CN109498652B

CN109498652B - Application of inducible regulatory T cell-derived exosome

Info

Publication number: CN109498652B
Application number: CN201811389855.2A
Authority: CN
Inventors: 郑颂国; 陈敬荣; 王菊华
Original assignee: Third Affiliated Hospital Sun Yat Sen University
Current assignee: Third Affiliated Hospital Sun Yat Sen University
Priority date: 2018-11-21
Filing date: 2018-11-21
Publication date: 2022-03-08
Anticipated expiration: 2038-11-21
Also published as: CN109498652A

Abstract

The invention relates to application of an inducible regulatory T cell-derived exosome (iTreg exo), in particular to the iTreg exo and application thereof in preparation of a medicament for treating rheumatoid arthritis, and belongs to the technical field of biological medicines. The iTreg exo can carry miR-449a-5p to inhibit proliferation and differentiation of Th1 and Th17 cells, reverse the imbalance of Treg (anti-inflammatory cells)/Th 17 (inflammatory cells) ratio in a mouse with rheumatoid arthritis, inhibit inflammatory reaction and relieve clinical symptoms, can be used as a new means for treating the rheumatoid arthritis, and has important clinical significance for further understanding the occurrence and development of exosomes in arthritis diseases and developing a new method for treating the arthritis.

Description

Application of inducible regulatory T cell-derived exosome

Technical Field

The invention belongs to the technical field of biological medicines, and relates to application of an inducible regulatory T cell-derived exosome (iTreg exo) in preparation of a medicine for treating rheumatoid arthritis.

Background

Treg cells (regulatory T cells) are a cell population for regulating and controlling the immune function of an organism, and can maintain the tolerance of an immune system to self components so as to keep the organism in immune steady state. The cells express Foxp3, CD25 and CD4 as cell phenotype characteristics. Over the last 20 years, studies have demonstrated that Treg cells have the effect of suppressing the pathophysiological immune response of various pathways in infections, tumors, organ transplants, allogeneic fetal immune related diseases. Treg cells can be divided into naturally occurring naturally regulatory T cells (nTregs) and induced adaptive regulatory T cells (astregs or iTregs). The applicant found for the first time that TGF- β can induce Foxp3 expression in peripheral naive T cells, differentiating them into induced Treg cells (iTreg). Phenotypically, iTreg and nTreg express similar molecules, such as CD25, CTLA-4, GITR, CCR4, CD62L and Foxp3, among others. nTreg and iTreg exhibit similar immunosuppressive functions.

Exosomes are discoid membrane vesicles (about 50-100nm in diameter) containing complex RNA and proteins, mainly derived from multivesicular bodies formed by invagination of intracellular lysosomal microparticles, and released into the extracellular matrix after fusion with the cell membrane via the multivesicular body outer membrane. Many of the exosomes released by immune cells and non-immune cells have recently been shown to have a range of immune system physiological properties, including antigen presentation, immunomodulation and programmed cell death. Many cells produce extracellular vesicles such as CD4+, CD8+ T cells, B cells, and Dendritic Cells (DCs). These cell-derived exosomes have been shown to mediate immune stimulation and immune modulation. Exosomes play an important role in intercellular communication, and proteins, mirnas, mrnas and cytokines can be transmitted and transferred between cells by endocytosis uptake or interaction of exosome membrane proteins with cell membrane proteins. Recent studies have shown that mouse-derived CD4+ CD25+ Foxp3+ T cells (Tregs) release exosomes and reduce proliferation of CD4+ T cells and cytokine expression, with immunosuppressive functions. T cell exosomes can transfer miRNAs leading to degradation of their target genes or to inhibit the function of regulatory receptor Antigen Presenting Cells (APCs). Recent studies have shown that Treg exosomes inhibit expression of effector T cells by one miRNA (Let-7 d).

Rheumatoid Arthritis (RA) is an autoimmune systemic, heterogeneous disease with symmetrical multiple facet joint involvement as the major clinical manifestation. Chronic progressive destruction of the synovial membrane, cartilage, tendons, etc. of the affected joints occurs. The prevalence rate of RA in China is 0.34%, the number of sick people exceeds 500 thousands, and the disability rate of RA patients who are not treated regularly in 3 years is as high as 75%. RA is common in young and middle-aged years and is the leading cause of labor loss. And patients almost receive medicine treatment for life, lack of cure means, and are expensive, which brings serious social and economic burden to the country and patients. Therefore, improving the diagnosis and treatment level of RA and reducing the disability rate of RA are important health and economic problems to be solved urgently in China and are also important ways for improving the life and life quality of patients, protecting social labor force and promoting social and economic development. The highest goal for treatment of RA and the like is to repair a specific immune deficiency or to modify the overall immune response to achieve a destructive effect that prevents an autoimmune response without destroying normal immune defenses.

At present, the clinical treatment method for RA is support and symptomatic treatment, and the adopted treatment method mainly comprises the following steps: the use of immunosuppressants, anti-inflammatory therapies, symptomatic treatments and the like cannot cure RA, and even serious side effects such as secondary infection, secondary tumor and the like can be brought to threaten the life of a patient. Although the biological agents which have been developed in recent years are widely used in clinical applications, such as tumor necrosis factor alpha inhibitors for cytokine targeting therapy, anti-CD 20 antibodies for antagonizing B cells, and biological agents for antagonizing IL-6 and IL-6 receptors for cytokines, have only a certain effect and efficacy, and there is an urgent need to develop novel drugs and techniques with definite efficacy and few toxic and side effects. The exosome is used as a drug carrier, is a complex and special protein and phospholipid bilayer structure different from a common liposome, and has unique advantages mainly represented by: (1) exosomes are produced by the human body, and have low immunogenicity; (2) exosomes are not easy to decompose and clear in a human body, and have long half-life in the human body; (3) good targeting and high efficiency of transporting goods to cells; (4) exosomes are small in diameter and can selectively penetrate to inflammatory or tumor sites using an enhanced osmotic retention effect. Therefore, it is possible to develop an inducible regulatory T cell-derived exosome (iTreg exo) for use in the preparation of a medicament for treating rheumatoid arthritis.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides application of an inducible regulatory T cell-derived exosome (iTreg exo) in preparing a medicament for treating rheumatoid arthritis.

In order to achieve the purpose, the invention adopts the technical scheme that: the invention provides application of iTreg exo in preparing a medicament for treating rheumatoid arthritis.

As a preferred embodiment of the application of the invention, the iTreg exo is the iTreg exo carrying miR-449a-5 p.

As a preferred embodiment of the application of the invention, the iTreg exo is an exosome encapsulating miR-449a-5p mimics or an exosome released by mmu-miR-449a-5p overexpression cells.

In the present invention, the iTreg exo is used to inhibit proliferation and differentiation of Th1, Th17 cells, reverse the imbalance of Treg/Th17 ratio, and inhibit inflammatory responses.

As a preferred embodiment of the application of the invention, the medicine has RNAi effect of miR-449a-5 p.

As a preferred embodiment of the application of the invention, the medicament for treating rheumatoid arthritis is a targeted medicament.

The invention also provides a medicament for treating rheumatoid arthritis, which comprises iTreg exo.

As a preferred embodiment of the medicament of the present invention, the medicament further comprises a pharmaceutically acceptable carrier.

Compared with the prior art, the invention has the beneficial effects that: the iTreg exo can carry miR-449a-5p to inhibit proliferation and differentiation of Th1 and Th17 cells, reverse the imbalance of Treg (anti-inflammatory cells)/Th 17 (inflammatory cells) ratio in a mouse with rheumatoid arthritis, inhibit inflammatory reaction and relieve clinical symptoms, can be used as a new means for treating the rheumatoid arthritis, and has important clinical significance for further understanding the occurrence and development of exosomes in arthritis diseases and developing a new method for treating the arthritis.

Drawings

Fig. 1 is a detection diagram of iTreg and an identification diagram of iTreg exo in example 1. Wherein A is Mouse

Flow assay images of Treg differentiation in vitro with or without TGF- β and IL-2 in CD 4; b is a transmission electron microscope image of the exosome; c is nanosight detection of the particle size distribution of the exosome; d is a strip image of exosome membrane surface markers Alix, CD63 and CD81Western blot; and E is a flow detection image of the exosome membrane surface molecules CD63 and CD 81.

FIG. 2 is a graph showing the cell proliferation assay of iTreg exo co-cultured with CD8+, CD4+ T cells in vitro in example 2. Wherein A is a flow detection image of mouse CD8+ and CD4+ T cell proliferation inhibited by exosome; b is a statistical chart representing 3 independent replicates of the a experiment.

FIG. 3 is a graph of the in vitro differentiation of Th1 and Th17 cells by iTreg exo in example 2. Wherein A is a flow detection image of exosome inhibiting in-vitro differentiation of Th1 and Th17 cells; b is a statistical chart representing 3 independent replicates of the a experiment.

Fig. 4 is a pathological examination of iTreg exo treated CIA mice in example 3. Wherein A is a change chart of the morphological sizes of spleen and lymph nodes of a CIA mouse after treatment; b is a statistic chart of arthritis pathological scores of CIA mice at different time points after treatment; c is a staining image of a section HE of a knee joint part obtained by sacrifice after treatment of the CIA mouse; d is an image of the leg paw of the treated CIA mouse obtained by killing the treated CIA mouse and performing micro-CT scanning.

FIG. 5 is a graph of the Th17/Treg ratio following iTreg exo treatment of CIA mice in example 3. Wherein A is a flow detection image of the ratio of Th17/Treg (CD 4+ cells in a circle) of a CIA mouse after treatment; b is a statistical plot of Th17/Treg representing 3 independent replicates of the A experiment.

Fig. 6 is a diagram of the iTreg exo miRNA sequencing and bioinformatic analysis in example 4. Wherein A is an exosome miRNA sequencing heatmap derived from Treg cells induced by in vitro addition or without adding TGF-beta; b is a miRNA KEGG enrichment analysis bar graph of iTreg exo relative to control exosome differential expression; c is a verification diagram of iTreg exo high expression miRNA-449a-5p qPCR.

FIG. 7 is a graph of miR-449a-5p-inhibitor-iTreg exo in example 5. Wherein A is

Flow detection images of CD4 in vitro differentiation experiments of TGF-beta and IL-2 and miR-449a-5p-inhibitor transfected Tregs; b is miR-449a-5p-inhibitor transfected in the presence of TGF-beta and IL-2

Fluorescence microscopy images after CD 42 d; c is a transmission electron microscope image of miR-449a-5p-inhibitor-iTreg exo.

FIG. 8 is a cell proliferation assay of miR-449a-5p-inhibitor-iTreg exo co-cultured in vitro with CD8+, CD4+ T cells in example 5. Wherein A is a flow detection image of mouse CD8+ and CD4+ T cell proliferation inhibited by exosome; b is a statistical chart representing 3 independent replicates of the a experiment.

FIG. 9 is a graph of miR-449a-5p-inhibitor-iTreg exo in example 5 on the in vitro differentiation of Th1 and Th17 cells. Wherein A is a flow result image of miR-449a-5p-inhibitor-iTreg exo in vitro differentiation experiments of Th1 and Th17 cells; b is a statistical chart representing 3 independent replicates of the a experiment.

FIG. 10 is a pathological examination map of miR-449a-5p-inhibitor-iTreg exo in example 6 after treatment of CIA mice. Wherein A is a change chart of the morphological sizes of spleen and lymph nodes of a CIA mouse after treatment; b is a statistic chart of arthritis pathological scores of CIA mice at different time points after treatment; c is a staining image of a section HE of a knee joint part obtained by sacrifice after treatment of the CIA mouse; d is an image of the leg paw of the treated CIA mouse obtained by killing the treated CIA mouse and performing micro-CT scanning.

FIG. 11 is a graph of the Th17/Treg ratio of miR-449a-5p-inhibitor-iTreg exo treated CIA mice in example 6. Wherein A is a flow detection image of the ratio of Th17/Treg (CD 4+ cells in a circle) of a CIA mouse after treatment; b is a statistical plot of Th17/Treg representing 3 independent replicates of the A experiment.

FIG. 12 is a graph showing the results of a dual-luciferase report experiment of miR-449a-5p and target gene notch1 in example 7. Wherein A is sequence alignment of potential notch 13' UTR target sites of miR-449a-5p and a mutation schematic diagram of the potential sites; b is a luciferase reporter gene result graph of 293T cells transfected by potential notch 13' UTR target site wild type and mutant plasmids of miR-449a-5p and miR-449a-5 p; c is a graph showing the results of the change of expression levels of noch 1 and NICD mRNA after 293T cells are transfected with miR-449a-5 p; d is a graph showing the result of the protein level of NICD after 293T cells are transfected with miR-449a-5 p; e is a mouse

Results plot of CD4 for changes in notch1 and NICD mRNA expression levels under Th1, Th17 polarization conditions, iTreg exo co-culture and miR-449a-5p transfection conditions, respectively; f is a mouse

Results of the NICD protein levels of CD4 under Th1, Th17 polarization conditions, iTreg exo co-culture and miR-449a-5p transfection conditions, respectively.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.

The in-vitro and in-vivo experiment is carried out on a CIA mouse by preparing an exosome (iTreg exo) from an inducible regulatory T cell, so that the cell balance of Treg/Th17 of an arthritic mouse is changed, the inflammatory response is relieved, and the effect of treating the rheumatoid arthritis of the mouse is achieved; the iTreg exo can regulate the cell balance of Treg/Th17, and the expression of genes Th1 and Th17 is regulated by iTreg-derived miR-449a-5p, so that the purpose of treating rheumatoid arthritis is achieved.

Mouse-derived exosomes released from CD4+ CD25+ Foxp3+ T cells (Tregs) can reduce the proliferation of CD4+ T cells and the expression of cytokines, and have an immunosuppressive function. Exosomes can transfer miRNAs leading to degradation of their target genes or inhibition of function of regulatory receptor Antigen Presenting Cells (APCs). According to the invention, through CIA mouse development in-vivo and in-vitro tests, the relationship between iTreg exo and T cell subgroups, particularly Th1 and Th17 cells is analyzed, and whether the iTreg exo-derived miR-449a-5p regulates Th17/Treg balance influences the pathogenesis and disease mechanism of arthritis is researched. Through the research, the role of the Treg in the generation and development of the arthritis disease can be further understood to have important significance, and the method has important clinical significance for the research of the etiology prevention and treatment new targets of the arthritis.

In the following examples, the technical means used are conventional means well known to those skilled in the art, and the reagents and materials of the present application are commercially available or otherwise publicly available, unless otherwise specified. In the present invention, Graph Pad software and SPSS software were used for all mapping and data analysis; statistical analysis was analyzed using the T-test, and P values between groups less than 0.05 were considered to be significantly different; all experiments in the present invention were repeated at least 3 times.

Example 1 preparation of iTregs and isolation and characterization of iTreg exo

1. Preparation of inducible Tregs

Firstly, magnetic bead sorting is carried out to obtain mice

CD4 cells, then cultured for 3 days in the presence of TGF-beta and IL-2, as shown in FIG. 1A, the induction of iTreg was confirmed by flow cytometry examining the proportion of CD4+ CD25+ Foxp3+ cells, and simultaneously, an iTreg induction was established

CD4 control without addition of TGF- β.

Preparation and characterization of iTreg exo

In the preparation process of the induced Treg, the establishment of the method

CD4 in the absence of TGF-beta under the control, using no exosome serum medium to culture cells, 3 days later harvesting cell supernatant, adding a certain proportion of Exo-quick incubation overnight to extract exosome. The exosome is identified by adopting a transmission electron microscope method, as shown in figure 1B,circular vesicles with a diameter of around 100nm with a bilayer membrane structure were detected. Western blot was also used to detect exosome surface markers, such as Alix, CD63, CD81 as shown in FIG. 1D. In addition, exosome surface markers CD63 and CD81 were also detected using flow cytometry, as shown in fig. 1E. Thus, the prepared exosomes were identified by the above method.

Example 2 detection of iTreg exo to inhibit proliferation and differentiation of T cells

Carboxyfluorescein diacetate succinimidyl ester (CFSE) is a fluorescent dye that can permeate cell membranes directly into cells, and once entering cells, its acetate group is removed by intracellular esterase, and CFSE with fluorescence is produced and is not easily released outside cells. Meanwhile, the succinimidyl ester of CFSE can be covalently bound to the primary amine group, and thus exist in the cell for a long time. As the cells divide, CFSE gradually distributes into daughter cells, halving each time, and thus, cell division can be easily detected using flow cytometry for several generations. We used CFSE labeling of mouse T cells to evaluate the effect of iTreg exo on T cell proliferation.

Figure 2 shows that iTreg exo significantly inhibited the proliferation of CD8+ and CD4+ cells, while exosomes secreted by control cells treated without TGF- β (i.e., Med exo) had no statistically different effect on the proliferation of CD8+ and CD4+ cells. Inflammatory cells play a key role in autoimmune diseases, particularly the central role of Th17 cells in rheumatoid arthritis, so we used in vitro Th1 and Th17 differentiation experiments to study the effect of iTreg exo on Th1 and Th17 differentiation, as shown in FIG. 3, iTreg exo can significantly inhibit the differentiation of Th1 and Th17 cells, while exosomes (i.e. Med exo) secreted by control cells without TGF-beta treatment have no significant inhibitory effect on the differentiation of Th1 and Th17 cells.

Example 3 verification of the therapeutic Effect of iTreg exo on rheumatoid arthritis

CIA is achieved by immunizing a gene-susceptible mouse after mixing type II collagen with freund's complete adjuvant (CFA). The basic principle is that some amino acid fragments in the middle of type II collagen exogenously injected into animals can be used as MHC type II molecules (such as IAq of mice) to be combined and core antigen peptide fragments recognized by a T Cell Receptor (TCR) to induce T cell activation and proliferation, and C II is a main protein in cartilage and is a target tissue of RA, so that autoimmune diseases of experimental animals can be induced. Taking a joint of a mouse, performing HE staining, or carrying out micro CT on the whole joint to measure the bone destruction degree, and observing the time difference of arthritis onset; severity of the disease; degree of osteoarticular destruction, etc. The results are shown in fig. 4, where iTreg exo-treated arthritic mice had delayed onset, decreased joint destruction, and reduced splenic and lymph node enlargement compared to normal arthritic mice, while Med exo control group had no therapeutic effect on arthritic mice. Furthermore, in the mouse pathogenesis peak period (35-40 days after C II injection), cells or regulatory T cells and the like in draining lymph nodes and/or spleen are extracted, lymph nodes and spleen of the two kinds of arthritis pathogenesis peak period mice are extracted, single cells are separated, and the number of proinflammatory cells and inflammation suppressor cells in vivo, phenotype difference and the like are detected by flow staining. The results are shown in fig. 5, the iTreg exo group Th17 cells are reduced compared with the control CIA mouse group, the tregs are increased, and have a significant difference compared with the model group, while the Med exo control group has no statistical difference compared with the model group, which indicates that the iTreg exo can reduce the proinflammatory cells and increase the inflammatory suppressor cells in the arthritis mouse.

Example 4 iTreg exo-carried miRNA sequencing detection and bioinformatic analysis

Exosomes contain many components and we explored iTreg exo for mirnas with differentially high expression relative to Med exo with RNA-seq. As shown in FIG. 6A, miR-5112, miR-7213-5p, miR-181d-3p, miR-99b-3p, miR-449a-5p, miR-92b-5p, miR-676-3p, miR-24-1-5p, miR-1954, miR-449a-3p, miR-8117, miR-7001-3p, miR-6900-5p, miR-196A-1-3p, miR-let-7c-1-3p is highly expressed relative to control cells. Next, for the miRNAs, we selected four tools of TargetScan, miRDB, miRTarBase, miRWalk to predict the target gene, and performed KEGG analysis on the target gene, as shown in fig. 6B. In the notch signal path and miR-449a-5p involved in the method, the verification of whether the iTreg exo sample is high-expressing miR-449a-5p is carried out by qPCR (quantitative polymerase chain reaction), and as shown in FIG. 6C, the iTreg is remarkably high-expressing miR-449a-5p relative to Med exo.

Example 5 detection of iTreg exo inhibition of T cell proliferation and differentiation by miR-449a-5p

The exosome is an important way for the exosome to play a role in regulating the proliferation and differentiation of cells by carrying miRNA to be taken up by target cells to influence the expression of target cell genes. To confirm that the iTreg exo inhibits the proliferation and differentiation of T cells by carrying miR-449a-5p, firstly, we harvested the supernatant after 2 days of miR-449a-5p-inhibitor transfection in the iTreg preparation process, extracted the exosomes and detected the exosome morphological size by electron microscopy, as shown in FIG. 7. Whether miR-449a-5p-inhibitor-iTreg exo can also inhibit the proliferation and differentiation of T cells in vitro is discussed later. FIG. 8 and FIG. 9 show that miR-449a-5p-inhibitor-iTreg exo loses its effect of inhibiting T cell proliferation and differentiation, indicating that iTreg exo inhibits T cell proliferation and differentiation by carrying miR-449a-5 p.

Example 6 validation of the therapeutic Effect of iTreg exo on rheumatoid arthritis by miR-449a-5p

To further confirm the immunoregulation effect of iTreg exo by carrying miR-449a-5p, we used a CIA mouse model for verification, as shown in FIG. 10, the iTreg exo group significantly delayed the onset degree and osteoarticular destruction of mice, whereas the iTreg-derived exosome (miR-449a-5p-inhibitor-iTreg exo) transfected by miR-449a-5p inhibitor treated group had a level of arthritis onset, joint destruction and splenic and lymph node enlargement close to those of the model group. Further, through the detection of the proportion of Th17 cells and Treg cells, as shown in FIG. 11, the effect of the iTreg exo on Th17/Treg is reversed after miR-449a-5p inhibitor transfection, namely compared with the iTreg exo group, the miR-449a-5p-inhibitor-iTreg exo group loses the regulation effect on Th17/Treg, which indicates that the iTreg exo inhibits the generation and development of CIA by carrying miR-449a-5 p.

Example 7 validation of iTreg exo targeting notch1 by carrying miR-449a-5p

To confirm that the iTreg exo exerts immunosuppressive functions by targeting notch1 via miR-449a-5p, we verified in vitro transfection of the notch1 dual luciferase reporter plasmid with 293T cells. First, weSequence alignment was performed to obtain the potential notch1 target site of miR-449a-5p, 2 sites as shown in FIG. 12A. The subsequent transfection results are shown in fig. 12B, the fluorescence intensity of the wild type + iTreg exo group reporter of notch1 is reduced, while the fluorescence intensity of the wild type + iTreg exo group reporter of notch1 is not reduced, so that it can be confirmed that the iTreg exo-derived miR-449a-5p has a targeting effect on the notch 1. To demonstrate the targeting effect of miR-449a-5p on notch1, we transfected 293T cells with miR-449a-5p and miR-449a-5p inhibitor, respectively, as shown in fig. 12C, 12D, miR-449a-5p significantly inhibited the expression of notch1 and NICD mRNA and protein levels. Further, adopt

CD4 was co-cultured with iTreg exo and transfected with miR-449a-5p under Th1 and Th17 polarization conditions, respectively, as shown in FIGS. 12E and 12F, miR-449a-5p-inhibitor-iTreg exo group had no inhibitory effect on notch1 and NICD relative to iTreg exo group, indicating that iTreg exo inhibits the expression of notch1 by carrying miR-449a-5 p.

Based on the results, the applicant finds that iTreg exo is transmitted to Th1 and Th17 cells through carrying miR-449a-5p to regulate expression of notch1 genes, and inflammatory response is inhibited. It follows from this that: the iTreg exo can change the balance of Treg/Th17 cells of a mouse with rheumatoid arthritis, inhibit inflammatory reaction and relieve clinical symptoms, and can be used as a new means for treating the rheumatoid arthritis.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. Application of iTreg exo carrying miR-449a-5p in preparation of medicines for treating rheumatoid arthritis.

2. The use according to claim 1, wherein the iTreg exo is an exosome encapsulating miR-449a-5p mimics or an exosome released by mmu-miR-449a-5p overexpressing cells.

3. The use according to claim 1 or 2, wherein the iTreg exo is used to inhibit proliferation and differentiation of Th1, Th17 cells, to reverse an imbalance in the Treg/Th17 ratio, and to inhibit inflammatory responses.

4. The use of claim 1 or 2, wherein the medicament has the effect of high expression of miR-449a-5 p.

5. The use of claim 1 or 2, wherein the medicament for the treatment of rheumatoid arthritis is a targeted medicament.

6. A medicament for treating rheumatoid arthritis, comprising iTreg exo carrying miR-449a-5 p.

7. The medicament of claim 6, wherein: the medicament also comprises a pharmaceutically acceptable carrier.