1 hour at room temperature. After thorough washing with TBST, the membrane was developed using an ECL chemiluminescence kit and images were acquired on a chemiluminescence imaging system. ImageJ software was used to perform semi-quantitative analysis of the grayscale values ​​of the bands. The experimental results showed that compared with the control group, IL-10 alone or in combination with LPS could induce an increase in the protein level of HO-1 in DCs. Figure 1 As shown in B.

[0079] (2) Detection of HO-1 high -tolDCs surface co-stimulatory molecule CD40 expression

[0080] Collect DCs from each group, wash the cells with PBS containing 1% BSA (FACS buffer), and resuspend the cells. Add Fc receptor blockers (such as anti-CD16 / 32 antibodies) and incubate for 10 minutes. Then add a fluorescently labeled antibody combination: CD11c-PE, MHC-II-APC, and CD40-FITC, and incubate on ice for 30 minutes in the dark. Wash the cells 2-3 times with FACS buffer, resuspend them, and detect them on a flow cytometer. Gating strategy: First, circle the living cells based on the forward scatter angle (FSC) and side scatter angle (SSC), and then circle CD11c among the living cells. + MHC-II + DCs were further analyzed for CD11c + MHC-II + The expression level of CD40 in the cell population (mean fluorescence intensity MFI or percentage of positive cells). Experiments have shown that the expression of CD40 in IL-10-induced tolDCs is significantly lower than that in LPS-induced mature DCs. Figure 2 shown.

[0081] (3) HO-1 high -tolDCs produce less inflammatory cytokines

[0082] Cells from each group were collected, and RNA was extracted and reverse transcribed according to the RT-qPCR method in 1.3(1). Specific primers were used to detect the mRNA expression levels of inflammatory cytokines (such as IL-6, TNF-α) and chemokines (such as MCP-1). The primer sequences are as follows:

[0083] IL-6: forward 5'-CTGCAAGAGACTTCCATCCAG-3',

[0084] reverse 5′-AGTGGTATAGACAGGTCTGTTGG-3′;

[0085] TNF-α: forward 5'-CCTGTAGCCCACGTCGTAG-3',

[0086] reverse 5′-GGGAGTAGACAAGGTACAACCC-3′;

[0087] MCP-1: forward 5'-TAAAAACCTGGATCGGAACCAAA-3',

[0088] reverse 5′-GCATTAGCTTCAGATTTACGGGT-3′;

[0089] β-actin: forward 5'-ACCTTCTACAATGAGCTGCG-3',

[0090] Reverse 5′-CTGGATGGCTACGTACATGG-3′.

[0091] Experiments have shown that IL-10 pre-treated DCs can effectively resist the production of inflammatory factors induced by LPS, such as Figure 3 As shown in AC.

[0092] (4) HO-1 high -tolDCs’ antioxidant function

[0093] Collect cells from each group and follow the instructions of the specific detection kit.

[0094] 1) Malondialdehyde (MDA) level detection: Use an MDA detection kit. Briefly, collect cells and add lysis buffer to prepare cell homogenate. Take an appropriate amount of homogenate and react with MDA detection reagent, measure the absorbance at a specific wavelength (such as 532nm), and calculate the MDA content based on the standard curve, expressed as nmol / mg protein. The results showed that IL-10 pretreatment can effectively resist the increase in MDA levels caused by LPS stimulation, such as Figure 3 As shown in D.

[0095] 2) Glutathione (GSH) level detection: Collect cells, add protein remover and centrifuge to obtain supernatant. Use GSH detection kit, react supernatant with detection reagent, measure absorbance at a specific wavelength (such as 412nm), calculate GSH content according to standard curve, and express in μmol / g protein or μmol / 10 6 The results showed that IL-10 pretreatment could effectively resist the decrease of GSH level caused by LPS stimulation. Figure 3 As shown in E.

[0096] (5)HO-1 high Chemotactic function of -tolDCs

[0097] 1) Detection of chemokine receptor CCR7 expression: Collect cells from each group, prepare single cell suspension, label with flow cytometry antibodies: CD11c-PE, MHC-II-APC and CCR7-FITC (or other fluorescently labeled CCR7 antibodies), and detect changes in the expression level of CCR7 on the surface of DCs by flow cytometry. The method is the same as 1.3 (2). The results of flow cytometry showed that the expression level of chemokine receptor CCR7 in IL-10-induced tolDCs was significantly lower than that in mDCs, such as Figure 4 As shown in A.

[0098] 2) Transwell migration assay: Transwell chambers (pore size 8 μm) were used to detect the migration ability of DCs. DCs treated with different methods were collected and resuspended in chemotaxis medium (RPMI-1640 containing 1% BSA and 25 mM HEPES) to adjust the cell density to 1×10 6 / mL. 100 μL of cell suspension was added to the upper chamber of Transwell, and 600 μL of chemotactic medium containing chemokine CCL19 (200 ng / mL, ligand of CCR7) was added to the lower chamber. The 24-well plate was placed in a 37°C, 5% CO2 incubator and cultured for 3-4 hours. After the culture was completed, the upper chamber was removed and the non-migrated cells on the surface of the upper chamber membrane were gently wiped off with a cotton swab. After washing the lower surface of the membrane with PBS, it was fixed with 4% paraformaldehyde for 10 minutes, and then stained with 0.1% crystal violet for 10-15 minutes. After thorough washing with PBS, it was dried. 5-10 high-power fields were randomly selected under an inverted microscope to take pictures and count the number of cells that migrated to the lower surface of the membrane. No chemokine was added to the lower chamber of the blank control group. The chemotactic ability was evaluated by comparing the number of migrated cells in each group. Chemotaxis experiments have shown that the chemotactic ability of tolDCs induced by IL-10 is also significantly lower than that of mDCs. Figure 4 As shown in B.

[0099] 2. Preparation and Identification of a Novel Multifunctional Dendritic Cell-Derived Exosome: HO-1 high -tolDex

[0100] 2.1HO-1 high Isolation of tolDCs-derived exosomes

[0101] Collect cell culture supernatant and culture in serum-free medium or exosome-depleted FBS for at least 24 hours. Isolate exosomes using differential ultracentrifugation: ① Centrifuge the collected supernatant at 2000g for 30 minutes at 4°C to remove cells and large cell debris, and collect the supernatant. ② Centrifuge the supernatant from step 1 at 10,000g for 20-30 minutes at 4°C to remove large vesicles and apoptotic bodies, and collect the supernatant. ③ (Optional) Filter the supernatant from step 2 through a 0.22μm filter to further remove residual large particles. ④ Ultracentrifuge the supernatant from steps 2 or 3 at 100,000g for 70-90 minutes at 4°C, discard the supernatant, and collect the precipitate (crude exosomes). ⑤ Resuspend the precipitate in pre-chilled PBS and ultracentrifuge again at 100,000g for 70-90 minutes at 4°C to wash. ⑥ Discard the supernatant and the precipitate is the purified exosomes. Resuspend the exosomes with an appropriate amount (e.g., 100-200 μL) of sterile PBS. Determine the exosome protein concentration using a BCA protein concentration assay kit, aliquot, and store at -80°C for later use. high -tolDex separation and preparation process as shown in Figure 5 As shown in A.

[0102] 2.2HO-1 high -Identification of tolDex

[0103] (1) Transmission electron microscopy (TEM) morphology observation:

[0104] Take about 10 μL of exosome suspension and drop it onto a copper mesh covered with a carbon support film. Let it stand for 1-2 minutes to allow the exosomes to adsorb. Use filter paper to absorb excess liquid. Use 2% phosphotungstic acid solution (pH = 7.0) or 1% uranyl acetate solution for negative staining for 1-2 minutes. Use filter paper to absorb excess dye again and let the copper mesh dry naturally at room temperature. Use transmission electron microscopy at an accelerating voltage of 80-120 kV to observe the morphological characteristics (typical "cup-and-saucer" double-layer membrane structure) and size of exosomes, such as Figure 5 As shown in B.

[0105] (2) Nanoparticle Tracking Analysis (NTA) to detect particle size distribution and concentration:

[0106] The nanoparticle tracking analyzer was used for detection. The instrument illuminates the sample with a laser and tracks the Brownian motion of individual particles, thereby calculating the hydrodynamic diameter and concentration of the particles. The particle size distribution curve and average particle size of the exosomes were recorded and analyzed. The results showed that the new exosomes prepared by us meet the particle size of standard exosomes, such as Figure 5 As shown in C.

[0107] (3) Western blot detection of exosome marker proteins and HO-1 content:

[0108] Take an appropriate amount of exosome sample (e.g. 10-20 μg protein) and perform Western blot according to the method in 1.3(1). Detect exosome positive markers such as CD81 and TSG101 and negative markers such as Calnexin. At the same time, detect whether HO-1 protein is enriched in exosomes. Parent cell lysate is used as a cell control. Western blot results show that the HO-1 protein we prepared is high -tolDex highly expresses exosome positive markers CD81 and TSG101, and does not contain negative marker Calnexin. More importantly, the new exosomes we prepared are significantly enriched in HO-1 protein, such as Figure 5 As shown in D.

[0109] 3. In vitro experiments showed that HO-1 high -tolDex effectively inhibits DCs overactivation and inflammatory response

[0110] 3.1HO-1 high -tolDex inhibits the expression of costimulatory molecule CD40 on the surface of DCs

[0111] Cultured imDCs (according to method 1.1) or LPS-induced mDCs (according to method 1.2) were used as recipient cells. Experimental groups: ①imDC / mDC: control group; ②imDC+tolDex: imDCs and HO-1 prepared above high -tolDex co-culture; ③mDC: DCs matured after stimulation with 100 ng / mL LPS; ④mDC+tolDex: mDCs co-cultured with the HO-1 prepared above high -tolDex co-culture. After 24 hours of co-culture, recipient DCs were collected and the expression levels of CD11c, MHC-II and co-stimulatory molecule CD40 on the surface of DCs were detected by flow cytometry according to the method described in 1.3(2). The results of flow cytometry showed that the expression level of surface molecule CD40 of DCs co-cultured with tolDex was significantly reduced, as shown in Figure 2. Figure 6 As shown in A.

[0112] 3.2HO-1 high -tolDex inhibits the production of inflammatory factors by mature DCs

[0113] The same experimental grouping and treatment as in 3.1 were used. After co-culture, the recipient cells were collected and the total RNA in the cells was extracted according to the method described in 1.3(3). The mRNA expression of inflammatory cytokines and chemokines was detected by reverse transcription and RT-qPCR. The results of RT-qPCR experiments showed that the levels of inflammatory factors such as IL-6, TNF-α and MCP-1 produced by DCs co-cultured with tolDex were significantly reduced. Figure 6 As shown in BD.

[0114] 4. In vivo experiments showed that HO-1 high -tolDex alleviates DCs hyperactivation, inflammation, and oxidative stress damage in lupus mice

[0115] The SLE mouse lupus model was induced by pristane. The mice were randomly divided into the following groups: ①WT group: healthy control group (C57BL / 6 mice); ②SLE model group: lupus model mice induced by pristane; ③tolDCs group: lupus mice were treated with tolDCs cells; ④tolDex group: lupus mice were treated with HO-1 high -tolDex injection treatment. Animal Ethics Statement: The animal experiments were reviewed and approved by the Animal Ethics Committee of our institution, Animal Ethics Number: xmsq2022-0792, and the execution process strictly adhered to the ethical requirements of experimental animal welfare.

[0116] 4.1HO-1 high -tolDex inhibits the overactivation of DCs in the spleen and lymph nodes of SLE model mice

[0117] The spleen and lymph node tissues of each group of mice were collected, ground on ice, and passed through a 70 μm cell sieve to prepare a single cell suspension. The spleen cell suspension was lysed with ACK buffer to remove red blood cells. The cells were washed with FACS buffer and resuspended. Fc receptor blocking and fluorescent antibody (CD11c-PE, MHC-II-APC, CD40-FITC) staining were performed as described in 1.3(2). CD11c in the spleen and lymph nodes was analyzed by flow cytometry. + MHC-II + The expression level of costimulatory molecule CD40 on the surface of DCs subsets. Flow cytometry analysis showed that the expression level of CD40 on the surface of DCs in the spleen and lymph nodes of mice in the tolDex treatment group was significantly downregulated. Figure 7 shown.

[0118] 4.2HO-1 high -tolDex inhibits the levels of inflammatory factors in the kidneys of SLE model mice

[0119] A piece of kidney tissue was taken from each group of mice, quickly frozen in liquid nitrogen, and stored at -80°C. According to the RT-qPCR method described in 1.3(3), total RNA was extracted from the kidney tissue, reverse transcribed into cDNA, and the changes in the mRNA expression levels of inflammatory factors such as IL-6, TNF-α and MCP-1 in the kidney tissue were detected. The results were normalized with internal reference genes (such as β-actin). The experimental results showed that both tolDCs and tolDex treatments could effectively inhibit the levels of inflammatory factors, but the effect of tolDex was more significant, as shown in Figure 2. Figure 8 As shown in A.

[0120] 4.3HO-1 high -tolDex inhibits renal oxidative stress in SLE model mice

[0121] Take another piece of kidney tissue from each group of mice and weigh it accurately. According to the method described in 1.3(4), use commercially available MDA and GSH detection kits to prepare kidney tissue homogenate and detect the MDA content and GSH level in the kidney tissue. The results are usually expressed as unit weight of tissue or unit protein amount. After tolDex treatment, the MDA content in the kidneys of lupus mice was effectively downregulated, and the GSH content was effectively increased, such as Figure 8 As shown in B and C.

[0122] 4.4HO-1 high -tolDex upregulates the content of HMOX1 / HO-1 in the kidneys of SLE model mice

[0123] 1) RT-qPCR detection of HMOX1 mRNA: Part of the kidney tissue was taken (same as 4.2), and the RT-qPCR method described in 1.3(1) was used to detect the changes in the expression level of HMOX1 mRNA in the kidney tissue. Compared with lupus mice, the expression of HMOX1 in the kidney of mice treated with tolDCs and tolDex was significantly increased, and the effect of tolDex on upregulating HMOX1 was more significant. Figure 9 As shown in A.

[0124] 2) Western blot detection of HO-1 protein: Part of the kidney tissue was taken (same as 4.2), and the total protein of the kidney tissue was extracted and quantified according to the Western blot method described in 1.3(1), and the expression level of HO-1 protein in the kidney tissue was detected. β-actin was used as an internal reference. Consistent with the above mRNA change trend, the expression of HO-1 in the kidney of mice treated with tolDCs and tolDex increased significantly, among which the effect of tolDex on upregulating HO-1 was more significant, as shown in Figure 2. Figure 9 As shown in B.

[0125] Through the above detailed experimental scheme, the present invention aims to systematically explain thehigh -tolDex's preparation process, identification standards, and its multiple therapeutic potentials, including immune tolerance, anti-inflammation, and antioxidant properties, demonstrated in in vitro and in vivo models, provide a solid experimental basis and technical support for the development of novel, efficient, and safe "cell-free" therapies for autoimmune diseases such as SLE.

[0126] In addition, the present invention also provides the use of exosomes prepared by the method for preparing dendritic cell-derived exosomes as described in the above technical solution in autoimmune diseases.

[0127] Compared to existing technologies, the technical solution of the present invention achieves a synergistic effect of multiple therapeutic effects by cleverly combining the immune regulatory potential of tolerant dendritic cells, the advantages of exosomes as cell-free carriers, and the efficient loading of the key anti-inflammatory and antioxidant molecule HO-1, thereby significantly overcoming the limitations of traditional therapies and natural exosomes in treating complex autoimmune diseases such as SLE. The specific effects are as follows:

[0128] (1) Collaboratively address the core pathological mechanism of SLE: the vicious cycle of inflammation, oxidative stress, and immune imbalance

[0129] Existing therapies often focus on immunosuppression or simple anti-inflammatory treatment, but fail to adequately intervene in the accompanying severe oxidative stress and its interaction with inflammation and immune activation. high -tolDex is a nano drug that combines immune tolerance induction, potent anti-inflammatory and antioxidant functions. high -tolDCs and their derived exosomes were verified by RT-qPCR and Western blot). Exosomes can directly or indirectly enhance the antioxidant defense capacity of target cells and lesion tissues (verified by MDA and GSH level determination). At the same time, HO-1 itself and its metabolites have significant anti-inflammatory effects (verified by the expression level determination of inflammatory factors such as IL-6, TNF-α, and MCP-1). In addition, inheriting the characteristics of IL-10-induced tolDCs, HO-1 high -tolDex can inhibit the overactivation of DCs (as demonstrated by reduced surface CD40 expression and weakened chemotactic ability) and the abnormal activation of autoreactive T cells (although this briefing document does not list detailed T cell function experiments, inducing tolerance is a core characteristic of tolDCs and is indirectly achieved by inhibiting antigen presentation), thereby helping to re-establish immune tolerance. This "three-in-one" functional model can simultaneously block the pathological process of SLE from multiple dimensions, breaking the vicious cycle of "inflammation-oxidative stress-immune activation" and achieving more comprehensive and effective disease control.

[0130] (2) Provide a safer, more stable and easier to scale up cell-free therapy strategy

[0131] Traditional tolDCs cell therapy faces problems such as poor in vivo stability of in vitro induced cells, easy reversal, complex preparation process, difficult storage and transportation, and safety risks associated with cell transplantation (such as immune rejection and uncertain proliferation). The present invention uses exosomes as carriers to effectively circumvent these shortcomings of cell therapy. Exosomes have good biocompatibility and low immunogenicity. They can exist stably in body fluids and are easy to separate and purify by standardized methods such as ultracentrifugation. They are convenient for quality control, storage and transportation (such as freezing), and can be administered by simple injection. As a cell-free product, HO-1 high -tolDex is safer, reduces the risk of potential cell transplant complications, and provides a more feasible solution for the clinical treatment of autoimmune diseases.

[0132] (3) Enhance the immunomodulatory efficacy and targeting potential of exosomes

[0133] The function of naturally secreted exosomes is limited by the state of the parent cells and may lack the ability to specifically aggregate to lesions. The present invention, by subjecting parent DCs to a specific IL-10 induction treatment, not only imparts tolerance characteristics but, more importantly, ensures the efficient enrichment of the key therapeutic molecule HO-1 in the prepared exosomes (verified by Western blot analysis of exosome markers and HO-1). This functional "engineering" significantly enhances the anti-inflammatory and antioxidant capacity of exosomes, making them more effective in addressing the severe pathological microenvironment of SLE, improving treatment efficiency and reducing off-target effects.

[0134] (4) Provide scientific basis for the development and targeted delivery of new drugs for autoimmune diseases

[0135] This study demonstrates a novel approach for preparing multifunctional nanomedicines by targeting the functional changes of parent cells and isolating functionally enriched exosomes. This approach is not only applicable to HO-1, but in principle, a similar strategy can also be used to package other molecules with therapeutic potential (such as immunosuppressive factors, antioxidant enzymes, and targeting ligands) into exosomes, allowing for customized designs targeting different diseases or different stages of the same disease. This provides an important theoretical foundation and technical support for the development of the next generation of efficient and safe targeted immunomodulators and nanodrug delivery systems.

[0136] In summary, the HO-1 of the present invention highThe tolDex technology solution demonstrates significant synergistic effects in immune tolerance induction, anti-inflammatory, and antioxidant capabilities, overcoming the shortcomings of traditional therapies and natural exosomes. It provides a new, efficient, and safe cell-free solution for drug design and development of autoimmune diseases such as SLE, with important scientific significance and huge potential for clinical translation.

[0137] Although existing technologies may overlap with the present invention in some aspects, there is currently no other technical solution that can simultaneously, effectively, and in the same carrier achieve the triple synergistic functions of "immune tolerance induction + potent anti-inflammatory + significant antioxidant" emphasized by the present invention, especially through the unique technical path of "directed induction of dendritic cells to highly express key molecules (such as HO-1) and use of exosomes derived from them as cell-free carriers" to achieve the purpose of the invention.

[0138] The following are some potentially relevant technical directions and their comparison with the present invention:

[0139] (1) Direct cell therapy using tolDCs

[0140] Similarities: Able to induce immune tolerance.

[0141] Differences and advantages of the present invention: As described in the technical background, tolDCs cell therapy has problems such as poor in vivo stability, susceptibility to reversal by inflammatory microenvironment, complex storage and transportation, potential immunogenicity and uncontrollable cell distribution. high -tolDex, as a cell-free preparation, overcomes these shortcomings and has better stability, safety and ease of use.

[0142] (2) Using natural, unmodified exosomes from imDCs (imDex)

[0143] Similarities: imDex may inherit some of the tolerance-inducing properties of imDCs.

[0144] Differences and advantages of the present invention: Natural imDC-Dex may have weak immunosuppressive ability, and its anti-inflammatory and antioxidant functions have not been optimized and enhanced. The content of key functional proteins such as HO-1 may not be sufficient to cope with the complex pathological microenvironment of SLE. The present invention induces tolDCs by IL-10 and ensures that they highly express HO-1, so that the produced HO-1 high -tolDex was significantly enhanced and optimized in terms of immune tolerance, anti-inflammation and antioxidant effects.

[0145] (3) Using exosomes from other sources and performing engineering modifications

[0146] Similarities: Exosomes derived from mesenchymal stem cells (MSCs): MSC-Exo, also have the potential for immune regulation and can be engineered.

[0147] Differences and advantages of the present invention: In terms of the specificity of parental cells, DCs are professional antigen-presenting cells that play a central role in the initiation and regulation of immune responses. The molecular profiles carried by exosomes produced by tolDCs may act more directly and specifically on immune cells, thereby more effectively inducing immune tolerance. However, MSC-Exo mainly acts through autocrine / paracrine mechanisms, and its mechanism and efficacy of inducing immune tolerance may be different from those of exosomes derived from professional antigen-presenting cells (such as tolDCs). Although MSC-Exo has anti-inflammatory and antioxidant effects, its natural HO-1 level or induction ability may not be as good as that of HO-1 that has been optimized. high The main advantage of MSC-Exo may be tissue repair and fibrosis inhibition, which is different from the core cycle of immune tolerance and inflammation / oxidative stress focused on in this invention.

[0148] (4) Direct use of HO-1 agonists or recombinant HO-1 protein for treatment

[0149] Similarities: Can exert the anti-inflammatory and antioxidant effects of HO-1.

[0150] Differences and advantages of the present invention: Simple HO-1 agonists or proteins mainly target inflammation and oxidative stress, and are unable to solve the core immune imbalance and autoantibody production problems of SLE. In terms of delivery and targeting, small molecule drugs or recombinant proteins may have problems such as low bioavailability, short half-life, and lack of targeting. The HO-1 of the present invention high -tolDex utilizes the natural carrier properties of exosomes, which may improve the delivery efficiency and enrichment of HO-1 at the lesion site. At the same time, tolDC confers it the advantage of immune tolerance.

[0151] Although there are other strategies to improve SLE, they often focus on a single or partial pathological link, or have deficiencies in safety, stability, multifunctional integration, etc. high The core innovation of the tolDex preparation method and its application lies in the ingenious integration of the immune tolerance properties of tolDCs, the advantages of exosomes as cell-free therapeutic carriers, and the anti-inflammatory and antioxidant functions enhanced by cell engineering.

[0152] The present invention demonstrates unique advantages and prospects in achieving multi-dimensional, efficient and safe intervention in the vicious cycle of "immune imbalance-inflammation-oxidative stress" in systemic lupus erythematosus (SLE), and is difficult to be completely replaced by existing single technologies or simple combinations to achieve the same comprehensive effect.

[0153] The specific description of the present invention in the above embodiments is only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Technical engineers in this field may make some non-essential improvements and adjustments to the present invention based on the contents of the above invention, which fall within the scope of protection of the present invention.

Claims

1. A method for preparing dendritic cell-derived exosomes, characterized by: The following steps are involved: (1) Inducing bone marrow-derived dendritic cells (DCs) to differentiate into tolerogenic dendritic cells (tolDCs) in vitro through interleukin-10 (IL-10) and ensuring that these tolDCs highly express the key antioxidant and anti-inflammatory protein: heme oxygenase-1 (HO-1); (2) From the above-mentioned HO-1 highly expressed tolDCs ( HO-1high-tolDCs ) were isolated and purified from the cell culture supernatant by ultracentrifugation: HO-1high-tolDex ; (3) The prepared HO-1high-tolDex Conduct comprehensive characterization of its physicochemical properties and biological functions, including its morphology, particle size, marker expression, HO-1 enrichment, and anti-inflammatory and antioxidant functions; (4) Through in vitro cell experiments and in vivo experiments in systemic lupus erythematosus (SLE) animal models, it was demonstrated that HO-1high-tolDex It can effectively inhibit the overactivation of target cells and inflammatory response, reduce immunopathological damage, renal inflammation and oxidative stress damage in model animals, and induce immune tolerance.

2. The method for preparing dendritic cell-derived exosomes according to claim 1, wherein: Tolerogenic dendritic cells that highly express HO-1 ( HO-1high-tolDCs ) preparation method, induction and culture of dendritic cells: 6-8 week old C57BL / 6 mice were taken and killed by cervical dislocation, and the femur and tibia were separated under sterile conditions; RPMI-1640 complete medium was drawn with a syringe to repeatedly flush the bone marrow cavity and collect the bone marrow cell suspension; red blood cell lysis buffer (ACK lysis buffer) was added to lyse the red blood cells for 5 minutes, and the cells were washed with phosphate buffered saline (PBS) and resuspended. 1×106 The cells were inoculated at a density of 100 cells / mL in RPMI-1640 complete medium containing 20 ng / mL recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and 10 ng / mL recombinant mouse interleukin-4 (rmIL-4) and placed at 37°C. 5% CO2 Culture in an incubator; on days 3 and 5, half of the medium was replaced and an equal amount of cytokines were supplemented; on days 6-7, suspension and semi-suspension cells were collected as immature dendritic cells (imDCs); The obtained imDCs were adjusted to a cell density of 1×106 / mL, and grouped for processing: ①Control (Ctrl) group (imDCs): immature DCs without any treatment; ②Lipopolysaccharide (LPS) group (mDCs): imDCs were stimulated and cultured with 100 ng / mL LPS for 24 hours to induce them to transform into mature dendritic cells (mDCs); ③IL-10 group ( HO-1high-tolDCs ): 40 ng / mL recombinant mouse IL-10 (rmIL-10) was added to imDCs and cultured for 48 hours to induce their differentiation into HO-1high-tolDCs ④IL-10+LPS group: imDCs were first treated with 40 ng / mL rmIL-10 for 48 hours, and then 100 ng / mL LPS was added for co-culture for 24 hours to detect the stability of IL-10-induced tolDCs under LPS stimulation.

3. The method for preparing dendritic cell-derived exosomes according to claim 2, wherein: Dendritic cell-derived exosomes: HO-1high-tolDex Preparation method, induction HO-1high-tolDex : Collect cell culture supernatant, culture in serum-free medium or exosome-depleted serum medium for at least 24 hours, and isolate exosomes by differential ultracentrifugation: ① Centrifuge the collected supernatant at 2000 g for 30 minutes at 4°C to remove cells and large cell debris, and collect the supernatant; ② Centrifuge the supernatant from step ① at 10,000 g for 20-30 minutes at 4°C to remove larger vesicles and apoptotic bodies, and collect the supernatant; ③ Filter the supernatant from step ② through a 0.22 μm filter to further remove residual larger particles; ④ Ultracentrifuge the supernatant from step ② or ③ at 100,000 g for 70-90 minutes at 4°C, discard the supernatant, and collect the precipitate to extract crude exosomes; ⑤ Resuspend the pellet in pre-chilled PBS and wash again by ultracentrifugation at 100,000 g for 70-90 minutes at 4°C. ⑥ Discard the supernatant and precipitate the purified exosomes. Resuspend the exosomes in 100-200 μL sterile PBS and determine the exosome protein concentration using a BCA protein concentration assay kit. Aliquot and store in a -80°C refrigerator until use.

4. The method for preparing dendritic cell-derived exosomes according to claim 3, wherein: HO-1high-tolDex Identification of: (1) Transmission electron microscopy (TEM) morphology observation; (2) Nanoparticle tracking analysis (NTA) to detect particle size distribution and concentration; (3) Western blot was used to detect the content of exosome marker proteins and HO-1.

5. Use of exosomes prepared by the method for preparing dendritic cell-derived exosomes according to any one of claims 1 to 4 in treating autoimmune diseases.

6. A method for treating autoimmune diseases using exosomes derived from dendritic cells, characterized in that: By leveraging the immunomodulatory potential of tolerant dendritic cells, the advantages of exosomes as cell-free carriers, and the efficient loading of the key anti-inflammatory and antioxidant molecule HO-1, we can achieve a synergistic effect of multiple therapeutic effects and collaboratively address the core pathological mechanism of SLE: the vicious cycle of inflammation, oxidative stress, and immune imbalance.

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